Review Article Production Methods for Hyaluronandownloads.hindawi.com/journals/ijcc/2013/624967.pdf · it hyaluronic acid [ ]. s s Hyaluronan from many di erent tissues of vertebrates

Hindawi Publishing CorporationInternational Journal of Carbohydrate ChemistryVolume 2013 Article ID 624967 14 pageshttpdxdoiorg1011552013624967

Review ArticleProduction Methods for Hyaluronan

Carmen G Boeriu12 Jan Springer1 Floor K Kooy13

Lambertus A M van den Broek1 and Gerrit Eggink1

1 Wageningen UR Food amp Biobased Research PO Box 17 6700 AAWageningen The Netherlands2 Universitatea ldquoAurel Vlaicurdquo Str E Dragoi nr 2 310330 Arad Romania3 Genencor International BV PO Box 218 2300 AE Leiden The Netherlands

Correspondence should be addressed to Carmen G Boeriu carmenboeriuwurnl

Received 29 November 2012 Accepted 21 January 2013

Academic Editor Thomas J Heinze

Copyright copy 2013 Carmen G Boeriu et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Hyaluronan is a polysaccharide with multiple functions in the human body being involved in creating flexible and protective layersin tissues and inmany signalling pathways during embryonic development wound healing inflammation and cancer Hyaluronanis an important component of active pharmaceutical ingredients for treatment of for example arthritis and osteoarthritis andits commercial value far exceeds that of other microbial extracellular polysaccharides Traditionally hyaluronan is extracted fromanimal waste which is a well-established process now However biotechnological synthesis of biopolymers provides a wealth of newpossibilities Therefore geneticmetabolic engineering has been applied in the area of tailor-made hyaluronan synthesis Anotherapproach is the controlled artificial (in vitro) synthesis of hyaluronan by enzymes Advantage of using microbial and enzymaticsynthesis for hyaluronan production is the simpler downstream processing and a reduced risk of viral contamination In this paperan overview of the different methods used to produce hyaluronan is presented Emphasis is on the advancements made in the fieldof the synthesis of bioengineered hyaluronan

1 Introduction

Hyaluronic acid also known as hyaluronan is a linear poly-saccharide composed of a repeating disaccharide unit of120573(14)-glucuronic acid (GlcUA)-120573(13)-N-acetylglucosamine(GlcNAc) (Figure 1) Both individual carbohydrate residuesin hyaluronan adopt the stable chair conformation whichdetermines the conformation of the polymer in solution thatis described as an overall random coil structure that mayhave also highly flexible regions Nevertheless in terms ofchemical structure hyaluronan is a simple linear polymerwith high molecular mass and exceptional rheological prop-erties Hyaluronan is a member of the glycosaminoglycansfamily that includes chondroitindermatan sulfate keratansulfate and heparinheparan sulfate each with a characteris-tic disaccharide-repeating structure of an amino sugar eitherglucosamine or galactosamine and a hexose either galactoseglucuronic acid or iduronic acid which can be carboxylatedor sulfated [1] Hyaluronan is the only glycosaminoglycan

member that is not sulfated and is not covalently bound toa proteoglycan core protein

Research on hyaluronan expands over more than onecentury (Table 1) The first report that can be linked tohyaluronan dates from 1880 when the French chemistPortes observed that the mucin in the vitreous body whichhe named ldquohyalomucinerdquo behaved differently from othermucoids in cornea and cartilage Fifty-four years later Meyerand Palmer described a procedure for isolating a novel glyco-saminoglycan from the vitreous of bovine eyes which theynamed hyaluronic acid based on hyaloid (vitreous) anduronic acid [3] Since in vivo the polymer exists in an ionizedform as polyanion it is generally referred to as hyaluronan

In living organisms hyaluronan is produced by hyaluro-nan synthase enzymes which synthesize large linear poly-mers of the repeating disaccharide 120573(14)-GlcUA-120573(13)-GlcNAc by alternate addition of GlcUA and GlcNAc tothe growing chain using their activated nucleotide sugars(UDP-glucuronic acid and UDP-N-acetylglucosamine) as

2 International Journal of Carbohydrate Chemistry

Table 1 Important events in research on hyaluronan products

Time Event

1880Portes reported that mucin from the vitreousbody differs from other mucoids in cornea andcartilage and named it hyalomucine [2]

1934Meyer and Palmer isolated and identified thepolysaccharide from the vitreous body and namedit hyaluronic acid [3]

30sndash50s

Hyaluronan from many different tissues ofvertebrates was isolated identified andcharacterized A few pathogenic bacteria werefound that produce hyaluronan and use it toencapsulate their cells

50s

The chemical structure of hyaluronan waselucidated by Karl Meyer and his team They usedhyaluronidase to produce overlappingoligosaccharides that were structurally analyzedby conventional techniques [4]Interest emerged to use hyaluronan in eye surgeryas a substitute of the vitreous body

40sndash70s

Extraction processes from animal tissues wereoptimized to remove protein and to minimizehyaluronan degradation First studies onhyaluronan production through bacterialfermentation and chemical synthesis wereinitiated

1979

First patent on ultrapure hyaluronan isolated fromrooster combs [5] This was the starting of theindustrial manufacturing of hyaluronan fromanimal sources for human applications In 1980using the methods of Balasz Pharmacia (Sweden)introduced Healon a product used in cataractsurgery

90sndash00s

Revival of studies on bacterial fermentation toproduce hyaluronan of high molecular weightEmphasis on controlling polymer size andpolydispersity

1993

The gene encoding for a single enzyme thatpolymerizes UDP-GlcNAc and UDP-GlcUA intohyaluronan is isolated by DeAngelis andcoworkers from Streptococcus pyogenesHyaluronan synthases from othermicroorganisms were identified and characterized[6 7]

1996The largest hyaluronan fragment an octamer waschemically synthesized through controlledaddition of disaccharide units [8]

2003Research on the enzymatic synthesis ofhyaluronan and monodisperse hyaluronanoligosaccharides with defined length [9 10]

substrates The number of repeat disaccharides 119899 in a com-pleted hyaluronan molecule is approximately 10000 butchains of hyaluronan with up to 25000 disaccharide unitshave been reported The size of the polymers (119872

119908from

5000Da to 20 million Da) in vivo varies with the type oftissue For example hyaluronan in the human umbilical cordhas a molecular mass of 3-4 million Da whilst the molecular

OH

O

NH

C

119899

OH

OH

O

O

OHOO

O

O

HO

Glucuronic acid 119873-Acetylglucosamine

Figure 1 Repeating unit of hyaluronan

mass of hyaluronan from human synovial fluid is 6 mil-lion Da However the reported molecular weight of isolatedhyaluronan polymers may be underestimated depending onthe isolation and analysis method used Hyaluronan is notmonodisperse in molecular weight and the experimentallydetermined polydispersity (119872

119908119872119899) of the polymer depends

as well on both the method of extraction and the method ofanalysis used For example the values of the polydispersityof a hyaluronan sample determined by high-performanceliquid chromatography varied with the type of column thedetection method and the flow rate used [11ndash13] This resultsin a big variation of the values reported for the 119872

119908and

polydispersity of hyaluronan between laboratories and showsthe importance for standardization of the methodologyfor characterization and reporting of hyaluronan molecularproperties

Hyaluronan is present in all vertebrates High concen-trations are found in the umbilical cord the synovial fluidbetween joints skin and the vitreous body of the eye Itwas estimated that in the body of a person of 70 kg about15 g hyaluronan is found in different tissues of which onethird is turned over every day The human skin containsover 50 of the hyaluronan in the body [14 15] Roostercombs contain the highest concentrations of hyaluronan(75mg gminus1) ever reported for animal tissues [16] Hyaluronanis also present in the capsule of a small number of micro-bial pathogens such as Pasteurella multocida and group Aand C streptococci among which Streptococcus pyogenes (ahuman pathogen) and the animal pathogens Streptococcusequi and Streptococcus uberis that quite likely pirated theenzymatic machinery from vertebrate hosts for its synthesisThese microorganisms use hyaluronan to encapsulate theircells forming a perfect disguise against the animal defensesystem and facilitating the adhesion and colonization of thebacterial cells [17] Since the hyaluronan polymer producedin animals and the aforementioned bacteria is identicalthe host immune defense is not triggered to repel thepathogenic bacteria contrary to other bacteria with a differentextracellular capsule Intriguingly the nonimmunogenic andnoninflammatory properties of bacterial hyaluronan benefitmammals currently too since bacterial hyaluronan formsan excellent source for medical grade hyaluronan Initiallyhyaluronan was believed to be an inert compound havingno specific interaction with other macromolecules However

International Journal of Carbohydrate Chemistry 3

Table 2 Biomedical and pharmacologic applications of hyaluronan

Area Application Example

Biomedical

Orthopedic surgery ArthritisRheumatology OsteoarthritisOphthamology Eye surgery

Otolaryngology Treatment of thevocal fold

Dermatology and plasticsurgery Dermal filler

Wound healing anddressing

Diabetic ulcer skinburns

Tissue engineeringPharmacology Drug delivery

from the discovery of the interaction of hyaluronan withcartilage proteoglycans by Hardingham andMuir [18] a largenumber of reports have been published on the role of hyaluro-nan in cellular activity migration mitosis inflammationcancer angiogenesis fertilization and so on (see for moreinformation [19 20])

In addition hyaluronan and its derivatives have beenlargely studied and applied in the biomedical field (Table 2)[21ndash23] Due to the high level of biocompatibility hyaluronanis extensively used in viscosurgery in the treatment ofarthritis to supplement the lubrication of arthritic joints asmicrocapsule for targeted drug delivery and in cosmetics as ahydrating and antiaging material

The biological functions of hyaluronan are stronglydepending upon its size High molecular weight hyaluronanpolymers (119872

119908gt 5times105Da) are space filling antiangiogenic

and immunosuppressive medium size hyaluronan chains(119872119908

between 2 times 104ndash105Da) are involved in ovulationembryogenesis and wound repair oligosaccharides with15ndash50 repeating disaccharide units (119872

119908between 6 times 103ndash

2 times 104Da) are inflammatory immuno-stimulatory andangiogenic while small hyaluronan oligomers (119872

119908from 400

to 4000Da) are anti-apoptotic and inducers of heat shockproteins [24] Lower molecular weight hyaluronan and smalloligosaccharides are produced by controlled depolymeriza-tion of high molecular weight hyaluronan using physicaltreatment (thermal treatment pressure) irradiation (electronbeam gamma ray microwave) acid treatment ozonolysismetal catalyzed radical oxidation and enzymatic hydrolysiswith hyaluronidase (EC 32135)

The commercial value of hyaluronan far exceeds thatof other microbial extracellular polysaccharides With anestimatedworldmarket value of $US 500million it is sold forup to $US 100000 per kilogram [25] On top of this severalgroups have reported effects of hyaluronan oligosaccharideson cellular behavior that could imply the use of thesemolecules in cancer treatment or in wound healing [26] It isclear that hyaluronan is much more than a space-filling inertcomponent of the extracellular matrix and will become moreand more important as a pharmaceutical component andindeed is amultifunctionalmegadalton stealthmolecule [27]

In this paper the different methods to produce hyaluronanwill be discussed as depicted in Figure 2

2 Biosynthetic Pathways for Hyaluronan inLiving Organisms

The overall reaction of hyaluronan synthesis is as follows

119899UDP-GlcUA + 119899UDP-GlcNAc

997888rarr 2119899UDP + [GlcUA + GlcNAc]119899

(1)

The reaction is catalyzed by a single enzyme utilizing bothsugar substrates to synthesize hyaluronan [28] In contrast toother glycoaminoglycans which are synthesized in the Golginetwork hyaluronan is synthesized at the plasma membrane[29 30] Hyaluronan synthases (EC 241212) are predictedto have multiple membrane spanning domains with a largeintracellular loop on the plasma membranersquos inner faceThe only known exception is the P multocida hyaluronansynthase which has a membrane attachment domain near thecarboxyl terminus Hyaluronan molecules are extruded intothe extracellular matrix in coordination with their synthesis[31] but they also exist intracellularly In the extracellularmatrix hyaluronan exists in a number of different formsFor example in vertebrates it can be intercalated within aproteoglycan complex referred to glycocalyx if it is a moredelicate pericellular matrix or it can be bound to membranereceptors of the cell surface It can interact with bindingproteins so-called hyaladherins which is also the case forintracellular hyaluronan [15] The mechanisms regulatingextracellular versus intracellular location of hyaluronan arenot known yet Recently the presence of hyaluronan cableshas been reported that was the result of the incorporation ofthe heavy chain of interalpha-trypsin inhibitor with hyaluro-nan [32] Depending on the type of tissue different hyaluro-nan concentrations and molecular weight evoke differentcell responses for example during embryonic developmenthealing processes inflammation and cancer How the cellresponse is influenced by the different molecular weight ofhyaluronan is still an intriguing question [15 24]

In 1996 the first reports were published concerning thecloning of mammalian hyaluronan synthases [33 34] Sincethen in human cells three hyaluronan synthase genes havebeen found encoding enzymes having distinct enzymaticproperties with respect to hyaluronan size and size distribu-tion [35] which are differentially inducible by growth factorsand cytokines [36 37]

In 1993 the first gene encoding the enzyme responsiblefor hyaluronan synthesis denoted HA synthase or HASwas identified from group A streptococci [6 7] This geneis part of an operon containing the hasA gene encodinghyaluronan synthase the hasB gene encoding UDP-Glucosedehydrogenase and the hasC gene encoding UDP-Glucose-pyrophosphorylase [38ndash40] A hydropathy plot of the hasAgene predicts three transmembrane segments which is inaccordance with the early findings of Markovitz and cowork-ers who showed that the hyaluronan synthase activity islocated in the cell membrane of streptococci [41] Cloning of


Enzymes from

Streptococcus pyogenesPasteurella multocida

Production methods for

hyaluronan

Extraction from

animal tissuesBacterial

productionIn vitro production

Streptococci

Non pathogenicmicro organisms

Energy and carbon resources

Process conditionsBatch versus chemostatStrain improvement

Rooster combsHuman umbilical cords

Bovine synovial fluidVitreous humor of cattle

Enterococcus faecalisEscherichia coliAgrobacterium spLactococcus lactisBacillus subtilis

Figure 2 Production methods for hyaluronan

the group A and later the group C streptococcal hyaluronansynthase genes [42] allowed researchers to confirm that onlyone gene product (the HA-synthase protein) is required forhyaluronan biosynthesis

The first vertebrate gene identified as being HA synthasewas the Xenopus laevis gene DG42 [43] Since then morevertebrate HA synthase genes were identified [44] and inaddition an HA synthase from an algal virus was discovered[17 45]The streptococcal vertebrate and viral HA synthasesshow protein sequence similarity and appear to have a singleglycosyl transferase 2 (GT2) family module [46] with somesimilarities to chitin synthaseThe last extension of organismsin which a HA synthase gene was identified is Cryptococcusneoformans The CPS1 gene from this pathogenic yeastencodes a HA synthase with high homology to the previouslymentioned HA synthase proteins [47] An HA synthase thatdiffers in protein sequence and in predicted topology from allother HA synthases was identified and cloned from type A Pmultocida an animal pathogen [48]

Unlike the other HA synthases (Table 3 grouped as ClassI HA synthases) which are integral membrane proteins andelongate hyaluronan chains at the reducing end (Class I-R) orat the nonreducing end [50] thePmultocidaHAsynthase hasa membrane attachment domain near the carboxyl terminushas two GT2 modules and elongates the HA chains at thenonreducing end It is therefore called a Class II HA synthase[17] So far the P multocida HA synthase is the only knownmember of the Class II HA synthases [49 51 52]

The regulation of hyaluronan synthesis is much morecomplex in vertebrates than in bacteria because hyaluronanfulfills various functions in the mammalian body depending

Table 3 Hyaluronan synthase classification system as proposed byWeigel and DeAngelis [49]

Class IReducing Nonreducing

Class II

Members

Streptococcuspyogenes Sequisimilis Suberis humansand mice

Xenopus laevisalgal virus

Pasteurellamultocida

Hyaluronanchaingrowth

Reducing end Nonreducing end Nonreducingend

on the tissue involved and the hyaluronan chain lengthrequired For the three mammalian HA synthase isozymesdifferent expression patterns are observed between adult andembryonic tissues [54 55] Several regulatory factors areknown to be involved in controlling HA synthase transcrip-tion such as morphogenesis cytokines growth factors andantisensemRNA [56 57] Furthermore hyaluronan synthesisseems to be controlled through translation regulation sincea latent pool of HA synthase exits within the cell interior inthe endoplasmic reticulum-Golgi compartments that uponinsertion in the cell membrane becomes activated [53] HAsynthase activity can bemodulated by posttranslationalmod-ification such as phosphorylation and N-glycosylation [58]Intermediate and small oligosaccharides are probably pro-vided through hyaluronan cleavage by hyaluronidases sincemammalian HA synthase isozymes synthesize hyaluronan


Peptidoglycan

Hyaluronan

Glycolysis

Glucose

Cell wallpolysaccharides

Fructose-6-P

Glucosamine-6-P

Glucosamine-1-P

UDP-N-acetylglucosamine

Glucose-6-P

Glucose-1-P

UDP-glucoseGlucosamine-1-P

UDP-glucuronic acid

Hexokinase (Phosphoglucoisomerase)

(Phosphoglucomutase) (Amidotransferase)

(Mutase)

(Acetyltransferase)

(Pyrophosphorylase)

(UDP glucose dehydrogenase)

(UDP-glucose pyrophosphorylase)hasC

hasB

Pgm GlmS

GlmM

hasD

hasD

hasE

hasAhasA (Hyaluronan synthase)

119873-Acetylglucosamine-1-P

Figure 3 Schematic overview of the hyaluronan synthesis pathway in S zooepidemicus adapted from [53] 2013 The American Society forBiochemistry and Molecular Biology

higher than 105Da [14] Due to the high complexity of theregulation of hyaluronan synthesis in vertebrates a cohesiveview on the relation between the regulatory components isnot available yet

Recently the metabolic route of hyaluronan formationin S zooepidemicus was elucidated The streptococcal HAsynthase is found in operons also encoding one or moreenzymes involved in biosynthesis of the activated sugars [59]The has operon in S zooepidemicus encodes for five genes(Figure 3) hyaluronan synthase (hasA) UDP-glucose dehy-drogenase (hasB) UDP-glucose pyrophosphorylase (hasC)a glmU paralog encoding for a dual function enzyme acetyl-transferase and pyrophosphorylase activity (hasD) and a pgiparalog encoding for phosphoglucoisomerase (hasE) [60]HasB and hasC are involved in UDP-GlcUA synthesis whilehasD and hasE are involved in the synthesis of UDP-GlcNAcThe hyaluronan biosynthesis is a costly process for bacteriain terms of carbon and energy resources and competes withcell growth because of the large pathway overlap with thecell wall biosynthesis (Figure 3) Other streptococci strainshave has operons that contain besides hyaluronan synthaseonly the hasB and hasC genesThis suggests that the superiorhyaluronan synthesis observed for S zooepidemicus relates tothe availability of UDP-sugar precursors Understanding themetabolic routes for hyaluronan synthesis had a significantrole in the optimization of the microbial production ofhyaluronan allowing the control of the polymer chain lengthand increasing the product yield

3 Production of Hyaluronan

Industrialmanufacturing of hyaluronan is based on twomainprocesses the extraction from animal tissues and micro-bial fermentation using bacterial strains Both technologiesproduce polydisperse high molecular weight hyaluronan(119872119908ge 1 times 106Da polydispersity ranging from 12 to 23)

for biomedical and cosmetic applications [12 61 62] Thefirst process to be applied at industrial scale was theextraction of hyaluronan from animal waste which is stillan important technology for commercial products but ishampered by several technical limitations One drawbackin the extraction process is the inevitable degradation ofhyaluronan caused by (a) the endogenous hyaluronidaseactivity in animal tissues breaking down the polymer chainthrough enzymatic hydrolysis and (b) the harsh conditionsof extraction Extraction protocols have been improved overthe years but still suffer from low yields due to the intrinsiclow concentration of hyaluronan in the tissue and from highpolydispersity of polymer products due to both the naturalpolydispersity of hyaluronan and to the uncontrolled degra-dation during extractionAs in any process for the productionof therapeutic compounds from animal sources there is apotential risk of contamination with proteins and virusesbut this can be minimized by using tissues from healthyanimals and extensive purification Nevertheless concernson viral (particularly avian) and protein (particularly bovine)contamination increased the interest in the biotechnologicalproduction of hyaluronan

Production of hyaluronan by bacterial fermentationevolved to a mature technology in the last two decades of the20th century In the early developmental stages of bacterialfermentation using group A and C streptococci optimizationof culture media and cultivation conditions along with strainimprovement were used to increase hyaluronan yield andquality Hyaluronan yields reached 6-7 g Lminus1 which is theupper technical limit of the process caused by mass transferlimitation due to the high viscosity of the fermentationbroth Bacterial fermentation produces hyaluronan with highmolecular weight and purity but risk of contaminationwith bacterial endotoxins proteins nucleic acids and heavymetals exists The identification of the genes involved inthe biosynthesis of hyaluronan and of the sugar nucleotide


Table 4 A comparative view of technologies for manufacturing hyaluronan

Advantages Disadvantages

Extraction from animalmaterials

(i) Well-established technology (i) Variation in product quality(ii) Available raw material at low costs (ii) Risk of polymer degradation(iii) Product with very high119872

119908up to

20MDa(iii) Risk of contamination with protein nucleic acids andviruses

(iv) Natural product (iv) Extensive purification needed(v) Low yield

Bacterial production(i) Mature technology (i) Use of genetically modified organism (GMO)

(ii) High yield (ii) Risk of contamination with bacterial endotoxinsproteins nucleic acids and heavy metals

(iii) Product with high119872119908(1ndash4MDa)

Enzymatic synthesis

(i) Versatile technology (i) Emerging technology in development stage(ii) Control of the119872

119908of the products

Products up to 055ndash25MDa and alsodefined oligosaccharides can beobtained

(ii) Technological and economic viability must still bedemonstrated

(iii) No risks of contamination(iv) Constant product quality

precursors in the 90s opened the way towards hyaluronanproduction using safe nonpathogenic recombinant strains

In the past decade a new technology emerged usingisolated HA synthase to catalyze the polymerization of theUDP-sugar monomers This novel enzymatic technology forhyaluronan synthesis is very versatile allowing producingboth high molecular weight hyaluronan and hyaluronanoligosaccharides with defined chain length and low polydis-persity Production of monodisperse hyaluronan oligosac-charides on mg-scale using two single-action mutants of theP multocida HA synthase was demonstrated by the group ofDeAngelis [9] but large scale production was not achievedyet A comparative analysis of established and emerging tech-nologies for high molecular weight hyaluronan production isgiven in Table 4

31 Extraction from Animal Tissues Extraction of hyaluro-nan from animal tissues was initially used for laboratoryinvestigations in order to identify and characterize the poly-mer and to elucidate its biological potential and biomedicalapplication Hyaluronan from almost all tissues of vertebratesincluding the vitreous body of the eye umbilical cordsynovial fluid pig skin the pericardial fluid of the rabbitand the cartilage of sharks was isolated and described [63]Recently the extraction of hyaluronan from fish eyes as analternative resource outside animal husbandry was reported[64] Nevertheless the most accessible sources for large scaleproduction high molecular weight hyaluronan are roostercombs (12 times 106Da) the human umbilical cords (34 times106Da) the vitreous humor of cattle (77 times 104ndash17 times 106Da)and the bovine synovial fluid (14 times 106Da)

Despite the numerousmethods for isolation and purifica-tion of hyaluronan reported in the early literature it was onlyin 1979 that a method became available for the production ofpharmaceutical grade hyaluronan by extraction from animal

tissues Balazs [5] developed an efficient procedure to isolateand purify hyaluronic acid from rooster combs and humanumbilical cords that set the basis of the industrial productionof hyaluronan from rooster combs for medical application

Hyaluronan is soluble in water but extraction of highlypure high molecular weight hyaluronan from animal tissuesis difficult since in biological materials hyaluronan is usuallypresent in a complex with other biopolymers including pro-teoglycans [65] Methods to release hyaluronan from thesecomplexes comprise the use of proteolytic enzymes (iepapain pepsin pronase and trypsin) hyaluronan ion-pairprecipitation (with eg cetylpyridinium chloride) precipita-tion with organic solvents nonsolvent precipitation deter-gents and so forth [21] Ultrafiltration and chromatographyare used to remove the degradation products and othercontaminants Sterile filtration is used to remove allmicrobialcells prior to alcohol precipitation drying and conditioningof the end product

Despite extensive purification animal hyaluronan can becontaminated with proteins and nucleic acids The quantityand nature of contaminants differ with the source as wasshown for hyaluronan isolates from human umbilical cordand bovine vitreous humor containing elevated protein andnucleic acid levels compared to those from rooster comb andbacterial capsule isolates [12] Diseases as bovine spongiformencephalopathy (BSE) made us aware of the risk of cross-species viral infection Consequently hyaluronan from ani-mal sources has to be extensively purified to get rid of thesecontaminants and extraction and purification processes arecontinuously improved to fulfill the high quality standardsfor medical applications To date animal waste is still themost important source for the industrial manufacturing ofhyaluronan formedical application andprovides up to severaltones of medical-grade hyaluronan preparations per yearPharmacia (Sweden) Pfizer (USA) Genzyme (USA) and


Diosynth (The Netherlands) were among the first companiesthat produced hyaluronan from animal waste at industrialscale Commercially available hyaluronan preparations fromanimal sources have a molecular weight ranging from severalhundred thousand up to 25 million Da [66]

32 Bacterial Production The development of hyaluronanproduction through bacterial fermentation started in the 60swhen it was acknowledged that animal derived hyaluronansources can contain undesired proteins causing possibleallergic inflammation responses [67] Since the hyaluronanpolymer produced in animals and bacteria is identicalbacterial hyaluronan is not immunogenic and therefore isan excellent source for medical grade hyaluronan Extractinghyaluronan from microbial fermentation broth is a relativelysimple process with high yields An additional and impor-tant advantage of microbial hyaluronan production is thatmicrobial cells can be physiologically andor metabolicallyadapted to produce more hyaluronan of high molecularweight Therefore microbial hyaluronan production usingeither pathogenic streptococci or safe recombinant hostscontaining the necessary hyaluronan synthase is nowadaysmore and more preferred

321 Production of Hyaluronan with Streptococci StrainsThe first reported hyaluronic acid isolation from group Ahemolytic streptococci resulted in 60ndash140mg Lminus1 hyaluronan[68] Since then many different attempts have been madeto increase the amount of hyaluronan including traditionaltechniques as optimizing the extraction process adapting theculture media and selecting strains with high hyaluronanproductivity In thirty years the hyaluronan yields usingbatch fermentation increased from 300ndash400mg Lminus1 [69] to 6-7 g Lminus1 [70] which is the practical limit due to mass transferlimitations [25] Group C streptococci which are not humanpathogens and have superior hyaluronan productivity arefrequently used for hyaluronan synthesis instead of GroupA streptococci or instead the animal pathogenic bacteriumP multocida The most used strains are S equi subsp equiand S equi subsp zooepidemicus From dairy food productsS thermophilus strains with high productivity of usefulexopolysaccharides including hyaluronan were isolated [71]offering a safe hyaluronan producing organism Recently arecombinant S thermophilus was constructed that was ableto produce 12 g Lminus1 hyaluronan and the average molecularweight was comparable with the wild type strain [72]

Streptococci strains for hyaluronan production generallyuse glucose as carbon sourceThe yield of hyaluronan on glu-cose under aerobic fermentation conditions varies between 5and 10 [25 73 74] which is significantly higher than typicalyields for complex polysaccharides in lactic acid bacteriaOther carbon sources like starch [75] lactose sucrose anddextrin [76] can be used for hyaluronan productivity similarto glucose The use of carbon sources like starch and lactosewhich are largely available at lower costs is important in viewof the process economics

Research on optimization of hyaluronan productionthrough fermentation of streptococci strains addressed the

improvement of the hyaluronan yield the increase of themolecular weight and the reduction of the polymer poly-dispersity parameters which are essential for hyaluronanapplications The main question was if the molecular weightcould be further regulated either through strain improve-ments optimized process conditions or through metabolicengineering These topics will be discussed below

Regulation within Bacterial Cells Energy and Carbon Re-sources Hyaluronan biosynthesis in streptococci requires alarge amount of energy and competes with the bacterialcell growth for glucose as energy provider or as UDP-sugarprecursor Indeed when unlimited glucose is available thehighest bacterial growth was observed at optimal cultivationconditions (pH 7 and 37∘C) whilst the highest hyaluronanproductivity and molecular weight were obtained at sub-optimal growth conditions since when cells are growingslowly the carbon and energy resources are available forother processes [77] Under glucose-limiting conditions firsthyaluronan productivity and then molecular weight declines[25] Decrease of the initial glucose concentrations from60 to 20 g Lminus1 decreased the hyaluronan concentration andmolecular weight from 420 g Lminus1 and 30 million Dalton to165 g Lminus1 and 265 million Dalton respectively [77]

Aerobic culturing conditions increased the hyaluro-nan productivity by 50 as well as hyaluronan molecularweight whereas cell growth was unaffected [77ndash79] Underaerobic fermentation conditions streptococci change theirmetabolism from producing lactate into producing acetateformate and ethanol This generates four ATP moleculesper hexose for the acetate production instead of two ATPmolecules formed in lactate production [80] It was assumedthat the increase of hyaluronan production is due to theincreased levels of ATP or to increased levels of NADHoxidase which removes the excess of NADH in the pres-ence of oxygen However studies designed to improve ATPformation by increased acetate production through maltosemetabolism [80] or increasing NADH oxidase levels bymetabolic engineering [81] revealed that hyaluronan synthe-sis was not limited by energy resources

Studies have shown that only a critical level of dissolvedoxygen of 5 is needed to increase the hyaluronan yieldduring cell growth above this value the yield is constant[79] Interestingly the expression ofHA synthase in S zooepi-demicus is nine times higher under aerobic conditions thanunder anaerobic conditions [74] explaining the increase ofhyaluronan synthesis Furthermore oxygen induces enzymesinvolved in the production of UDP-GlcNAc (hasD) andacetoin recycling which offers an additional ATP moleculeand acetyl-CoA that can be used in hyaluronan production[82]

Based on these results we can conclude that elevated HAsynthase concentrations and UDP-GlcNAc availability seemto be themain reason for the increase in hyaluronan synthesisunder aerobic conditions

Influence of Process Conditions on Hyaluronan Yield andMolecular Weight Streptococcal fermentation to producehyaluronan is like other fermentations influenced by


medium composition pH temperature aeration and agi-tation Especially aerobic conditions and the initial glucoseconcentration had a large influence on hyaluronan yield andmolecular weight as was discussed above Both agitationand fermentation modus have a rather complex effect onhyaluronan production and are therefore in greater detaildescribed below

The effect of agitation on the yield and the molecu-lar weight of hyaluronan in Streptococcus fermentation iscomplex Increasing agitation increases the hyaluronan yieldunder both anaerobic and aerobic conditions most probablydue to an enhanced mass transfer induced by the reducedviscosity of the broth [77 79 83 84] However hyaluronanmolecular weight increases at moderate impeller speed dueto mass transfer enhancement but decreases at high impellerspeed most probably due to degradation by the reactive oxy-gen species that are formed under aerobic conditions at highimpeller speed [85] Oxidative degradation of hyaluronan canbe prevented by the addition of oxygen scavengers such assalicylic acid in the culture media resulting in an increase ofthe hyaluronan molecular weight [85 86]

Batch versus Chemostat In batch fermentation the hyaluro-nan production is limited to 6-7 g Lminus1 due to high viscosityof the broth and mass transfer constraints Further improve-ment of the hyaluronan yield is therefore not possible throughhigh-cell-density fermentation or through a high-yield strainA continuous culture is the best strategy to improve thevolumetric productivity of hyaluronan synthesis for fourreasons First cell growth of streptococci can be maintainedat the exponential phase Extension of the exponential phasecould lead to an increased amount of hyaluronan since itwas shown that hyaluronan synthesis stops in the stationaryphase [40 87] and the excretion of theHA synthase and othercell wall proteins which takes place in the stationary phaseis prevented [88] Second suboptimal growth conditions canbe imposed by nutrient limitation in order to decrease theresource competition between cell growth and hyaluronansynthesis [77 89] Third a turnaround phase is avoided byusing a continuous culture [90] Finally the viscosity of thebroth can be controlled by controlling the hyaluronan con-centration thus enhancing the mass transfer in the reactorChemostat cultivation of S zooepidemicus by controlling themedium conditions has been reported [75 90 91] and thisopens the way for further improvement of the hyaluronanproduction process

Strain Improvement Improved hyaluronan producing strainshave been obtained by random mutagenesis and selection ofmutant strains oriented to the reduction of the hyaluronan-depolymerising enzyme responsible for the decrease ofmolecular weight of hyaluronan during fermentation (hyalu-ronidase-negative strains) and to the reduction of strep-tolysin the exotoxin responsible for the 120573-hemolysis (non-hemolytic strains) Fermentation of nonhemolytic andhyaluronidase-free mutants produced hyaluronan with amolecular weight varying from 35 to 59 million Da andyields around 6-7 g Lminus1 [70 76 92] Randommutagenesis hasalso resulted in Streptococci strains that produce hyaluronan

of extraordinary high molecular weight ranging from 6 to 9millionDa andwithmoderate yields of 100 to 410mg Lminus1 [93]

Further improvement of strains requires genetic engi-neering of the producer microorganisms to engineer thehyaluronan metabolic pathway Metabolic engineering inS zooepidemicus and in recombinant hosts has demonstratedthat the ratio between UDP-GlcNAc and UDP-GlcUA andthe ratio between HA synthase and substrates are the mostimportant factors to influence hyaluronan molecular weightChen et al individually overexpressed each of the five genesin the has operon in S zooepidemicus They discovered thatoverexpression of hasA involved in hyaluronan biosynthesisand hasB and hasC involved in UDP-GlcUA biosynthesisdecreased molecular weight while overexpression of hasEinvolved in UDP-GlcNAc biosynthesis greatly enhancedmolecular weight Overexpression of hasD had no effectbut molecular weight was further increased when combinedwith hasE overexpression [60] Upregulation of hasD in Szooepidemicus has been observed at aerobic conditions [82]but also in the presence of an empty plasmid [94] bothaccompanied by a production of higher 119872

119908hyaluronan

Marcellin et al [94] suggested that regulation of hasD is at thetranslational or posttranslational level and that the plasmidburden is sufficient to remove the hasD encoded step as alimiting step In summary these results indicate that witha proper balance between the synthesis pathways to UDP-GlcNAc and UDP-GlcUA the hyaluronan molecular weightcan be further increased and controlled

In addition to the ratio between the two precursors theimportance of the ratio between precursor UDP-GlcUA andHA synthase also influences hyaluronan molecular weightThis was demonstrated for the heterologous host L lactis twoplasmids were constructed containing either hasA or hasBfrom S zooepidemicuswith the inducible expression promot-ers NICE and lacA Hyaluronan molecular weight increasedsignificantly at hasAhasB ratios below 2 indicating thathigher UDP-GlcUA availability per HA synthase enhanceshyaluronan molecular weight [95]

To conclude production of hyaluronan by fermentationof streptococci strains is a mature technology affordinghigh molecular weight highly pure polymers suitable formedical pharmaceutical and cosmetic applications Severaltons per year of bacterial hyaluronan are currently producedfor medical and cosmetic applications Companies that pro-duce hyaluronan through streptococcal fermentation are forinstance Q-Med (Uppsala Sweden) Lifecore Biomedical(Chaska USA) and Genzyme (Cambridge USA)

322 Hyaluronan from Recombinant Nonpathogenic Mi-croorganisms To avoid the risk of exotoxin contamina-tion in hyaluronan products from pathogenic streptococcistrains safe organisms have been genetically engineered intohyaluronan producers by introducing HA synthase enzymesfrom either streptococci or Pmultocida Using this approachhyaluronan producing strains of Enterococcus faecalis [7]E coli [7 48 96 97] Bacillus subtilis [98] Agrobacteriumsp [99] and L lactis [95 100ndash102] were obtained andexploited for hyaluronan production Heterologous expres-sion ofHA synthase (hasAgene in streptococci) was sufficient


to induce production of hyaluronan Since the hyaluronanbiosynthesis has partially a common pathway with cell wallbiosynthesis (Figure 3) production of hyaluronan by the hostgoes however on the expense of cell growth due to thedepletion of sugar precursors Better hyaluronan producingheterologous strains with improved intracellular availabilityof sugar precursors were obtained by coexpression of theHA synthase hasA gene derived from S equi or P multocidawith hasB homologue (UDP-glucose dehydrogenase) or hasB+ hasC homologues (UDP-glucose dehydrogenase + UDP-glucose pyrophosphorylase) from E coli A recombinantE coli strain obtained using this strategy produced 2 g Lminus1hyaluronan and the yield was increased to 38 g Lminus1 when theculture media was supplemented with glucosamine [99]

Using a similar strategy namely the coexpression of theHA synthase hasA gene from P multocida with hasB homo-logue (UDP-glucose dehydrogenase) from E coli into thefood-grade microorganisms Agrobacterium sp [99] and Llactis [101] these strains were able to produce hyaluronan atlevels up to 03 g Lminus1 for engineered Agrobacterium sp and065 g Lminus1 for the recombinant L lactis Although hyaluronanproduction yields were low the hyaluronan production fromthese food-grade microorganisms has high potential for foodand biomedical applications

Without overexpression of hasB or an analog encodingforUDP-glucose dehydrogenaseUDP-GlcUA levels are oftenlimiting in heterologous hosts [7 97 98 101] whereas UDP-GlcNAc seems to be adequately available in the hosts sinceit is a main component for bacterial cell wall synthesisAttempts to overexpress both synthetic pathways to UDP-GlcNAc and UDP-GlcUA in heterologous hosts have been sofar unsuccessful but overexpression of hasA hasB and hasChas led to considerable increases in hyaluronan synthesisvarying between 200 and 800 depending on the host [96100]

A process for the production of ultrapure sodium hya-luronate by the fermentation of a novel nonpathogenicrecombinant strain of B subtiliswas developed by the biotechcompany Novozymes [103] A major advantage of using Bsubtilis as host microorganism is that it is cultivable at largescale does not produce exo- and endotoxins and does notproduce hyaluronidase To build up the recombinant Bacillusstrain that produces hyaluronan expression constructs uti-lizing the hasA gene from S equisimilis in combination withoverexpression of one or more of the three native B subtilisprecursors genesmdashtuaD (hasB homologue encoding UDP-glucose dehydrogenase) gtaB (hasC encoding UDP-glucosepyrophosphorylase) and gcaD (hasD encoding UDP-N-acetyl glucosamine pyrophosphorylase) The recombinant Bsubtilis strain was able to produce up to 5 g Lminus1 of hyaluronanwith a molecular weight of 1ndash12 million Da when cultivatedon a minimal medium based on sucrose at pH 7 and 37∘C[94] The hyaluronan is secreted into the medium and is notcell-associated which simplifies the downstream processingsignificantly making the use of water-based solvents possiblefor hyaluronan recovery The Bacillus hyaluronan showsvery low levels of contaminants (ie proteins nucleic acidsmetal ions and exo- and endotoxins) and thus has a high

safety profile including no risks of viral contamination or oftransmission of animal spongiform encephalopathy

As an alternative to prokaryotic HA production hyaluro-nan can be produced by infecting green algae cells of thegenus Chlorella with a virus [45 104] although the reportedyields were low (05ndash1 g Lminus1)

33 In Vitro Production of Hyaluronan Using Isolated HASynthase Synthesis of hyaluronan using isolated HA syn-thase becomes relevantwhen hyaluronan polymers of definedmolecular weight and narrow polydispersity are neededIsolatedHA synthase is able to catalyze in vitro at well-definedconditions the same reaction as it catalyzes in vivo namelythe synthesis of hyaluronan from the nucleotide sugars UDP-GlcNAc andUDP-GlcUA Preparative enzymatic synthesis ofhyaluronan using the crude membrane-bound HA synthasefrom S pyogenes was demonstrated although the yield waslow around 20 [105]The hyaluronan yield was increased to90 when the enzymatic hyaluronan synthesis was coupledwith in situ enzymatic regeneration of the sugar nucleotidesusing UDP and relatively inexpensive substrates Glc-1-P andGlcNAc-1-P in a one-pot reaction The average molecularweight of the synthetic hyaluronan was around 55 times 105Dacorresponding to a degree of polymerization of 1500

High molecular weight monodisperse hyaluronan poly-mers with 119872

119908up to 2500 kDa (sim12000 sugar units) and

polydispersity (119872119908119872119899) of 101ndash120 were obtained by enzy-

matic polymerization using the recombinantPmultocidaHAsynthase PmHAS overexpressed in E coli [106] PmHASuses two separate glycosyl transferase sites to addGlcNAc andGlcUAmonosaccharides to the nascent polysaccharide chainHyaluronan synthesis with PmHASwas achieved either by denovo synthesis from the two UDP-sugars precursors (1) andby elongation of an hyaluronan-like acceptor oligosaccharidechain by alternating repetitive addition of the UDP-sugars asfollows

119899UDP-GlcUA + 119899UDP-GlcNAc + z[GlcUA-GlcNAc]119909

997888rarr 2119899UDP + [GlcUA + GlcNAc]119909+119899

(2)

The control of the chain length and polydispersity ofthe hyaluronan polymer is determined by the intrinsicenzymological properties of the recombinant PmHAS (i)the rate limiting step of the in vitro polymerization appearsto be the chain initiation and (ii) in vitro enzymaticpolymerization is a fast nonprocessive reaction Thereforethe concentration of the hyaluronan acceptor controls thesize and the polydispersity of the hyaluronan polymer inthe presence of a finite amount of UDP-sugar monomers[106] Using this synchronized stoichiometrically-controlledenzymatic polymerization reaction low molecular weighthyaluronan (sim8 kDa) with narrow size distribution wassynthesized One important feature of the PmHAS is thatchain elongation occurs at the nonreducing end of thegrowing chain and this makes the use of modified accep-tors as substrates possible and consequently the synthesisof hyaluronan polymers with various end-moieties Theelongation of a hyaluronan tetrasaccharide labeled at thereducing end with the fluorophore 2-aminobenzoic acid


using PmHAS and the quantitative formation of fluores-cent hyaluronan oligomers and polymers was demonstrated[10 107]

These studies have shown the high potential of the in vitroenzymatic synthesis of hyaluronan polymers and oligomerswith low polydispersity but for the large scale applicationfurther developments are needed Technological bottlenecksthat must be resolved are (a) production of robust enzymesfor hyaluronan synthesis (b) selective separation of the UDPbyproduct from the reaction mixture to prevent enzymeinactivation and to allow UDP recycling for the synthesisof UDP-sugar substrates (c) development of efficient pro-cesses for the production of the hyaluronan-like templatefor elongation and (d) the development of simple low costtechnologies for the synthesis of sugar nucleotide substratesstarting from bulk carbohydrates Since sugar nucleotides areexpensive substrates their regeneration is a crucial step for thedevelopment of an economic process for the production ofhyaluronan

For in vitro production of hyaluronan the Class I HAsynthases are less suitable due to the fact that they are integralmembrane proteins Research with these HA synthases ismostly performed on membrane fractions but this system isless suitable for larger scale production An alternative couldbe the immobilization of the enzyme in the cell wall of forinstance yeast Production of various humanoligosaccharideshas shown to be feasible with yeast cells in which glycosyltransferases are expressed and anchored in the cell wallglucan [108]

A far more promising enzyme for in vitro production ofhyaluronan is the P multocida Class II HA synthase Thisenzyme is not an integral membrane protein and the analysisof the subcellular location and enzyme activity has shown thata recombinant truncated version of the protein lacking a 216carboxy terminal amino acids retained HA synthase activitybut was located in the cytoplasm when expressed in E coli[109] Furthermore it was shown that two distinct transferaseactivities are located on the P multocida HA synthase eachof which can be inactivated by mutations in the DXD aminoacid motif present in the active site while retaining the othertransferase activity [110]

UDP-Recycling and Sugar Nucleotide Synthesis (EnzymaticCascade Bacterial Production)The general metabolic routesfor incorporation of sugar units into glycosaminoglycansare their prior conversion into sugar nucleotides such asUDP-GlcA and UDP-GlcNAc for hyaluronan synthesis Thebiosynthetic reactions are known as LeLoir pathways andthese pathways can be different in microorganisms [111] Forexample the pathways for the synthesis of UDP-GlcNAc ineukaryotes and prokaryotes are different Production of sugarnucleotides needs also cofactor regeneration for preparativeapplications because the cofactors are too expensive to beused as stoichiometric agents Nowadays several cofactorscan be effectively regenerated using enzymes for exampleNAD+ by glutamate dehydrogenase with 120572-ketoglutarateor whole cell based methods for example Corynebac-terium ammoniagenes that converts orotic acid into UTP[112ndash115]

4 Future Perspectives

Hyaluronan with its exceptional properties andmultiple anddiverse applications has proved to be an exceptional mate-rial for medical pharmaceutical and cosmetic applicationsTremendous developments have been achieved in the pastdecades in all hyaluronan-related fields covering the wholechain from the production of hyaluronan polymers andoligomers to the development of advancedmaterials for clin-ical applications Latest accomplishments in hyaluronan pro-duction and in particular the elucidation of the biosyntheticpathways in hyaluronan producing microorganisms opensnewpremises for the further optimization of biotechnologicalprocess for hyaluronan production with safe hosts Furtherunderstanding of the in vivo regulation of hyaluronanmolec-ular weight will benefit the biotechnological production ofdefined hyaluronan products with narrow polydispersity Webelieve that bacterial fermentation using nonpathogenic safeheterologous hosts will become the main source of highmolecular weight hyaluronan and metabolic engineeringwill be used to improve and control molecular weightStimulated by the developments of production techniquesand the growing knowledge on the biological function ofhyaluronan research to enhancing existing medical productsand to validating new concepts in medical therapies will beset forth

Abbreviations

BSE Bovine spongiform encephalopathyHA synthase or HAS Hyaluronan synthase119872119908 Weight average molecular mass119872119899 Number average molecular mass

GlcAU Glucuronic acidGT Glycosyl transferaseUDP Uridine diphosphateGlcNac N-acetyl glucosamineGMO Genetically modified organism

References

[1] P LDeAngelis ldquoGlycosaminoglycan polysaccharide biosynthe-sis and production today and tomorrowrdquo Applied Microbiologyand Biotechnology vol 94 no 2 pp 295ndash305 2012

[2] H G Garg and C A Hales Chemistry and Biology of Hyaluro-nan Elsevier 2004

[3] K Meyer and J W Palmer ldquoThe polysaccharde of the vitreoushumorrdquo The Journal of Biological Chemistry vol 107 no 3 pp629ndash634 1934

[4] B Weissmann and K Meyer ldquoThe structure of hyalobiuronicacid and of hyaluronic acid from umbilical cordrdquo Journal of theAmerican Chemical Society vol 76 no 7 pp 1753ndash1757 1954

[5] E A Balazs ldquoUltrapure hyaluronic acid and the use thereofrdquoUS Patent US4141973 1979

[6] P L DeAngelis J Papaconstantinou and P H Weigel ldquoMolec-ular cloning identification and sequence of the hyaluronansynthase gene from group A Streptococcus pyogenesrdquo TheJournal of Biological Chemistry vol 268 no 26 pp 19181ndash191841993


[7] P L DeAngelis J Papaconstantinou and P H Weigel ldquoIso-lation of a Streptococcus pyogenes gene locus that directshyaluronan biosynthesis in acapsular mutants and in heterolo-gous bacteriardquoThe Journal of Biological Chemistry vol 268 no20 pp 14568ndash14571 1993

[8] G Blatter and J C Jacquinet ldquoThe use of 2-deoxy-2-trichloroacetamido-D-glucopyranose derivatives in synthesesof hyaluronic acid-related tetra- hexa- and octa-saccharideshaving amethyl120573-D-glucopyranosiduronic acid at the reducingendrdquo Carbohydrate Research vol 288 pp 109ndash125 1996

[9] P L DeAngelis L C Oatman and D F Gay ldquoRapid chemoen-zymatic synthesis of monodisperse hyaluronan oligosaccha-rides with immobilized enzyme reactorsrdquo The Journal of Bio-logical Chemistry vol 278 no 37 pp 35199ndash35203 2003

[10] F K Kooy Enzymatic Production of Hyaluronan Oligo- andPolysaccharides Wageningen University Wageningen TheNetherlands 2010

[11] B Porsch R Laga and C Konak ldquoBatch and size-exclusionchromatographic characterization of ultra-high molar masssodiumhyaluronate containing low amounts of strongly scatter-ing impurities by dual low angle light scatteringrefractometricdetectionrdquo Journal of Liquid Chromatography and Related Tech-nologies vol 31 no 20 pp 3077ndash3093 2008

[12] A Shiedlin R Bigelow W Christopher et al ldquoEvaluation ofhyaluronan from different sources streptococcus zooepidemi-cus rooster comb bovine vitreous and human umbilical cordrdquoBiomacromolecules vol 5 no 6 pp 2122ndash2127 2004

[13] R Mendichi and L Soltes ldquoHyaluronan molecular weight andpolydispersity in some commercial intra-articular injectablepreparations and in synovial fluidrdquo Inflammation Research vol51 no 3 pp 115ndash116 2002

[14] R Stern ldquoDevising a pathway for hyaluronan catabolism are wethere yetrdquo Glycobiology vol 13 no 12 pp 105Rndash115R 2003

[15] M Erickson and R Stern ldquoChain gangs new aspects ofhyaluronan metabolismrdquo Biochemistry Research Internationalvol 2012 Article ID 893947 9 pages 2012

[16] T C Laurent and J R E Fraser ldquoHyaluronanrdquo The FASEBJournal vol 6 no 7 pp 2397ndash2404 1992

[17] P L DeAngelis ldquoHyaluronan synthases Fascinating glycosyl-transferases from vertebrates bacterial pathogens and algalvirusesrdquo Cellular and Molecular Life Sciences vol 56 no 7-8pp 670ndash682 1999

[18] T E Hardingham and H Muir ldquoThe specific interaction ofhyaluronic acid with cartilage proteoglycansrdquo Biochimica etBiophysica Acta vol 279 no 2 pp 401ndash405 1972

[19] V C Hascall ldquoHyaluronan a common threadrdquo GlycoconjugateJournal vol 17 no 7ndash9 pp 607ndash616 2000

[20] N Afratis C Gialeli D Nikitovic et al ldquoGlycosaminoglycanskey players in cancer cell biology and treatmentrdquo FEBS Journalvol 279 no 7 pp 1177ndash1197 2012

[21] C Schiraldi A La Gatta and M De Rosa ldquoBiotechnologicalproduction and application of hyaluronanrdquo in Biopolymers MElnashar Ed pp 387ndash412 InTech Rijeka Croatia 2010

[22] F Freitas V D Alves and M A M Reis ldquoAdvances in bac-terial exopolysaccharides from production to biotechnologicalapplicationsrdquo Trends in Biotechnology vol 29 no 8 pp 388ndash398 2011

[23] B D Ulery L S Nair and C T Laurencin ldquoBiomedicalapplications of biodegradable polymersrdquo Journal of PolymerScience B vol 49 no 12 pp 832ndash864 2011

[24] R Stern A A Asari and K N Sugahara ldquoHyaluronanfragments an information-rich systemrdquo European Journal ofCell Biology vol 85 no 8 pp 699ndash715 2006

[25] B F Chong L M Blank R Mclaughlin and L K NielsenldquoMicrobial hyaluronic acid productionrdquo Applied Microbiologyand Biotechnology vol 66 no 4 pp 341ndash351 2005

[26] S Ghatak S Misra and B P Toole ldquoHyaluronan oligosac-charides inhibit anchorage-independent growth of tumor cellsby suppressing the phosphoinositide 3-kinaseAkt cell survivalpathwayrdquo The Journal of Biological Chemistry vol 277 no 41pp 38013ndash38020 2002

[27] J Y Lee and A P Spicer ldquoHyaluronan a multifunctionalmegaDalton stealth moleculerdquo Current Opinion in Cell Biologyvol 12 no 5 pp 581ndash586 2000

[28] P L DeAngelis and P H Weigel ldquoImmunochemical confir-mation of the primary structure of streptococcal hyaluronansynthase and synthesis of high molecular weight product by therecombinant enzymerdquo Biochemistry vol 33 no 31 pp 9033ndash9039 1994

[29] P Prehm ldquoHyaluronate is synthesized at plasma membranesrdquoBiochemical Journal vol 220 no 2 pp 597ndash600 1984

[30] A A E Chavaroche L A M van den Broek and G EgginkldquoProductionmethods for heparosan a precursor of heparin andheparan sulfaterdquo Carbohydrate Polymers vol 93 no 1 pp 38ndash47 2013

[31] N Itano ldquoHyaluronan biosynthesis a multifaceted processrdquoConnective Tissue vol 33 no 3 pp 221ndash226 2001

[32] C A de la Motte and J A Drazba ldquoViewing hyaluronanimaging contributes to imagining new roles for this amazingmatrix polymerrdquo Journal of Histochemistry and Cytochemistryvol 59 no 3 pp 252ndash257 2011

[33] N Itano and K Kimata ldquoMolecular cloning of human hyaluro-nan synthaserdquo Biochemical and Biophysical Research Communi-cations vol 222 no 3 pp 816ndash820 1996

[34] A P Spicer M L Augustine and J A McDonald ldquoMolecularcloning and characterization of a putative mouse hyaluronansynthaserdquo The Journal of Biological Chemistry vol 271 no 38pp 23400ndash23406 1996

[35] N Itano T Sawai M Yoshida et al ldquoThree isoforms ofmammalian hyaluronan synthases have distinct enzymaticpropertiesrdquoThe Journal of Biological Chemistry vol 274 no 35pp 25085ndash25092 1999

[36] P Heldin ldquoMolecular mechanisms that regulate hyaluronansynthesisrdquoGeneTherapy andMolecular Biology vol 3 pp 465ndash474 1999

[37] A Jacobson J Brinck M J Briskin A P Spicer and P HeldinldquoExpression of human hyaluronan syntheses in response toexternal stimulirdquo Biochemical Journal vol 348 no 1 pp 29ndash352000

[38] B A Dougherty and I Van de Rijn ldquoMolecular characterizationof hasB from an operon required for hyaluronic acid synthesisin group A streptococci Demonstration of UDP-glucose dehy-drogenase activityrdquoThe Journal of Biological Chemistry vol 268no 10 pp 7118ndash7124 1993

[39] B A Dougherty and I Van de Rijn ldquoMolecular characterizationof hasA from an operon required for hyaluronic acid synthesisin group A streptococcirdquo The Journal of Biological Chemistryvol 269 no 1 pp 169ndash175 1994

[40] D L Crater B A Dougherty and I Van de Rijn ldquoMolec-ular characterization of hasC from an operon required for


hyaluronic acid synthesis in group A streptococci Demonstra-tion of UDP-glucose pyrophosphorylase activityrdquoThe Journal ofBiological Chemistry vol 270 no 48 pp 28676ndash28680 1995

[41] AMarkovitz J A Cifonelli andADorfman ldquoThebiosynthesisof hyaluronic acid by group A Streptococcus VI Biosynthesisfrom uridine nucleotides in cell-free extractsrdquo The Journal ofBiological Chemistry vol 234 pp 2343ndash2350 1959

[42] K Kumari and P H Weigel ldquoMolecular cloning expressionand characterization of the authentic hyaluronan synthase fromGroup C Streptococcus equisimilisrdquo The Journal of BiologicalChemistry vol 272 no 51 pp 32539ndash32546 1997

[43] P L DeAngelis and A M Achyuthan ldquoYeast-derived recom-binant DG42 protein of Xenopus can synthesize hyaluronan invitrordquo The Journal of Biological Chemistry vol 271 no 39 pp23657ndash23660 1996

[44] P H Weigel V C Hascall and M Tammi ldquoHyaluronansynthasesrdquoThe Journal of Biological Chemistry vol 272 no 22pp 13997ndash14000 1997

[45] P L DeAngelis W Jing M V Graves D E Burbank and JL Van Etten ldquoHyaluronan synthase of chlorella virus PBCV-1rdquoScience vol 278 no 5344 pp 1800ndash1803 1997

[46] P M Coutinho E Deleury G J Davies and B HenrissatldquoAn evolving hierarchical family classification for glycosyltrans-ferasesrdquo Journal ofMolecular Biology vol 328 no 2 pp 307ndash3172003

[47] A Jong C H Wu H M Chen et al ldquoIdentification and char-acterization of CPS1 as a hyaluronic acid synthase contributingto the pathogenesis of Cryptococcus neoformans infectionrdquoEukaryotic Cell vol 6 no 8 pp 1486ndash1496 2007

[48] P L DeAngelis W Jing R R Drake and A M AchyuthanldquoIdentification and molecular cloning of a unique hyaluronansynthase from Pasteurella multocidardquo The Journal of BiologicalChemistry vol 273 no 14 pp 8454ndash8458 1998

[49] P H Weigel and P L DeAngelis ldquoHyaluronan synthasesa decade-plus of novel glycosyltransferasesrdquo The Journal ofBiological Chemistry vol 282 no 51 pp 36777ndash36781 2007

[50] S Bodevin-Authelet M Kusche-Gullberg P E Pummill P LDeAngelis and U Lindahl ldquoBiosynthesis of hyaluronan direc-tion of chain elongationrdquo The Journal of Biological Chemistryvol 280 no 10 pp 8813ndash8818 2005

[51] P E Pummill T A Kane E S Kempner and P L DeAngelisldquoThe functional molecular mass of the Pasteurella hyaluronansynthase is amonomerrdquoBiochimica et Biophysica Acta vol 1770no 2 pp 286ndash290 2007

[52] P L DeAngelis ldquoMicrobial glycosaminoglycan glycosyltrans-ferasesrdquo Glycobiology vol 12 no 1 pp 9Rndash16R 2002

[53] K Rilla H Siiskonen A P Spicer J M T Hyttinen M ITammi and R H Tammi ldquoPlasma membrane residence ofhyaluronan synthase is coupled to its enzymatic activityrdquo TheJournal of Biological Chemistry vol 280 no 36 pp 31890ndash318972005

[54] M Nardini M Ori D Vigetti R Gornati I Nardi and RPerris ldquoRegulated gene expression of hyaluronan synthasesduring Xenopus laevis developmentrdquo Gene Expression Patternsvol 4 no 3 pp 303ndash308 2004

[55] J Y L Tien and A P Spicer ldquoThree vertebrate hyaluronansynthases are expressed during mouse development in distinctspatial and temporal patternsrdquo Developmental Dynamics vol233 no 1 pp 130ndash141 2005

[56] M Suzuki T Asplund H Yamashita C H Heldin and PHeldin ldquoStimulation of hyaluronan biosynthesis by platelet-derived growth factor-BB and transforming growth factor-1205731

involves activation of protein kinase Crdquo Biochemical Journalvol 307 no 3 pp 817ndash821 1995

[57] H Chao and A P Spicer ldquoNatural antisense mRNAs tohyaluronan synthase 2 inhibit hyaluronan biosynthesis and cellproliferationrdquoThe Journal of Biological Chemistry vol 280 no30 pp 27513ndash27522 2005

[58] D Vigetti A Genasetti E Karousou et al ldquoModulation ofhyaluronan synthase activity in cellular membrane fractionsrdquoThe Journal of Biological Chemistry vol 284 no 44 pp 30684ndash30694 2009

[59] L M Blank P Hugenholtz and L K Nielsen ldquoEvolution ofthe hyaluronic acid synthesis (has) operon in Streptococcuszooepidemicus and other pathogenic streptococcirdquo Journal ofMolecular Evolution vol 67 no 1 pp 13ndash22 2008

[60] W Y Chen E Marcellin J Hung and L K NielsenldquoHyaluronan molecular weight is controlled by UDP-N-acetylglucosamine concentration in Streptococcus zooepidemi-cusrdquo The Journal of Biological Chemistry vol 284 no 27 pp18007ndash18014 2009

[61] L Soltes RMendichi D LathMMach andD Bakos ldquoMolec-ular characteristics of some commercial high-molecular-weighthyaluronansrdquo Biomedical Chromatography vol 16 no 7 pp459ndash462 2002

[62] P S Harmon E P Maziarz and X M Liu ldquoDetailed character-ization of hyaluronan using aqueous size exclusion chromatog-raphy with triple detection and multiangle light scatteringdetectionrdquo Journal of Biomedical Materials Research B vol 100no 7 pp 1955ndash1960 2012

[63] E U Ignatova and A N Gurov ldquoPrinciples of extraction andpurification of hyaluronic acidrdquo Khimiko-FarmatsevticheskiiZhurnal vol 24 no 3 pp 42ndash46 1990

[64] I Amagai Y Tashiro and H Ogawa ldquoImprovement of theextraction procedure for hyaluronan from fish eyeball and themolecular characterizationrdquo Fisheries Science vol 75 no 3 pp805ndash810 2009

[65] M OrsquoRegan I Martini F Crescenzi C De Luca and MLansing ldquoMolecular mechanisms and genetics of hyaluro-nan biosynthesisrdquo International Journal of Biological Macro-molecules vol 16 no 6 pp 283ndash286 1994

[66] G Kogan L Soltes R Stern and P Gemeiner ldquoHyaluronicacid a natural biopolymer with a broad range of biomedical andindustrial applicationsrdquo Biotechnology Letters vol 29 no 1 pp17ndash25 2007

[67] J C Thonard S A Migliore and R Blustein ldquoIsolationof hyaluronic acid from broth cultures of Streptococcirdquo TheJournal of Biological Chemistry vol 239 pp 726ndash728 1964

[68] F E Kendall M Heidelberger and M H Dawson ldquoA sero-logically inactive polysaccharide elaborated by mocoid strainsof group A hemolytic Streptococcusrdquo The Journal of BiologicalChemistry vol 118 no 1 pp 61ndash69 1937

[69] B Holmstrom and J Ricica ldquoProduction of hyaluronic acid bya Streptococcal strain in batch culturerdquo Applied EnvironmentalMicrobiology vol 15 no 6 pp 1409ndash1413 1967

[70] J H Kim S J Yoo D K Oh et al ldquoSelection of a Streptococcusequi mutant and optimization of culture conditions for theproduction of high molecular weight hyaluronic acidrdquo Enzymeand Microbial Technology vol 19 no 6 pp 440ndash445 1996

[71] N Izawa T Hanamizu R Iizuka et al ldquoStreptococcus ther-mophilus produces exopolysaccharides including hyaluronicacidrdquo Journal of Bioscience and Bioengineering vol 107 no 2pp 119ndash123 2009


[72] N Izawa M Serata T Sone T Omasa and H OhtakeldquoHyaluronic acid production by recombinant Streptococcusthermophilusrdquo Journal of Bioscience and Bioengineering vol 111no 6 pp 665ndash670 2011

[73] H J Gao G C Du and J Chen ldquoAnalysis of metabolic fluxesfor hyaluronic acid (HA) production by Streptococcus zooepi-demicusrdquoWorld Journal of Microbiology and Biotechnology vol22 no 4 pp 399ndash408 2006

[74] X J Duan H X Niu W S Tan and X Zhang ldquoMechanismanalysis of effect of oxygen on molecular weight of hyaluronicacid produced by Streptococcus zooepidemicusrdquo Journal ofMicrobiology and Biotechnology vol 19 no 3 pp 299ndash3062009

[75] J Zhang X Ding L Yang and Z Kong ldquoA serum-freemedium for colony growth and hyaluronic acid production byStreptococcus zooepidemicus NJUST01rdquo Applied Microbiologyand Biotechnology vol 72 no 1 pp 168ndash172 2006

[76] J H Im J M Song J H Kang and D J Kang ldquoOptimizationof medium components for high-molecular-weight hyaluronicacid production by Streptococcus sp ID9102 via a statisticalapproachrdquo Journal of IndustrialMicrobiology and Biotechnologyvol 36 no 11 pp 1337ndash1344 2009

[77] D C Armstrong M J Cooney and M R Johns ldquoGrowth andamino acid requirements of hyaluronic-acid producing Strep-tococcus zooepidemicusrdquoAppliedMicrobiology and Biotechnol-ogy vol 47 no 3 pp 309ndash312 1997

[78] M J Cooney L T Goh P L Lee and M R Johns ldquoStruc-tured model-based analysis and control of the hyaluronic acidfermentation by Streptococcus zooepidemicus physiologicalimplications of glucose and complex-nitrogen-limited growthrdquoBiotechnology Progress vol 15 no 5 pp 898ndash910 1999

[79] W C Huang S J Chen and T L Chen ldquoThe role ofdissolved oxygen and function of agitation in hyaluronic acidfermentationrdquo Biochemical Engineering Journal vol 32 no 3pp 239ndash243 2006

[80] B F Chong and L K Nielsen ldquoAerobic cultivation of Strep-tococcus zooepidemicus and the role of NADH oxidaserdquoBiochemical Engineering Journal vol 16 no 2 pp 153ndash162 2003

[81] B F Chong andLKNielsen ldquoAmplifying the cellular reductionpotential of Streptococcus zooepidemicusrdquo Journal of Biotech-nology vol 100 no 1 pp 33ndash41 2003

[82] T F Wu W C Huang Y C Chen Y G Tsay and C SChang ldquoProteomic investigation of the impact of oxygen onthe protein profiles of hyaluronic acid-producing Streptococcuszooepidemicusrdquo Proteomics vol 9 no 19 pp 4507ndash4518 2009

[83] M R Johns L T Goh and A Oeggerli ldquoEffect of pH agitationand aeration on hyaluronic acid production by Streptococcuszooepidemicusrdquo Biotechnology Letters vol 16 no 5 pp 507ndash512 1994

[84] S Hasegawa M Nagatsuru M Shibutani S Yamamoto and SHasebe ldquoProductivity of concentrated hyaluronic acid using aMaxblend (R) fermentorrdquo Journal of Bioscience and Bioengineer-ing vol 88 no 1 pp 68ndash71 1999

[85] X Zhang X J Duan andW S Tan ldquoMechanism for the effect ofagitation on the molecular weight of hyaluronic acid producedby Streptococcus zooepidemicusrdquo Food Chemistry vol 119 no4 pp 1643ndash1646 2010

[86] M Cazzola F Orsquoregan and V Corsa ldquoCulture medium andprocess for the preparation of highmolecular weight hyaluronicacidrdquo Fidia Advanced Biopolymers SRL 2003

[87] I Van De Rijn ldquoStreptococcal hyaluronic acid Proposedmech-anisms of degradation and loss of synthesis during stationary

phaserdquo Journal of Bacteriology vol 156 no 3 pp 1059ndash10651983

[88] A Mausolf J Jungmann H Robenek and P Prehm ldquoSheddingof hyaluronate synthase from streptococcirdquo Biochemical Jour-nal vol 267 no 1 pp 191ndash196 1990

[89] D C Ellwood C G T Evans G M Dunn et al ldquoProductionof hyaluronic acidrdquo Fermentech Medical Limited 1996

[90] W C Huang S J Chen and T L Chen ldquoProduction ofhyaluronic acid by repeated batch fermentationrdquo BiochemicalEngineering Journal vol 40 no 3 pp 460ndash464 2008

[91] L M Blank R L McLaughlin and L K Nielsen ldquoStableproduction of hyaluronic acid in streptococcus zooepidemicuschemostats operated at high dilution raterdquo Biotechnology andBioengineering vol 90 no 6 pp 685ndash693 2005

[92] H Y Han S H Jang E C Kim et al ldquoMicroorganism pro-ducing hyaluronic acid and purification method of hyaluronicacidrdquo p 26 2004

[93] S Stahl ldquoMethods and means for the production of hyaluronicacidrdquo US6090596 2000

[94] E Marcellin W Y Chen and L K Nielsen ldquoUnderstandingplasmid effect on hyaluronic acid molecular weight producedby Streptococcus equi subsp zooepidemicusrdquo Metabolic Engi-neering vol 12 no 1 pp 62ndash69 2010

[95] J Z Sheng P X Ling X Q Zhu et al ldquoUse of inductionpromoters to regulate hyaluronan synthase and UDP-glucose-6-dehydrogenase of Streptococcus zooepidemicus expressionin Lactococcus lactis a case study of the regulation mechanismof hyaluronic acid polymerrdquo Journal of Applied Microbiologyvol 107 no 1 pp 136ndash144 2009

[96] H Yu and G Stephanopoulos ldquoMetabolic engineering ofEscherichia coli for biosynthesis of hyaluronic acidrdquo MetabolicEngineering vol 10 no 1 pp 24ndash32 2008

[97] Z Mao H D Shin and R Chen ldquoA recombinant E colibioprocess for hyaluronan synthesisrdquo Applied Microbiology andBiotechnology vol 84 no 1 pp 63ndash69 2009

[98] B Widner R Behr S Von Dollen et al ldquoHyaluronic acidproduction in Bacillus subtilisrdquo Applied and EnvironmentalMicrobiology vol 71 no 7 pp 3747ndash3752 2005

[99] Z Mao and R R Chen ldquoRecombinant synthesis of hyaluronanby Agrobacterium sprdquo Biotechnology Progress vol 23 no 5 pp1038ndash1042 2007

[100] S B Prasad G Jayaraman and K B RamachandranldquoHyaluronic acid production is enhanced by the additional co-expression of UDP-glucose pyrophosphorylase in LactococcuslactisrdquoAppliedMicrobiology and Biotechnology vol 86 no 1 pp273ndash283 2010

[101] L J Chien and C K Lee ldquoHyaluronic acid production byrecombinant Lactococcus lactisrdquo Applied Microbiology andBiotechnology vol 77 no 2 pp 339ndash346 2007

[102] S B Prasad K B Ramachandran and G Jayaraman ldquoTran-scription analysis of hyaluronan biosynthesis genes in Strepto-coccus zooepidemicus and metabolically engineered Lactococ-cus lactisrdquo Applied Microbiology and Biotechnology vol 94 no6 pp 1593ndash1607 2012

[103] A Sloma et al ldquoRecombinant expression of bacterial hyaluro-nan synthase operon genes in Bacillus and hyaluronic acidproductionrdquo p 218 2003

[104] M V Graves D E Burbank R Roth J Heuser P L Deangelisand J L Van Etten ldquoHyaluronan synthesis in virus PBCV-1-infected chlorella-like green algaerdquo Virology vol 257 no 1 pp15ndash23 1999


[105] CDeLucaM Lansing IMartini et al ldquoEnzymatic synthesis ofhyaluronic acid with regeneration of sugar nucleotidesrdquo Journalof the American Chemical Society vol 117 no 21 pp 5869ndash58701995

[106] P L DeAngelis ldquoMonodisperse hyaluronan polymers synthesisand potential applicationsrdquo Current Pharmaceutical Biotechnol-ogy vol 9 no 4 pp 246ndash248 2008

[107] F K Kooy M Ma H H Beeftink G Eggink J Tramperand C G Boeriu ldquoQuantification and characterization ofenzymatically produced hyaluronan with fluorophore-assistedcarbohydrate electrophoresisrdquoAnalytical Biochemistry vol 384no 2 pp 329ndash336 2009

[108] Y I Shimma F Saito FOosawa andY Jigami ldquoConstruction ofa library of human glycosyltransferases immobilized in the cellwall of Saccharomyces cerevisiaerdquo Applied and EnvironmentalMicrobiology vol 72 no 11 pp 7003ndash7012 2006

[109] W Jing and P L De Angelis ldquoDissection of the two transferaseactivities of the Pasteurella multocida hyaluronan synthase twoactive sites exist in one polypeptiderdquoGlycobiology vol 10 no 9pp 883ndash889 2000

[110] W Jing and P L DeAngelis ldquoAnalysis of the two active sitesof the hyaluronan synthase and the chondroitin synthase ofPasteurella multocidardquoGlycobiology vol 13 no 10 pp 661ndash6712003

[111] S Milewski I Gabriel and J Olchowy ldquoEnzymes of UDP-GlcNAcbiosynthesis in yeastrdquoYeast vol 23 no 1 pp 1ndash14 2006

[112] H Zhao and W A Van Der Donk ldquoRegeneration of cofactorsfor use in biocatalysisrdquo Current Opinion in Biotechnology vol14 no 6 pp 583ndash589 2003

[113] A Ruffing and R R Chen ldquoMetabolic engineering of microbesfor oligosaccharide and polysaccharide synthesisrdquo MicrobialCell Factories vol 5 p 25 2006

[114] W Liu and P Wang ldquoCofactor regeneration for sustainableenzymatic biosynthesisrdquo Biotechnology Advances vol 25 no 4pp 369ndash384 2007

[115] C A G M Weijers M C R Franssen and G M VisserldquoGlycosyltransferase-catalyzed synthesis of bioactive oligosac-charidesrdquo Biotechnology Advances vol 26 no 5 pp 436ndash4562008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


Table 1 Important events in research on hyaluronan products

Time Event

1880Portes reported that mucin from the vitreousbody differs from other mucoids in cornea andcartilage and named it hyalomucine [2]

1934Meyer and Palmer isolated and identified thepolysaccharide from the vitreous body and namedit hyaluronic acid [3]

30sndash50s

Hyaluronan from many different tissues ofvertebrates was isolated identified andcharacterized A few pathogenic bacteria werefound that produce hyaluronan and use it toencapsulate their cells

50s

The chemical structure of hyaluronan waselucidated by Karl Meyer and his team They usedhyaluronidase to produce overlappingoligosaccharides that were structurally analyzedby conventional techniques [4]Interest emerged to use hyaluronan in eye surgeryas a substitute of the vitreous body

40sndash70s

Extraction processes from animal tissues wereoptimized to remove protein and to minimizehyaluronan degradation First studies onhyaluronan production through bacterialfermentation and chemical synthesis wereinitiated

1979

First patent on ultrapure hyaluronan isolated fromrooster combs [5] This was the starting of theindustrial manufacturing of hyaluronan fromanimal sources for human applications In 1980using the methods of Balasz Pharmacia (Sweden)introduced Healon a product used in cataractsurgery

90sndash00s

Revival of studies on bacterial fermentation toproduce hyaluronan of high molecular weightEmphasis on controlling polymer size andpolydispersity

1993

The gene encoding for a single enzyme thatpolymerizes UDP-GlcNAc and UDP-GlcUA intohyaluronan is isolated by DeAngelis andcoworkers from Streptococcus pyogenesHyaluronan synthases from othermicroorganisms were identified and characterized[6 7]

1996The largest hyaluronan fragment an octamer waschemically synthesized through controlledaddition of disaccharide units [8]

2003Research on the enzymatic synthesis ofhyaluronan and monodisperse hyaluronanoligosaccharides with defined length [9 10]

substrates The number of repeat disaccharides 119899 in a com-pleted hyaluronan molecule is approximately 10000 butchains of hyaluronan with up to 25000 disaccharide unitshave been reported The size of the polymers (119872

119908from

5000Da to 20 million Da) in vivo varies with the type oftissue For example hyaluronan in the human umbilical cordhas a molecular mass of 3-4 million Da whilst the molecular

OH

O

NH

C

119899

OH

OH

O

O

OHOO

O

O

HO

Glucuronic acid 119873-Acetylglucosamine

Figure 1 Repeating unit of hyaluronan

mass of hyaluronan from human synovial fluid is 6 mil-lion Da However the reported molecular weight of isolatedhyaluronan polymers may be underestimated depending onthe isolation and analysis method used Hyaluronan is notmonodisperse in molecular weight and the experimentallydetermined polydispersity (119872

119908119872119899) of the polymer depends

as well on both the method of extraction and the method ofanalysis used For example the values of the polydispersityof a hyaluronan sample determined by high-performanceliquid chromatography varied with the type of column thedetection method and the flow rate used [11ndash13] This resultsin a big variation of the values reported for the 119872

119908and

polydispersity of hyaluronan between laboratories and showsthe importance for standardization of the methodologyfor characterization and reporting of hyaluronan molecularproperties

Hyaluronan is present in all vertebrates High concen-trations are found in the umbilical cord the synovial fluidbetween joints skin and the vitreous body of the eye Itwas estimated that in the body of a person of 70 kg about15 g hyaluronan is found in different tissues of which onethird is turned over every day The human skin containsover 50 of the hyaluronan in the body [14 15] Roostercombs contain the highest concentrations of hyaluronan(75mg gminus1) ever reported for animal tissues [16] Hyaluronanis also present in the capsule of a small number of micro-bial pathogens such as Pasteurella multocida and group Aand C streptococci among which Streptococcus pyogenes (ahuman pathogen) and the animal pathogens Streptococcusequi and Streptococcus uberis that quite likely pirated theenzymatic machinery from vertebrate hosts for its synthesisThese microorganisms use hyaluronan to encapsulate theircells forming a perfect disguise against the animal defensesystem and facilitating the adhesion and colonization of thebacterial cells [17] Since the hyaluronan polymer producedin animals and the aforementioned bacteria is identicalthe host immune defense is not triggered to repel thepathogenic bacteria contrary to other bacteria with a differentextracellular capsule Intriguingly the nonimmunogenic andnoninflammatory properties of bacterial hyaluronan benefitmammals currently too since bacterial hyaluronan formsan excellent source for medical grade hyaluronan Initiallyhyaluronan was believed to be an inert compound havingno specific interaction with other macromolecules However




Biomedical















119908from 400








(1)





Enzymes from



hyaluronan

Extraction from



Streptococci














Class II

Members








Peptidoglycan

Hyaluronan

Glycolysis

Glucose


Fructose-6-P

Glucosamine-6-P

Glucosamine-1-P


Glucose-6-P

Glucose-1-P


UDP-glucuronic acid



(Mutase)

(Acetyltransferase)

(Pyrophosphorylase)



hasB

Pgm GlmS

GlmM

hasD

hasD

hasE















119908up to






Enzymatic synthesis
































119908hyaluronan



















(2)










Abbreviations



References




































































































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




Biomedical















119908from 400








(1)





Enzymes from



hyaluronan

Extraction from



Streptococci














Class II

Members








Peptidoglycan

Hyaluronan

Glycolysis

Glucose


Fructose-6-P

Glucosamine-6-P

Glucosamine-1-P


Glucose-6-P

Glucose-1-P


UDP-glucuronic acid



(Mutase)

(Acetyltransferase)

(Pyrophosphorylase)



hasB

Pgm GlmS

GlmM

hasD

hasD

hasE















119908up to






Enzymatic synthesis
































119908hyaluronan



















(2)










Abbreviations



References




































































































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Enzymes from



hyaluronan

Extraction from



Streptococci














Class II

Members








Peptidoglycan

Hyaluronan

Glycolysis

Glucose


Fructose-6-P

Glucosamine-6-P

Glucosamine-1-P


Glucose-6-P

Glucose-1-P


UDP-glucuronic acid



(Mutase)

(Acetyltransferase)

(Pyrophosphorylase)



hasB

Pgm GlmS

GlmM

hasD

hasD

hasE















119908up to






Enzymatic synthesis
































119908hyaluronan



















(2)










Abbreviations



References




































































































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Peptidoglycan

Hyaluronan

Glycolysis

Glucose


Fructose-6-P

Glucosamine-6-P

Glucosamine-1-P


Glucose-6-P

Glucose-1-P


UDP-glucuronic acid



(Mutase)

(Acetyltransferase)

(Pyrophosphorylase)



hasB

Pgm GlmS

GlmM

hasD

hasD

hasE















119908up to






Enzymatic synthesis
































119908hyaluronan



















(2)










Abbreviations



References




































































































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






119908up to






Enzymatic synthesis
































119908hyaluronan



















(2)










Abbreviations



References




































































































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




















119908hyaluronan



















(2)










Abbreviations



References




































































































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of








119908hyaluronan



















(2)










Abbreviations



References




































































































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of















(2)










Abbreviations



References




































































































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of









Abbreviations



References




































































































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






























































































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



























































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

Documents

Review Article Production Methods for Hyaluronandownloads.hindawi.com/journals/ijcc/2013/624967.pdf · it hyaluronic acid [ ]. s s Hyaluronan from many di erent tissues of vertebrates