Microbially Derived Pectinases Review

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IOSR Journal of Pharmacy and Biological Sciences (IOSRJPBS) 
ISSN: 2278-3008 Volume 2, Issue 2 (July-August 2012), PP 01-05 
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Microbially Derived Pectinases: A Review 
 
Apoorvi Chaudhri, & Vuppu Suneetha 
School Of Biosciences and Technology, VIT University, Vellore-632014, India 
 
Abstract: In recent Biotechnology Microbially derived pectinases have Industrial Exploitation and market 
Potential. Pectinase are enzymes breaking pectic substances by hydrolysis of pectin and breakdown of complex 
polysaccharides in plant tissues into simpler molecules with extra ordinary specificity, catalytic power and 
substrate specificity. Pectinases are produced from a wide variety of microorganisms including bacteria, fungi, 
actinomycetes, yeast. They are classified into polysaccharide hydrolases, polysaccharide lysases and 
carbohydrate esterases. The production of pectinase from microorganisms involves screening and 
Characterization of microorganisms, the downstream processes depend on the purity required. Enzyme assays 
are performed, and the biochemical properties studied. Pectinases have wide applications; they find use in pulp 
industry, in textile industry, in food industry and in water treatment, in friut industries; Clarification. The cock 
tail preparation of pectinase enzyme in Industrial exploitation can hence be discussed. 
Keywords: esterases, hydrolases, lyases 
 
I. Introduction 
Pectins are complex polysaccharides consisting of partially methyl esterified α- (1,4) linked 
homogalacturonic acid backbone and branched neutral sugar side chains. They are important components of cell 
wall and middle lamella, and can be found in fruits and vegetables. Enzymes cleaving pectic substances are 
called as pectinolytic enzymes or pectinases[1]. Food processing enzymes including Pectinases account for 45 
percent of enzyme usage. Pectinases are phytopathogenic substances [2]. Microbially derived Pectinases find 
more use due to their advantage over plant and animal derived pectinases. The reasons being cheap production, 
easier gene manupilations, faster product recovery, further microbial enzymfes are usually free of harmful 
substances. Table I shows microorganisms producing different types of pectinase. Pectinases based on the 
mechanism used to attack the galacturonan backbone are classified as polysacchride hydrolases, Polysaccharide 
lysase and Carbohydrate esterases. These include Endopolygalacturonases( EC 3.2.1.1.5), 
Exopolygalacturonases( EC 3.2.1.67) , Pectate lyases (EC 4.2.2.2), Pectin lyases( EC 4.2.2.10) and Pectin 
methyl esterases( EC 3.1.11). Endo-PG catalyses random hydrolysis of α-1,4 glycosidic linkage between two 
non methylated acid residues. Endo PG s are produced by bacteria, fungi, yeast, and come under glycosyl 
hydrolase family[1]. Exo PG are glycoproteinas and degrade in a terminal fashion. They are of two types: exo 
PG EC(3.2.1.67) and exo PG EC(3.2.1.82) , which cleave α-(1,4) glycosidic bonds of GAlA residues[3]. Pectin 
lyases degrade pectin polymer by β-elimination mechanism so as to form 4,5 unsaturated 
oligogalacturonides[2]. Pectin methyl esterases catalyse the esterification of pectin which is methylated 
polygalacturonic acid resulting in the formation of de esterified pectin which releases hydrogen ions and 
methanol[4]. Pectin lyases catalyze the trans-eliminative cleavage of the galacturonic acid polymer[5]. Fig I 
shows the mode of action of the various pectinase. PME is found in peel and core of tissue prints, cellular 
subdivision of juice vesicles and in stigma cells. Pectin methyl esterases have highest levels in orange and lime 
but lowest levels in lemon[6]. PME has isoforms PME I, PME II, PME III as a result of their elution order in a 
heparin sepharose column [6].The optimum pH of the three PME isoforms having molecular weight 42 KDa is 
6.5-9.0[4]. PME I is more thermo resistant to thermal treatment than the other two forms. Further study is 
required to find out the reason for the existence of three different isoforms of PME [7,4] if its due to different 
post translational modifications or due to different genes[4]. 
 
 
Microbially Derived Pectinases: A Review 
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Fig I. Mode of action of Pectinases; PMG is polymethylgalacturonase, PG is polygalacturonase, PL is pectin 
lyase, PE is pectinesterase. R group is H in case of PG, CH3 in case of PGL. R group is CH3 in case of PL, H in 
case of PGL[8]. 
 
I. Pectinases Produced By Reported Microorganisms[9] 
 
II. Pectinase Production 
The production of pectinase from microorganisms involves the following steps -isolation and 
screening of the microorganism, growth of microorganism on a culture medium, fermentation, purification 
steps. The production of the enzyme can either be by Solid State fermentation as in the case of Pectinase 
production from Aspergillus awamori[10,11], production from B.subtilis[12], production from Penicillium 
viridicatum[13] or by Submerged fermentation as in the production of xylano-pectinolytic enzymes from 
 Pectin methyl 
esterase 
Pectin lyase Pectate lyase Endo PG Exo PG 
 
 
 
 
 
 
 
 
 
Reported 
pectinase 
producing 
microorganis
ms 
 
Aspergillus 
fumigatus 
Af293 
 
Aspergillus niger 
 
 
Bacillus 
alcalophilus 
 
Alternaria 
alternata 
 
Aspergillus 
niger 
Aspergillus 
aculeatus 
 
 
Colletotrichum 
gloeosporioides f. 
sp. malvae 
 
Bacillus 
licheniformis 
Botryotinia 
fuckeliana 
 
 
Aspergillus 
tubingensis 
Bacillus sp. 
NRRL B-
14911 
 
 
Emericella 
nidulans 
 
Campylobacter 
concisus 13826 
 
Colletotrichum 
gloeosporioides f. 
sp. malvae 
 
Botryotinia 
fuckeliana 
Botryotinia 
fuckeliana 
 
Dictyostelium 
discoideum AX4 
 
Cellvibrio 
japonicus 
Diaporthe 
helianthi 
 
 
Emericella 
nidulans 
 
 
Aspergillus 
oryzae 
 
Glomerella 
cingulata 
Globodera 
rostochiensis 
Emericella 
nidulans 
 
Vibrio 
vulnificus 
YJ016 
 
Clostridium 
acetobutylicu
m ATCC 824 
 
Pectobacterium 
carotovorum 
 
Klebsiella 
oxytoca 
 
Gibberella 
circinata 
 
Yersinia 
pestis 
Antiqua 
 
Emericella 
nidulans 
 
Penicillium 
griseoroseum 
 
Meloidogyne 
javanica 
 
Kluyveromyces 
marxianus 
 
 
Penicillium 
viridicatum 
Microbially Derived Pectinases: A Review 
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B.pumilus[14], however there are a number of advantages of using solid state process over submerged 
fermentation[15]. Pectinase production by B.subtilis was found to be extracellular since the pectinase enzyme 
was present in culture supernatant and no pectinase activity was observed in cell lysate or in cell washings of 
intact cells, At high inoculums levels enzyme production is decreased due to a competition for nutrients among 
bacterial population[12,11]. The enzyme produced is purified by unit operations of ultrafiltration processes and 
low pressure liquid chromatography. 
 
III. Factors Affecting Pectinase Production 
The selection of microbial source (wild type, recombinant, mutagenized) alongwith various parameters; 
pH, metal ions, Temperature,affect the pectinase production. Surfactants such as tween-20, twea-80 increase the 
enzyme production due to favourable effect on cell membrane permeability which leads to secretion of the 
enzyme. Pectinase synthesis is inhibited by SDS PAGE because of the denaturation of enzyme.The degrading 
ability of the enzyme is enhanced by agitation [12]. 
 
3.1. Substrate 
The culture medium varies from organism to organism; grape pomace is nutrient medium for 
A.awamori[10,16], potato dextrose agar media for Mucor flavus[17] , yeast extract and wheat bran for Bacillus 
subtilis[12] , sabouraud dextrose agar for P.viridicatum RFC3[13]. Maximum pectinase yield was obtained in 
the media with PGA (230 U/ml)[2,11], then with wheat bran(190 U/ml), cotton seed cake(160U/ml), whereas 
glucose shows least enzyme production and acts as a repressor. Carbon sources also effect the producion of 
pectinase. As observed PGA, lactose, pectin increased pectinase production [18]. In Aspergillus fumigates 
sucrose yielded maximum pectinase production [7]. Among the nitrogen sources for pectinase production the 
maximum yield of pectinase was shown by yeast extract. Pectinase production is inhibited by glycine, urea, 
ammonium nitarate while wheat bran, peptone, ammonium chloride, yeast extract enhance pectinase production 
[18]. The induction of pectate lyases is higher with pectin than with polygalacturonic acid [11] The PGL activity 
increases as concentration of reducing sugars in the culture broth decreases [17]. 
 
3.2. PH 
Except for exo PGase from Fusarium oxysporum and endo PGase from Bacillus licheniformis, all 
PGase have acidic optimum pH between 3.3-7. Xylano-pectinolytic enzymes find a wide application in 
biobleaching industry, the optimal efficiency is at pH 8.5[14]. The optimum pH of the three PME isoforms that 
is 6.5-9.0 depends on the salt concentration, salts, mask the carboxylic charged groups from those involved in 
the enzyme substrate recognition thus effecting the PME activity[4]. Pectate lyases have an optimum pH of 
8.5[11]. Fungi and yeast produce PGase with acidic pH. For pectinase production by Bacillus subtilis, the 
highest pectinase production is observed at pH 9.5. The optimum pH for growth and pectinase production for 
most of the bacteria is 7-10[12]. Some bacterial strains; Streptomyces QG-11-3 and Aspergillus aculeatus 
produced PGase active at pH 3.0[19]. 
 
3.3. Temperature 
The activity of pectinases depends on thermal stability. In fruit juice industries [1, 16] and wine 
processing the property of PGase of M.rouxii being efficient at 20°C and sensitive to 30°C is used [1]. Pectinase 
production by Bacillus subtilis the optimum temperature was found to be 37°C [12]. Pectate lyase optimal 
temperature under standard assay was 70°C [11]. Pectinase from Streptomyces sp QG-11-3 have optimal 
activity at 60°C. PGase from fungi have optimum activity at 50°C while from yeast the temperature varies from 
40°C to 60°C [19]. 
 
3.4. Metal ions 
The activity of Endo PGase is reduced due to Cu
2+
and Hg
2+
[1]. Metal ions Hg 
2+
 , Zn 
2+
, Mg
2+
 inhibit 
enzyme production due to inhibition by thiol group blocking agents as there is possible involvement of the thiol 
group in the enzyme s active site[12,16]. Mn
2+
 increases the PGase activity, however Li
2+
, Fe
2+
, Rb
2+
 have no 
effect on the activity[12]. At high concentration of metal ions the enzyme production is low due to blockage of 
secretion of protein into external medium. Bacterial pectate lyases need Ca 
2+
 for growth[11], fungal pectinase 
do not need Ca
2+
. 
 
IV. Assay Methods 
Enzyme assays are performed for characterization of enzymatic activity. One such method is 
ruthenium dye assay, other methods are DNS(dinitro salicylic acid) , cannon ferske routine viscosimeter[10]. 
 
 
 
Microbially Derived Pectinases: A Review 
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4.1. Polygalacturonase 
Amount of reducing sugar is estimated using 3,5 dinitrosalicylae and arsenomolybdate copper method, 
viscosity reduction methods also used[5]. Estimation also done by Interaction of ruthenium red dye with a 
polyuronic acid. 
 
4.2. Pectate lyase 
Observing the increase in the absorbance at 235 nm in spectrophotometer is one such assay method. 
Viscosity reduction method is also used [5]. Reduction in viscosity is given as, 
% Reduction in viscosity = T1-T2/T1-T3 
Here T1, T2 and T3 represent reaction mixture without enzyme, test mixture and water[5]. 
 
4.3. Pectin Methyl Esterase 
The increase in the binding of RR to pectin as the number of methyl esters attached to pectin decreases 
is the basis for pectin methyl esterase activity[1]. Another PME essay is spectrophtometric assay where pectin is 
the substrate and is titrated with 0.05 M NaOH. 
The PME activity is thus given as, 
{(V-V1)*M*1000}/ (V*t) 
Here v is ml of NaOH, V1 is NaOH to titrate blank, M is molarity of NaOH, V is the ml of enzyme 
used, t is incubation time in min). PGA or Pectin act as substrates for the activity of pectinase [4] . 
The prescence of protein can be determined by protein assays; Lowry’s method, coomassie blue plus assay. 
Study of the biochemical properties of enzymes can be done by sodium dodecyl sulphate polyacrylamide gel 
electrophoresis (SDS-PAGE), the molecular mass can be determined by mass spectrometry, and isoelectric 
focusing determines the pI values. 
 
V. Applications 
Pectinases are used in the textile industry as they are capable of depolymerising the pectin breaking it 
into low molecular water soluble oligomers improving absorbency and whiteness of textile material and 
avoiding fiber damage [12]. Pectinase is used in juice clarification. There is use of xylano-pectinolytic enzymes 
in paper and pulp industry. Pectinases are effective in biobleaching of mixed hard wood and bamboo kraft pulp, 
As pretreatment of kraft pulp with xylano-pectinolytic enzymes from same alkalo thermotolerant isolate 
produced pulp with superior quality facilitating adaptation of environment friendly technology in paper pulp 
industry [14]. Pectinase also find application in the degumming of plant fibres, retting of plant fibres, pectinase 
from Bacillus species are used in waste water treatment, Pectinase are further used in coffee and tea 
fermentation by breaking pectins present in tea leaves, oil extraction by avoiding emulsification formation, 
improvement of chromaticity and stability of red wines [9], pectinase improve wine characeristics of colour and 
turbidity, biscouring of cotton. Pectinases posse biological applications in protoplast fusion technology and plant 
pathology [20]. 
 
5.1. Endo PG 
Endo PGA-1 is applied in the food industry as it decreases the viscosity by 77% and increases the 
transmittance of apple juice by 84% [21]. Since the plant cell wall is made of pectin hence the prescence of 
pectin increases the viscosity of the juice, pectinases thus break pectin aiding in clarification process further use 
is in fruit softening and plant infection processes [3]. Acidic Endo PG fined use in food industry for clarification 
of juice, wine by degrading pectin. Alkaline Endo PG s are used in paper, textile industries, treatment of pectic 
wastewater [21]. 
 
5.2. Pectin lyases 
Role in microbial phytopathogenesis and in fruit juice industries as they degrade pectin without 
disturbing the ester group (aroma) and do not lead to methanol formation, they are also used in degumming and 
retting of natural fibres[2]. Since pectin lyases degrade complex plant biomass into fermentable sugars hence it 
finds wide application in second generationbiofuel technology. The alkaliphilicity and high thermostability of 
pectin lyases may find implications in fibre degumming [22, 7]. Attention to clone pectin lyases gene from other 
organisms should be given [2]. 
 
5.3. Pectin methyl esterases 
Involved in synerasis which is the formation of calcium pectate from free carboxyl group (produced 
due to PME) and calcium ions. They are of great importance In food industry for optimizing heat treatment of 
fruits and vegetables, they are also involved in control of complex cell wall metabolism [4]. 
Pectinases produced from psychrophiles or thermophilic microorganisms are of high interest [1, 16, 11]. Further 
study should be done on the functions of pectinase in pests [4]. 
Microbially Derived Pectinases: A Review 
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VI. Acknowledgment 
The authors want to express their thanks to DST (Department of Science and Technology) India For 
Financial assistance under young scientist scheme, Dr.Rambir Singh, Scientist for constant encouragement and 
VIT University for providing infrastructural lab facilities for carrying out further research studies. 
 
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