Behaviour of pectinases under process conditionsG.S.N. Naidu, T. Panda

Abstract Hydrolysis of pectic substances by pectinaseswere carried out under process conditions. Poly-methylgalacturonase, polygalacturonase and pectinlyaseare three enzymes studies in this regard. Pectin is used asthe substrate for polymethylgalacturonase and pectinlyasewhereas polygalacturonic acid was used as the substratefor polygalacturonase. The initial concentration was variedbetween 1.0 and 5.0 kg/m3 for polymethylgalacturonaseand polygalacturonase and between 4.0 and 8.0 kg/m3 forpectinylase. Hydrolysis experiments were carried out byvarying initial substrate to enzyme ratio and reducinggroups formed are measured. The behaviour of pectinasesduring hydrolysis are studied from the plot of (reducinggroups formed)/initial substrate vs. initial substrate toenzyme ratio. This study is helpful in predicting theamount of substrate and enzyme required to produce therequired amount of reducing groups within a stipulatedtime.

1IntroductionPectic substances and other polysaccharides contribute tothe ®rmness and structure of plant cells. Aspergillus nigeris used for industrial production of pectolytic enzymes.This fungus synthesizes polymethylgalacturonase (PMG),polygalacturonase (PG), pectinlyase (PL) and pectinester-ase (PE) [1].

Pectolytic enzymes play a vital role in food processingindustries, viz., in the production of fruit juices, softdrinks, and liquors [2]. These enzymes are also instru-mental in maceration, liquefaction and in extraction ofvegetables tissues [3±6]. They also help in reduction inviscosity of fruit pulp which in turn help ®ltration, clari-®cation of fruit juices and wood preservation [7±8]. Sincepectolytic enzymes are widely used in food processing andbeverage industries, it is necessary to study their perfor-mance during hydrolysis.

An extensive literature is available on the hydrolysis ofpectic substances by pectinases. Most of the work wasrelated to the characterization of pectinases, determination

of mode of enzymatic action and inhibition studies [9±11].In general, enzyme assays are carried out for short inter-vals of time. In practical situations, the enzymes are ex-posed for longer time duration. The rates of hydrolysisdecline with the time due to depletion of substrate,product inhibition and enzyme deactivation [12]. Hence,the study of performance of the enzymes under processconditions become necessary.

In this communication, we tried to understand the be-haviour of pectinases during hydrolysis of pectic sub-stances under process conditions.

2Materials and methods

2.1MicroorganismAspergillus niger NCIM 548 was obtained from the Na-tional Chemical Laboratory, Pune, India. The organismwas maintained on potato dextrose agar slants containing(kg/m3): potato, 200; dextrose, 25; agar, 20. Slants wereincubated at 30 °C for 72 h.

2.2ChemicalsGalacturonic acid (Sigma Chemical Co., St. Louis, MO,USA) was used as the standard for calculating activities ofPMG and PG. Polygalacturonic acid (Sigma Chemical Co.,St. Louis, MO, USA) was used as the substrate for PG. Theother chemicals of analytical grade were procured in India.

2.3Development of InoculumThe spores from a three day old culture were dispersedinto 10 cm3 of sterile water. One cm3 of this suspension,containing approximately 105±107 spores, was used as in-oculum to 500 cm3 Erlenmeyer ¯ask containing 100 cm3

production medium.

2.4Production of pectolytic enzymesSubmerged culture was used for the production of pecto-lytic enzymes. The medium was autoclaved at 121 °C for 20minutes and inoculated after cooling to 30 °C. The enzymeproduction medium contained (kg/m3): corn, 20.97; glu-cose, 34.6; KH2PO4, 2.0; and ammonium sulphate, 8.42[13]. The initial pH of the medium was maintained at 4.5.The culture was incubated at 30 °C for 6 days on a rotatoryshaker maintained at 160 rpm. The culture was harvested

Bioprocess Engineering 21 (1999) 397±400 Ó Springer-Verlag 1999

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Received: 19 November 1998

G.S.N. Naidu, T. Panda (&)Biotechnology Research Centre,Indian Institute of Technology ± Madras,Chennai ± 600 036, India

on sixth day, centrifuged and the supernatant was storedin sterile container at 10 °C until further use.

2.5Experimental methodologyIn our laboratory, the physical (pH of the substrate andtemperature of incubation) and chemical parameters(volume of enzyme and substrate) were optimized duringhydrolysis of pectic substances under assay conditions(unpublished result). The optimum results are shown inTable 1. Based upon these values the ratio of initial sub-strate to enzyme are selected for study under processconditions. The different substrate concentrations andinitial substrate to volume ratio considered for experi-ments are shown in Tables 2±4 for PMG, PG and PL re-spectively. At each initial substrate concentration theinitial substrate to enzyme ratio was varied and hydrolysisreaction was carried out at optimum conditions of pH and

temperature (unpublished result). Experiments were car-ried out with different combinations as in Tables 2±4. Thesamples were collected at regular intervals of time and thereducing groups formed are measured. The amount ofreducing groups formed becomes saturated after certaintime and that value was considered for plots. A plot of(reducing groups formed/initial substrate) vs. initial sub-strate to enzyme ratio was made to study the behaviour ofthese enzymes during hydrolysis under process condi-tions.

2.6Measurement of reducing groups

2.6.1Measurement of reducing groups by the actionof PMG and PGThe hydrolysis reaction was terminated by adding 0.5 cm3

of copper reagent to 0.5 cm3 of the sample. The resultingsolution was placed in vigorously boiling water bath for10 minutes. After cooling to 30 °C, 1 cm3 of ar-senomolybdate reagent was added and the absorbance wasread on a spectrophotometer at 500 nm against suitableblank. The end product is oligomers of galacturonic acid.Oligomers as high as pentamer give the same absorbanceof monomer. Hence, galacturonic acid was used as thestandard [14]. Pure galacturonic acid were prepared insuitable concentration range and a standard plot wasprepared to estimate the reducing group.

The measurement of reducing groups by the action ofPG was measured in the same manner as discussed forPMG using polygalacturonic acid as the substrate.

2.6.2Measurement of reducing groups by the action of PLThe hydrolysis reaction was stopped by adding 3.5 cm3 of0.5 M HCl to 1.5 cm3 of sample and contents were esti-mates spectrophotometrically (Shimadzu UV ± 1601 PC)for 4,5 ± unsaturated galacturonosyl residues at 235 nmversus suitable blanks. A molar extinction coef®cient of5550 M)1 cm)1 was used to calculate the reducing group[15].

3Results and discussionThe plot of (reducing groups formed/initial substrate) vs.initial substrate to enzyme ratio was made to study theperformance of these enzymes, Figs. 1±3 for PMG, PG andPL respectively.

Table 1. Optimum values of chemical (volume of substrate andenzyme) and physical parameters (pH and temperature) obtainedduring hydrolysis of pectic substances under assay conditions(unpublished result)

Enzyme Volume ofsubstrate(cm3)

Volume ofenzyme(cm3)

pH Temperature(°C)

PMG 0.2 0.074 5.3 30PG 0.4 0.086 6.3 26PL 0.63 0.7 4.85 35

The initial substrate concentration maintained during hydrolysisforPMG: 2.4 kg/m3

PG: 2.4 kg/m3

PL: 5.0 kg/m3

Table 2. Experimental plan for hydrolysis of pectin by PMGunder process conditions

Pectinconcentration(kg/m3)

1.0 2.0 3.0 4.0 5.0

Volume ratioof substrate toenzyme

0.5 1.0 2.0 3.0 4.0

The conditions maintained are:pH: 5.3; Temperature: 30 °C; shaker speed: 160 rev/min

Table 3. Experimental plan for hydrolysis of polygalacturonicacid by PG under process conditions

Polygalactur-onic acidconcentration(kg/m3)

1.0 2.0 3.0 4.0 5.0

Volume ratioof substrate toenzyme

2.0 3.0 4.0 5.0 6.0

The conditions maintained are:pH: 6.3; Temperature: 26 °C; shaker speed: 160 rev/min

Table 4. Experimental plan for hydrolysis of pectin by PL underprocess conditions

Pectinconcentration(kg/m3)

4.0 5.0 6.0 7.0 8.0

Volume ratioof substrate toenzyme

0.4 0.8 1.0 2.0 3.0

The conditions maintained are:pH: 4.85; Temperature: 35 °C; shaker speed: 160 rev/min

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In the case of PMG, maximum hydrolysis took placewhen the initial substrate concentration is 2.0 kg/m3 andinitial substrate to enzyme ratio was 3.0. The optimumratio of substrate to enzyme obtained under assay condi-tion was 2.7 which is close to that obtained in this study.There was decrease in the reducing groups formed whenthe initial substrate to enzyme ratio was above 3.0 kg/m3.This is probably due to substrate inhibition.

In the case of PG, maximum hydrolysis took place atinitial substrate to enzyme ratio of 4.0 and at a substrateconcentration of 2.0 kg/m3. The hydrolysis was found todecrease beyond these conditions. When the initial sub-strate concentration is more than 3.0 kg/m3 the optimumsubstrate to enzyme ratio was found to be 5.0 otherwisethe ratio was 4.0. This clearly indicates that initial sub-strate concentration and initial substrate to enzyme ratio

has signi®cant effect on the hydrolysis of polygalacturonicacid. In this case also the substrate inhibition was ob-served.

The optimum substrate to enzyme ratio obtained for PLis 1.0 which was found close to that obtained under assayconditions. The maximum hydrolysis took place when theinitial substrate concentration was at 5.0 kg/m3 beyondwhich there was decrease in hydrolysis. At high substrateconcentrations (more than 5.0 kg/m3) the decrease inhydrolysis is very less when compared with lower con-centrations. Substrate inhibition was also found in PL.

4ConclusionUnderstanding the behaviour of pectinases under processconditions was necessary apart from the similar studyunder assay conditions. In assay conditions, the substrateconcentration is held constant and the reactions are al-lowed for shorter duration. In process conditions it wasobserved that initial substrate concentration and initialsubstrate to enzyme ratio have signi®cant effect on thehydrolysis of pectic substances. The kind of plots drawn inthis study are helpful in predicting the amount of substrateand enzyme required to produce the required amount ofreducing groups within a stipulated time.

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Fig. 1. Plot of (reducing groups formed/initial substrate) vs.initial substrate of enzyme ratio during hydrolysis of pectinby PMG

Fig. 2. Plot of (reducing groups formed/initial substrate) vs.initial substrate of enzyme ratio during hydrolysis of polygalac-turonic acid by PG

Fig. 3. Plot of (reducing groups formed/initial substrate) vs.initial substrate of enzyme ratio during hydrolysis of pectin by PL

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G.S.N. Naidu, T. Panda: Behaviour of pectinases under process conditions

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