Bioresource Technology 77 (2001) 215227

Review paper

Applications of pectinases in the commercial sector: a review

D.R. Kashyap a, P.K. Vohra a, S. Chopra a, R. Tewari b,*
a
b

Department of Microbiology, Punjab University, Chandigarh 160014, India

Department of Biotechnology, Punjab University, Chandigarh 160014, India
Accepted 10 August 2000

Abstract
Pectinases are one of the upcoming enzymes of fruit and textile industries. These enzymes break down complex polysaccharides
of plant tissues into simpler molecules like galacturonic acids. The role of acidic pectinases in bringing down the cloudiness and
bitterness of fruit juices is well established. Recently, there has been a good number of reports on the application of alkaline
pectinases in the textile industry for the retting and degumming of ber crops, production of good quality paper, fermentation of
coee and tea, oil extractions and treatment of pectic waste water. This review discusses various types of pectinases and their
applications in the commercial sector. 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Pectinase; Industrial applications

1. Introduction

2. Structure of pectin

Pectinases were some of the rst enzymes to be used

in homes. Their commercial application was rst observed in 1930 for the preparation of wines and fruit
juices. Only in the 1960s did the chemical nature of plant
tissues become apparent and with this knowledge, scientists began to use a greater range of enzymes more
eciently. As a result, pectinases are today one of the
upcoming enzymes of the commercial sector. Primarily,
these enzymes are responsible for the degradation of the
long and complex molecules called pectin that occur as
structural polysaccharides in the middle lamella and the
primary call walls of young plant cells. Pectinases are
now an integral part of fruit juice and textile industries
as well as having various biotechnological applications.
The estimated value of sales of all industrial enzymes in
1995 was $1 billion, of which some $75 million was assessed for pectinases. By 2005, the whole world market
for industrial enzymes is expected to be $1.72 billion
(Godfrey and West, 1996). The main emphasis of this
article is on the types of pectinases and their applications in industries.

Chemically, pectic substances are complex colloidal

acid polysaccharides, with a backbone of galacturonic
acid residues linked by a (14) linkages. The side chains
of the pectin molecule consist of L -rhamnose, arabinose,
galactose and xylose. The carboxyl groups of galacturonic acid are partially esteried by methyl groups and
partially or completely neutralized by sodium, potassium or ammonium ions. Based on the type of modications of the backbone chain, pectic substances are
classied into protopectin, pectic acid, pectinic acid and
pectin (Be Miller, 1986).
Protopectin. This is a parent pectic substance and
upon restricted hydrolysis yields pectin or pectinic acid.
Protopectin is occasionally a term used to describe the
water-insoluble pectic substances found in plant tissues
and from which soluble pectic substances are produced
(Kilara, 1982).
Pectic acids. These are the galacturonans that contain
negligible amounts of methoxyl groups. Normal or acid
salts of pectic acid are called pectates.
Pectinic acids. These are the galacturonans with various amounts of methoxyl groups. Pectinates are normal
or acid salts of pectinic acids (Kilara, 1982). Pectinic
acid alone has the unique property of forming a gel with
sugar and acid or, if suitably low in methyl content, with
certain other compounds such as calcium salts.

Corresponding author. Tel.: +172-541441 ext. 4085; fax: +172541409.

E-mail address: shaktit@glide.net.in (R. Tewari).

0960-8524/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved.
PII: S 0 9 6 0 - 8 5 2 4 ( 0 0 ) 0 0 1 1 8 - 8

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D.R. Kashyap et al. / Bioresource Technology 77 (2001) 215227

Pectins. A generic name for the mixture of widely

diering compositions containing pectinic acid as the
major component. Pectin in native form is located in the
cell wall and it may be interlined with other structural
polysaccharides and proteins to form insoluble protopectin.
In an unripe fruit, pectin is bound to cellulose microbrils in the cell wall. Such pectin is insoluble and
hence confers rigidity on cell walls. However, during
ripening the structure of pectin is altered by naturally
occurring enzymes in the fruits. These alterations involve the breakdown of the pectin chain or of side
chains attached to the units, which make up the main
chain. In either case, the result is that the pectin becomes
more soluble and its grip on the surrounding cell walls is
loosened and the plant tissue softens.
The pectic substances account for about 0.54% of
the weight of fresh material. When the tissue is ground,
the pectin is found in the liquid phase (soluble pectin)
causing an increase in viscosity and the pulp particles,
whereas other pectin molecules remain bound to cellulose brils by means of side chains of hemicellulose and
thus facilitate water retention (Pieri et al., 1989). Mechanical crushing of pectin-rich fruit yields a fruit juice
with high viscosity, which remains bound to the pulp in
the form of a jellied mass. It is dicult to extract this
juice by pressing or using other mechanical methods.
With the addition of pectinases the viscosity of the fruit
juice drops, the pressability of the pulp improves, the
jelly structure disintegrates and the fruit juice is easily
obtained and with higher yields.
The raw press juice is rich in insoluble particles,
which are mainly made up of pectic substances. These
particles are known as `cloud particles'. In these particles, a protein nucleus with a positive surface charge is
coated by negatively charged pectin molecules (Pilnik
and Voragen, 1993). This negative charge causes the
pectin molecules to repel one another. Pectinases degrade this pectin and expose part of the positively
charged protein beneath, thus reducing electrostatic repulsion between cloud particles which causes these
particles to aggregate to larger particles. These larger
particles eventually settle out, but to improve the process, occulating agents (nings) such as gelatin, tannin
or bentonite (a type of clay) can be added. Yeasts and
other microbes, which may have contaminated the juice,
are also precipitated by nings. What is left is a transparent but by no means clear juice. A second centrifugation and subsequent ltration are needed to give the
clear juice that many consumers prefer.
3. Classication of pectic enzymes
Pectinases are classied under three headings according to the following criteria: whether pectin, pectic

acid or oligo-D -galacturonate is the preferred substrate,

whether pectinases act by trans-elimination or hydrolysis and whether the cleavage is random (endo-, liquefying of depolymerizing enzymes) or endwise (exo- or
saccharifying enzymes). The three major types of pectinases are as follows.
3.1. Pectinesterases (PE)
Pectinesterases also known as pectinmethyl hydrolase, catalyzes deesterication of the methoxyl group of
pectin forming pectic acid. The enzyme acts preferentially on a methyl ester group of galacturonate unit next
to a non-esteried galacturonate unit.
3.2. Depolymerizing enzymes
These are the enzymes:
3.2.1. Hydrolyzing glycosidic linkages
They include:
3.2.1.1. Polymethylgalacturonases (PMG). Catalyze the
hydrolytic cleavage of a-1,4-glycosidic bonds. They may
be:
1. Endo-PMG: causes random cleavage of a-1,4-glycosidic linkages of pectin, preferentially highly esteried
pectin.
2. Exo-PMG: causes sequential cleavage of a-1,4-glycosidic linkage of pectin from the non-reducing end of
the pectin chain.
3.2.1.2. Polygalacturonases (PG). Catalyze hydrolysis of
a-1,4-glycosidic linkages in pectic acid (polygalacturonic
acid). They are also of two types:
1. End-PG: also known as poly (1,4-a-D -galacturonide)
glycanohydrolase, catalyzes random hydrolysis of a1,4-glycosidic linkages in pectic acid.
2. Exo-PG: also known as poly(1,4-a-D -galacturonide)
galacturonohydrolase, catalyzes hydrolysis in a sequential fashion of a-1,4-glycosidic linkages on pectic
acid.
3.2.2. Cleaving
Cleaving a-1,4-glycosidic linkages by trans-elimination, which results in galacturonide with an unsaturated
bond between C4 and C5 at the non-reducing end of the
galacturonic acid formed. These include:
3.2.2.1. Polymethylegalacturonate lyases (PMGL).
Catalyze breakdown of pectin by trans-eliminative
cleavage. They are:
1. Endo-PMGL: also known as poly(methoxygalacturonide) lyase, catalyzes random cleavage of a-1,4-glycosidic linkages in pectin.

D.R. Kashyap et al. / Bioresource Technology 77 (2001) 215227

2. Exo-PMGL: catalyzes stepwise breakdown of pectin

by trans-eliminative cleavage.
3.2.2.2. Polygalacturonate lyases (PGL). Catalyze
cleavage of a-1,4-glycosidic linkage in pectic acid by
trans-elimination. They are also of two types:
1. Endo-PGL: also known as poly (1,4-a-D -galacturonide) lyase, catalyzes random cleavage of a-1,4-glycosidic linkages in pectic acid.
2. Exo-PGL: also known as poly(1,4-a-D -galacturonide)
exolyase, catalyzes sequential cleavage of a-1,4-glycosidic linkages in pectic acid.
3.3. Protopectinase
This enzyme solubilizes protopectin forming highly
polymerized soluble pectin.
On the bases of their applications, pectinases are
mainly of two types: acidic pectinases and alkaline
pectinases. The important producers of these pectinases
as reported in the literature are given in Table 1.

4. Application of pectinases
4.1. Acidic pectinases
Acidic pectic enzymes used in the fruit juice industries
and wine making often come from fungal sources, es-

217

pecially from Aspergillus niger. The juices produced by

these industries commercially include: (A) Sparkling
clear juices, (apple, pear and grape juices, (B) Juices with
clouds (citrus juices, prune juices, tomato juice and
nectars), and (C) Unicellular products where the intent
is to preserve the integrity of the plant cells by selectively
hydrolyzing the polysaccharides of the middle lamella.
The objectives of adding enzymes dier in these three
types of fruit and vegetable juices.
4.1.1. Sparkling clear juices
In the case of sparkling clear juices enzymes are
added in order to increase the juice yield during pressing
and straining of the juice and to remove suspended
matter to give sparkling clear juices (free of haze). Some
examples of such juices and their further uses are described below.
4.1.1.1. Apple. Apple juice is manufactured as natural,
unltered and unclaried, juice containing a high percentage of pulp; as a hazy juice that has been centrifuged
to remove coarse particles but not ltered; and nally as
ltered clear and amber-colored juice prepared by enzymatic treatment (Kilara, 1982). Although pectinases
that can depolymerize highly esteried pectin are the
major types of enzymes used in apple juice processing
they are by no means the only enzymes used for this
purpose. A combination of pectinases and cellulases has
been reported to give a juice yield up to 100% (Alkorta

Table 1
Characterization of microbial pectinases
Producer
Acidic pectinases
Aspergillus niger CH4
Penicillium frequentans
Sclerotium rolfsii
Rhizoctonia solani
Mucor pusilus
Cloctridium thermosaccharolyticum
Alkaline pectinases
Bacillus sp. RK9
Bacillus sp. NT-33
Bacillus polymyxa
Bacillus pumilis
Amucola sp.
Xanthomonas compestris
Bacillus No. P-4-N
Bacillus stearothermophillus
Penicillium italicum CECT 22941
Bacillus sp. DT 7
Bacillus subtilis
Pseudomonas syringae pv. Glycinea

Type of pectinase

Opti. pH
for activity

Opti. Temp. for

activity (C)

Reference

Endo-pectinase,
Exo-pectinase

4.56.0

Below 50

Acuna-Arguelles et al., 1995

3.55.0
4.54.7

50

Borin et al., 1996

3.5
4.8
5.0
5.57.0

55
50
40
3040

Channe and Shewal, 1995

Marcus et al., 1986
Al-Obaidi et al., 1987
Rijssel et al., 1993

10.0
10.5
8.49.4
8.0-8.5
10.25
9.5
1010.5
9.0
8.0
8.0
8.5
8.0

75
45
60
70
2530
65
70
50
60
6065
3040

Fogarty and Kelly, 1983

Cao et al., 1992
Nagel and Vaughn, 1961
Dave and Vaughn, 1971
Bruhlmann et al., 1994
Nasumo and Starr, 1967
Horikoshi, 1990
Karbassi and Vaughn, 1980
Alana et al., 1990
Kashyap et al., 2000
Chesson and Codner, 1978
Magro et al., 1994

Endopolygalacturonase (Endo-PG)
Endo-PG
Endo-PG
PG
Polygalacturonate
hydrolase
PGL
PG
PG
PATE
Pectate lyase (PAL)
PATE
PG
PATE
Pectin lyase
Pectin lyase
PAL
PAL

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D.R. Kashyap et al. / Bioresource Technology 77 (2001) 215227

et al., 1998). Another potential contributor to the haziness is starch. Unripe apples may contain up to 15%
starch. This can be broken down using an amylase
(strictly speaking, an amyloglucosidase) active at the pH
of apple juice, added at the same time as the pectinases.
Juice extraction. The initial steps in the extraction of
juice from apples include washing, sorting and crushing
of apples in a mill. Although pectinases are often added
at this stage, better results are achieved if the apple
pulp is rst stirred in a holding tank for 1520 min so
that enzyme inhibitors (polyphenols) are oxidized by
naturally occurring poly-phenol oxidase present in the
fruit. As these polyphenols in the pulp inhibit pectic
enzymes, addition of microbial polyphenol oxidases to
the fruit juices has also been suggested. An alternative
way to remove polyphenols in the fruit juices is the
addition of polyvinyl pyrolidone (PVP) which oxidizes
the phenolic substances hence creating an environment
for the action of pectic enzymes (De Vos and Pilnik,
1973).
Pectic enzymes are used in apple juice preparation to
facilitate pressing or juice extraction and to aid in the
separation of a occulent precipitate by sedimentation,
ltration or centrifugation. Schols et al. (1990) described
a new pectinase called rhamnogalacturonase and its role
in the maceration of apple tissue. This enzyme was
found in Aspergillus aculeatus initially, but it seems also
to be produced by other Aspergillus spp. Treatment with
pectinase takes anything from 15 min to 2 h depending
upon the exact nature of the enzyme and how much is
used, the reaction temperature and the variety of apple
chosen (Kilara, 1982). Some varieties, such as Golden
Delicious, are notoriously dicult to breakdown. During incubation the pectinase degrades soluble pectin in
the pulp, which would otherwise hamper juice extraction
in two ways. First, these slimy pectin particles become
saturated with juice, which is then dicult to extract
from the pulp and second, they block drainage channels
in the pulp through which the juice must pass. It is also
important that the pulp is not broken down too much,
as it is then becomes dicult to press. Enzyme treatment
is particularly eective with mature apples and those
from cold storage (Rombouts and Pilnik, 1986).
Clarication. Clarication is aected by pH temperature, contact time and enzyme concentration. A
juice with low pH will clarify more readily than one
with a higher pH, and as the temperature increases the
rate of clarication also increases as long as the temperature is below denaturation temperature for the
enzyme 4060C (Kilara, 1982). In general, the time
required to obtain clarication is inversely proportional
to the concentration of enzyme used at constant temperature over the range of 550C and treatment time
of 216 h.
If a cloudy product is required, the juice is pasteurized immediately after pressing, to denature any residual

enzymes. Centrifugation then removes large pieces of

debris, leaving most of the small particles in suspension.
For a clear juice these suspended particles have to be
removed. A dose of commercial enzyme e.g., `Rapidase
pomaliq', which contains pectinases, hemicellulases, and
cellulases is the accepted way of removing suspended
particles (Grassin and Fauquembergue, 1996). This enzyme is supplied by GistBrocades and is used at 200
600 g per ton of apples while stirring the pomace at 40
60 rpm for approximately 3 h at 50C. The liquied
pomace is centrifuged. Fig. 1 shows the signicant steps
involved in production of apple juice.
Yamaski et al. (1964) showed that clarication of
apple juice could be obtained by a mixture of PG and
PME alone without the presence of contaminating
enzymes from the apples. Recent work has shown that
puried pectin lyase (PL) used at pH 34 can clarify
apple juice (Ishii and Vokotsuka, 1971, 1973). This
research has also shown that apple juice containing
9192% esteried pectin can be easily claried by
pure PL.
Clear apple juice can sometimes develop a haze during storage, especially refrigerated storage. This defect is
termed `After Haze' and results from juice processed at

Fig. 1. Production of apple juice (Grassin and Fauquembergue, 1996).

D.R. Kashyap et al. / Bioresource Technology 77 (2001) 215227

temperature higher than storage temperature. The haze

may result from polymerization of polyphenols and
oxidation of proanthocyanidins during milling and
pressing. Another appearance defect called `Starch
Haze' may sometimes develop in apple juice prepared
from early-season apples (contain up to 15% starch).
Starch haze can be removed by fungal amylases (diastase) added to juices heated to 77C to gelatinize the
starch and then cooled to 52C before diastase addition.
Pectic enzyme is added at the same time as the diastase.
In conclusion, advantages of the use of pectinases in
apple juice are the following: their availability allows the
producer to diversify the type of products, i.e., cloudy,
clear juices, clear concentrates, etc., and to increase the
value of this raw material. In practice, the total time for
the juice extraction is shorter than the classical process.
Enzymes also help in production of juices and concentrates that are very stable and have a good taste with a
reduced pomace waste, and reduced production cost
(higher yield, less equipment, less labor in a concentration process). Finally, they also permit the processing of
dierent fruits by the same process.
Cider. Use of pectinases assists in the production of
French ciders of high and consistent quality from cider
apple varieties that are bitter, sweet or sour (Drilleau,
1985). Traditionally, the fermentation of apple juice was
very slow (a few weeks to 3 months ) and with some
residual sugars at the end, depending on the type of cider produced (dry, semi-sweet and sour). The cider was
aromatic but with low acidity and sometimes with a
bitter taste because of lactic, acetic acids and other
contaminations.
In cider production, the rst step is the preparation
of apple, followed by clarication using commercial
fungal pectinases. Pectic enzymes enable the cider producer to control and accelerate the mechanism of coagulum (a gel of pectinates) formation. The coagulum
entraps pectateprotein and tannins, which are removed
from the juice. The high quality of the juice thus obtained encourages good fermentation and produces an
aromatic cider.
4.1.1.2. Pear. Juice processing. The composition of pear
pulp and the changes during ripening and post-harvest
storage are similar to those described for apple. There is
a decrease in protopectin content and viscosity, and
increase in pectate, during pear ripening (Rahunard and
Neukon, 1964). During maturation of pears phenolic
compounds are oxidized and form insoluble compounds
(lignin). The presence of scleroids or stone cells in the
pulp results in a lower textural quality (Ranadive and
Haard, 1973). These stone cells are lignocellulosic and
contain approximately 18% lignin and 82% carbohydrate, which makes ltration or ultralteration dicult.
Araban, hemicellulose and cellulose contents are higher
than in apple. This is why it is important to use pec-

219

tinases with strong arabanase and arabinosidase activities. The process is similar to that for apples. Pears are
crushed and enzymes are added to the pulp in maceration step at about 75 g ton1 and left for 3060 min at
ambient temperature. The pectinases PL, PME, PG and
arabanases improve the extraction and yield of juice.
After pressing, the juice is depectinized with pectinases
containing high arabanase activities for about 1 h at
4550C. The juice is then ned and ltered or ultraltered and concentrated. Pears can also be processed to
purees or nectars.
Fruit nectars are pulpy fruit juices blended with sugar
syrup and citric acid to produce a ready-to-drink beverage. They come from diluted puree or pulpy juice
processing. The most important quality factor of these
nectars is that the cloud should be stable. After mixing
with water, sugar and acid, the cloud particles tend to
settle down and sometimes form a gel with a clear supernatant layer: this separation is a serious defect which
reduces the attractiveness of the product (Askar et al.,
1990).
The use of exogenous enzymes can improve the cloud
stability in nectars, and make puree concentration easier. Enzyme preparations like Rapidase LIQ (Gist
Brocades), Pectinex Ultra SP (Novo Nordisk), or Rohapect TF (Rohm) which have high PG and PL activities combined with cellulases and hemicellulases have
been found to decrease the viscosity, keep cloud stability, and improve the ability to concentrate the product.
The resulting puree is concentrated, deaerated, heated to
a temperature of 8595C, sweetened with sugar syrup
and acidied with citric acid so as to maintain constant
sugars: acid ratio at 1516 Brix. The resultant nectar is
canned or bottled, seal processed at 100C for 23 min
and marketed.
4.1.1.3. Grape juice and wine. Grape juice is not consumed in large amounts because it is too sweet (about
200 g l1 sugars) or too acid (up to 10 g l1 tartaric
acid). It is mixed with other fruit juices such as apple.
Pederson (1980) and Luh and Kean (1975) have reviewed the technology of grape juice processing. With
the high pectin content (510 g l1 ) grapes are dicult to
crush and to press. They are destemmed, crushed and
heated to 60C or 80C to release colour (in case of
black grapes) from the skins and destroy the endogenous polyphenoloxidase. Then enzymes such as Cytolase PCL5 (GistBrocades) or Ultrazyme (Novo
Nordisk) are added at approximately 50 g ton1 to
macerate the berries and increase the yield. The free
running juice is separated from the solids by dierent
kinds of lters (rotary vacuum, earth) and/ or by centrifugation. The ltered juice is cooled to 0C to prevent
fermentation and then depectinized, at approximately
200 ppm over about 2 weeks. Pectinases, mainly PG,

220

D.R. Kashyap et al. / Bioresource Technology 77 (2001) 215227

and hemicellulases like arabinogalactanases, allow insoluble particles to occulate. At the same time, cold
storage of the juice encourages removal of tartarate and
reduces the total acidity to acceptable levels. The juice is
then ltered with diatomaceous earth and concentrated,
pasteurized and bottled.
Pectic enzymes from fungi, typically Aspergillus niger,
Penicillium notatum, or Botrytis cinerea are useful in
wine making. Although juices of berries, peaches, apples, pears, and other fruits are employed (Robertson,
1977; Pilnik, 1982; Fogarty and Kelly, 1983), grape
wines are produced in greatest volume and hence will be
considered here.
Pectic enzymes are used to reduce haze or gelling of
grape juice at any one of three stages: at the rst stage,
while the grapes are being crushed; at the second stage,
which involves the must (free-run juice) before its fermentation or after; or at the last stage, once the fermentation is complete, when the wine is ready for
transfer or bottling. The addition of pectinases at the
rst stage is preferred since it increases the volume of
free-run juice and reduces pressing time. The better release of anthocyanins of the red grapes into the juice
achieved by pectic enzymes is another advantage of
these enzymes, which has received attention from redwine manufacturers also (Pilnik and Voragen, 1993).
Enzyme treatment of the juice at the second stage
before or during the fermentation, settles out many
suspended particles and often some undesirable microorganisms. Finally, addition of pectic enzymes to the
fermented wine at the last stage increases ltration rate
and clarity, but the level of enzyme supplemented must
be adjusted to allow for the inhibitory eect of alcohol
on pectinases.
4.1.1.4. Strawberry, raspberry, blackberry. The production of clear juices and concentrates from strawberries,
raspberries or blackberries requires enzymatic depectinization. The juices from these fruits contain high
amounts of pectin that remains as `colloidally dissolved
residue' making the juices viscous (Will and Dietrich,
1992). The clarication, ltration, and concentration of
these juices therefore become dicult. These residual
pectins and hemicelluloses also bind to phenolic substances and protein during the processing and storage
and result in the formation of irreversible complexes
that enzymes cannot breakdown. An additional problem
is contamination of strawberries and raspberries by B.
cinerea (Grassin and Fauquembergue, 1996). This fungus secretes a b-1,31,6-linked glucan in the berries,
which forms gum and hence reduces the lterability and
the clarity of the juice. These glucans can be hydrolyzed
by b-glucanases.
The addition of enzymes such as Rapidase BE (Gist
Brocades) to the berries at 100150 g ton1 during the
maceration step makes extraction, ltration, and con-

centration of these juices easier. After pasteurization the

juices are cooled down to 3035C and if necessary,
treated with pectinase to remove any residual pectin still
remaining in these juices. The juices are then ltered and
concentrated. They are stored at low temperature and in
dark conditions.
4.1.2. Cloudy juices
Pectin enzymes containing high levels of polygalacturonase activity are added to fruit juices to stabilize the
cloud of citrus juices, purees and nectars. Some examples are described below.
4.1.2.1. Orange. In contrast to apple pectin, which is
highly methylated, pectin from orange is only partly
methylated. This is because orange juice naturally contains large amounts of pectin esterase: an enzyme which
strips methoxyl groups from pectin molecules. In the
presence of calcium ions, insoluble calcium pectate is
formed in orange juice leading to the undesirable precipitation of haze particles. Two methods are widely
employed to prevent this cloud loss: the rst is to denature the pectin esterase by heating the juice. Unfortunately this spoils the juice avor. An alternative is to
freeze the juice concentrate, thereby holding the enzyme
in an inactive state. Orange juice is often sold like this,
but the drawback is that frozen concentrates are expensive to store and transport.
In the juice extraction process from oranges pectinases can be used at two stages of the process. They can
be added either at the end of the pulp wash extraction at
0.52.0 g per 100 kg pulp at room temperature, to reduce the viscosity or can be added after the rst nisher
at the same dose at room temperature and left for about
30 min (Rebeck, 1990). This gives a better extraction of
sugars and soluble solids, resulting in a higher yield and
therefore a lower viscosity. Enzymatic treatment increases cloud stability. In this treatment, degradation of
the pectin is limited to lower the viscosity without attacking the insoluble pectin that maintains the stability
of cloud. It is very important to use pectinases with the
lowest activity of PME as possible, to avoid clarication
of the pulp wash. The ideal enzyme would be a pure PL.
On the other hand, Rapidase Citrus 2000 (supplied by
GistBrocades) contains side activities, which decrease
the bitterness of the pulp wash, and improve its taste
and avor level.
4.1.2.2. Lemon. The traditional method of clarifying
lemon juice relies upon the natural pectin esterase content of the fruit. The major products e.g., cloudy peel
products are obtained principally from citrus peel. Peels
and pulp are ground to particle size of 35 mm and
mixed with water (1:1), heated at 95C to destroy the
citrus PME, and cooled down at 50C. The mixture so
obtained contains a high content of pectin, polysac-

D.R. Kashyap et al. / Bioresource Technology 77 (2001) 215227

charides, glycoproteins, waxes and essential oils. It is

treated with pectinases or pectinases plus cellulases.
After 1 h, the mixture goes through a nisher and the
extract liquid is further depectinized with enzyme if
necessary. It is centrifuged, pasteurized, concentrated,
bottled and marketed.
Recovery of peel oils. Essential oils are located in
special cells in the avedo of citrus fruits. They contain
hydrocarbons (terpenes and sesquiterpenes), oxygenated
compounds (aldehydes, esters, alcohols, ketones, phenols) and non-volatile residues (waxes, avonoids, fatty
acids). After juice extraction, the peel particles and the
oilwater emulsion are separated. The nished emulsion
is passed through a sand cyclone and is fed to a centrifuge to produce an oil-rich emulsion, which is sent to a
polisher that recovers the clear oil (Sterger, 1979). The
oil is then concentrated up to 6080%. In the second
stage, the polisher removes the remaining water and the
very ne particles. A cold storage allows the oil to be
dewaxed for a better purication.
Enzymes can improve the process time, yield of oil
from the emulsion, and the quality of the nal product.
Better separation of the oilwater emulsion needs pectinases and proteases. By hydrolyzing the pectinprotein
complexes, oil is released more easily into the aqueous
phase.
Citrus salads. Citrus salads can be prepared by
treating peeled citrus fruits with pectinases to digest
residual albedo still bound to the segments. These
materials are immersed in pectinase solution e.g., 1
2% Rapidase citrus 2000 (GistBrocades), for 3060
min at room temperature. The products are then
pasteurized for storage or chilled to be used for citrus
salads.
Dried animal feed. Peels, seed residues and pulp
ejected by the citrus fruit extractor can be used to make
dried animal feed. The mixture is lime treated to increase
the pH to 8.0 to take advantage of the citrus PME. As
this endogenous enzyme has optimal enzyme activity at
these pH values, it causes the demethylation of citrus
pectin (about 7080%). Enzymes such as cytolase M 102
(GistBrocades) are used to improve the extraction of
fermentable sugars, and formation of calcium pectate,
increase the rmness of the mixture, and allow better
yields of extracted liquor. The pomace is then dried to
810% moisture and this is used as an alternative or a
supplement to corn as a source of concentrated feed
nutrient for dairy cattle and sheep.
4.1.2.3. Mango. Mango is processed as a puree or nectar,
juice and various juice drink blends. A typical mango
nectar is one with 2030% puree, pH around 3.5 and
titrable acidity 0.20.3%. Exogenous enzymes are used
to increase the concentration of mango puree or to
produce clear juice and concentrate. Pectinases, hemi-

221

cellulases and cellulases are important activities used to

degrade mango pulp. Enzymatic treatment of mango
mash provides a yield of around 80% of total mango
juice present in the fruit.
4.1.2.4. Apricot. Apricot nectar exhibits a phenomenon
of cloud loss, which can be avoided by grinding the pulp
very nely (Siliha and Pilnik, 1985). These cloud particles of apricot nectar are composed of 86% individual
apricot cells, 5% broken cells, 4% cell wall fragments
and other aggregates. Cells in the fruit pulp contains
only primary cell walls that consist of approximately
90% polysaccharide and 10% protein (Weiss and Samann, 1972). Addition of pectolytic enzymes separately,
or in combination with homogenization, leads to a
cloud-stable apricot nectar, whereas homogenization
alone fails to achieve cloud stability.
4.1.2.5. Guava. The major producers of guava are South
Africa, India and Hawaii. It is eaten fresh or made into
juice, nectar, puree, jam and jelly. Guava puree is reprocessed into nectars, juice drink blends, syrups, jams
and jellies. Fruit pulps are treated with exogenous enzymes to improve juice extraction (Chen Chin et al.,
1984).
Guava pericarp, like pear, has two main cellular
types: parenchyma and stone cells. Stone cells are woody
materials that are strongly lignied and resistant to degradation by pectinases. The mesocarp portion of the
guava contains 90% of the total cell wall material of
guava pulp 77% of which are stone cells. The dominant
structural feature in parenchyma cell walls is cellulose
with side chains of glucans and xyloglucans and highly
branched arabinans. Therefore a combination of enzymes such as pectinesterase, arabanase, hemicellulase,
tannase and cellulase are used to improve the clarication of the guava juice.
4.1.2.6. Papaya. Papaya puree is prepared by conversion
of papaya esh into a semi-liquid product. As the puree
is viscous even if the fruit contains endogenous PME
and PG, it is necessary to depectinize it before concentration. Pectinases such as Rapidase (GistBrocades)
can be added for this purpose. The depectinized puree is
then concentrated from 2.4 to 3 fold.
4.1.2.7. Pineapple. The processing of pineapple mainly
involves the fruit being sliced or cubed and then canned.
A cloudy or clear juice concentrate can be processed and
is used to cover the slices and cubes in the cans. Pineapple juice results from the surplus juice drained from
the eradicator, juice drained during crushed-pineapple
preparation, or from fruits which are too small for
slicing (these are byproducts of the canning factory).
This juice is pasteurized, cooled down to produce
cloudy juice or depectinized at 50C for 2030 min or at

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D.R. Kashyap et al. / Bioresource Technology 77 (2001) 215227

20C for a few hours to produce clear concentrate.

Enzymes such as Rapidase pineapple (GistBrocades),
pectinex Ultra SP (Novo nordisk) or Rohapect BIL
(Rohm) are used to process a clear concentrate (Grassin
and Fauquembergue, 1996).
4.1.2.8. Banana. Banana is widely appreciated for its
avor and aroma (isoamylacetate) either as banana juice
or a mixture with other juices. However, banana juices
are too pulpy and too viscous to yield beverage juices
easily.
Banana can be processed at the green stage, mainly
for alcohol production, or allowed to mature. Dierent
enzymatic treatments are required to produce puree or
clear concentrate juice. Ripe bananas have high sugar
content, which increases the temperature of starch gelatinization. This is why exogenous amylases like thermostable bacterial amylase (BATs) are used after
heating the pulp to 100C for 10 min for starch gelatinization. After cooling to 45C and pH correction with
organic acid to 4.5, Hazyme (GistBrocades) is added at
15 g per 100 kg over 23 h to produce fermentable
sugars, which can be used for ethanol production.
Yellow, fully ripe, bananas are blanched at 85C for
23 min after peeling and grinding (this is done to inhibit banana polyphenol oxidase and prevent browning), or treated with 100 ppm sulfur dioxide. Banana pH
is 4.54.8 and optimal for exogenous pectinases. Blanched pulp is macerated using pectic enzymes, pressed
and the juice is claried or centrifuged before concentration. Enzyme treatment gives reduced viscosity, high
yield and concentration of the juices. Alcohol can also
be produced from these juices.
4.1.3. Unicellular products
Unicellular products are those products which are
formed by the transformation of organized tissues into a
suspension of intact cells, resulting in products that can
be used as base material for pulpy juices and nectars, as
baby foods, as ingredients for the dairy products such as
puddings and yogurt and as protoplasts for various
biotechnological applications. The enzymes used for this
purpose are referred to as ``macerases'' and this process
is referred to as maceration. It is likely that the best
enzyme preparation for maceration contains cellulases
and hemicellulases in addition to the pectic enzymes.
Some examples are described below.
4.1.3.1. Maceration of plant tissue. Maceration is a
process by which organized tissue is transformed into a
suspension of intact cells, resulting in pulpy products
used as base material for pulpy juices and nectar, as
baby foods and an ingredient for diary products such as
puddings and yogurts. Enzymatic degradation of pectin
after a mild mechanical treatment often improves
product properties if the process is carried out to leave

as many cells as possible intact, and the aim of enzymatic treatment is the transformation of tissue into a
suspension of intact cells (Bock et al., 1983). Pectin degradation should aect only the middle lamella of the
plant tissues.
Before maceration, the endogenous PE must be inactivated, and this is important for the maceration of
many products (Dongowski and Bock, 1980). Exogenous pectinases for fruit juice extraction and clarications are based on the destruction of pectin and other
dietary compounds of the fruits. Enzymatic maceration
leads to limited pectin degradation, therefore preventing
starch leaking out of the cells and avoiding the quality
defect of gluiness of the reconstituted product. The
process is often used for carrots (Bock et al., 1984) and
dried instant potato mash (Bock et al., 1979).
4.1.3.2. Liquefaction and sachharication of biomass.
Agricultural resources of fermentable carbohydrates
such as sugar beat, sugarcane, wheat, rice, and potatoes
must be processed in order to obtain fermentable sugars.
Large quantities of plant cell-wall materials become
available as byproducts in the beet-sugar and potato
starch industries. A large spectrum of enzymes (pectinases, cellulases, hemicellulases) is needed for the production of fermentable sugars from polysaccharides and
for the disruption of the cell wall matrix, so giving liquefaction of the material and the release of intracellular
carbohydrates (Beldman et al., 1984).
4.1.3.3. Isolation of protoplasts. In plant breeding programmes, many desirable combinations of characters
cannot be transmitted through conventional methods of
genetic manipulation. A process other than the sexual
cycle has now become available for higher plants and
can lead to genetic recombination. This non-conventional genetic procedure, involving fusion between isolated somatic protoplasts (walless, naked cells) under in
vitro conditions and subsequent development of their
product (heterokaryon) to a hybrid plant, may be a
useful tool for the induction of genetic variability and
combination of traits which do not exist in nature
(Gleba, 1978).
Methods of protoplast isolation from the plants are
classied into three main groups: (a) mechanical (nonenzymatic), (b) sequential enzymatic (two steps), and (c)
mixed enzymatic (simultaneous) procedures. Mechanical isolation, which includes cutting of plasmolysed tissue with a sharp-edged knife and release of protoplasts
by deplasmolysis, is now only of historical importance,
because of the low yield of the protoplasts obtained by
this method. Therefore, enzymatic processes involving
pectinases and cellulases mixture have been tried for this
purpose.
Takebe et al. (1968) employed a sequential or twostep procedure for isolating protoplasts using commer-

D.R. Kashyap et al. / Bioresource Technology 77 (2001) 215227

cial preparations of enzymes. This approach involves

initial incubation of macerated plant tissues with pectinases. The tissues are then converted into protoplasts
by a cellulase treatment. However, Power and Cocking
(1969) mixed two enzymes together (simulaneous
procedure) and isolated protoplasts in one step. In this
mixed enzyme approach, plant tissues are plasmolysed
in the presence of a mixture of pectinases (0.5% (w/v)
macerozyme, Kinki Yakult Manufacturers, Nishinomiya, Japan, or 0.5% (w/v) pectinase, Sigma Chemicals, St. Louis MO, USA; or 5% (w/v) pectinol R10,
Rohm and Hass, Philadelphia PA, USA) and cellulase
(5% (w/v), Onozuka P1500 from Trichoderma viride,
Meiji Seika Kaisha, Tokyo, Japan) in 20% (w/v) sucrose
adjusted to pH 5.6 with 2 N HCl.
The use of commercially available enzymes of fungal
as well as bacterial origin has enabled the isolation of
protoplasts from practically every plant tissue in which
cells have not acquired lignication.
4.2. Alkaline pectinases
Alkaline pectinases are mainly used in the degumming and retting of ber crops and pretreatment of
pectic wastewater from fruit juice industries. These enzymes come mostly from bacterial sources. In the industrial sector, alkaline pectinases, mainly from Bacillus
spp. are applied for the following purposes.
4.2.1. Retting and degumming of ber crops
Pectinolytic enzymes are involved in the retting and
degumming of jute, ax, hemp, ramie, kena (Hibiscus
sativa), and coir from coconut husks (Chesson, 1980;
Bruhlmann et al., 1994). Retting is a fermentation process in which certain bacteria (e.g., Clostridium, Bacillus)
and certain fungi (e.g., Aspergillus, Penicillium) decompose the pectin of the bark and release ber (Sharma
and Robinson, 1983). Commercially, retting is done by
one of the two basic forms. In dew retting (an aerobic
process) plant straw is thinly spread on the ground and
exposed to the action of the fungi and aerobic bacteria
for 210 weeks. Species of Cladosporium, Penicillium,
Aspergillus and Rhodotorula have been isolated from
dew-retted plants (Fogarty and Ward, 1972). Dew retting may produce ber of a lower quality than the alternative anaerobic process. Anaerobic retting is
achieved by submerging straw sheaves in water pits,
concrete tanks or in running fresh water (Chesson,
1980). Tank and other stagnant retts rapidly become
depleted of dissolved oxygen encouraging the development of an anaerobic ora (Chesson, 1978). Species of
Clostridia, especially Clostridium butyricum and Cl.
felsineum are considered as the major retting agents
(Hellinger, 1953; Vonzyakovskaya et al., 1974); Bacillus,
Pseudomonas and Micrococcus spp. have been isolated
from retting ax (Rosemberg and de Franca, 1967), jute

223

(Ahmed, 1963), sisal and coir (Jayasankar et al., 1967)

and have been shown to macerate straw ber in the
laboratory.
During water retting of ax, ber separation is
brought about by a pectinase enzyme produced by
bacteria (Chesson, 1978). In a series of experiments
conducted by Sharma (1987), a mixture of pectinase and
Ultrazyme and an enzyme extracted from culture of
Ceraceomyces sublaevis has been found to be the most
suitable enzyme preparation for deploymerizing noncellulosic material present on dew retted bers (at 45C,
pH 5.06.0). When characterized, the commercial enzymes used in this investigation contained a wide range
of polysaccharide degrading enzymes (polygalacturonase, PL, xylanase and traces of cellulase). Pectinol AC
(pectinase preparation) contained PG, xylanase and
traces of PL. Ultrazyme contained xylanase as the main
component of the preparation along with traces of PG.
Ramie bers are an excellent natural textile, but
decorticated ramie bers contain 2035% ramie gum,
which mainly consists of pectin and hemicellulose; hence
it is necessary to degum bers for meeting the requirement for textiles. In a classical degumming process, this
gum is removed by treatment of decorticated bers with
hot alkaline solution (1220% NaOH solution) with or
without application of pressure (Cao et al., 1992). In
addition to the high consumption of energy, this process
also results in serious environmental pollution. The degumming with microbes and their enzymes is a likely
means to solve this problem. A combined microbial and
enzymatic process has been proposed to reduce the
consumption of chemicals and energy (Paul and Bhattacharya, 1979; Gurucharanam and Deshpande, 1986).
Although degumming with neutrophilic bacteria has
been reported (Cao et al. (1992), only a few reports of
degumming with alkalophilic bacteria have been published.
For degumming mostly neutrophillic microbes have
been studied so far (Gurucharanam and Deshpande,
1986), but due to diculties in prevention of heterogenous contamination and some other disadvantages,
these strains have not been applied on an industrial
scale. Deshpande and Gurucharanam (1985) reported
that pretreating the decorticated ramie bers with alkali could accelerate the degumming process. In a
series of experiments carried out by Cao et al. (1992),
they have reported the isolation of a Bacillus sp.
(strain NT-33) which could grow well at pH 10.0,
which is of great advantage to the productivity and
activity of degumming enzymes. Moreover, fermentation at high pH values also has the advantage of
preventing contamination and thus an open fermentation process can be developed. This strain has been
reported to remove more than 70% ramie gun after 24
h fermentation under alkaline conditions. The quality
of degummed bers produced by this technique sat-

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D.R. Kashyap et al. / Bioresource Technology 77 (2001) 215227

ised the textile industry standards. Pectinolytic enzymes from actinomycetes have also shown good
correlation between the pectate lyase activity and the
degumming eects, resulting in good separation of the
bast ber.
4.2.2. Treatment of pectic wastewater
The wastewater from the citrus-processing industry
contains pectinaceous materials that are barely decomposed by microbes during the activated-sludge
treatment (Tanabe et al., 1986). Tanabe et al. (1987)
have tried to develop a new wastewater treatment
process by using an alkalophillic microorganism. Their
soil isolate of an alkalophilic Bacillus sp. (GIR 621),
produces an extracellular endopectate lyase in alkaline
media at pH 10.0. Treatment with this strain has
proved to be useful in removing pectic substances from
the wastewater.
For treatment of wastewater from citrus processing
industries various processes have been investigated,
which include: physical dewatering, spray irrigation,
chemical coagulation, direct activated sludge treatment
and chemical hydrolysis followed by methane fermentation (Tanabe et al., 1986). These processes have some
defects, such as low eciency due to chemical resistance
of the pectic substances, high treatment cost, long
treatment periods and complexity of the process.
A soft-rot pathogen, Erwinia carotovora (FERM P7576), which secrets endo-pectate lyase, has been reported to be useful in the pretreatment of pectinaceous
wastewater (Tanabe et al., 1986). However, because of
the danger from its phytopathogenecity, indirect pretreatment by the pectolytic enzyme produced from the
bacterium has also been compared and reported to
solubilize almost all the pectic substances contained in
the wastewater.
4.2.3. Production of Japanese paper
Alkaline pectinase produced by Bacillus sp. and Erwinia carotovora, due to its strong macerating activity,
has been used for retting of Mitsumata bast (Tanabe
and Kobayashi, 1987). These retted basts have been
used for the preparation of Japanese paper (Horikoshi,
1990). The strength of the pulp from bacterial retting is
as high as that obtained by the conventional soda-ash
cooking method. The paper sheets prepared from this
pulp are very uniform and soft to touch.
4.2.4. Paper making
Pulp and paper mills are beginning to use enzymes
to solve problems in their manufacturing processes.
Papermaking is essentially a continuous ltration process in which a dilute suspension of bers, ber fragments (nes), and inorganic ller particles, such as clay
or CaCO3 , is formed into sheets. The need for water
drainage leads to use of a lter fabric with holes large

enough to allow passage of the nes and ller particles.

In modern papermaking, retention aids are added to
pulp to keep nes and ller particles in paper sheets
and to speed the drainage of water. Cationic polymers
of various structures are commonly used as retention
aids (Horn and Linhart, 1996). Alkaline peroxide
bleaching of pulps solubilizes polysaccharides, which
are troublesome interfering substances (Holbom et al.,
1991). Prominent among these polysaccharides are
pectins, or polygalacturonic acids. The ability of polygalacturonic acids to complex cationic polymers
(cationic demand) depends strongly on their degree of
polymerization, monomers, dimers, and trimers of
galacturonic acid do not cause measurable cationic
demand, but hexamers and long chains have high cationic demand (Thornton et al., 1994). Pectinase can
depolymerize polymers of galacturonic acids, and subsequently lower the cationic demand of pectin solutions
and the ltrate from peroxide bleaching (Reid and
Ricard, 2000).
4.2.5. Oil extraction
Oils from rape seed (Canola), coconut germ, sunower seed, palm, kernel and olives are traditionally
produced by extraction with organic solvents. The most
commonly used solvent is hexane, which is a potential
carcinogen. Cell-wall-degrading enzymes, including
pectinase, may be used to extract vegetable oil in an
aqueous process by liquefying the structural cell wall
components of the oil-containing crop.
Recently, the plant cell-wall-degrading enzyme preparation has begun to be used in olive oil preparation.
The enzymes are added during grinding of the olives,
thereby the oil is released easily in the subsequent separation techniques (West, 1996). Enzyme treatment
hence causes an increase in yield of olive oil. The increase in the yield depends upon pH, temperature, and
dosage of the enzyme used.
Olivex, an enzyme preparation derived from A.
aculeatus, that contains pectinolytic activity besides
hemicellulases and cellulolytic activity has been shown
to give good oil extraction and better stability when
stored. The oil also shows increased content of polyphenols and vitamin E, which stabilizes the oil against
rancidity.
4.2.6. Coee and tea fermentation
Pectinases play an important role in coee and tea
fermentation. Fermentation of coee using pectinolytic
microorganisms is done to remove the mucilage coat
from the coee beans. Pectic enzymes are sometimes
added to remove the pulpy layer of the bean, threefourths of which consists of pectic substances. Cellulases
and hemicellulases present in the enzyme preparation
aid the digestion of the mucilage. A diluted commercial
enzyme preparation is sprayed on to the beans at a dose

D.R. Kashyap et al. / Bioresource Technology 77 (2001) 215227

of 210 g per ton at 1520C. The fermentation stage of

coee processing is accelerated and reduced from 40 to
80 h to about 20 h by enzyme treatment. Since largescale treatment of coee with commercial pectinases is
costly and uneconomical, inoculated waste mucilage is
used as a source of microbial pectic enzymes. The fermentation liquid is washed, ltered and then sprayed on
to the beans (Amorim and Amorim, 1977; Carr, 1985;
Godfrey, 1985).
Fungal pectinases are also used in the manufacture of
tea. Enzyme treatment accelerates tea fermentation, although the enzyme dose must be adjusted carefully to
avoid damage to the tea leaf. The addition of pectinase
also improves the foam-forming property of instant tea
powders by destroying tea pectins (Carr, 1985).

5. Commercial pectinases

Supplier

Location

Brand
name

C.H. Boehringer
Sohn
Ciba-Geigy, A.G.

Ingelheim,
West Germany
Basel,
Switzerland
Aarthus,
Denmark
Tokyo, Japan

Panzym

Grinsteelvaeket
Kikkoman
Shoyu, Co.
Schweizerische
Ferment, A.G.
Societe Rapidase,
S.A.
Wallerstein, Co.
Rohm, GmbH

Basel,
Switzerland
Seclin, France
Des Plaines,
USA
Darmstadt,
West Germany

Ultrazyme
Pectolase
Sclase
Pectinex
Rapidase,
Clarizyme
Klerzyme
Pectinol,
Rohament

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