1

CHAPTER 1

INTRODUCTION

1.1 ENZYMES FOR INDUSTRY

The nature’s only catalysts are considered to be the enzymes (Fariha

Hasan et al 2005). Enzymes offer the advantages of mild conditions of

reactions, specificity, and reduced wastage of chemicals. It is required in

0.1 % - 1.0 % of the substrate, minimal waste treatment cost and contribution

to the BOD, and the industries utilizing enzymes in their processes require

less cost to set up the facility. Most of the enzymes are from bacterial origin

and are very useful and stable than plant or animal origin. The advantages of

bacterial enzymes are consistent quality and availability throughout the year,

unlike the seasonal variation in plant sources. A total of only 2 % of the

microorganism in the world have been tested as enzyme sources. The

bacterial origin offer very high activities of enzymes, mostly are of neutral or

alkaline pH optimum, and thermostable. The catalytic properties of enzymes,

secreted by microorganisms, in the production of food products like cheese,

sourdough, vinegar, and in leather making, linen etc., are in use from 18th

century. The production of these microbial enzymes is cheaper, easier and

safer to produce by fermentation route and with the advent of recombinant

DNA technology it is produced with better or improved properties. The

recombinant gene technology offers many benefits to the enzyme industry.

This technology allows the use of safe, well documented organisms that are

easy to grow in fermentors, enhance the productivity, purity, activity and

stability. Genetic and environmental manipulation to increase the yield of

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cells (Demain 1971), to increase the enzyme activity of the cells by making

the enzyme of interest constitutive, or by inducing it, or to produce altered

enzymes (Betz et al 1974), may be employed easily using microbial cells

because of their short generation times, their relatively simple nutritional

requirements, and since screening procedures for the desired characteristic are

easier. The advent of recombinant DNA technology has led to

commercialization of many enzymes which could not be previously produced

in large quantities. The lowering of economic activity world-wide has not

crippled the pharmaceutical and biocatalyst enzymes industry. The increased

demand on industrial enzymes is focussed to reach $7 billion by the end of

2013. A new area of biocatalysis which is expanding dramatically is the

biotransformation for organic and fine chemical synthesis. Lipids form a

major composition of the earth’s biomass and to breakdown and transfer of

these lipids, lipolytic enzymes play a major role. Lipases are the principal

enzymes that are at the forefront of this development and are being used to

resolve the racemic mixtures, synthesis of chiral building blocks for

pharmaceuticals, agrochemicals and pesticides.

1.2 LIPASES - OCCURRENCE AND INDUSTRIAL

IMPORTANCE

Lipases (triacylglycerol hydrolases EC 3.1.1.3) are fat-splitting

‘ferments’ (Verger 1997) or lipid-hydrolysing enzymes that catalyse the

hydrolysis of water-insoluble esters of glycerol and long chain fatty acids.

The credit to the discovery of lipase goes to Claude Bernard, who in 1856 was

the first to report the existence of lipase in pancreatic juice which hydrolyzed

insoluble oil droplets into soluble products. The definition of lipase is just not

a carboxy - esterase that hydrolyses acyl glycerols but it is defined in terms of

kinetic terms based on interfacial phenomenon as a highly efficient enzyme

hydrolyzing substrates having a carboxylic ester group aggregated in water,

the property distinguishing lipases from esterases. These enzymes occur

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widely in nature and are found in most organisms from the microbial, plant

and animal kingdoms. The interest in microbial lipases was developed due to

a shortage of pancreas and the difficulties in the collection methods of other

sources of lipases. However lipases from the microbial flora comprising

bacteria, fungi and yeast have a wide-ranging commercial significance (Jaeger

and Reetz 1998) and the factors contributing to their industrial importance are

that they are i) stable in organic solvents, ii) do not require cofactors,

iii) possess a broad substrate specificity and iv) exhibit a high

enantioselectivity. The synthesis of biopolymers and biodiesel,

pharmaceutical products with enantiopurity, agrochemicals and flavour

compounds are the novel biotechnological applications that are carried out

with lipases. The Table 1.1 summarizes the sources of triacylglycerol lipases.

Table 1.1 Sources of triacylglycerol lipases

Sources Name

Mammalian Human Pancreatic Lipase

Horse Pancreatic Lipase

Pig Pancreatic Lipase

Guinea Pig Pancreatic Lipase

Fungal Rhizomucor meihei

Pencillium cambertii

Humicola lanuginose

Rhisopus oryzae

Candida rugosa

Candida antarctica lipase A

Candida antarctica lipase B

Aspergillus niger

Geotrichium candidum

Bacterial Chrombacterium viscosum

Pseudomonas cepacia

Pseudomonas aeruginosa

Pseudomonas fluorescens

Pseudomonas fragi

Bacillus thermocate nulatus

Staphylococcus hyicus

Staphylococcus aereus

Staphylococcus epidermidis

Courtesy: TIBTECH (1998)

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1.3 BIOCHEMICAL FEATURES OF LIPASES

The lipase family of enzymes exhibits a high degree of structural

and functional similarity. The three dimensional structure of lipases belong to

a characteristic folding pattern known as the hydrolase fold (Schrag and

Cygler 1993). The lipase structure is composed of a core of upto eight parallel

beta strands, connected and surrounded by alpha helices. The active site is

formed by a catalytic triad consisting of a serine residue as the nucleophile,

histidine as base and aspartic (or glutamic) acid as the acidic residue. The

active site residues are placed inside a hydrophobic pocket termed as

‘nucleophilic elbow’ and the pocket is covered by a lid like structure,

composed of one or two amphiphillic alpha helices. The activation of lipases

requires the opening up of the pocket by the displacement of the lid and this

process is known as interfacial activation

The catalytic mechanism of lipase hydrolysis consists of four

subsequent steps: i) the absorption and activation of the lipase at the interface

between aqueous and organic phase and then binding of the ester substrate

within the hydrophobic pocket; ii) in the second step, the nucleophilic oxygen

of the serine side chain attacks the carbonyl carbon atom of the ester bond

leading to the formation of a tetrahedral intermediate and this is stabilized by

one or two hydrogen bonding with amide nitrogen atom of the amino acid

residues located in the region called as ‘oxyanion hole’. iii) The ester bond is

then cleaved liberating an alcohol and leaves behind the acyl-enzyme

complex. In the last step, the acyl-enzyme is hydrolysed, when a water

molecule (sometimes another alcohol) enters the active site, thereby liberating

the free fatty acid (or a new trans-ester with the alcohol) and the enzyme is

regenerated. Example of lipase catalysis mechanism is given in Figure 1.1.

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Figure 1.1 Mechanism of lipase catalysis

Courtesy: Mats Holmquist (2000)

A wide range of assay protocols have been developed for the

estimation of lipase activity (Beisson et al 2000). Based on the general

triacylglycerol hydrolysis reaction, the lipase activity is assayed by the release

of either fatty acids or glycerol from triacylglycerols or fatty acid ester.

Various lipase assay methods and its principle are given in the Table 1.2.

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Table 1.2 Screening and analytical assays for lipase activity

Method and Materials Principle Remarks

i) Plate assay:Agar plates supplemented withTAGs such as tributyrin andtriolein (olive oil).

Lipolysis of TAGs producesa clear halo or colour

change of Phenol Red / Victoria Blue/ Nile Blue Sulphate, or fluorescencewith Rhodamine B under UV lightdue to dye-FFAs complexation.

Convenient for rapid screeningbut false-positives occur due tomedium acidification fromacidic metabolites

ii) Titrimetry:

pH stat (emulsified TAGs).

Simple titration using fats andoils, TAGs with / without pHindicators.

Potentiometric determination ofFFAs liberated upon hydrolysis.

Neutralization reaction of FFAs withNaOH to constant end-point pH.

Can generate kinetic constantsand needs expensive equipment.

Process is extremely timeconsuming and laborious.

iii) Spectrophotometry:Carboxylic esters of p-nitrophenol or 2, 4 dinitro- phenol.

Colorimetry: Fatty acidconjugates of -naphthol.

Turbidimetry: Tweens

Estimation of the hydrolysed yellow-coloured p-nitrophenol at 420 nm and2, 4-dinitrophenol at 360 nm.

Red colour of -naphthol monitoredat 505 nm, after complexation withdiazonium salt.

Precipitation of FFAs with calciumor copper and measurement ofturbidity at 500 nm.

Very simple procedure butassayed only for relatively purelipases.

The esters are not stable atextreme pH and are not lipasespecific substrates.

Simple, reproducible, sensitiveand generally used for plateassays

Synthetic dilauryl glycerolresourfin ester

Resourfin released by lipasehydrolysis, measured for absorbanceat 575 nm.

Not straightforward to use sinceit requires separation of reactantsand products.

iv) Fluorescence:Fluorogenic substrates such as

–linked pyrenic acyl-glycerolderivatives.

Non-flourescent 4-methylumbelliferyl oleate.

Lipolytic activity quantified in termsof increasing fluorescence intensitywith time.

Highly fluorescent 4 - methylumbelliferone released after lipaseaction and is therefore quantified.

Rapid assay but expensivesubstrates limit its usage.

Most sensitive assay butexpensive substrates.

v) Chromatographic procedures:TLC / GC / HPLC, and usingradiolabelled TAGs incase ofquantitative analysis

Analysis and quantification of theproduct or residual substrate throughspecific columns.

Time-consuming and not suitedfor routine analysis.

vi) Interfacial tensio-metrymethods

Monomolecular film technique:Wilhelmy Plate method

Atomic forcemicroscopy:Infrared spectroscopy:

Lipolysis at lipid / water interfaceand so interfacial surface pressureregulated by substrate supply.

TAG bilayers supported on micahydrolyzed by lipases and regions ofthese indentations detected andstudied as function of time with AFMtip.

Lipase activity of TAGs in reversemicelles, monitored by recordingfourier-transform IR spectroscopyand products quantified using themolar extinction coefficients.

Extensive setup but providesprecise kinetic data.

Suited for kinetic modeling oflipase action but requiressophisticated instruments.

Expensive and sophis-ticatedinstruments involved.

Courtesy: Biotechnol. Appl. Biochem (2003)

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1.4 CANDIDA ANTARCTICA LIPASES

Among the widely used enzymes for biocatalytical purposes are the

lipases produced by different strains of genus Candida sp. In the late 1960’s

the yeast strain Candida antarctica was isolated from Lake Vanda in

Antarctica and was found to produce two different lipases (CALA and

CALB). The lipases produced by Candida antarctica are stable when

immobilized and the enzymes retain their activity at higher temperatures for

longer durations without significant loss in the enzyme activity. The two

lipases of C. antarctica were characterized and the amino acid and the DNA

sequences encoding these lipases were sequenced at Novo Nordisk A/S, by

Patkar, Hoegh and others. The lipase B was later crystallized and its structure

was also determined by Uppenberg et al (1994).

C. antarctica lipase B is found to exhibit varying physio-chemical

characteristics and is summarized in Table 1.3 (Kirk and Christensen 2002).

The lipase B has become one of the most prominent enzymes, in organic

synthesis, especially for the kinetic resolution of racemates. Currently, lipase

B is the widely targeted enzyme for protein engineering so as to improve and

optimize its substrate specificity and enantioselectivity (Lutz 2004).

Table 1.3 Characteristics of CALB

Characteristics CALB

Molecular weight (kDa)

Isoelectric point (pI)

pH optimum

Thermostability at 70°C

pH stability

33

6.0

7

15

7 - 10

Courtesy: Ole Kirk and Wurtz Kristensen (2002)

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As both C. antarctica lipases have gained significant commercial

importance and as the expression levels in the native organism are too low,

recombinant over-expression is needed for the large-scale production of these

biocatalysts.

1.5 PICHIA PASTORIS RECOMBINANT EXPRESSION SYSTEM

In the production of recombinant proteins, yeasts have offered

advantages over both bacterial and mammalian systems. The eukaryotic yeast

offers the capacity to post-translationally modify the secreted protein and to

glycosylate at lesser costs incurred in using mammalian system for

expression. The first yeast selected for production of heterologous eukaryotic

proteins was Saccharomyces cerevisiae, due to the accumulated knowledge of

its genetics and physiology. It is generally regarded as safe (GRAS) for

human use through experience with the organism in brewing and baking

industry. However, in the 1970s, the methylotrophic yeasts gained significant

importance, due to its rather unique ability to anabolise methanol to very high

cell mass and was used as a cattle feed as single cell protein. Later in the

following decade, Phillips Petroleum, together with Salk Institute

Biotechnology / Industrial Associates (SIBIA, CA) developed the

methylotrophic yeast Pichia pastoris into an efficient recombinant protein

production system. Pichia pastoris belonging to the yeast family are easy to

manipulate genetically as E. coli or Saccharomyces cervisiae. They have a

relatively high growth rate than higher eukaryotes and can be grown to higher

cell densities than bacteria, producing a 10 – 100 fold higher heterologous

protein.

Pichia pastoris has become an ideal host for the expression of

recombinant proteins (Cereghino and Cregg 2000) due to the contribution of

following factors: it can be easily manipulated at the molecular genetic level

(e.g. gene targeting, high-frequency DNA transformation, cloning by

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functional complementation); it can express proteins at high levels due to its

highly efficient and tightly regulated promoter from the alcohol oxidase I

gene (AOX1); and it can perform many ‘higher eukaryotic’ protein

modifications, such as glycosylation, disulfide-bond formation, and

proteolytic processing.

1.5.1 Genetics and Properties

P. pastoris, a methylotropic yeast is one of approximately a dozen

yeast species representing four different genera that are capable of

metabolizing methanol as its sole carbon source. The other genera include

Candida , Hansenula and Torulopsis. The methanol utilization pathway, of

these microbial genera is highly inducible and involves several unique

enzymes that take place initially in the peroxisomes where methanol is

oxidized to formaldehyde and hydrogen peroxide using molecular oxygen

from the AOX1 gene (Tschopp et al 1987b). The subsequent metabolic steps

take place in the cytoplasm. The first enzyme of the pathway, alcohol oxidase

(AOX1 and AOX2), can account for up to 35 % of the total protein in cells

when grown on methanol, whereas it is undetectable in cells grown on

glucose, glycerol or ethanol (Sreekrishna et al 1997). This highly inducible

and stringently regulated alcohol oxidase gene (AOX1) promoter has been

used to express heterologous proteins in P. pastoris.

There are three phenotypes of P. pastoris strains with regard to

methanol utilization and these phenotypic characteristics are an important

factor during P. pastoris cultivation process and protein production

(Macauley-Patrick et al 2005). The methanol utilization plus phenotype, or

Mut+ grows on methanol at the wild-type rate and require high feeding rates

of methanol in large-scale fermentations. A maximum of 3 % methanol is

used to express protein cloned in Mut+ strains. The MutS, or methanol

utilization slow phenotype, have a disruption in the AOX1 gene. The cells

then rely on the weaker secondary alcohol oxidase gene AOX2 for methanol

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metabolism and slower methanol utilization strain is produced. A maximum

of 1 % methanol is used to express protein cloned in MutS strains. The Mut ,

or methanol utilization minus phenotype, are unable to grow on methanol,

since these strains have both AOX genes deleted. Deletion of AOX genes does

not affect the strains ability to induce expression at high levels from the AOX1

promoter (Chiruvolu et al 1997). The different Mut phenotypes have

exhibited varied effects for the production of recombinant proteins. The lower

growth and protein production of MutS on methanol is particularly preferable

for production of recombinant proteins where the folding is rate-limited.

Though Mut+ expression is higher than that with cells of the MutS phenotype

when higher methanol concentrations were used, the former strains may get

oxygen limited at high methanol concentrations, leading to cell lysis.

Recombinant P. pastoris strains are obtained by transforming the

host strains with the constructed plasmid, which on electroporation gets

integrated into the chromosome at a specific locus and generates a genetically

stable transformants / clone. Chromosomal integration is highly desirable than

the use of episomal plasmid expression systems as episomal plasmids tend to

have low copy number, which affects the amount of product expressed.

Integration into the genome can occur via homologous recombination when

the vector / expression cassette contains regions that are homologous to the

P. pastoris genome and the integration can occur via gene insertion or gene

replacement. Integration by gene insertion can result in tandem multiple

integration events due to repeated recombination events at a rate of 1 – 10 %

of transformants. Integrations are usually site-directed at the HIS4 gene or at

the primary alcohol oxidase (AOX1) locus. Transformations that target gene

replacement generally result in single copy transformants; however, gene

replacement transformants are usually more genetically stable. Figure 1.2 and

Figure 1.3 illustrate the gene integration and gene replacement event that

occur in Pichia genome by homologous recombination.

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Figure 1.2 Integration of expression cassette by gene replacement in

Pichia genome

Figure 1.3 Integration of expression cassette by gene insertion in Pichia

genome

(Courtesy: Invitrogen life technologies, Multi-Copy Pichia Expression Kit, Manual,

p. 63, p. 66)

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1.5.2 P. pastoris AOX system

Methylotrophic yeast, Pichia pastoris can utilize methanol as the

sole source of carbon and energy (Figure 1.4). Methanol induces a specific

methanol utilization pathway leading to expression of key enzymes under the

control of tightly regulated promoters. One of these key enzymes, alcohol

oxidase (AOX), catalyses the oxidation of methanol to formaldehyde and

hydrogen peroxide. Since hydrogen peroxide is very toxic to the cells, the

reaction takes place in specialized organelles called peroxisomes, where

hydrogen peroxide is degraded into oxygen and water by the activity of the

enzyme catalase (Cereghino et al 2000). AOX is encoded by two genes in

P. pastoris namely, AOX1 and AOX2, former being responsible for the

majority of alcohol oxidase activity in the cell (Cregg et al 1989). The AOX1

gene expression is controlled at the level of transcription and the presence of

methanol is essential to induce high levels of transciption. 5 % of RNA

isolated from methanol grown cells is from the AOX1 gene, whereas AOX1

message is undetectable in cells grown on any other carbon source (Tschopp

et al 1987a). In a bioreactor, cultures with methanol feeding at growth

limiting rates, AOX1 transcription levels can be as high as 30 % of total

soluble protein. Like the S. cerevisiae GAL1 gene, AOX1 gene is under the

control of two mechanisms, repression / derepression and induction. The

repressing carbon source can be any other carbohydrate other than methanol,

does not result in transcription of AOX1 (Higgins and Cregg 1998).

1.5.3 P. pastoris expression strains

Pichia pastoris strains are now available with a wide variety of

genotypes. All P. pastoris expression strains are derived from NRRL - Y

11430 (Northern Regional Research Laboratories, Peoria, IL). The strains

have one or many auxotrophic mutations which help in selection of

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Figure 1.4 The methanol pathway in P. pastoris

Courtesy: Michael M. Meagher (2000)

1:alcohol oxidase, 2: catalase, 3: formaldeyde dehydrogenase, 4:

formate dehydrogenase, 5:dihydroxyacetone synthase,

6:dihydroxyacetone kinase, 7: fructose 1,6-biphosphate

aldolase, 8: fructose 1,6-biphosphatase (Cereghino et al 2000).

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expression vectors having the selectable marker gene when transformed. All

of these strains with the mutations grow on complex media but require

supplementation with the appropriate nutrient(s) like histidine for growth on

minimal media after transformation.

P. pastoris strains KM71, GS115 and SMD1168 are defective in the

histidine dehydrogenase gene (his4). These strains allow the transformants to

be selected based on their ability to grow in non - histidine containing agar

media. The Pichia strains X–33 and GS115 are differentiated from other

strains based on their ability to utilize methanol as they have a functional

copy of the alcohol oxidase 1 gene (AOX1) responsible for approximately

85 % of the utilization of methanol by the alcohol oxidase enzyme and are

designated as Mut+ phenotype. P. pastoris strains KM71 and KM71H have a

partial insertion in AOX1 gene and thus rely only on AOX2 for methanol

utilization. This strain grows slower than wild type strains on methanol

showing MutS phenotype (methanol utilization slow phenotype).

The Pichia strains such as SMD1163 (his4 pep4prb1), SMD1165

(his4 prb1) SMD1168 (his4 pep4) are protease deficient strains, and they have

been shown to be effective in reducing degradation of some foreign proteins

(Brierley et al 1998, White et al 1995). These strains have a disruption in the

genes encoding proteinase A (PEP4) and / or proteinase B (PRB1)

(Sreekrishna et al 1997). Proteinase A is a vacuolar aspartyl protease

necessary for the activation of vacuolar proteases, such as carboxypeptidase Y

and proteinase B. Proteinase B has about half the activity of the processed

enzyme before being activated by proteinase A. Therefore, pep4 mutants

eliminate the activity of proteinase A and carboxypeptidase Y, and partially

reduce proteinase B activity. The prb1 mutants eliminated activity of

proteinase B, whereas pep4 prb1 double mutants showed a significant

reduction or elimination of all three of these protease activities (Jahic et al

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2003). Various P. pastoris strains used for heterologus protein production are

given in Table 1.4 with their features.

Table 1.4 Genotype and Phenotype of P. pastoris strains

Strain Genotype Phenotype Reference

GS115 his4 Mut+, His- (Cregg et al 1985)

KM71his4, aox1:

ARG4;arg4Muts, His-

X-33 Wild type — (Li et al 2001)

SMD1168 His4, pep4 Mut+, His-, pep4- (White et al 1995)

SMD1165 his4, prb1 Mut+, His-, prb1- (Abdulaev et al 1997)

SMD1163his4, prb1,

pep4

Mut+, His-,

pep4, prb1- (Sreekrishna et al 1997)

(Courtesy: Invitrogen life technologies, Multi-Copy Pichia Expression Kit, Manual)

1.5.4 Alternative promoters

Although the AOX1 promoter has been successfully used to express

numerous foreign genes, there are circumstances in which this promoter may

not be suitable. Alternative promoters to the AOX1 promoter are the

P. pastoris GAP, FLD1, PEX8, and YPT1 promoters. P. pastoris

glyceraldehyde 3 - phosphate dehydrogenase (GAP) gene promoter provides

strong constitutive expression on glucose at significant level (Waterham et al

1997). Since the GAP promoter is constitutively expressed, it is not a good

choice for the production of proteins that are toxic to the yeast. The FLD1

gene encodes a glutathione - dependent formaldehyde dehydrogenase, a key

enzyme required for the metabolism of certain methylated amines as nitrogen

sources and methanol as a carbon source (Shen et al 1998). The FLD1

promoter can be induced with either methanol as a sole carbon source (and

ammonium sulfate as a nitrogen source) or methylamine as a sole nitrogen

source (and glucose as a carbon source). The FLD1 promoter offers the

flexibility to induce high levels of expression using either methanol or

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methylamine, an inexpensive nontoxic nitrogen source. For proteins where

there are limitations in the post translational machinery, the P. pastor is PEX8

and YPT1 promoters may be of use. The PEX8 gene encodes a peroxisomal

matrix protein that is essential for peroxisome biogenesis (Sears et al 1998). It

is expressed at a low but significant level on glucose and is induced modestly

when cells are shifted to methanol. The YPT1 gene encodes a GTPase

involved in secretion, and its promoter provides a low but constitutive level of

expression in media containing glucose, methanol, or mannitol as carbon

sources

1.5.5 Glycosylation

Glycosylation is the most common post - translational modification

of proteins prior to protein secretion. Approximately 0.5 – 1.0 % of the

translated proteins in eukaryotic genomes are glycoproteins. Glycosylation

occurs in the lumen of the endoplasmic reticulum after protein translation. It

is thought that since many mammalian native proteins are glycosylated, it

must be necessary to have the correct glycosylation patterns on recombinant

proteins to ensure their biological activity. In yeast the glycosylation pattern is

different that of mammalian systems. In N - linked glycosylation of proteins

the mannose units added are in excess compared to mammalian systems.

Since the glycosylated gene products from P. pastoris generally have much

shorter of 10 to 12 mannose subunits than those expressed in S. cerevisiae

which will have 20 – 50 mannose units, it is much more attractive host for the

expression of recombinant proteins (Bretthauer et al 1999). P. pastoris is

capable of forming both O - and N - linked glycosylation to the secreted

proteins (Goochee et al 1991). Pichia - derived glycosylated proteins have the

potential to trigger inappropriate immune responses if used as

pharmaceuticals. The immunogenicity of Pichia - derived proteins is an issue

that has attracted interest in the literature with regard to humanizing the

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glycosylation patterns (Asami et al 2000, Choi et al 2003). Candida

antarctica lipase B gene was cloned in E. coli and in Pichia pastoris and the

function of non glycosylated lipase B produced is similar to that of wild type

Pichia pastoris, indicating glycosylation is not required for the activity (Gaub

et al 2006, Hult et al 2008).

1.5.6 Gene dosage

Gene copy number has been identified as a ‘rate - limiting’ step in

the production of recombinant proteins from P. pastoris (Clare et al 1991).

Increasing the number of copies of the expression cassette generally has the

effect of increasing the amount of protein expressed (Clare et al 1991,

Romanos et al 1995, Vassileva et al 2001).

A number of researchers have had significant success with

increasing gene dosage utilizing both the AOX1 and GAP promoters (Clare

et al 1991, McGrew et al 1997, Vassileva et al 2001), therefore, before

considering increasing the gene copy number as an optimization strategy for

recombinant protein production from P. pastoris, the identity of the promoter

must be considered in advance. Depending on the type of protein, upto certain

gene copy number there is proportional effect in the recombinant protein

production after which there is no increase in productivity. Increasing the

gene copy number might reasonably be expected to exert a knock - on effect

on transcription and translation, both of which may become rate - limiting due

to a lack of resources, such as precursors and energy (Hohenblum et al 2004).

However, in Pichia it has been proposed that it is more likely that any

limitations are due to post - translational events, such as folding within the

endoplasmic reticulum, membrane translocation and signal sequence

processing.

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1.5.7 Codon Usage

In most of the expression studies in P. pastoris the gene containing

higher percentage of AT rich regions are poorly expressed due to improper

transcription termination of mRNA. For better expression, these genes have to

be modified with respect to Pichia preferred codon. The frequency of codon

usage from highly expressed proteins is given in Table 1.5. The optimal GC

content for good expression of protein is around 40 - 45 % and this is

achieved by altering the codon to most Pichia preferable one.

1.5.8 Protein secretion

One of the most valuable option in recombinant Pichia pastoris

expression system is the availability of secretion signals that can be flanked to

the protein of interest, causing the protein to be secreted out of the cell into

the medium used for growth.

A variety of secretory signal sequences are used such as the

P. pastoris acid phosphatase signal (PHO), yeast invertase signal (SUC2)

(Chang et al 1986; Payne et al 1995), factor secretion signal etc, (including

native signals sequence present in the parent organism to secrete the protein)

have been used with success. During the recombinant protein expression, the

endopeptidase sequence (Glu-Lys-Arg*) separates the signal sequence from

the protein coding gene and the yeast Kex2 signal peptidase cleaves the signal

peptide at this sequence and the mature protein is secreted in the culture

medium. Each signal has its particular advantage and there is no common rule

by which the most effective sequence can be used. The S. cerevisiae MAT

prepro signal peptide has found most success and is the one of most

frequently incorporated into P. pastoris expression vectors (Sreekrishna et al

1997). The factor, a yeast peptide pheromone consisting of 13 amino acid

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Table 1.5 Frequency of codon usage in highly expressed Pichia pastoris

genes

Amino acid Codon Number Fraction Amino acid Codon Number Fraction

Gly GGG 0.00 0.00 Trp TGG 39.00 1.00

Gly GGA 59.00 0.22 End TGA 0.00 0.00

Gly GGT 197.00 0.74 Cys TGT 35.00 0.83

Gly GGC 9.00 0.03 Cys TGC 7.00 0.17

Glu GAG 112.00 0.58 End TAG 1.00 0.20

Glu GAA 80.00 0.42 End TAA 4.00 0.80

Asp GAT 56.00 0.32 Tyr TAT 18.00 0.12

Asp GAC 118.00 0.68 Tyr TAC 128.00 0.38

Val GTG 10.00 0.05 Leu TTG 120.00 0.52

Val GTA 8.00 0.04 Leu TTA 21.00 0.09

Val GTT 107.00 0.50 Phe TTT 24.00 0.19

Val GTC 87.00 0.41 Phe TTC 104.00 0.81

Ala GCG 1.00 0.00 Ser TCG 6.00 0.03

Ala GCA 25.00 0.10 Ser TCA 14.00 0.07

Ala GCT 147.00 0.60 Ser TCT 89.00 0.47

Ala GCC 71.00 0.29 Ser TCC 71.00 0.37

Arg AGG 2.00 0.01 Arg CGG 2.00 0.01

Arg AGA 111.00 0.79 Arg CGA 0.00 0.00

Ser AGT 8.00 0.04 Arg CGT 26.00 0.18

Ser AGC 3.00 0.02 Arg CGC 0.00 0.00

Lys AAG 145.00 0.79 Gln CAG 31.00 0.34

Lys AAA 38.00 0.21 Gln CAA 59.00 0.66

Asn AAT 18.00 0.13 His CAT 11.00 0.13

Asn AAC 119.00 0.87 His CAC 77.00 0.88

Met ATG 60.00 1.00 Leu CTG 35.00 0.15

Ile ATA 0.00 0.00 Leu CTA 7.00 0.03

Ile ATT 93.00 0.56 Leu CTT 43.00 0.18

Ile ATC 72.00 0.44 Leu CTC 7.00 0.03

Thr ACG 5.00 0.03 Pro CCG 0.00 0.00

Thr ACA 8.00 0.05 Pro CCA 97.00 0.57

Thr ACT 86.00 0.50 Pro CCT 66.00 0.39

Thr ACC 74.00 0.43 Pro CCC 7.00 0.04

Courtesy: http://www.kazusa.or.jp/codon/

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residues plays an important role in the process of mating initiation. It is

known that factor is initially synthesized as a larger precursor,

prepro- -factor (Brake et al 1984). Prepro factor consists of a signal

sequence, prosegment and the repeats of spacer peptides followed by mature

factor sequences. This leader sequence is popular for its high level of

expression in the yeast system (Brake et al 1984, Hashimoto et al 1998a, b).

In certain cases the recombinant protein expression have been tried with its

native signal sequence of the protein and found to produce more stable

recombinant protein than using factor signal sequence (Koganesawa et al

2001). The Saccharomyces cerevisiae secretion signal peptide ( -factor) was

used with AOX1 promoter for the production of Yarrowia lipolytica and very

high activity was produced in fed – batch cultivation (Tan et al 2007). This

showed the possibility that there is some influence in the secretion of

recombinant protein with its secretion signal sequence.

1.5.9 Fermentation of Pichia pastoris

A property of the P. pastoris system is the ease with which

expression strains can be scaled - up from shake flask to high cell density

fermenter cultures. Efforts have gone into the optimization for the production

of heterologous proteins which have led to availability of detailed fed-batch

and continuous culture protocols (Stratton et al. 1998). The advantage of

P. pastoris relative to S. cerevisiae is that it prefers a respiratory pathway

rather than a fermentative mode of growth. In high cell density cultures of

S. cereviseae, ethanol (the product of S. cerevisiae anaerobic fermentation)

rapidly builds to toxic levels which limit further growth and foreign protein

production. Since Pichia pastoris prefers respiratory growth, it can be

cultured to high cell densities (500 - 600 OD600) in the controlled environment

of the fermenter with little risk of `pickling' itself. Most importantly, it is only

21

in fermenter, where parameters such as pH, aeration and carbon source feed

rate can be controlled, that it is possible to achieve high expression of foreign

protein.

The high salts / high cell density fermentation protocol was first

reported by Brierley et al (1990) using P. pastoris which expressed bovine

lysozyme C2 as a model system and resulted in an expression of up to

600 mg/L of the recombinant lysozyme. Currently, the three-stage process is

typically utilized for the production of foreign proteins in fermenter cultures

of P. pastoris. In the first stage, the recombinant strain is batch-cultured in a

simple defined medium with a non-fermentable carbon source such as

glycerol to accumulate biomass. The second stage is a fed - batch transition

phase in which glycerol is fed to the culture at a growth - limiting rate to

further increase the biomass concentration and to prepare (derepress) the cells

for induction. The third stage, induction phase, is started by adding methanol

to the culture at a slow rate, which facilitates the culture’s acclimation to

methanol and initiates the synthesis of the recombinant protein. The methanol

feed rate is then adjusted upwards periodically until the desired growth rate is

reached.

The methanol feeding strategy during the induction phase is one of

the most important factors for maximizing heterologous protein production

(Cos et al 2006). Monitoring and control of methanol concentration is a key

parameter in P. pastoris expression system and involves different strategies

such as using on-line methanol sensors either for liquid or gas phase, by

dissolved oxygen level, or by constant feeding rate to optimize the induction

phase. Moreover the methanol induction phase also depends on the optimum

conditions (eg: temperature, pH and culture medium), phenotype and specific

characteristics of the heterologous protein product.

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The methanol feeding strategy differs with regard to the methanol -

utilising ability of P. pastoris strains. For the Mut+ strains, strategies based on

the pO2 of the culture have led to high yields of recombinant proteins with

respect to the amount of methanol fed and to the biomass formed (Invitrogen

2002). This method is based on modifying the methanol feeding rates in order

to avoid methanol exhaustion as indicated by a sharp increase in dissolved

oxygen. A rational feeding strategy proposed by Zang et al (2000) which is

based on the predetermined exponential rate to maintain a constant desired

growth rate has also shown to be more efficient in the recombinant protein

production. Generally it has been observed that when the cells are fed with

methanol at a growth-limiting rate, the induction of protein expression is

almost 3 to 5 times higher than in cells growing in excess of methanol (Trinh

et al 2003).

The fermentation strategy for MutS KM71 strain was the same as

with Mut+ GS115 strains. The methanol feed rate was lesser in order to

maintain methanol at a concentration between 0.2 % and 0.8 % in the

fermentor. The methylotrophic yeast strains when grown under limiting

concentrations of methanol leads to the formation of alcohol oxidase and

catalase in their peroxisomes. The breakdown product of methanol by alcohol

oxidase, hydrogen peroxide is degraded by catalase. The supplementation of

the media with hydrogen peroxide during induction phase along with

methanol increases oxidation of methanol (Duff 1990). However, in some

reports it was suggested that feeding multicarbon substrates in addition to

methanol show increased productivities (Zang et al 2003). The most used

co-substrate has been glycerol, but it is important to optimize the ratio of

methanol to glycerol in the substrate feeding rate, as excess glycerol represses

the heterologous protein production. Other carbon sources such as sorbitol,

mannitol, alanine and lactic acid have also been used as co-feeding substrates

(Xie et al 2005). These substrates have the advantage that they are non AOX1

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repressive carbon sources and have shown increased protein productivities but

their biomass yields are lower than glycerol.

A major problem that has hampered the protein productivities

during Pichia pastoris fermentations has been with proteolysis. The secreted

recombinant proteins are proteolytically degraded in the culture medium by

the proteases, secreted or released from the lysed cells (Sinha et al 2004). The

use of protease-deficient strains has been found to enhance the yield and the

quality of various heterologous proteins but these strains are not as vigorous

as wild strains and have lower viability (Cereghino and Cregg 2000).

Nitrogen limitations have also been found to increase protease activity

(Kobayashi et al 2000) and maintaining the ammonium requirements as well

as addition of amino-acid rich supplements (eg. peptone, casamino acids) to

the culture medium (Ohya et al 2002) has been observed to reduce

proteolysis.

Another major problem associated with the production of

recombinant proteins using Brierleys medium composition is that a lipid layer

is formed during fermentation, may be due to the increased lysis of cells. So

the fermentation strategy was shifted to reduced concentration of the medium

composition suggested by Brierley et al to half (Brady et al 1998). The

reduction of salt concentration reduced the lipid layer formation which

hindered in downstream processing.

The yields of some recombinant protein have also been influenced

by lower cultivation temperature (Hong et al 2002) and lowering of pH values

(Jahic et al 2002) possibly due to poor stability of these proteins, folding

problems at higher temperatures, and release of more proteases from dead

cells at higher pH. Therefore, achieving good expression levels using Pichia

pastoris recombinant expression systems involves an optimum combination

of the cultivation strategies.

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1.6 OBJECTIVES

The current work is an attempt to clone and express Candida

antarctica lipase B using recombinant DNA technology. The CALB has a

very broad range of substrate specificity and the main factor which limits the

industrial use of CALB is the high price of the immobilized enzyme product.

In an attempt to reduce the cost factor of the immobilized enzyme, different

strains of Pichia pastoris have been used for transformation. To attain this

goal the objectives of the current work was designed as follows:

Cloning of CALB in pPICZ B vector, transforming into Pichia

pastoris GS115 and KM71 strains and checking the level of

lipase activity.

Increasing the copy number of CALB in pPICZ B (Multicopy)

and pPIC9k (High copy), transforming into Pichia pastoris

GS115 strain and checking the level of lipase activity.

High cell density fermentation of all the Pichia clones to

maximize productivity of lipase B.