Polaina J., MacCabe A.P. (ed.). Industrial Enzymes. Structure, Function and Applications

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MICROBIAL XYLANOLYTIC CARBOHYDRATE ESTERASES 91

an inactive mutant of AnFaeA (S133A) in complex with O-{5-O-[(E)-feruloyl]--

L-arabinofuranosyl}-(1→3)-O--D-xylopyranosyl-(1→4)-D-xylopyranose (FAX

)

and observed that the ferulic acid moiety of the substrate was visible in the electron

density map showing interactions through its OH and OCH

groups with the

hydroxyl groups of Tyr80. However, the remaining groups of the substrate (i.e. the

arabinose and the two xylose units) were not visible. Accordingly, in the structure

of the XynZ FAE in complex with FAX

determined by Schubot et al. (2001), the

ferulic moiety was clearly visible in the active site while the carbohydrate parts

of the substrate were not, suggesting that tight binding of the carbohydrate is not

required for catalysis. These results are in agreement with the synthetic ability of

StFaeC in non-conventional media where the esterase seems to be able to esterify

a broad spectrum of sugars showing specificity only to the ferulic moiety (Vafiadi

et al., 2005). In contrast to FAEs, determination of the crystal structure of a family

10 xylanase from Thermoascus aurantiacus complexed with xylobiose containing

an arabinofuranosyl-ferulate side-chain, revealed that the distal glycone subsite of

the enzyme makes extensive direct and indirect interactions with the arabinose

side-chain, while the ferulate moiety is solvent-exposed (Vardakou et al., 2005).

4. USE OF ACETYL XYLAN ESTERASES AND FERULIC ACID

ESTERASES AS BIOSYNTHETIC TOOLS

There is an increasing demand for “green” (environmentally friendly) production

processes for biodegradable polymers with modified hydrophobic and rheological

characteristics. Enzymatic acylation of oligo- and polysaccharides is more environ-

mentally friendly than classical chemical synthesis. AcXE from S. commune,a

member of CE family 1 catalyses acetyl group transfer to methyl -D-xylop-

yranoside and other substrates (Biely et al., 2003). This work was the first published

example of reverse reactions by AcXE.

Various hydroxycinnamic acids (ferulic, p-coumaric, caffeic, sinapinic) have

widespread industrial potential by virtue of their antioxidant properties. Generally,

such natural antioxidants are partially soluble in aqueous media, limiting their

usefulness in oil-based processes and that has been reported to be a serious disad-

vantage if an aqueous phase is also present. The modification of these compounds

via esterification with aliphatic alcohols results in the formation of more lipophilic

derivatives. The direct esterification of natural phenolic acids including the above

mentioned hydroxycinnamic acids with aliphatic alcohols catalysed by various

lipases in organic media has been reported, albeit with low reaction rate and yield.

Several authors have demonstrated that the lipase-inhibiting effect of electron-

donating substituents conjugated to the carboxylic groups in hydroxylated deriva-

tives of cinnamic acids like ferulic, p-coumaric, sinapinic and caffeic acid, is strong

(Figueroa-Espinoza and Villeneuve, 2005). Esterification can be carried out by

lipases only if the aromatic ring is not para-hydroxylated and the lateral chain is

saturated. Thus, the enzymatic esterification of cinnamoyl substrates can be obtained

using only FAEs as biocatalysts.

92 TOPAKAS AND CHRISTAKOPOULOS

Transesterification of phenolic acids was catalysed by using a type A FAE

from F. oxysporum (FoFaeA) trapped in a n-hexane/1-propanol/water surfactantless

microemulsion (Topakas et al., 2003a). Greater synthetic activity was observed in

ternary water-organic mixtures having a lower water content. The synthetic activity

of esterases follows a pattern similar to their hydrolytic activity against various

methyl esters of cinnamic acids. FoFaeA shows a preference for the hydrolysis of

methoxylated substrates (Topakas et al., 2005b) while conversion to butyl esters

was greater with ferulic and sinapinic acids. Type B esterases from F. oxysporum

(FoFaeB) (Topakas et al., 2003b) and S. thermophile (StFaeB) showed preference

for the hydrolysis of hydroxylated substrates (Topakas et al., 2005b) and the

conversion to butyl esters was enhanced with p-coumaric and caffeic acids (Topakas

et al., 2003b, 2004). The type-C FAE StFaeC from S. thermophile demonstrated

maximum hydrolytic activity against methyl ferulate (Topakas et al., 2005b).

Optimal yields were achieved producing butyl esters with ferulic acid (Topakas

et al., 2005a). Furthermore, it was reported that the same enzyme catalysed the

transfer of the feruloyl group to L-arabinose (Fig. 2) in a ternary water-organic

mixture consisting of n-hexane, t-butanol and water system, achieving a conversion

of about 40% of L-arabinose to a feruloylated derivative (Topakas et al., 2005a).

This work was the first example of sugar esterification with unsaturated arylaliphatic

acids, like methoxylated or hydroxylated derivatives of cinnamic acids (such as

ferulic acid). Lipases are not able to catalyse such a reaction due to electronic and/or

steric effects (Otto et al., 2000).

Phenolic acid sugar esters have demonstrable antitumoural activity and the

potential to be used to formulate antimicrobial, antiviral and/or anti-inflammatory

agents. As esters based on unsaturated arylaliphatic acids such as cinnamic acid

and its derivatives are known to display anticancer activity, specific FAEs could be

employed in the tailored synthesis of such pharmaceuticals.

The potential use of FAEs for the synthesis of feruloylated oligomers or polymers

using feruloyl esterases opens the door for the design of modified biopolymers with

new properties and bioactivities.

Figure 2. Transesterification of methyl ferulate with L-arabinose by StFaeC

MICROBIAL XYLANOLYTIC CARBOHYDRATE ESTERASES 93

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CHAPTER 7

STRUCTURAL AND BIOCHEMICAL PROPERTIES

OF PECTINASES

SATHYANARAYANA N. GUMMADI

∗

, N. MANOJ AND D. SUNIL KUMAR

Department of Biotechnology, Indian Institute of Technology-Madras, Chennai, India

∗

gummadi@iitm.ac.in

1. INTRODUCTION

Pectin and other pectic substances are complex polysaccharides, which contribute

firmness and structure to plant tissues as a part of the middle lamella. The

basic unit in pectic substances is galacturonan (-D-galacturonic acid). Pectic

substances are classified into two types; homogalacturonan and heterogalacturonan

(rhamnogalacturonan). In homogalacturonan, the main polymer chain consists

of -D-galacturonate units linked by 1 → 4 glycosidic bonds, whereas in

rhamnogalacturonan, the primary chain consist of 1 →4 linked -D-glacturonates

and with about 2–4% L-rhamnose units that are 1 →2 and 1 → 4 linked to

D-galacturonate units (Whitaker, 1991). The side chains of rhamnogalacturonans

usually consist of L-arabinose or D-galacturonic acid units. In plant tissues, about

60–70% of the galacturonate units are esterified with methanol and occasionally

with ethanol. Based on the degree of esterification, pectic substances are classified

into protopectin, pectinic acid, pectin and polygalacturonic acid (Table 1). Molecular

size, degree of esterification and weight distribution of polygalacturonic acid

residues are important factors that contribute to heterogeneity in pectic substances.

Relative molecular masses of pectic substances isolated from various sources such

as citrus fruits, apple and plums, range from 25 to 350 kDa.

Pectinases are a complex and diverse group of enzymes involved in the

degradation of pectic substances. The diversity of forms of pectic substances in plant

cells probably accounts for the existence of various forms of these enzymes. Pecti-

nases are classified depending on their substrate and mode of enzymatic reaction

(Fig. 1). Pectinases act as carbon recycling agents in nature by degrading pectic

substances to saturated and unsaturated galacturonans, which are further catabolized

J. Polaina and A.P. MacCabe (eds.), Industrial Enzymes, 99–115.

100 GUMMADI ET AL.

Table 1. Different types of pectic substances

Pectic substances Structural description Properties

Protopectin Galacturonate units linked by -1,4-glycosidic

linkages. The carboxyl groups are highly esterified

with methanol. Polymer is highly cross-linked with

or with other polysaccharides

Insoluble in water.

Degree of esterification

> 90%

Pectin Galacturonate units linked by -1,4-glycosidic

linkages. The carboxyl groups are esterified with

methanol

Soluble in water. Degree

of esterification at least

75%

Pectinic acid Galacturonate units linked by -1,4-glycosidic

linkages. The carboxyl groups are slightly esterified

with methanol

Soluble in water. Degree

of esterification varies

between 0 and 75%

Pectic acid Galacturonate units linked by -1,4-glycosidic

linkages.

Soluble in water. Degree

of esterification 0

Rhamnogalacturonan Galacturonate units linked by -1,4-glycosidic

linkages with rhamnose units lined by -1,2 and

-1,4 linkages. The side chains are homogeneous

polymers of galacturonic acid and arabinose

Soluble in water.

Apart from those mentioned in the table, oligomers of galacturonate and methyl galacturonate are

present.

to 5-keto-4-deoxy-uronate and finally to pyruvate and 3-phosphoglyceraldehyde

(Vincent-Sealy et al., 1999). Pectinases from phytopathogenic fungi such as

Aspergillus flavus, Fusarium oxysporum and Botrytis cinerea are also known to play

a vital role in plant pathogenicity or virulence by degrading pectic compounds

present in cell wall (Lang and Dörenberg, 2000; DiPietro and Roncero, 1996; Ten

Have et al., 1998). Pectinases, especially polygalacturonase, is known to play a

major role in pectin breakdown during the final stages of fruit ripening (Sozzi-

Quiroga and Fraschina, 1997; Chin et al., 1999). Polymethylgalacturonase (PMG),

polygalacturonase (PG), pectin lyase (PL), polygalacturonate lyase (PGL) and

pectinesterase (PME) are industrially important pectinases discussed in this chapter.

2. BIOCHEMICAL CHARACTERISTICS OF PECTINASES

2.1. Polymethylgalacturonase

PMG activity can be determined by measuring the reducing sugars formed due to

the hydrolysis of glycosidic bond or by measuring the reduction in viscosity of the

substrate. Highly esterified pectin is the best substrate for PMG whereas pectic acid

and pectate derivatives do not react with PMG. Few reports are available on the

biochemical characteristics of the enzyme due to following reasons: (a) the enzyme

has not been purified to homogeneity and characterized and (b) the activity of this

enzyme has not been demonstrated in the absence of other pectic enzymes and in

the presence of 100% methylated pectin. Hence, researchers need to be very careful

in reporting the activity of PMG. Aspergillus was found to be a major producer

STRUCTURAL AND BIOCHEMICAL PROPERTIES OF PECTINASES 101

Figure 1. Classification of different pectinases based on their reaction with different pectic substances

of PMG followed by species belonging to Penicillium, Botrytis and Sclerotium.

PMG from A. niger showed optimum pH between 4 and 7 and highly esterified

pectin (95%) was the best substrate (Koller and Neukom, 1967). The analysis of

hydrolyzed products suggests Aspergillus produced only endo-PMG. Exo PMG has

not been reported so far. A highly acidic pectinase (optimum pH 2.3) from A. niger

has also been reported (Naidu and Panda, 1998b). The isolation of PMG and its

biochemical characteristics need to be explored further for industrial applications.

2.2. Polygalacturonases

PG hydrolyzes the glycosidic linkages of polygalacturonates (pectates) by both

exo and endo splitting mechanisms. Endo PGs act on the homogalacturonan

backbone and break it into oligogalacturonates whereas exo-PGs break down

polygalacturonates to di- and mono–galacturonates. PG activity can be determined

by measuring the reducing sugars formed due to hydrolysis or by viscosity reduction

method. However, the viscosity reduction method is less sensitive for exo PGs as

the decrease in viscosity is relatively low. Cup plate method can also be used for

estimating PG activity by viewing the clearing zones after staining with ruthenium

red (Dingle et al., 1953; Truong et al., 2001). Endo PGs are widely distributed

among fungi, bacteria and yeast. Endo PGs often occur in different forms having

molecular weights in the range of 30–80 kDa and pI ranging between 3.8 and 7.6.