PROTEASES FROM CELL CULTURE OF Bromelia hemisphaerica ...

FSB1 – 2004
Food Science and Biotechnology in Developing Countries
PROTEASES FROM CELL CULTURE OF Bromelia hemisphaerica
1b Barrera-Badillo G., 1a Cruz y Victoria M. T. and 2a Oliver-Salvador M. C.
1 Departamento de Graduados e Investigación en Alimentos, Escuela Nacional de
Ciencias Biológicas, IPN. 2 Departamento de Bioprocesos, Unidad Profesional
Interdisciplinaria de Biotecnología, IPN. Av. Acueducto s/n Barrio La Laguna
Ticomán, México, D. F., C.P. 07340. coliver@acei.upibi.ipn.mx
Keywords: Bromelia hemisphaerica, Bromeliaceae, plant cell culture, cysteine
proteases.
Abstract:
The juice of fruits of Bromelia hemisphaerica contains a high level of cysteine proteases. Callus
culture initiated from stem segments of B. hemisphaerica has also been shown to contain cysteine
protease(s) by proteolytic activity. Induction of callus has been shown to vary with the hormonal
composition of the Murashige-Skoog medium. Callus and cell suspension culture of B.
hemisphaerica synthesized both extracellular and intracellular protease(s).
INTRODUCTION
Plants have been for a long time of great importance not only as food sources, but
also as supply of a wide variety of chemicals including pharmaceutical,
insecticides, flavors, colorants and fragrances. Generally, the plant products of
interests are the secondary metabolites. The development of plant cell culture as
an alternative source of secondary products has been encouraged by a number of
factors which include independence from seasonal variation and pests and
diseases, a defined production system with products available when and where
required, a consistent quality and quantity, on the other hand freedom from political
restraints. Recent estimates from a number of sources give values of $300 - $500
dollars per kilo for such products, and therefore it is clear that the yield of this
product must be higher for a plant cell culture process to be profit. Plant derived
enzymes such as papain, bromelain and ficin are used in some industrial
processes such as brewing, meat tenderization, modification of functional
properties of proteins, clotting of soft cheese, etc. Some of the plant proteinase
that are synthesized in vivo by species of Caricaceae (Carica papaya, Jacaratia
mexicana); Moracea (Ficus carica) and Compositae (Cynara cardunculus) have
been obtained through plant cell culture (1-4).
The Bromeliaceae contains hundreds of species and many have been found to
contain considerable protease activity in their fruits. Bromelia hemisphaerica is one
of them and this plant grown in tropical and subtropical regions of Mexico (5, 6).

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Food Science and Biotechnology in Developing Countries
The aim of this study was to establish the callus and cell suspension culture of B.
hemisphaerica in order to investigate the production of protease(s) under in vitro
conditions. And investigate the effects of the auxins and cytokinins in the medium
on the initiation and growth of callus culture.
Abbreviations: 2,4-dichlorophenoxyacetic acid (2,4-D), naphtalenacetic acid (NAA), Indolacetic acid
(IAA), 4-amino-3,5,6-Trichloropiridin-2-carboxilic acid (Picloram) and Bencilaminopurine (BAP).
MATERIALS AND METHODS
Plant germination
Seeds were obtained from a specimen of B. hemisphaerica maintained at Centro
de Desarrollo de Productos Bióticos, IPN Experimental Field. Surface-sterilized
seeds were placed on Knop medium with 0.5% (w/v) of agar. Cultures were
maintained at 25 °C in darkness for three weeks and then under photoperiod of 16
h light, 8 h darkness for four weeks.
Callus initiation
The calli were induced with stem explants from B. hemisphaerica plants obtained
under aseptic conditions in Murashige and Skoog (MS) ¼ basal medium with 20 g
l -1 sucrose and 6 g l -1 agar. The medium also were supplemented with four auxins
(2, 4-D, NAA, IAA and Picloram) and two cytokinins (Kinetin and BAP) in different
proportions giving twelve combinations (MS1 to MS12). And two auxins
concentrations (1.0 y 0.5 mg/L) were used. The pH was adjusted to 5.7 before
autoclaving at 121 °C for 20 min. The cultures were carried out at 30 + 3 °C and
under photoperiod as described above. After that the calli were induced, they were
transferred to fresh medium each two weeks. The proteolytic activity was
determined in the medium where the callus was growing.
Suspension culture
Cell suspension cultures were established by suspending 3-5 g fresh weight of calli
into 50 ml of MS medium containing 0.5 mg l -1 Picloram, 0.5 mg l -1 BAP and 4 %
(w/v) of sucrose in 125 ml Erlenmeyer flasks. Medium culture was subcultured
every week. The cell suspension cultures were carried out at 27 + 2 °C and under
13 hours of light and 11 hours of darkness conditions and shaken at 150 rpm. The
proteolytic activity was determined every week in the culture medium.
Protease assay
The proteolytic activity was measured both in callus and cell culture medium by
Kunitz method, modified by Ortega and del Castillo (8) according with the protocol
described by Badillo et al., (2), using 1% casein in sodium phosphate buffer 0.05
M pH 7.6 as substrate at 37°C.

RESULTS AND DISCUSSION
FSB1 – 2004
Food Science and Biotechnology in Developing Countries
Plants grown in aseptic conditions were obtained after to 50 days with germination
of 80 %. Callus induction were obtained with several combination of BAP or Kinetin
and Picloram or 2,4-D concentration in agar medium. With 1.0 mg l -1 of auxins
callus induction was observed in the mediums MS 4, MS 5, MS 8 and MS 12 after
to 60 days. Response was better with 0.5 mg l -1 of auxins because callus induction
was observed in the mediums MS 1, MS 5, MS 8, MS 9 and MS 12 after to 50 days
(Figure 1 and Table 1). In some cases organogenesis was obtained. The organs
were formed with low concentration of both ANA or AIA and BAP, especially with
1.0 mg/L of ANA and without cytokinins roots were induced (Table 1).
Table 1. The effect of auxins concentration on callus induction
MEDIUM COMPOSITIÓN RESULTS
(MS) AUXINS CYTOKININS
(mg l -1 )
AUXIN 1.0 mg l -1 AUXIN 0.5 mg l -1
MS 1 2,4-D - Oxidation Callus
MS 2 ANA - Roots Oxidation
MS 3 AIA - Oxidation Oxidation
MS 4 Picloram - Organogenesis Oxidation
MS 5 2,4-D 0.25 Kinetin Callus Callus
MS 6 ANA 0.25 Kinetin Organogenesis Oxidation
MS 7 AIA 0.25 Kinetin Organogenesis Organogenesis
MS 8 Picloram 0.25 Kinetin Callus Callus
MS 9 2,4-D 0.5 BAP Oxidation Callus
MS 10 ANA 0.5 BAP Organogenesis Organogenesis
MS 11 AIA 0.5 BAP Oxidation Oxidation
MS 12 Picloram 0.5 BAP Callus Callus
In this study we get green to brown callus with hard to soft texture, callus approx.
0.5 -2 cm thick after 60 days of culture (Figure 1).
A B C D
Figure 1. Callus induction: (A) callus inducted in MS 8 medium with 0.5 mg l -1 Picloram. (B) callus inducted in
MS 9 medium with 0.5 mg l -1 2,4-D. (C) callus inducted in MS 12 medium with 0.5 mg l -1 Picloram. (D) callus
inducted in MS 12 medium with 0.5 mg l -1 Picloram.

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Food Science and Biotechnology in Developing Countries
The specific protease activity in the callus medium (extracellular enzyme) was
different for each growth-regulator combination (Figure 2). The maximal value of
specific activity of callus was obtained with Picloram and BAP 0.5 mg l -1 . And these
calli were selected for transfer to liquid medium to form cells suspension cultures.
The results indicated that callus and biomass from cell suspension culture contain
higher concentration of intracellular proteases than extracellular proteases (Figure
2). However the extracellular protease is more attractive with a view to
biotechnological production. The extracellular protease activities of free cell culture
medium are shown in the figure 3.
The enzyme activity directly correlated with the biomass production (date not
shown). In addition the extracellular protease reached the maximal value after 13
weeks decreasing subsequently to lower value (Figure 3).
Proteolytic activity was showed in agar medium were calli grown (Figure 2).
UT/L
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
MS4 MS5 MS8 MS12 CALLUS
Murashige & Skoog Medium
Figure 2. Extracellular and intracellular (calli in MS12) protease activity of calli of
B. hemisphaerica grown in different growth regulators proportion, Auxins 0.5 mg l -1 .
Activity expressed in terms of tyrosine (UT/L).
Cell suspension culture medium had extacellular proteolytic activity of 2000 –
20000 UT/L (Figure 3).

UT/L
25000
20000
15000
10000
5000
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Food Science and Biotechnology in Developing Countries
.
0
0 2 4 6 8 10 12
Weeks
14 16 18 20 22 24
Figure 3. Extracellular protease of cell suspension cultures of B. hemisphaerica.
MS 12: Picloram and BAP 0.5 mg l -1 . Activity expressed in terms of tyrosine (UT/L).
CONCLUSION
In our opinion callus and cell suspension cultures of B. hemisphaerica show
promising potential for the production of cysteine protease that might be suitable
for the transformation of foods.
Acknowledgements
This work was supported by Mexican National Polytechnic Institute (IPN) Project CGPI: 20030510.
The authors would like to thank: 1a,2a SIBE-COFFA-IPN, 1b PIFI-IPN,

References:
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Food Science and Biotechnology in Developing Countries
1. Mel G.P., M edora R.S. and Bilderback D.E. (1979). Substrate specificity of the enzym es
from papaya ca lus culture. Z. Pflanzelphysiol. Bd. 91 S: 279-282
2. Badilo C. J.A. Cruz M . A. Garibay O. C. y O liver S. M.C. 2002. Cultivo de células de
Jacaratia mexicana. Enzimas proteolíticas. Informe de proyecto de investigación, UPIBI, IPN.
3. Cormier F., Charest C. and Dufresne C. (1989). Partial purification and properties of
proteases from fig (Ficus carica) ca lus culture. Biotech. Le ters, 11(11): 797-802.
4. Figueiredo A. C., Fevereiro, P., Cabral, J. M. S., Fonseca, M. M. R., Novais, J. M. and Pais,
M. S. S. (1987). Calus and suspension cultures of biom ass production of Cynara
cardunculus (Com positae). Biotechnology Le ters, 9(3): 213-218.
5. Tam er, M. and M avituna, F. (1997). Protease from freely suspended and im m obilized
Mirabilis jalapa. Process Biochem istry, 32(3): 195-200.
6. Cruz V. M.T. 1993. Aislam iento y caracterización parcial de la enzima proteolítica
“hem isfericina” obtenida de Brom elia hem isphaerica. Tesis de m aestría en ciencia
(Alim entos) ENCB. IPN. México.
7. Briones M. R. Flores C. G. Oliver S. C. Cruz L. A. Bazaldúa M. C. Solano N. A. y Cortés V.
M.I. 2002. Estudios de estabilidad de preparaciones refinadas de hem isfericina. Memoria
CEPROBI-IPN: 1-6.
8. Ortega, M. L. and del Castilo, L. M. (1966). Actividad de la mexicaína en presencia de altas
concentraciones de urea. Ciencia Mexicana, 24: 247-251.

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Food Science and Biotechnology in Developing Countries
Properties of glucose oxidase immobilised in liposomes
Berenice Ordóñez, Angélica Delgadillo, Elizabeth Contreras, Judith Jaimez,
José Manuel Rodriguez-Nogales*
Chemistry Research Centre. University of Hidalgo State..Carretera Pachuca-
Tulancingo, Km 4.5. CP 42070. Pachuca. Hidalgo. México.
* rjosem@uaeh.reduaeh.mx
ABSTRACT
Glucose oxidase was encapsulated in liposomes by the dehydration-rehydration
method. Some characteristics of the liposomal and free glucose oxidase were
compared. The enzyme encapsulated shows an apparent inhibition by glucose with a
apparent Km value higher than that of the free enzyme. The Vmax of liposomal enzyme
decreased by a factor of 0.35. The optimum temperature of the both enzyme forms
remained similar but the liposomal enzyme showed maximal activity at a more acid
pH.
KEY WORDS
Antimicrobial enzymes; DRV; glucose oxidase; liposome.
INTRODUCTION
Antimicrobial enzymes are ubiquitous in nature, playing a significant role in the
defence mechanisms of living organisms against infection by bacteria and fungi. 1
Emerging preservation techniques, such as glucose oxidase and lactoperoxidase
system, have received particular attention. 2 The antimicrobial activity of the glucose
oxidase is due to the cytoxicity of the H2O2 formed. 2
One of the major problems with the use of glucose oxidase is achieving its temporal
protection against microbial infection. The major factors responsible for this behaviour
are the uncontrolled and high production rate of hydrogen peroxide and the high
reactivity of hydrogen peroxide. 3 To obtain slow release of hydrogen peroxide at least
one of the components of the hydrogen peroxide generating system must be

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Food Science and Biotechnology in Developing Countries
immobilized. This can be the enzyme (glucose oxidase) and/or the substrate
(glucose).
The immobilization of glucose oxidase in liposome may be useful for the sustained
release of hydrogen peroxide. In this context, the present paper reports on several
attempts to gain knowledge of the immobilization of glucose oxidase in liposomes.
The effects of microencapsulation on the catalytic efficiency of the enzyme compared
to those of its native counterpart were investigated.
MATERIALS AND METHODS
Liposomes were prepared by the dehydratation-rehydratation vesicle method (DRV)
described by Kirby and Gregoriadis. 4 Enzymatic activity of free and liposomal glucose
oxidase were determined using D-glucose as substrate. The quantity of H2O2
liberated, as a result of oxidation of glucose, was determined spectrophometrically
using peroxidase in combination with o-dianisidine. 5
The apparent kinetic constants of Michaelis for the free and liposomal glucose
oxidase were determined by measuring the enzymatic reaction rates at different
substrate concentration ranging from 0.06 to 1.39 mol dm -3 at 25°C and at pH 6.0.
Michaelis constants were calculated by analysing the data according to the Leonora
program, an application specially designed for the analysis of enzyme kinetic. 6
The effect of pH and temperature of both free and microencapsulated glucose
oxidase activity was studied using a response surface design. 7 . The design of the
statistical experiments and the evaluation were performed using the computer
program Statgraphics® Plus for Windows 4.0 (Statistical Graphics Corp., Rockville,
MD 20852-4999 USA).
All experiments were carried out in triplicate under identical conditions and at least
three replicated samples were used in analytical determination. Mean values and
standard deviations are present in figures and tables.

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Food Science and Biotechnology in Developing Countries
RESULTS AND DISCUSSION
It has been assumed that the enzyme inside the vesicle follows Michaelis-Menten
kinetics identical to the behavior outside of the vesicle. 8 In order to study the kinetic
effect of microencapsulation of glucose oxidase in liposomes, the initial rates of
glucose oxidase reaction by the free and liposomal enzyme were measured at
various glucose concentrations (Fig. 1). At glucose concentration higher than 0.28
mol dm -3 , liposomal glucose oxidase did no follow pure Michaelis-Menten kinetics and
the form of the plot is typical of enzymes exhibiting substrate inhibition 20 . This
behaviour was reaffirmed fitting the experimental data to the reaction equation for
substrate inhibition. The data was also fitted to reaction equation for product inhibition
and the fit was not good. All of the above considerations indicate that the liposomal
glucose oxidase suffer an apparent inhibition by glucose accumulating inside the
vesicles with a substrate inhibition constant of the 0.95 ± 0.12 mol dm -3 .
The Km and Vmax values were (i) 0.14 ± 0.02 mol dm -3 and 5.70 ± 0.52 U cm -3 ,
respectively, using soluble enzyme and (ii) 0.19 ± 0.02 mol dm -3 and 2.01± 0.13 U
cm -3 , respectively, when the entrapped enzyme was assayed. The apparent Km of the
encapsulated enzyme was higher than of the enzyme in solution suggesting that the
lipid vesicles limited the permeation rate of substrate through the semi-permeable
vesicle membrane. This difference could be also due to chemical and/or
conformational changes in the enzyme structure provoked by an association of
glucose oxidase with the lipid vesicles.

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Food Science and Biotechnology in Developing Countries
V (U cm -3 )
6
5
4
3
2
1
0
Free
0.0 0.5 1.0 1.5
Figure 1. Initial rate vs. substrate concentration plots for glucose oxidase free
and entrapped in liposomes.
The decrease in the Vmax value caused by microencapsulation is considered to result
from the lipid membrane, which can offer significant resistance to the transport of
substrates. Therefore, the microencapsulation procedure limited accessibility of
glucose molecules to the active sites of the enzyme and caused a decrease in the
maximum reaction rate.
1.0
0.8
0.6
0.4
0.2
0.0
Liposome
0.0 0.5 1.0 1.5
Cs (mol dm -3 ) Cs (mol dm -3 )
Differences in the activity of free and liposomal glucose oxidase as a function of pH
and temperature were investigated. Figure 2 indicates that pH and temperature
promote different behaviour in the native enzyme in comparison with the entrapped
one. Thus, native enzyme exhibits a maximum at pH 6.5 and 25ºC while liposomal
enzyme shows a maximum a pH 5.2 and 26ºC. In the case of entrapped glucose
oxidase, the effect of the temperature is more important to acid values of pH.

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Food Science and Biotechnology in Developing Countries
Figure 2. Effect of pH and temperature on enzymatic activity for glucose oxidase
free and entrapped in liposomes.

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Food Science and Biotechnology in Developing Countries
REFERENCIAS
1. Gould GW. 1996. Industry perspectives on the use of natural antimicrobials
and inhibitor for food applications. J Food Prot suppl:82-86
2. Fuglsang CC, Johansen C, Christgau S and Adler-Nissen J. 1995.
Antimicrobial enzymes: applications and future potential in the food industry.
Trends Food Sci Technol (6):390-396.
3. De Jong SS, De Haan BR and Tan HS. 1998. Longterm antimicrobial activity
obtained by sustained release of hydrogen peroxide. US Patent 5747078.
4. Kirby CJ and Gregoriadis G. 1998. Dehydratation-rehydration vesicles: a
simple method for high yield drug entrappement in liposomes. Bio/Technol (2):
979-984.
5. Jones MN, Hill KJ, Kaszuba M and Creeth JE. 1998. Antibacterial reactive
liposomes encapsulating coupled enzyme systems.. Int J Pharm (162):107-
117.
6. Cornish-Bowden A. 1995. Practical aspects of kinetic studies, in Fundamentals
of Enzyme Kinetics, ed by Cornish-Bowden A. Portland Press Ltd, London, pp
55-72.
7. D.C. Montgomery. 2000. Response surface methodology, in Design and
Analysis of Experiments, John Wiley & Sons, New York, p. 224.
8. Blocher M, Walde P, Dunn P. 1999. Modeling of enzymatic reactions in
vesicles: the case of chymotrypsin. Biotechnol Bioeng (62):36-43.

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Food Science and Biotechnology in Developing Countries
EFFECT OF INVERTASE PRODUCTION OF Aspergillus niger Aa 20 IN SUBMERGED
CULTURE
Cuitláhuac Aranda-Vásquez 1 , Octavio Loera-Corral 2 , Raul Rodríguez-Herrera 1 ,
Juan Carlos Contreras-Esquivel 1 and Cristóbal Noé Aguilar 1 *.
1 Food Research Dept. School of Chemistry. Universidad Autónoma de Coahuila. Saltillo, Coahuila,
México. *Corresponding author: cag13761@mail.uadec.mx
2 Department of Biotechnology. Universidad Autónoma Metropolitana. Iztapalapa, México, D.F.
ABSTRACT
Invertase production kinetics were studied in submerged culture with or without an enzymatic
inducer (sucrose) or repressor (glucose). The maximum production of the enzyme (2170.8
U/Lmin) was reached in experiments with 50g/L of inducer in 60 hours. Maximal invertase
production (37639.58 U/L*min).was obtained with 6.25 g/L of glucose and 50 g/L of sucrose in
72 hours of culture.
Key words: invertase, glucose, sucrose, submerged culture.
INTRODUCTION
The invertase (called sucroase or β-fructofuranosidase), is an enzyme widely used in food
industry over all in confectionery. Action of this enzyme is breaking the sucrose in glucose and
fructose for obtain glucoside and fructoside syrup, which are more expensive that the sucrose
itself, for this reason is important the production and utilization of the enzyme (Romero-Gomez
et al., 2000).
The microbial metabolism in particular the fungus, allows to synthesize great variety of proteins
or enzymes, but do not make all in the same time. Only if an inducer is in contact with the
microorganism the transcript gene level is up, this make that the fungus begin to synthesize the
indicated enzyme for metabolize the inducer molecule.
In the same form that a microorganism synthesize a enzyme which can metabolize a near
molecule, too avoid synthesize enzymes in which case end product is available free in the
culture media, this kind control is called enzymatic repression for feed-back or for end product
disposition. There are other form of enzymatic repression which consist in the prevention of the
contact between the microorganism with the molecule that actives the corresponding operón in
this form the structural gene levels stay in her minimum value and enzyme neither is
synthesized, this type of repression is called for feed-forward or for absence of initial activator.
The technologies for enzyme production in submerged culture had their best age in the First
World War; in that moment the penicillin production in great quantities was priority for the
companies, the major part of investigation that was available in moment was with respect to
liquid culture for this reason used this technique for satisfied the enormous demand of penicillin
and drugs. After of First World War the companies follow produced enzymes with the same

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Food Science and Biotechnology in Developing Countries
begin operation technique, for this reason the major of companies now uses the liquid
fermentation in their production processes. However, some physiological aspects of enzyme
production in this kind of processes have not been studied to detail, still.
In this study the effect of glucose and sucrose concentration on invertase production of
Aspergillus niger Aa-20 in submerged culture was evaluated.
MATERIAL AND METHODS
Microorganism and inoculum preparation
Spores of Aspergillus niger Aa-20 stored at -20ºC were used in all experiments. Inoculum of
microorganism was prepared on potato dextrose agar and spores were harvested with tween 80
(0.01%).
Induction experiments
All experiments were carried out at the same conditions. Volume, 1 liter of culture media
Czapek-Dox: NaNO3 0.765%, KH2PO4 0.304%, MgSO4 0.152%, KCl 0.152, and several initial
sucrose concentrations of sucrose (6.25, 12.5, 25, 50 and 100g/L). It is important consider that
the rest of components were adjusted for maintain the ratio C/N at 10. Incubating temperature
of 30ºC for 72 hours, with a monitoring each 12 hours, inoculation level of 1*10 8 spore/L and
shaking at 200 r.p.m. were conditions used for experiments.
Repression experiments
Repression experiments were carried out at same conditions of volume, temperature, time,
monitoring, shaking and inoculation level that those experiments of induction, In this set of
experiments, a fixed concentration of inducer (sucrose at 50 g/L) and several concentration of
enzymatic repressor (glucose: 6.25, 12.5, 25, 50, 100g/L) were added.
Enzymatic assay for invertase
Invertase activity was assayed following the DNS-method reported by Ashokkumar et al (2001).
Total sugars determination:
Total sugars content was evaluated with phenol-sulfuric acid method as reported by Aguilar
(2000).
Reducing sugar determination
Reducing sugar content was determined with the DNS method (Miller, 1959).
Biomass produced determination
Biomass concentration was evaluated gravimetrically.

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Food Science and Biotechnology in Developing Countries
RESULTS AND DISCUSSION
Induction experiments: Figure 1 shows the results obtained for enzymatic activities at several
inducer concentrations through 72 hours of culture.
Activity (U/L*min)
2500
2000
1500
1000
500
0
Conc 6.25 Conc 12.5 Conc 25
Conc 50 Conc 100
0 12 24 36 48 60 72
Tme (Hours)
Figure 1. Invertase production at several sucrose concentrations.
Maximal invertase production was reached in cultures with 50 g/L at 60 hours. Results obtained
were higher than those reported by Ashokkumar et al (2001).
Repression experiments: Figure 2 shows the results obtained for enzymatic activities using
the same inducer concentration (50 g/L) and several glucose concentrations through 72 hours
of culture.
Activity (U/L*min)
45000
40000
35000
30000
25000
20000
15000
10000
5000
conc 0 conc 6,25 conc 12,5
conc 25 conc 50 conc 100
0
0 12 24 36 48 60 72 84 96
Time (Hours)
Figure 2. Invertase production at several glucose concentrations and 25 g/L of sucrose.

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Food Science and Biotechnology in Developing Countries
It is important to make a mention that experiments were called as “repression experiments”
however, it is very clear that in this study, glucose has a double role on invertase expression,
because at lowest initial concentrations it acts like activator of biomass production and it has a
direct effect on invertase production. In contrast, at higher than 12.5 g/L of glucose, this
molecule acts as repressor of the invertase production.
To evaluate the degree of modification on invertase production by two molecules used, it was
decided to stablish a induction or repression ratio. Both ratios were calculated dividing the
maximal invertase level at each experiment by the basal activity obtained in experiments with
glucose as sole carbon source at 30 g/L.
Figure 3 shows the results of induction and repression ratios for the invertase in different initial
concentrations of inductor and repressor enzymatic.
Repretion Ratio
250,0
200,0
150,0
100,0
50,0
0,0
R.R. I.R.
0 6.25 12.5 25 50 100
Initial Concentration (g/L)
Figure 3. Induction ratio at several sucrose concentrations (white rhombus)
and repression ratio at several glucose concentrarions and 25 g/L of sucrose
(black squares).
Use of glucose in culture medium stimulated significantly the invertase production; however, this
increasing capacity of glucose in enzyme activity is reverted at higher initial concentration of
50g/L. Cultures with inducer and glucose at initial concentration lower than 50 g/L favored the
invertase expression in comparison with those results obtained with sole inducer. This behavior
has been described previously by Aguilar et al (2001) using as study model the tannase
enzyme produced by the same fungus.
The best initial concentration of enzymatic inducer was 50g/L, obtain maximum activity at 60
hours with 2170.8U/L*min and the best initial concentration of repressor was at 6.25g/L with an
15
10
5
0
Induction Ratio

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Food Science and Biotechnology in Developing Countries
activity of 37640U/L*min in 72 hours. These results show that addition of an enzymatic
repressor at low levels have positive effect in enzyme production, the activity up 17.34 times
with relation at only inductor production.
REFERENCES
Aguilar, C.N. 2000. Inducción y represión de la síntesis de la tanasa de Aspergillus níger Aa-20
en cultivo en medios líquido y sólido. Tesis de doctorado. Universidad Autónoma Metropolitana.
Unidad Iztapalapa. México, D.F.
Aguilar, C.N., Augur, C., Favela-Torres, E. and Viniegra-González, G. 2001. Induction and
repression patterns of fungal tannase in solid state and submerged cultures. Process
Biochemistry. 36(6), 571-578.
Ashokkumar, B., Kayalvizhi, N. and Gunasekaran, P. 2001. Optimization of media for βfructofuranosidase
production by Aspergillus niger in submerged and solid state fermentation.
Process Biochemistry. 37, 331-338.
Miller, G.L. 1959. Use of Dinitrosalicylic acid reagent for determination of reducing sugar.
Analytical Chemistry. 31, 426-428.
Romero-Gomez, S.J., Augur, C. and Viniegra-Gonzalez, G. 2000. Invertase production by
Aspergillus niger in submerged and solid state fermentation. Biotechnology Letters. 22, 1255-
1258.

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PRODUCTION, PARTIAL PURIFICATION AND CHARACTERIZATION OF A FUNGAL TANNASE
PRODUCED BY SOLID STATE FERMENTATION.
Mario Cruz-Hernández, Juan Carlos Contreras-Esquivel,
Raúl Rodríguez-Herrera and Cristóbal Noé Aguilar*
Food Research Dept. School of Chemistry. Universidad Autónoma de Coahuila. Saltillo, Coahuila,
México. *Corresponding author: cag13761@mail.uadec.mx
ABSTRACT
Production and partial characterization of a fungal tannase was studied. Aspergillus niger GH1 was
selected by its capacity for tannase production in the solid state culture (SSC). Several culture
conditions were evaluated. A protocol for tannase pre-purification was developed. It included
concentration by polyethilenglycol (PEG), ionic exchange chromatography (IEC) and isoelectric
focusing Rotofor ® . Two tannase isoforms were found in the culture broth.
Key words: tannase, production, partial purification, partial characterization, solid state fermentation.
INTRODUCTION
Tannase or tannin acyl hydrolase (EC, 3.1.1.20) catalyzes the hydrolysis reaction of the ester bonds
present in the hydrolysable tannins and gallic acid esters. Its production at industrial level is by
microbial way using SmC, where the activity is expressed mainly of intracellular form, implying
additional costs in its production (Lekha and Lonsane, 1994). At the moment, the main commercial
applications of the tannase are given in the elaboration of instantaneous tea or of acorn liquor and in
the production of the gallic acid (Coggon et al., 1975; Chae and Yu, 1983; Pourrat et al., 1985; García-
Najera et al., 2002), which is an important intermediary compound in the synthesis of the antibacterial
drug, trimetroprim, used in the pharmaceutical industry (Sittig, 1988) and also in the food industry;
gallic acid is a substrate for the chemical or enzymatic synthesis of the propylgallate, a potent
antioxidant. Also, the tannase is used as clarifying agent in some wines, juices of fruits and in
refreshing drinks with flavour to coffee (Lekha et al., 1993).
Several studies have reported interesting advantages between the tannase produced by SSC in
relation with that produced by SmC. On this topic there are few reports, but they are clearly interesting
(Chaterjee et al., 1996; Lekha and Lonsane 1997; García-Peña et al., 1999; Ramírez-Coronel et al.,
1999; Aguilar et al., 1999; Aguilar et al., 2001a, 2001b and 2002a; Van de Lagemaat and Pyle, 2001).
In these, attractive advantages indicated, are the high production titles (up to 5.5 times more than in
SmC), the nature extracellular of the enzymes and the stability to wide pH and temperature ranges
(Lekha and Lonsane, 1994).
This study was aimed to evaluate several culture conditions for production partial purification and
characterization of fungal tannase
MATERIAL AND METHODS
Microorganism
The strain of Aspergillus niger GH1 (DIA/UAdeC-Collection) was selected after several comparative
studies between tannin-degrading strains (Cruz-Hernández et al., 2002). Spores were maintained at -
20ºC under a crio-protector system.

FSB1 – 2004
Food Science and Biotechnology in Developing Countries
Inoculum and culture medium
Inoculum was prepared by transferring the spores to flasks with Potato Dextrose Agar and incubating
at 30°C for 3-5 days. The spores were then scraped into a 0.01% Tween 80 solution and counted in a
Neubauer chamber. Culture medium for tannase production included (g/L): KH2PO4 (2.19),
(NH4)2SO4 (4.38), MgSO4.7H2O (0.44), CaCl2.7H2O (0.044), MnCl2.6H2O (0.009), NaMoO4.2H2O
(0.004), FeSO4.7H2O (0.06) and tannic acid (12.5) as sole carbon source (Aguilar et al. 2001).
Culture conditions
Two culture systems were used to produce tannase enzyme. Submerged culture (SmC) and solid
state culture (SSC). SmC conditions included agitation at 250 rpm, temperature 30ºC, initial pH 5.5
and several incubation times. Inoculum level was 1 x10 7 spores per ml of culture medium. The solid
support used in SSC was polyurethane foam (PUF) pulverized. An additional culture condition in SSC
was: 70 % initial humidity. SmC and SSC were kinetically monitored. Crude enzymatic extract from
SSC was obtained by compression of the fermented material.
Tannase assay
The tannase activity was evaluated by the method reported by Sharma (2000). One tannase unit was
defined as enzyme amount that release one µmol of gallic acid per min under assay conditions.
Substrate degradation
Total sugar content was determined by the method reported by Dubois (1956). Whilst reducing sugar
content was evaluated by the DNS method.
Concentration and partial purification of tannase
Tannase was concentrated using two methods with acetone and polyethylenglycol (PEG) and partially
purified through ionic exchange chromatography (IEC) using three different columns and
isoelectrofocusing using a Rotofor ® . Tannase activity and protein content were evaluated in each step
of protocol. Electrophoresis gels (SDS-PAGE) were analyzed at the end of each step.
RESULTS AND DISCUSSION
Evaluating the two culture systems as observed in the table 1. It is possible to observe that the strain
of A. niger GH1 in the solid culture, presented a higher production of the tannase enzyme.
For the fermentation time, to a high concentration of substrate the strain consumes the majority in the
first hours of the fermentation (Figure 1) and the tannase enzyme production of is observed at 24 h
(Figure 2). These results favored the purification of the enzyme therefore as can be observed in the
Figure 3 the production of the proteases shoot up after the 24 h of the fermentation and this would be
able to affect the tannase activity since Aguilar et al. (2000) showed that the protease activity
influences in the production of the tannase activity causing the decrease of enzyme activity.
The incubation time required for the production of the tannase enzyme by A. niger GH1 in solid state
culture is lowest than the time reported by Sharma (1999), that describes for A. niger Van Tieghem a
time of incubation for the production of the tannase enzyme of 120 h for which the reduction of the
time is favorable for the case from A.niger GH1.

FSB1 – 2004
Food Science and Biotechnology in Developing Countries
TANIC (g/L)
14
12
10
8
6
4
2
0
Table 1. Comparison in the production of tannase in liquid
and solid cultures at 30º C (30 h).
Strain
Cultures
Kind
GH1 SmC
GH1 SSC
*SmC: Submerged Culture.
*SSC: Solid State Culture.
0 8 16 24 32 40 48 56 64 72 80 88
TIME (h)
Figure 1. Substrate uptake by A.
niger at 30° C in SSC with 12.5g/L
of tannic acid.
Proteasse Activity U/ L*min
10
9
8
7
6
5
4
3
2
1
0
Tannase U/L Total Tannase U/L
537 537
2291 2291
Tannase Activity (U/L)
1800
1600
1400
1200
1000
800
600
400
200
0 6 12 18 24 30 36 42 48
Time (hours)
Figure 3. Protease activity of GH1
strain at 30° C in SSC with
concentrations of 25(♦) y 50 (■) g/L
of tannic acid.
0
0 6 12 18 24 30 36 42 48
hours
Figure 2. Tannase enzyme expression
by the GH1 strain at 30° C in SSC
with concentrations of 25(♦) and 50
(■) g/L of tannic acid.

FSB1 – 2004
Food Science and Biotechnology in Developing Countries
Therefore according to the results, the optimum values of production of the tannase enzyme by A.
niger GH1 and for the partial purification are: 30º C of incubation for 24 h in solid state culture and with
a substrate concentration of 50 g/L of tannic acid.
For the partial phase of purification was carried out the evaluation of two methods of purification of the
tannase enzyme, besides two proteins concentration methods. From the two methods evaluated to
concentrate proteins, the one that it was obtained greater performances was of polietilenglicol (PEG)
and dialysis membrane reported by Sharma (1999) since was obtained almost four times more of
tannase activity that with the acetone method (Table 2).
Table 2. Comparison of proteins precipitation methods evaluating the tannase activity and the
extract of the protein obtained in the production at preparative scale by A. niger GH1.
Technique
Activity Tannase
(U/L)
Protein
(mg/L)
Specific Activity
(U/mg)
Total Activity
(U)
PEG 2327.88 1198.07 2.0 11.63
Acetone 1:1 620.76 780.76 0.8 3.90
Acetone 1:2 0 767.30 0 0
Acetone 1:3 0 740.38 0 0
Results obtained of IEC showed that it is possible to separate the tannase from the rest of proteins,
however two isoforms of tannase were revetaled (figure 4). These results are not according with the
data reported by Barthomeuf et al., (1993), due they detected and purified one isoform of tannase by
this kind of chromatography.
Results obtained for isoelectrofocusing with the Rotofor ® revelated two isoforms of tannase (figure 4),
one of these showed a isoelectric point already to 7 and other one at 4. It is important to note that
results obtained in this study are according with the results published by Garcia-Peña et al (1999)
using the same technique.
This study permits to establish the first bases for the purification of tannase produced by Aspergillus
niger GH1 by solid state culture using polyurethane foam as inert support.

FSB1 – 2004
Food Science and Biotechnology in Developing Countries
kD
250
150
100
75
50
37
25
10
Ec PEG 15 ANX DEAE Q K
Figure 4. Comparison of purification steps on electrophoresis gels (SDS-PAGE). (Ec) crude
extract, (PEG) concentrated with polyethylenglycol, (15) fraction 15 from Rotofor ® , (ANX)
fraction from ANX column (DEAE) fraction from DEAE column (Q) fraction from Q column, (K)
tannase of reference (from Kikkoman).
Finally, it is important to note that tannase enzyme was produced at high levels at 30ºC for 24 hours of
culture with 50 g/L of inducer (tannic acid) Two isforms were reveled with pre-protocol developed.
Both isoforms presented molecular weights of 75 and 100 kDa.
Acknowledgements
This work is part of the research project on tannase and tannins economically financed by SEP-
CONACYT program and FOMIX COAH-CONACYT.
REFERENCES
Aguilar,C. Augur, C. Viniegra-González, G. and Favela, E. (1999). A Comparasion of Methods to
Determine Tanin Acyl Hidrolase Activity. Brazilian Archives of Biology and tecnology, 42, 355-361.
Aguilar C. N., Favela–Torres, E., Viniegra-Gonzáles G. and C. Augur. (2000). Culture conditions
dictete the production of proteases by Aspergillus niger Aa-20 reporte interno UAM1.
Aguilar y Gutiérrez-Sánchez, (2001). Review: Sources, Properties, Applications and Potencial uses of
Tannin Acyl Hidrolase. Food Science Technology Int. 7;(5) 373-382.
Aguilar, C. Augur C. Favela, E. Viniegra- González, G. (2001a) Induction and repression patterns of
fungal tannase in solid-state and submerged cultures. Process Biochemistry, 36, 565-570.
Aguilar y col., (2001b). Production of tannase by Aspergillus niger Aa-20 in sumerged and solid state
fermentation: influence of glucose and tannic acid. Journal of Industrial Microbiological and
Biotechnology. 26. 296-302.
Barthomeuf, C. Regerat, F., Pourrat, H. (1994). Improvement in tannase recovery using enzymatic
disruption on micelium in combination with reserve mycellar enzyme extraction. Biotechnol Tech 8:
137- 142.
Chae, S. Yu, T. (1983). Experimental manufacture of a com wine by fungal tannase. Hanguk Sipkum
Kwahakoechi 15: 326- 332.

FSB1 – 2004
Food Science and Biotechnology in Developing Countries
Chaterjee R. Dutta A. Banerjee R. y Bhatacharyya B. 1996. Production of tanase by solid state
fermentation. Bioprocess. 14, 159-162.
Coggon, P., Graham, N.H. and Sanderson G.W. (1975). U.K. Pat. 1,380,135.
Cruz-Hernandez, M.A., Rodriguez, R., Contreras-Esquivel, J.C., Lara, F. y C. N., Aguilar. (2002).
“Aislamiento y Caracterización Morfologica de Cepas Degradadoras de Taninos”. Tesis presentada
como requisito parcial para obtener el título de Químico Fármacobiólogo. Universidad Autónoma de
Coahuila.
Dubios, M. Gilles, K.A., Hamilton, J.K., Rebers, P.A y Smith, F. (1956). Colorimetric method for
determination of sugars and related substances. Anal. Chem. 28. 530.
García-Nájera J.A., Aguilar C.N., Rodríguez Herrera R., Reyes Vega ML, Prado Barragán A., Barajas
Bermúdez L., Contreras esquivel J.C. (2002). “Producción fúngica de tanasa y ácido gálico a partir de
taninos hidrolizables”. Tesis presentada como requisito parcial para obtener el título de Químico
Fármacobiólogo. Universidad Autónoma de Coahuila.
García- Peña, I (1996). Producción, purificación y caracterización de tanasa producida por
Aspergillus niger en fermentación en medio sólido. Tesis de maestría. Universidad Autómoma
Metropolitana, Iztapalapa. México.
Lekha P., Ramakrishna M. and Lonsane B. 1993. Strategies for isolation of potent culture capable of
producing tannin acyl hydrolase in higher titres. Chem. Mikrobiol Technol. Lebensmitt. 15, 5-10
Lekha, P., Lonsane, B. (1994). Comparative titres, location and properties of tannin acyl hydrolase
produced by Aspergillus niger PKL 104 in solid-state, liquid surface and submerged fernentations.
Proc Biochem 29: 497-503.
Lekha, P. and B. Lonsane (1997). Production and aplication of tannin acyl hydrolase: state of the art.
Adv Appl Microbiol 44: 215-260.
Pourrat, H., Regerat, F., Pourrat, A. And D. Jean (1985). Production of gallic acid from tara tannin by
a strain of A. niger. J Ferment Technol 63: 401-403.
Ramírez-Coronel, A, Viniegra-González, G. & Augur, C. (1999). Purification of a tanasa taken place by
Aspergillus niger Aa-20, in fermentation between solid. Proceedings of the VIII Mexican Congress and
IV Latin American Congress of Biotechnology and Bioingeniería. Huatulco, Oax., Mexico.
Sharma, S., Bhat, T. K. and R. K. Dawra. (1999). Isolation, Purification and Propieties of Tannase
From Aspergillus niger van Thieghem. Microbiology and Biotechnology 15: 673-677.
Sharma S. Bhat, T.K. and Dawra, R. (2000) A spectrophotometric method for assay of tannase using
rhodanine. Analytical Biochemistry 279. 85-89.
Sittig, M. 1988. Trimethoprim. In pharmaceutical manufacturing encyclopedia. New Jersey: Noyes
Publication. 282-284.
Van de Lagenmaat, J. and Pyle, D.L (2001). Solid-state fermentation and bioremediation:
development of a continuous process for the production of fungal tannase. Chemical Engineering
Journal 84: 115-123.

FSB1 – 2004
Food Science and Biotechnology in Developing Countries
PRODUCTION AND STABILITY TO pH AND TEMPERATURE OF TANNASE FROM
Aspergillus niger GH1
Crystela Quintero Flores, Cristóbal Noé Aguilar*,
Raúl Rodríguez-Herrera and Juan CarlosvContreras-Esquivel.
Food Research Departament. School of Chemistry. Universidad Autónoma de Coahuila. Saltillo,
Coahuila, México. *Corresponding author, e-mail: cag13761@mail.uadec.mx
ABSTRACT
Tannase is an enzyme with a high potential for use in food industry. In this study, tannase from
Aspergillus niger GH1 was produced by solid state fermentation using polyurethane foam as support.
Enzyme was concentrated by dialysis and its stability to several values of pH and temperature was
evaluated. Time of tanase assay was also, studied. Obtained results demonstrated that this fungal
enzyme had an optimal pH of 5, an optimal temperature of 30ºC and a time of reaction of 10 minutes.
Keywords: tannase, stability, pH, temperature, solid state fermentation.
INTRODUCTIÓN
Tannase or tannin acyl hydrolase (E.C. 3.1.1.20), catalyzes the hydrolysis of ester bonds present in
hydrolysable tannins molecules like tannic acid and gallate esters. It is a glycoprotein formed by a
mixture of one esterase and one despídase Several studies carried out in submerged fermentation
have reported that it has a pH of stability between 3,5 - 8,0, an optimum pH of 5,5 - 6.0; the
temperature of stability is between 30 and 60°C, and the optimum value at 30 - 40°C; Its iso-electric
point is around 4,0 - 4.5; and the reported molecular weight varies de186 to 300 kDa, depending on the
source (Lekha and Lonsane, 1997; Aguilar and Gutierrez, 2001).
Tannase can be obtained from vegetal, animals and microbial sources. Last source is the way where
this enzymes is more stable, and the microorganisms can produce it in great amounts. The
microorganisms more studied are the filamentous fungi, between which are the strains of Ascochyta,
Aspergillus, Chaetomuum, Mucor, Neurospora, Rhizopus, Trichothercium and Penicillum. Tannase is
commercially used in the chemical, pharmaceutical, feed, drinks and foods industries. The main
commercial application of this enzyme is the hydrolysis of the gallotannins for gallic acid production, an
intermediary product required for the synthesis of the antibacterial drug trimethoprim (Belmares-Cerda
et al., 2004).
Aspergillus niger GH1 has been described as a fungal microorganism with a high potential to degrade
tannins. Tannase produced by this strain has been studied recently and preliminary studies on its
puritication have been recently described by Cruz-Hernández, (2004).
In this study, the objective was to evaluate the temperature and pH stability of the tannse produced by
A. niger GH1 in solid state fermentation. Also, time of enzyme reaction has been studied..

FSB1 – 2004
Food Science and Biotechnology in Developing Countries
MATERIALS AND METHODS
Microorganism and inoculum preparation
Spores of Aspergillus niger GH1 (DIA-UAdeC collection) stored at -20ºC were used in all experiments.
Inoculum of microorganism was prepared on potato dextrose agar and spores were harvested with
tween 80 (0.01%).
Solid state fermentation (SSF)
For the SSF process, Erlenmeyer flasks (250 mL) were used like reactor. These flasks contain dried
pulverized polyurethane foam (PUF) like solid support, moisturized with inoculated liquid medium
(inoculation level 2x10 7 spores/L). The reactors are incubated at 30ºC and the samples were taken at
24 hours of culture).
Enzymatic assay for tannase
Tannase activity was assayed following the Rodanine-method reported by Sharma et al (2000). It is
important to state that time of enzyme reaction was determined under conditions used.
pH and Temperature stability
Dialyzed extracts were conditioned at several values of pH (3 -7) and temperature (20 – 80ºC) and its
tannase activity determined.
RESULTS AND DISCUSSION
In this study, tannase from the strain Aspergillus niger GH1 was produced by solid state fermentation
using polyurethane foam as support. After 24 hours of culture, fermented material was compressed to
obtain the crude enzyme extract, which wwas dialyzed against buffer at 4ºC.
Obtained results demonstrated that this fungal enzyme is thermo-labile with a stability of 30 at 40ºC,
and it had a maximum temperature value of 30ºC (Figure 1). To values higher than 50ºC the tannase
activity is seriously affected. It is important to note that to values lower than 30ºC enzyme is also, highly
sensible to temperature. This result is according with the literature.
Results obtained of the effect of pH to enzyme activity showed that the optimal pH is 5, and the
dialyzed tannase extract is stable to pH values from 4 to 7. Values higher than pH / presented several
inconvenient to analyze due the hydrolysis of substrate by alkali condition.

FSB1 – 2004
Food Science and Biotechnology in Developing Countries
Tannase Activity (U/L)
Tannase Activity (U/L)
5000
4500
4000
3500
3000
2500
2000
1500
1000
500
Figure 1. Effect of temperature on tannase activity.
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
0
20 30 40 50 60 70 80
Temperature (°C)
3 4 5 6 7
pH
Figure 2. Effect of pH on tannase activity.

FSB1 – 2004
Food Science and Biotechnology in Developing Countries
Under conditions used for tannase activity assay , the time of reaction found was 10 min. Original
method recommend a reaction time higher than this value, and this could provoke mistakes in the
tannase activity determination. Figure 3 shows the results obtained for tannase activity at several times
evaluated.
REFERENCES
Tannase activity (U/L)
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
0 5 10 15 20 25
Time (min)
Figure 3. Tannase activity at several times of reaction.
Lekha, P. and Lonsane, B. 1997. Production and aplication of tannin acyl hydrolase: state of the art.
Advances in Applied Microbiology. 44, 215-503.
Aguilar, C.N., Gutiérrez-Sánchez, G. 2001. Sources, properties, and potential uses of tannin acyl
hydrolase (3.1.1.20). Food Science and Technology International. 7(5):373-382.
Belmares, R., Contreras-Esquivel, J.C., Rodríguez, R., Ramírez-Coronel, A. and Aguilar, C.N. (2004).
Microbial production of tannase: an enzyme with potencial use in food industry. Lebensmittel-
Wissenschaft und-technologie Food Science and Technology. Aceptado. In press.
Cruz-Hernández, M.A. 2004. Producción y purificación parcial de la enzima tanasa de Aspergillus niger
GH1. M.Sc. thesis. Universidad Autónoma de Coahuila. Saltillo, Coahuila, México.

Food Science and Biotechnology for Developing Countries.2004
PURIFICATION AND CHARACTERIZATION OF A NEW CYSTEINE PROTEASE
FROM THE LATEX OF Jacaratia mexicana
1 Oliver-Salvador M. C., 1 Hernández-Pérez K. 2 Briones-Martínez R., 2 Cortes-Vázquez
M. I. y 3 Soriano-García M. 1 Unidad Profesional Interdisciplinaria de Biotecnología,
IPN. 2 Centro de Desarrollo de Productos Bióticos, IPN. 3 Instituto de Química, UNAM.
Av. Acueducto s/n, Barrio La Laguna, Ticomán, 07340. México, D. F.
coliver@acei.upibi.ipn.mx.
ABSTRACT. A cysteine protease (P-III) was isolated from the latex of Jacaratia
mexicana (Caricaceae) by hydrophobic interaction and cation exchange
chromatography. It was determined the P-III molecular mass for both samples by
vertical electrophoresis in SDS-polyacrilamide gel and the P-III cationic fraction was
assayed for specific absorptivity by the dry weight method.
KEY WORDS: cationic protease, cysteine enzyme, Jacaratia mexicana
INTRODUCTION. The major class of proteolytic enzymes present in plants belong
primarily to the cysteine class (1). This group of proteases is relevant to Plant
Physiology studies and specially in Food technology. Caricacea family is
characterized by plants producing great quantities of cysteine proteases, Jacaratia
mexicana is one of them. A clear example of this kind of proteases is papain which
has been widely used in several food and industrial processes, like meat tenderizing,
beer clarification, modification of food protein functional properties, production of
protein hydrolizates and bakery.
OBJECTIVE. To purify and charaterize a new cysteine protease from the latex of
Jacaratia mexicana.
METHODS. The latex from J. mexicana was collected at the Centro de Desarrollo de
Productos Bióticos (Biotic Products Development Center) an institution of the
National Polytechnical Institute (IPN) at Yautepec, Morelos, the latex was
precipitated two times with 18.7% NaCl (w/v), the resuspended solution (2X extract)
from J. mexicana (2) was injected into a 5.0 ml strong cation exchange column
(Econo-Pac High S). The fractions containing the new protease were gathered and
named as “protease P-III”, subsequently, a half portion was recirculated under the
same conditions and the rest was injected into a 5.0 ml column of hidrophobic
interaction (Econo-Pac Methyl). The cationic fraction obtained from the cation

Food Science and Biotechnology for Deveoping Countries. 2004
exchange column, P-III, was assayed for specific absorptivity by the dry weight
method (3). It was determined the P-III molecular mass for both samples by vertical
electrophoresis in SDS-polyacrilamide gel (4), the one from cationic exchange and
that from hidrophobic interaction chromatography, The pH effect on proteolytic
activity with 1% casein as substrate was carried out by Kunitz method, modified by
Ortega and del Castillo (5), at a pH range from 6.0 to 11, at 35 ºC, the pH effect on
enzyme activity with (10.0 mM in 0.1 M phosphate buffer) BAPNA was determined as
Dubois et al. (1988), at 4.0 a 9.0 pH values with Cys and 25 mM EDTA at 40 °C.
Temperature effect on proteolytic activity was measured by Kunitz method modified
by Ortega and del Castillo (5), with 1% casein in 0.1 M phosphate buffer pH 7.0, at
temperatures from 30 °C to 80 °C. Temperature effect on enzyme activity with
BAPNA was made by Dubois et al. (6) method, with 10.0 mM BAPNA in 0.1 M pH 7.0
phosphate buffer, Cys and 25 mM EDTA; at different temperature values from 30 °C
to 80 °C. The pH stability asay was made by incubating the enzyme at pH values
ranging from 3.0 to 10.0, for 2, 12 and 24 hours, the residual activity percentage was
determined by (5) at 20 °C. with 1% casein in 0.05 M pH 7.6 phosphate buffer at 35
°C.
RESULTS AND DISCUSION. Purification started with strong cationic exchange
chromatography of the J. mexicana 2X extract, which rendered 5 peaks, with
proteolytic activity (Figure 1); the peak III was marked as Protease III (P-III), and it
was passed separately through two kinds of chromatography: a) cationic exchange,
with a profile that shows peak III accompanied by P-II and P-IV fraction remains, the
peak with the greater proteolytic activity and hidrophobic interaction was the peak III,
with three components, from which the main peak presented the proteolytic activity.
Compared to b) cation exchange column fractions, these peaks are better defined, as
ocurred for the performed purification of P-IV and P-V with this chromatography (7).
Specific absorptivity for P-III, was 3.2 g -1 L cm -1 ; a value within the limit of the specific
absorptivity highest values found for another cysteine proteases from plant sources.
The results from SDS-polyacrilamide electrophoresis of P-III are showed in figure 2, it
can be seen clearly a single band of protein for P-III, so we can tell that the
purification was sufficient to make the biochemical characterization studies.

Food Science and Biotechnology for Developing Countries.2004
2
8
0
n
m
A
b
s
I II
III
IV
V
No. Fracción
No. Fraction
FIGURE 1.
Chromatography profile of the elution of 2X
extract
of J. mexicana in Econo-pac® High S, 0.05 M
phosphate
buffer , pH 6.3 and NaCl 0.0-1.0 M linear gradient.
1
.0
N
a
C
l M 0
.0
-
m
n
0
8
2
s
A
III
Fracción
No. Fracción
No.
FIGURE 2.
12.5% Poliacrilamide gel electrophoresis of P-III:
lane 1, P-III in Econo-pac Methyl, lane 2: P-III
rec in Econo-pac High S, lane 3: P-IV
M
.0
-0
.5
O
2S
4) 4
1
O
2S
4) 4
1
H
(N

Food Science and Biotechnology for Deveoping Countries. 2004
Fig. 3. Gel polyacrilamide 12.5% electrophoresis. Lane 1 :P-III (Econo-Pac Methyl) Lane 2: P-III-rec
( Econo Pac High S), lane 3: P-IV
The maximum amidase or hydrolytic activity for P-III was found at pH 9.0 (Fig. 4) and at 7.0 with
BAPNA, and the enzyme showed a classical bell shape similar to that of papain, besides there was a
reduction in activity in both cases near the optimal values. With BAPNA again, the maxima values for
temperature/proteolytic activity were found at 60 °C and 55 °C .
U/m g
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
5.5 6.0 6.5 7.0 7.5 8.0 8.5
pH
9.0 9.5 10.0 10.5 11.0
Fig. 4. pH effect on P-III enzyme activity with 1% casein-0.05M phosphate buffer, pH 7.6 and 20 mM
Cys at 35°C.

Food Science and Biotechnology for Developing Countries.2004
U/m g
6.4
5.6
4.8
4.0
3.2
2.4
1.6
0.8
0.0
30 35 40 45 50 55 60
T (°C)
65 70 75 80 85 90
Fig. 5. Temperature effect on P-III enzyme activity with 1% casein- 0.05 M phosphate buffer,
pH 7.6 and 20 mM Cys at 35 °C.
The above values are in the range of optimal pH and temperature reported for
another cysteine proteases of plants and are similar to that from another proteases
isolated from the same latex source. The studies of pH stability for P-III showed that
this enzyme maintains 100% of proteolytic activity within a 2-24 hs period along the
studied range of pH (4.0-9.0). The results here described show an enzyme with great
stability to pH, as occurs for other cysteine proteases obtained from J. mexicana
latex, i.e. P-IV and PV (pH 3.0-10.0) (7) and that produced by C.papaya (pH 3.0-
10.0) (8).
CONCLUSIONS.
• The described strategy was appropiate to achieve the purification of P-III
protease, the third in abundance in the latex from J. mexicana.
• Econo Pac® Methyl column of hydrophobic interaction showed a greater
separation capacity compared to the strong cationic exchanger of Econo Pac ®
High S.
• The specific absorptivity by the dry weight method rendered a value of 3.2 g -1 L
cm -1 for P-III
• P-III molecular mass was 24.84 kDa determined by electrophoresis

Food Science and Biotechnology for Deveoping Countries. 2004
• The optimal pH for the enzyme activity of P-III was 9.0 with casein and 7.0 over
BAPNA.
• Optimal temperature for the activity of P-III was at 60 °C with casein and 55 °C
over BAPNA.
• The enzyme presented a great stability within practically all the studied range of
pH over 2- 24 hours at 20 °C with casein.
BIBLIOGRAFIA.
1. Priolo, N., Morcelle, del V. S., Arribére, M. C., López, L. and Caffini, N. (2000). Isolation
and Characterization of a Cysteine Protease from the Latex of Araujia hortorum Fruits.
Journal of Protein Chemistry 19: 39-49.
2. Oliver, S. M. C. (1999). Purificación, Caracterización y Cristalización de la Proteasa
Cisteínica del látex del Pileus mexicanus: Mexicaína. Tesis de Doctor en Ciencias
(Alimentos). Escuela Nacional de Ciencias Biológicas-IPN, México.
3. Glazer, A. N. and Smith E. L. (1961). Phenolic hydroxyl ionization in papain. Journal of
Biological Chemistry 236: 2951-2984.
4. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 227: 680-685.
5. Ortega, M. L., Del Castillo, L. M. (1966). Actividad de la mexicaína en presencia de altas
concentraciones de urea. Ciencia Mexicana 24: 247-251.
6. Dubois, T., Jacquet, A., Scheneck, A. G. and Looze, Y. (1988). The thiol proteinases
from the latex of Carica papaya L. I. Fractionation Purification and Preliminary
Characterization. Biological Chemistry Hoppe-Seyler. 369: 733-740.
7. Fabián, M. J. C. (2003). Purificación y caracterización de la proteasa V del látex de
Jacaratia mexicana. Tesis de Licenciatura. UPIBI-IPN.
8. Barret, A.J, Rawlings, N.D. and Woessner, J.F. (1998). “Handbook of proteolytic enzymes”,
London, UK: Academic Press.

FSB1 – 2004
Food Science and Biotechnology in Developing Countries
Enzymatic Modification of Pectin to Produce Tailored Functional Stabilizer
L. Wicker and Y. Kim
Dept. Food Science and Technology, University of Georgia, Athens, GA, USA
lwicker@uga.edu
Abstract
Isozymes of citrus pectinmethylesterase (PME) de-esterified pectin from 73% degree of
esterification (DE) to ~61% DE. The modified pectins retained molecular weight, were calcium sensitive
and formed gels. Zeta potential for surface charge and interactions with milk proteins indicated that the
charge-modified pectins were unique. Modified pectins with tailored functionality may enhance utilization
of pectins from novel sources.
Keywords: de-esterification, charge distribution, protein interactions, stabilizer
Main Text
Pectins are important structural polysaccharides in the cell walls of many plants, which are of
considerable interest as gelling or stabilizing agent in the food industry (1). The degree of methyl
esterification (DE) implies a specific gelling mechanism. Besides the methoxy content, the distribution
pattern of free and esterified carboxyl groups and the length of unsubstituted galacturonan backbone
affect gelling. Chemical de-esterification is a random de-esterification process that results in decreased
molecular weight due to de-polymerization of pectin backbone by β-elimination. However, enzymatic deesterification
results in a blockwise distribution and undesired depolymerization of pectins are reduced (2;
3). Plant pectinmethylesterase can create a calcium sensitive pectin (CSP) in which HMP can gel in the
presence of Ca without the addition of sucrose as long as blocks of de-esterified pectin are present (4).
Willats et al. (5) found that the degree and pattern of methyl-esterification affects the elasticity of the gels
as well as their response to compressive strain. Ralet et al. (6) also showed that the blocks formed by
plant-PME treatment allowed blocks to form calcium-pectinate precipitates for high DE even though these
blocks were not long enough to induce abnormal polyelectrolyte behaviour. The objective of this research
was to characterize the calcium sensitivity of Valencia PME modified pectin by direct measure of viscous
and gelling properties in the presence of CaCl2. In addition, the effect of calcium on the surface charge of
the modified pectins was estimated by measuring the ζ-potential. This research has application to the
development of tailored pectins for specific functional properties using PMEs of known mechanism of
action.
Materials and Methods
Valencia PMEs were isolated as previously developed (Fig. 1) and used to de-esterify high methoxyl
pectin (73% DE) at 30°C to a target 63% DE. Pectin was precipitated and washed in ethanol and stored
at -20°C until use. Modified pectins were denoted at O-Pec, B-Pec, and U-Pec for original pectin, first
modified pectin and second modified pectin. Analytical ion exchange chromatography (IEX) was used to
estimate charge distribution using a 5 mL Q column (Bio Rad, Hercules, CA, USA), equilibrated in 0.5M
acetate, pH 5.0, and then eluted through the gradient of 0.5 to 1.3 M acetate buffer, pH 5.0. Molecular
weights (Mw) were determined using an HPSEC-multi angle light scattering system consisting of a
Waters P515 pump with an in-line degasser (Waters, Milford, MA) and two in-line filters (0.22 and 0.10
mm pore size, Millipore, Bedford, MA). Measurement of ζ- potential was performed using a Particle Size
Analyzer adding the BI-Zeta option (90 Plus, Brookhaven Inst., Holtsville, NY) with a 50 mV diode laser
(90 angle) and a BI-9000AT correlator. Gelling was evaluated after a 500 mM CaCl2 solution was added
to 2% pectin dispersion at 60°C to a final concentration of 35 mM. The mixture was immediately mixed
by a vortex, stored 24 h at 4°C. After the gels were tempered to room temperature, the texture profile
was measured (TA-XT2i, Texture Technologies Corp, Scarsdale, NY, fitted with a 25 kg load cell). The
particle size distribution was determined by laser diffraction using a Mastersizer S, (Malvern Instruments,
MA).

FSB1 – 2004
Food Science and Biotechnology in Developing Countries
Results and Discussion
The DE of pectin was reduced to about 62-63% DE by PME. Based on uronic acid results after IEX, the
elution profile of the unmodified pectin was polydisperse (Figure 1). The main component eluted at low
salt concentrations and smaller fraction eluted at higher ionic strength. The elution profile of pectins
modified by either of the PME fractions eluted near the same ionic strength as the second, smaller
fraction of the unmodified pectin. The elution profile of the modified pectins was similar, even though the
PME peptide fraction was different. The molecular weight of the modified pectins was not different from
the original pectin and was near 134,000 (Figure 3). There was greater variation in the pectins at low
molecular weight of the modified pectin and this is reflected in the higher polydispersity values (Table 1).
All pectins were negatively charged as measured by the zeta potential and the modified pectins were
more negatively charged than the original pectins (Table 1). The gelling profile as evaluated by TPA
indicated that the two modified pectins gelled in the presence of calcium even though at 63 %DE, the
pectins are high methoxyl pectins (Table 2). Typically, only pectins with %DE less than ~50% DE gel with
calcium. The original pectin did not gel in the presence of 35mM CaCl2 at 2% pectin. There was no
significant difference in hardness between the two modified pectins.
Interactions of original and modified pectins with isolated milk proteins indicated that there were unique
effects of pectins depending on the proteins. A mixture of milk caseins with any of the pectins reduced
particle size from over 100 µm to about 5-6 µm (Fig. 4). Based on the comparison with dispersion
systems of individual casein fractions, αS1,2 -, β-, and κ-casein, in the presence of charge modified
pectins, each casein fractions interacted uniquely depending on modified pectins (Fig. 5). In most cases,
pectins increased particle size and modified pectins increased particle size more than original pectins, but
particle size distribution was more monodisperse.
Conclusions
Slight modification of pectin charge can dramatically changed functional properties of pectins.
Ultimately, interactions of charge modified pectins with cations in dispersed systems gives an idea for the
development of tailored pectins as gelling and stabilizing agents.
References
1. Voragen A.G.J., Pilnik W., Thibault J-F., Axelos M.A.V., and Renard C.M.G.C. (1995) Pectins. pp. 287-
339 In A.M. Stephen (Ed.), Food polysaccharides and their applications. Marcel Dekker, New York.
2. Hotchkiss, A.T., Savary B.J., Cameron, R.G., Chau, H.K., Brouillette, J., Luzio, G.A., and
Fishman, M.L. (2002) Enzymatic modification of pectin to increase its calcium sensitivity while preserving
its molecular weight. Journal of Agricultural and Food Chemistry 50: 2931-2937.
3. Limberg G., Korner R., Buchholt H.C., Christensen T.M.I.E. Roepstroff P., and Mikkelsen J.D. (2000)
Analysis of different de-esterification mechanisms for pectin by enzymatic fingerprinting using endopectin
lyase and endopolygalacturonase II from A. Niger, Carbohydrate Research 327 (3): 293-307
4. Joye D.D. and Luzio G.A. (2000) Process for selective extraction of pectins from plant material by
differential pH. Carbohydrate Polymers 34: 337-342.
5. Willats W.G.T., Orfila C., Limberg G., Buchholt H.C., Van Alebeek G-J. W.M., Voragen A.G.J., Marcus
S.E., Christensen T.M.I.E., Mikkelsen J.D., Murry B.S., and Knox J.P. (2001) Modulation of the degree
and pattern of methyl-esterification of pectic homogalacturonan in plant cell walls. The Journal of
Biological Chemistry. 276 (22): 19404-19431.
6. Ralet M.C., Crėpeau M.J., Buchholt H.C., and Thibault J.F. (2003) Polyelectrolyte behaviour and
calcium binding properties of sugar beet pectins differing in their degrees of methylation and acetylation.
Biochemical Engineering Journal. 16: 191-201.

FSB1 – 2004
Food Science and Biotechnology in Developing Countries
PME Chromatography (Valencia pulp)
Bound PME (BP++)
Heparin
(Bound PME: hep)
(Bound PME: Con A)
30-70% Am. sulfate cut
Crude PME
SP Cation exchange column
Unbound PME
SP Cation exchange column
Bound PME (BP+) Unbound PME (UBP-)
Figure 1. Model flow diagram of PME purification. UBP- and BP+ represented U PME and B PME,
respectively.
Uronic Acid (mg/ml)
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0 5 10 15 20 25 30
Elution Time (min)
Figure 2. Analytical ion exchange chromatography elution of unfractionated pectin samples. (O-Pec):
original pectin. (B-Pec): SP-unbound and HP bound PME modified pectin. (U-Pec): SP-unbound and HP
unbound PME modified pectin. (O-Pec): original pectin; (U-Pec): UBP- PME; (B-Pec): BP+.

FSB1 – 2004
Food Science and Biotechnology in Developing Countries
Cumulative Weight Fraction (W)
1.2
1
0.8
0.6
0.4
0.2
0
1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07
Molecular Weight (g/mol)
O-Pec
U-Pec
B-Pec
Figure 3. Cumulative weight fraction plotted against molecular weight of unfractionated pectin samples.
(O-Pec): original pectin. (B-Pec): SP-unbound and HP bound pectin. (U-Pec): SP-unbound and HP
unbound pectin.
Table 1. The summary of chemical properties of O-Pec, B-Pec, and U-Pec by Valencia PME a .
% DE a
Mw
(g/mol)
Polydispersity
(Mw/Mn)
ζ-potential
(mV)
O-Pec 73 134,000 ± 3,439 1.96 -21.36 ± 0.14
B-Pec 63 132,250 ± 3,889 2.13 -30.10 ± 1.62
U-Pec 61 133,850 ± 3,748 2.11 -39.67 ± 1.05
a % DE from NMR spectra. Coefficient of variation ranged from 0.23 to 3.96% for 2 to 4 replicates. Mw:
weight average molecular, Mn: number average molecular weight

FSB1 – 2004
Food Science and Biotechnology in Developing Countries
Table 2. Texture profile analysis of pectin gels in the presence of 35 mM CaCl2 at 2% pectin solution.
Hardness
(N)
Cohesiveness
Springiness
(S)
Gumminess
(N)
Chewiness
(N·S)
B-Pec 0.80 a 0.67 a 1.52 a 0.53 a 0.81 a
U-Pec 0.88 a 0.65 a 2.07 a 0.57 a 1.20 a
a Mean values with different superscript in the same column are not significantly different at p < 0.05. (B-
Pec): SP-unbound and HP- bound PME modified pectin. (U-Pec): SP-unbound and HP- unbound PME
modified pectin. O-Pec and other fractionated pectins did not gel in the presence of 35mM CaCl2.
Volume (%)
7
6
5
4
3
2
1
0
0.01 0.1 1 10 100 1000 10000
Diameter (um)
Figure 4. Particle size distribution of 1% dispersion of casein with pectins (10:1) in acetate buffer, pH 3.8.
Percentage transmittance at 650nm values given in legend.
(CO): casein with O-Pec, (CB): casein with B-Pec, (CU): casein with U-Pec
Casein
CO
CU
CB

FSB1 – 2004
Food Science and Biotechnology in Developing Countries
Volume (%)
5
4
3
2
1
0
Volume (%)
Volume (%)
0.01 0.1 1 10 100 1000 10000
Diameter (um)
8
7
6
5
4
3
2
1
AC
ACO
ACB
ACU
0
0.01 0.1 1 10
Diameter (um)
100 1000 10000
6
5
4
3
2
1
KCO
KCU
KCB
BC
BCO
BCB
BCU
0
0.01 0.1 1 10 100 1000 10000
Diamter (um)
Figure 5. Particle size distribution of 1% dispersion of casein fractions with pectins (10:1) in acetate
buffer, pH 3.8. (a): AC : αS1,2 -Casein, ACO: AC in O-Pec, ACB: AC in B-Pec, ACU: AC in U-Pec (b): BC
: β-Casein, BCO: BC in O-Pec, BCB: BC in B-Pec, BCU: BC in U-Pec (c): KC : κ-Casein, KCO: KC in O-
Pec, CB: KC in B-Pec, KCU: KC in U-Pec. KC can’t measure the particle size.