Industrial applications of pectic enzymes a review

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Process Biochemistry Vol. 33, No. I, 21-28, 1998 pp. 
0 1998 Elsevier Science Ltd 
All rights resewed. Printed in Great Britain 
0032.9592/98 $19.00+0.00 
PII: SOO32-9592(97)00046-O 
Industrial applications of pectic enzymes: a 
review 
Itziar Alkorta,” Carlos Garb& Maria J. Llama and Juan L. Serra 
Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias, Universidad de1 Pais Vasco, Apdo. 644, 48080 Bilbao, 
Spain 
(Received 9 April 1997; accepted 19 April 1997) 
Abstract 
Although pectic enzymes have long been used to increase the yield and clarity of fruit juices, it is only 
recently that technological innovations, such as the use of immobilization supports and continuous-flow 
systems, have been considered to optimize these fruit processing procedures. To our knowledge, this is the 
first review to focus on the benefits brought to the field by these new technologies and their potential for 
commercial applications. 0 1998 Elsevier Science Ltd 
Keywords: pectic enzymes, immobilization, membrane reactors. 
Introduction 
Pectic suhstunces 
Pectic substances are glycosidic macromolecules of 
high molecular weight that are widespread in the plant 
kingdom. They form the major components of the 
middle lamella, a thin layer of adhesive extracellular 
material found between the primary walls of adjacent 
young plant cells. In addition, they constitute an 
important part of the primary plant cell wall. 
The American Chemical Society classified pectic 
substances into four main types: protopectins, pectinic 
acids, pectins and pectic acids [l]. Whereas protopec- 
tins are water-insoluble, the other three are either 
totally or partially soluble in water [2]. 
Chemically, pectic substances are essentially 
branched heteropolysaccharides containing between a 
few hundred and about one thousand building blocks 
per molecule, with a backbone consisting of galac- 
turonic acid residues part of which are methyl- 
esteritied. The pectin molecule (Fig. 1) is generally 
agreed to consist of a chain structure of axial-axial 
E-( 1,4)-linked D-galacturonic acid units, containing 
blocks of I.-rhamnosc rich regions, with mainly arabi- 
nose, galactose and xylose as side chains. The carboxyl 
*TO whom correspondence should be addressed 
groups of the galacturonic acid are partly esterified by 
methyl groups, and partly or completely neutralized by 
sodium, potassium or ammonium ions. Some of the 
hydroxyl groups on CZ and C, may be acetylated [3]. 
Although, owing to their great importance in many 
fields, pectic substances have been intensively studied 
for more than six decades a better knowledge of the 
chemical structure of industrial pectins is still required 
if we are to understand and improve their techno- 
logical applications. Curiously, and most likely due to 
their great complexity, despite many reports on the 
applications of pectic substances, mainly as food addi- 
tives, relatively little work dealing with the chemical 
composition and structure of industrial pectins has 
been published [4]. Here, it must be clearly stated that 
there is no such thing as a uniform pectic substance 
since they present numerous variations in molecular 
mass; in degree of esterification and acetylation; in 
content and type of binding of neutral sugars: and in 
the distribution of substituents and non-uronide com- 
ponents [5]. 
Apart from their function as ‘lubricating’ or 
‘cementing’ agents in the cell walls of higher plants. 
pectic substances are also involved in the interactions 
between plant hosts and their pathogens [6] and in the 
texture of fruits and vegetables during growing, 
ripening and storage [7]. Traditionally, pectic sub- 
stances have found application in the food industry 
22 Itziar Alkorta et al. 
Fig. 1. Structure of the pectin molecule. Only one chain of the major component of pectins - galacturonic acid partially 
methvlesterified - is renresented here. Side chains of galactose, arabinose and xylose residues are not included in the figure. 
Taken from Serra et al. [2j 
most often as gelling components [8], while nowadays 
they are also used as nutritional fibre [5]. 
Although research in the field of ‘pectic substances’ 
has been carried out for many decades by scientists and 
technologists from variety of disciplines, at present, 
interest in these substances is still continuing 
worldwide. 
the glycosidic bonds of either pectate (endo- and exo- 
polygalacturonate lyase) or pectin (endopoly- 
methylgalacturonate lyase). Polymethylgalacturonate 
lyases (all of which are endo-acting enzymes) are the 
only pectinases proven to hydrolyze pectin [5]. 
Pectic enzymes 
Pectic substances are naturally degraded by ‘pectic 
enzymes’. The classification of pectic enzymes is based 
on their attack on the galacturonan backbone of the 
pectic substance molecule. Basically three types of 
pectic enzymes exist: de-esterifying enzymes (pectines- 
terases), depolymerizing enzymes (pectinases: hydro- 
lases and lyases) and protopectinases. 
Finally, protopectinases, protopectin-solubilizing 
enzymes, which liberate water-soluble and highly poly- 
merized pectin from protopectin, are classified into two 
types: one type reacts with the polygalacturonic acid 
region of protopectin (A-type), the other with the poly- 
saccharide chains that may connect the polygalac- 
turonic acid chain and cell wall constituents (B-type) 
Fl. 
Table 1 and Fig. 2 summarize the classification of 
pectic enzymes and the enzymic mode of action of the 
most frequent depolymerases on the pectin molecule, 
respectively. 
The first ones catalyze the de-esterification of the 
methoxyl group of pectin forming pectic acid, and are 
produced by fungi, bacteria, yeast and higher plants [5]. 
These pectinesterases are present in practically all 
commercial pectolytic enzyme preparations and may be 
involved in changes in the pectic substances of fruits 
and vegetables during ripening, storing and processing 
[71. 
The depolymerases split the a-(1,4)-glycosidic bonds 
between galacturonic monomers in pectic substances 
either by hydrolysis (hydrolases) or by J-elimination 
(lyases). The hydrolases have been divided into four 
groups [9]: those preferring pectate were called polyg- 
alacturonases, while those preferentially degrading 
pectin were called polymethylgalacturonases. The pre- 
fixes endo- and exo- used in connection with either 
these names denoting a random- or terminal-action 
pattern, respectively. 
Most commercial preparations of pectic enzymes are 
obtained from fungal sources [lo]. In fact, although for 
obvious economical reasons it is very difficult to find 
reliable information about the commercial production 
of pectic enzymes, probably all producer strains are 
Aspergillus species [5]. Nevertheless, in our laboratory, 
after studying the growth and extracellular production 
of pectin lyase by a group of 16 fungi obtained from 
the Spanish Type Collection Culture (Table 2) we con- 
cluded that Penicillium italicum and Penicillium 
expansum were the best producers [l 1,121. 
Pectic enzymes and fruit juice industry 
Endopolygalacturonases are produced by a wide 
variety of organisms such as numerous fungi and 
bacteria, a few yeasts, higher plants and some plant- 
parasitic nematodes. Exopolygalacturonases, however, 
have been shown to occur in different fruits and veget- 
ables as well as fungi and some bacteria [5]. 
Pectic substances are responsible for the consistency, 
turbidity and appearance of fruit juices [13]. In fact, 
the presence of pectic substances in fruit juices causes 
a considerable increase in their viscosity, thereby 
impeding the processes of filtration and subsequent 
concentration [2]. 
In relation to polymethylgalacturonases, although 
there are some articles describingtheir catalytic activity 
[9], the existence of these enzymes appears to be ques- 
tionable since it is possible that polygalacturonase 
preparations, contaminated with pectinesterases, have 
been mistaken for polymethylgalacturonase-containing 
preparations. 
Pectic enzymes have long been used to increase 
juice yield and to clarify juices [14]. Actually, most 
pectic enzyme preparations are used in the fruit pro- 
cessing industry. Considering that pectic enzymes alone 
account for about one-quarter of the worlds food 
enzyme production [15], one can safely conclude that 
we are ‘dealing with a huge market that could benefit 
immensely from the application of technological inno- 
vations designed to reduce economical costs and 
increase the productivity of the system. 
Lyases, which are also called trans-eliminases, split Fruit juice clarification, a process required to facili- 
Industrial applications of pectic enzymes: a review 23 
Table 1. Classification of pectic enzymes acting on pectins or pectic acids 
EC suggested name Common name 
De-esterifying enzymes 
EC number Substrate Action pattern 
Polymethylgalacturonate esterase (PMGE) 
Depolymerizing enzymes 
Pectinesterase 3.1.1.11 Pectin Random 
Hydrolases 
Endopolygalacturonase (Endo-PC) 
Exopolygalacturonase 1 (Exo-PGl) 
Exopolygalacturonase 2 (Exo-PG2) 
Endopolymethylgalacturonase (Endo-PMG) 
Exopolymethylgalacturonase (Exo-PMG) 
Lyases 
Endopolygalacturonate lyase (Endo-PGL) 
Exopolygalacturonate lyase (Exo-PGL) 
Endopolymethylgalacturonate lyase (Endo-PMGL) 
Exopolymethylgalacturonate lyase (Exo-PMGL) 
Polygalacturonase 
Polygalacturonase 
Polygalacturonase 
Pectin hydrolase 
Pectin hydrolase 
Pectate lyase 
Pectate lyase 
Pectin lyase 
Pectin lyase 
3.2.1.15 
3.2.1.67 
3.2.1.82 
4.2.2.2 
4.2.2.9 
4.2.2.10 
Pectate Random 
Pectate Terminal 
Pectate Penultimate bonds 
Pectin Random 
Pectin Terminal 
Pectate Random 
Pectate Penultimate bonds 
Pectin Random 
Pectin Terminal 
Pectic enzymes acting on ohgogalacturonates have not been included in this table because they are not very abundant and of 
little interest for industrial pectin degradation. Enzymes have been classified and named according to the Enzyme Commission 
(EC) (IUPAC-IUB recommendations). 
tate filtration and remove turbidity, is the oldest use of viscosity as well as the flocculation of the micelles 
pectic enzymes [15]. The presence of pectic substances present [16], allows these particles to be separated by 
that suspend toward insoluble (pulp) particles leads to sedimentation or filtration. 
problems in the clarification of fruit juices. The addi- Another important application of pectic enzymes in 
tion of pectic enzymes, which results in a rapid drop in fruit processing is their role in fruit juice extraction. 
COO-CH 1 ’ OH 
‘“\, 
PMGL 
, OH coo- I OH YOO~ 
i1 
PMGE 
OH 
Fig. 2. Enzymatic mode of action of the most frequent depolymerases on the pectin molecule. PMGL: polymethylgalacturonate 
lyase (pectin lyase); PMG: polymethylgalacturonase (pectin hydrolase); PMGE: polymethylgalacturonate csterase (pectines- 
tcrase); PGL: polygalacturonate lyase (pectate lyase); PC: polygalacturonase (pectate hydrolase). Taken from Serra et al. 121 
24 Itziar Alkorta et al. 
Table 2. Production of pectin lyase by several fungi after 63 h 
of growth” 
soluble enzymes are used for the clarification of fruit 
juices. 
Strain Final pH Dry wt 
of cultures (ms/ml) 
Pectin 
lyase 
Wmg 
protein) 
Aspergillus carbonatius 
(CECT 2086) 
2.6 3.3 0,9 
A. niger 
(CECT 2088) 
3.9 7,6 0.9 
Penicillium enpansum 
(CECT 2275) 
3.5 2.1 4.7 
Most studies dealing with immobilized pectic 
enzymes can be classified into two groups [22]: (a) 
those concerning the immobilization of the whole 
complex of pectic enzymes, and (b) those showing the 
immobilization of separate purified pectic enzymes 
which were further studied for the effect of immobili- 
zation on their properties and mode of degradation of 
polymeric substrates. 
l? italicum 
(CECT 2294) 
3.7 3.1 12.0 
“Liquid Czapek Dox (modified) medium containing 05% 
(wt/vol) citrus pectin was inoculated with 5 x 10“ spores per 
ml in all cases. After 63 h of growth, aliquots were sampled 
and pH, biomass, and pectin lyase activity were determined. 
During this time, no pectin lyase activity was detected in 
culture filtrates of Saccharomyces fibuligera (CECT 1238), A. 
niger (CECT 277.5, 2090 and 2091), A. oryzae (CECT 2094 
and 2095) Rhizopus arrhizus (CECT 2339 and 2340), R. nigri- 
cans (CECT 2344) R. rhizopodiformis (CECT 2763), R. stob- 
nifer (CECT 2672), and R. thailandensis (CECT 2774). 
Again, degradation of pectin by these enzymes facili- 
tates pressing and ensures high yields. After crushing 
soft fruits such as blackcurrants, raspberries and black 
cherries, these crude juices are often very viscous and 
the remaining solids very difficult to separate from the 
juice [5]. The activity of pectic enzymes causes a drop 
in viscosity making it possible to extract juice by 
pressing. 
Pectic enzymes are also applied in the maceration 
and solubilization of fruit tissues to retain integrity of 
the cell wall in a process called liquefaction of tissues. 
Another relatively recent use of pectic enzymes is 
the selective treatment of citrus concentrates with 
endopolygalacturonase to give limited hydrolysis of the 
pectins so as to reduce viscosity (citrus juices tend to 
gel when concentrated) [14]. 
Different endopolygalacturonases have already been 
succesfully immobilized on various supports: endopoly- 
galacturonase of Aspergillus sp. was immobilized by 
adsorption onto porous powdered poly(ethylenetere- 
phthalate) and covalently bound via amino groups on 
poly(2,6-dimethyl-p-phenyleneoxide) and poly(6-capro- 
lactame) [23-261. Rexova-Benkova and Mrackova- 
Dobrotova [27] also studied the immobilization of an 
endo-o-galacturonase by covalent binding to (hydrox- 
yalkyl) methacrylate gel. Similarly, other materials such 
a y-alumina and various types of macromolecular sup- 
ports have been utilized to immobilize endopoly- 
galacturonase and pectinesterase for the treatment of 
fruit juices [19,28-311. In fact, these two enzymes have 
been covalently co-immobilized into a glycophase- 
coated controlled pore-glass to compare their 
behaviour to that shown by the enzymes immobilized 
separately [13,32]. These same authors [l&33] 
immobilized a commercial preparation of pectolytic 
enzymes onto a nylon-polyethyleneimine copolymer 
and studied their potential application for the claritica- 
tion of apricot juice. Porous glass particles were also 
utilized by Weibel et al. [34] to covalently immobilize 
pectinesterase. Finally, in our laboratory, pectin lyase 
purified from Penicillium itulicum was immobilized by 
covalent binding to nylon in order to evaluate its pos- 
sible application in fruit juice processing [ 16,351. 
Immobilized pectic enzymes and fruit juice 
clarification 
Traditionally, the industrial utilization of pectic 
enzymes in fruit juice processing has been conducted in 
conventional batch reactors using soluble enzymes [17]. 
Unfortunately, after each cycle of operation the 
enzymes can not be recovered for further use, and are 
inevitably present in the final product altering organo- 
leptic properties. In this context, the immobilization of 
pectolytic enzymes has proven to be very advantageous 
for continuous industrial use [18]. 
From all these papers, and others not mentioned 
here, two things can be noted: (a) the most frequent 
methods for immobilization of pectic enzymes are 
covalent binding to insoluble supports and adsorption 
on a solid support; and (b) although pectin lyase and 
pectinesterase have also been immobilized, the most 
frequentimmobilized enzyme has been endopolygalac- 
turonase. Both facts can also be concluded from the 
survey of the main data on immobilized pectolytic 
enzymes carried out by Lozano et ul. [33]. 
However, relatively little research has been carried 
out on the immobilization of pectic enzymes [19]. 
Several authors [20,21] have already reported the dif- 
ferent advantages that the immobilization of pectic 
enzymes offers in comparison with processes where 
In spite of this amount of research on immobilized 
pectic enzymes, the procedures proposed still show low 
immobilization yields, high support costs and methods 
too elaborate to be easily applied on an industrial 
scale. In fact, Alkorta et al. [16] in their studies on 
nylon immobilized pectin lyase from P italicurn found a 
relatively low percentage of activity, probably due to 
the existence of diffusional constraints that impeded 
both substrate access to the enzyme and the release of 
the product into the bulk phase where it was actually 
being monitored. In addition, immobilization itself 
Industrial applications of pectic enqmes: a review 2s 
could partly hinder the conformational changes for the 
activity of the enzyme. 
These results were not surprising since although 
over the last decade there has been increased interest 
in the preparation of immobilized pectic enzymes for 
the clarification and depectinization of fruit juices 
using a wide variety of carriers and methods, satisfac- 
tory results without any limitations have rarely been 
achieved. The coupling of the enzyme usually results in 
a decrease in enzyme activity and, in some cases, some 
other characteristics of the enzyme action mode are 
also altered [ 16,361. 
Nonetheless, there are in the literature some posi- 
tive results suggesting the vast potential of immobilized 
pectic enzymes for the clarification of fruit juices and 
other similar applications. For instance, Alkorta et al. 
[16] reported that, in spite of the low yield values, the 
nylon-immobilized pectin lyase was capable of pro- 
ducing a 36% reduction in viscosity in a pectin solution 
(compared to a 46Y o viscosity reduction found with the 
free soluble enzyme), a value considered acceptable for 
the commercial application of this immobilized system 
in fruit juice processing. Omelkova et al. [26] also 
found a slower decrease in viscosity when using an 
immobilized endopolygalacturonase as compared to 
the soluble enzyme. Lozano et al. [33] point out that 
the viscosity-reducing activity is clearly affected by 
immobilization because the steric constraints produced 
by the support mean that only the more external bonds 
of the pectin molecule are accesible to the immobilized 
pectolytic enzymes. 
Alkorta et al. [16] also examined the effect of 
immobilized pectin lyase on the viscosity of melon, 
apricot and other fruit juices. In all cases, a reduction 
in viscosity of at least 25% was observed after just 
60 min of incubation. Moreover, in the case of peach 
juice, the decrease in viscosity was similar to that 
obtained when the soluble enzyme was used. This may 
be due to the fact that the immobilized enzyme had a 
lower optimum pH of activity (closer to the pH of fruit 
juices) than its soluble counterpart. This shift to lower 
pH values is commonly found when immobilizing 
enzymes on supports actually possessing a positive net 
charge due to alteration of the proton distribution 
between the bulk phase and the surroundings of the 
immobilized enzyme [31,37,38]. Since many fruit juices 
show acidic pH values, this shift toward lower pH is an 
important benefit when considering the possible appli- 
cation of immobilized systems for the treatment of fruit 
juices. 
Alkorta rt al. [16] showed that the immobilization of 
pectin lyase on nylon caused a marked increase in the 
temperature stability of the enzyme. This higher 
thermal stability could be due to a restriction on its 
degrees of freedom caused by the immobilization itself, 
consequently hindering heat denaturation. Although it 
is well-known that an enzyme, when immobilized, 
sometimes turns out to bc more resistant to heat and 
chemicals than the soluble enzyme [39], the current 
state-of-the-art does not provide any direct answer to 
the question of what the stability of a given enzyme 
would be after immobilization [40]. In this respect, 
Ginalska et al. [41] found that endopolygalacturonase 
purified from A. niger was less sensitive to the action of 
metal ions after immobilization than was its soluble 
counterpart. 
Reactors for immobilized pectic enzymes 
The activity yields of immobilized systems using pectic 
enzymes appear to be negatively influenced by the col- 
loidal characteristics of pectin solutions and by the high 
molecular weight of pectic substances [42]. These diffu- 
sional limitations can be overcome, at least partly, 
thanks to the development of membrane reactors. 
Cross-flow membrane reactors have certain advantages 
when used to treat polymeric cloudy and viscous sub- 
strates [18]. For a review on membrane reactors and 
ultrafiltration, see Heath and Belfort 1431 and Zeman 
[44], respectively. 
Membrane reactors combine the advantages of con- 
ventional bioreactors and membrane technology by 
bringing together a continuous reaction and the simul- 
taneous separation of products from the reaction 
mixture. In addition to permitting the continuous 
removal of the resulting small-sized products from the 
reaction mixture containing the enzyme and the 
remaining large substrate, membrane reactors show, in 
principle, higher degrees of conversion than other 
types of bioreactors [45]. 
Recently. Lozano et al. [IX] reported the kinetics 
and operational behaviour of an immobilized pectolytic 
enzyme derivative in a membrane cross-flow reactor. 
Although ultrafiltration has been frequently used for 
fruit juice clarification [46,47], to our knowledge. only 
a few studies [ 17,18,48-501 that comhinc the USC of 
this technology and pectic enzymes have been 
reported. 
Alkorta et al. [17] designed and optimized a mem- 
branc reactor for the continuous /j-trans-elimination of 
citrus pectin using pectin lyasc from P italicurn. Figure 
3 shows the membrane reactor system used in this 
work. All four variables studied (i.c. enzyme and sub- 
strate concentrations, filtrate flow-rate, and reaction 
volume) had an effect on pectin convcrsion. Although 
a gradual decay in reactor activity was obscrvcd over 
time. the system was capable of maintaining a viscosity 
decrease of 55% for approximately 50 h. After that 
time. the enzyme lost part of its activity probably due 
to sheer stress [ 171. Lozano ct ~1. [I81 also found that 
after reaching a maximum conversion value. the system 
became progressively less cfticicnt. Ncverthcless. it 
seems likely that the system stability may dcpcnd on 
the nature of the enzyme under study. 
Alkorta et 01. [SO] cxamincd the decrease in viscosity 
26 Itziar Allvxta et al. 
Ultrafiltration I : - Valve 
/I \ 
Reactor 
Water bath 
u module 
I I - gauge 
Fig. 3. Scheme of the continuous-flow membrane 
system. Taken from Alkorta et al. [17] 
reactor 
of several fruit juices caused by pectin lyase in a batch 
and confined in a continuous-flow ultrafiltration mem- 
brane reactor. Although a similar viscosity reduction 
was achieved in both reactors (batch vs. continuous- 
flow membrane reactor) (Fig. 4), at this point, the 
intrinsic advantages of using continuous sytems should 
be taken into consideration. 
Conclusions on future applications of pectic enzymes 
Although pectic enzymes have long been used for fruit 
processing, it has only been recently that technological 
innovations such as the use of immobilization supports 
and continuous-flow systems have been considered in 
this context.Despite the fact that satisfactory results in the appli- 
cation of immobilized pectic enzymes without any 
limitations have rarely been achieved, the present data 
suggest that the immobilization of these enzymes 
appears to be of potential interest for the clarification 
of fruit juices. Once certain problems, such as diffu- 
sional constraints and the decrease in the enzyme 
activity usually found after immobilization, are over- 
(4 Batch reactor (b) Membrane reactor 
._ 
> o Melon 
20 - ??Purple grape 
I I 1 I I / I I 
0 10 20 30 40 0 120 240 360 480 
Operation time (min) 
Fig. 4. Decrease of viscosity of melon and purple grape juices 
catalyzed by I? italicurn pectin lyase in (A) a batch reactor 
and (B) a continuous-flow membrane reactor. The reaction 
mixture (20 ml in A and 100 ml in B) contained 0.2 U/ml 
pectin lyase and was incubated at 40°C and at the pH 
imposed by the juice. One hundred per cent viscosity values 
corresponded to 2.5 and 4.1 cSt for melon and grape juice, 
respectively. Modified from Alkorta et al. [SO] 
come the field will benefit greatly from the 
advantages intrinsic to immobilized systems. 
Similarly, although much research, mainly 
several 
on the 
optimization and design of a suitable reactor, is still 
required before considering a future commercial appli- 
cation, continuous-flow reactors and, in particular, 
membrane reactors show a great potential for their 
utilization in fruit juice processing. 
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