Biochemical Engineering Journal 63 (2012) 22–30

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Biochemical Engineering Journal

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Biochemical properties and thermal behavior of pectate lyase produced by

Pectobacterium carotovorum subsp. carotovorum BR1 with industrial potentials
V.B. Maisuria, A.S. Nerurkar ∗
Department of Microbiology and Biotechnology Centre, Faculty of Science, M. S. University of Baroda, Vadodara 390002, Gujarat, India

a r t i c l e i n f o a b s t r a c t

Article history: An extracellular pectate lyase (PL, EC 4.2.2.2) produced by Pectobacterium carotovorum subsp. carotovorum
Received 31 July 2011 BR1 was purified and characterized with respect to its biochemical properties including the thermo-
Received in revised form 15 January 2012 dynamic parameters of substrates hydrolysis and thermal behavior. The PL showed higher affinity for
Accepted 25 January 2012
polygalacturonic acid (Km , 0.4 g l−1 ) as compared to 70% methylated pectin (Km , 0.77 g l−1 ). Nonlinear
Available online 1 February 2012
thermal inactivation curves of PL at 50 and 60 ◦ C, fitted to a three-fraction first-order kinetic model. Fur-
thermore, the thermodynamic parameters of thermal deactivation of purified PL viz. H*, S*, Ed and
Keywords:
G* were determined over pH range 6–10 and in presence of different cations. Alkaline pH and Ca2+ ions
Pectate lyase
Enzyme biocatalysis
individually influenced the thermostability of PL at elevated temperatures 60 and 70 ◦ C, based on the
Kinetic parameters thermodynamic parameters. The studies suggested that this alkaline PL can be considered as potential
Enzyme deactivation candidate for various applications like pectic waste management, degumming of fibers, food, paper and
Thermodynamic parameters textile industries.
Enzyme technology © 2012 Elsevier B.V. All rights reserved.

1. Introduction carotovora subsp. carotovora (Ecc) reclassified or renamed as Pecto-

Microbial pectolytic enzymes specifically degrade the pectin,
which constitutes the middle lamella of plant cell-wall. Pectolytic
enzymes differ in their substrate preference (polygalacturonic acid
and low-methylated pectin or high-methylated pectin), in their
reaction mechanism (␣/␤-elimination or hydrolysis), and mode
of attack (terminal or in random) on the pectin polymer (exo
or endo) [1]. Among pectolytic enzymes, pectate lyase (PL, EC
4.2.2.2) catalyzes cleavage by ␤-eleminative mechanism of the
␣-1,4-glycosidic bond of polygalacturonic acid, which produces
4,5-unsaturated galacturonosyl at the non-reducing end [2]. Cal-
cium was found to be essential for the activity of pectate lyases from
both bacterial and fungal origin. It plays an important role in the
decomposition of plant residues, during infection caused by Erwinia
Abbreviations: PL, pectate lyase; PG, polygalacturonase; PNL, pectin lyase; Pcc,
Pectobacterium carotovorum subsp. carotovorum; Ecc, Erwinia carotovora subsp. caro-
tovora; Eca, E. carotovora subsp. atroseptica; Ech, E. chrysanthemi; w/v, weight per
volume; H# , enthalpy of substrate hydrolysis; S# , entropy of substrate hydrol-
ysis; G# , free energy changes during substrate hydrolysis; GE#–S , free energy
changes of enzyme–substrate complex; GE#–T , free energy changes of transition
state formation; Ea , activation energy; Q10 , temperature quotient; Kd , deactiva-
tion rate constant; t1/2 , half-life time; H*, enthalpy of thermal inactivation; S*,
entropy of thermal inactivation; G*, free energy change during thermal inactiva-
tion; Ed , thermal inactivation energy.
∗ Corresponding author. Tel.: +91 265 2794396; fax: +91 265 2792508.
E-mail address: anuner26@yahoo.com (A.S. Nerurkar).
bacterium carotovorum subsp. carotovorum (Pcc). Pcc also produces
other pectolytic enzymes viz. polygalacturonase (PG), pectin lyase
(PNL), and pectin methyl esterase (PME) for penetration into the
plant cell wall leading to maceration of plant tissues [2].
Microbial alkaline pectinases have wide-range of biotech-
nological applications in food processing, treatments of pectic
wastewaters, textile processing, paper making, degumming of
plant bast fibers, coffee and tea fermentations [3] also for plant
protoplast isolation and enzymatic solubilization of potato pulp
in dietary fiber extraction processes and agro-industrial wastes
management [4–6]. There are several reports of fungal alkaline
PLs being extensively used in degumming of ramie fibers, jute
retting, textile and paper industries [3], but scarcity of informa-
tion available on bacterial alkaline PL for industrial applications.
Biotechnological applications of these enzymes are highly depen-
dent on parameters such as pH, salt concentration and thermal
stability at high temperature, since their application in industrial
processes requires reactions to be conducted at high temperatures
in order to improve productivity [7]. Microbial sources of alkaline
pectinases is restricted to very few bacterial genera i.e. Bacillus
sp., Pseudomonas sp., Xanthomonas sp., and also fungi and acti-
nomycetes [3], among them a small number of microbial strains
such as Debaryomyces nepalensis [8], Aspergillus niger [9], and P.
carotovorum subsp. carotovorum (Pcc) [2], are known to produce
both PL and PNL in appreciable amounts. A Pcc strain was found
to produce PL that demonstrated properties which were found
worth exploring for their biotechnological potential. Studies like

1369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2012.01.007
 V.B. Maisuria, A.S. Nerurkar / Biochemical Engineering Journal 63 (2012) 22–30
purification and characterization of PL and heterologous expression
of pel gene from Pcc strain has been reported [10–12]. However,
there is sparse information regarding enzyme kinetic properties,
thermostability, inactivation kinetics and thermodynamic param-
eters studies of the PL produced by Pcc. This, to our knowledge is
first report of extensive characterization of purified PL from Pcc
BR1, with respect to biochemical properties, thermal inactivation
kinetics, pH and cation dependent thermal behavior. Based on these
studies it is proposed that PL from Pcc BR1 possesses biotechnolog-
ical potential.
2. Materials and methods
2.1. Microorganism and culture conditions
The pectolytic bacterium used in this study was isolated from
macerated tissue of Brinjal fruit (Solanum melongena var. esculen-
tum), and was identified and renamed as P. carotovorum subsp.
carotovorum (Pcc) BR1 (NCBI-GenBank accession no. FJ187821,
earlier reported as E. carotovora subsp. carotovora BR1). The prop-
agation was done on nutrient agar medium (Himedia Lab. Pvt. Ltd.,
India) containing 0.5% (w/v) pectin and incubated overnight at 30 ◦ C
[13].
2.2. Production and purification of PL
Pectolytic enzyme production was carried out in 500 ml of liq-
uid medium containing 0.5% (w/v) pectin, and at the end of the
growth phase culture supernatant was precipitated with chilled
acetone [13]. The purification of PL was followed with some mod-
ifications as per Basu et al. [7] and Kamiyama et al. [14]. The
precipitates were centrifuged at 15,880 × g for 15 min, after stor-
ing at 4 ◦ C for 4 h, dissolved in a 20 mM Tris–HCl buffer (pH 8.0),
dialyzed against the same buffer, and then chromatographed on a
CM-cellulose column (0.8 cm × 12 cm) at a flow rate of 0.5 ml min−1
using a linear gradient of 0–1 M NaCl in 20 mM Tris–HCl buffer (pH
8.0). Protein molecular weight markers (PMWH; Banglore GenieTM ,
India): carbonic anhydrase, 29 kDa; ovalbumin, 43 kDa; bovine
serum albumin, 66 kDa; myosin-rabbit muscle, 205 kDa were run
along the samples in the SDS–PAGE using a 12% polyacrylamide
gel [15], and the gel was stained using silver salts after the elec-
trophoresis [16]. The activity staining assay of the PL was modified
and performed using 12% polyacrylamide gel containing polygalac-
turonic acid as the substrate [17]. After gel-electrophoresis, the
gel was incubated overnight at 50 ◦ C in a 50 mM Tris–HCl buffer
(pH 8.5) containing 1 mM CaCl2 and then stained with 0.01% (w/v)
Toluidine blue-O instead of Ruthenium red.
2.3. Enzyme activity assay of PL
The PL activity was determined by measuring oligosaccharides
released as a result of the cleavage of polygalacturonic acid using a
thiobarbituric acid (TBA) reagent [7]. A reaction mixture containing
500 ␮l of 0.5% (w/v) polygalacturonic acid (PGA) in 50 mM Tris–HCl
buffer (pH 8.5) containing 1 mM CaCl2 and 100 ␮l of the appropri-
ately diluted enzyme solution was incubated at 50 ◦ C for 1 h and the
final absorbance of the reaction products was measured at 550 nm.
One unit of PL activity was defined as the amount of enzyme that
caused a change in the absorbance of 0.01 per hour at 50 ◦ C and pH
8.5.
2.4. Effect of temperature and pH on PL activity
The temperature optima and activation energy (Ea ) were deter-
mined by incubating an appropriate amount of the enzyme with
0.5% polygalacturonic acid in 50 mM Tris–HCl buffer (pH 8.5) at
23
various temperatures ranging from 20 to 70 ◦ C for 1 h, the sub-
strate being pre-incubated for 5 min at each temperature. The Ea
was determined using Arrhenius plot and temperature quotient
(Q10 ) was estimated as reported earlier [13]. The effect of the pH
on the PL was determined by measuring the activity at 50 ◦ C for 1 h
using different buffers (50 mM) containing 1 mM CaCl2 , viz. phtha-
late buffer (pH 2.6–3.0), Na-acetate buffer (pH 4.0–5.4), phosphate
buffer (pH 6.0–7.5), Tris–HCl buffer (pH 7.5–8.5) and glycine–NaOH
buffer (pH 9.0–11.0).
2.5. Estimation of enzyme kinetics and thermodynamic
parameters
The kinetic parameters, Km , Vmax and Kcat were determined by
measuring the PL activity reaction rates (as in Section 2.4 assay
conditions) at different substrate concentrations ranging from 0.01
to 0.5% (w/v) with a fixed amount of the enzyme in a 50 mM
Tris–HCl buffer (pH 8.5) with 1 mM CaCl2 at 50 ◦ C for 1 h. The Km
and Vmax values were obtained by analysis of data according to
Lineweaver–Burk equation, allowing the catalytic efficiency i.e., the
ratio Vmax /Km , and kinetic constants (Kcat /Km ) to be determined
[13]. The substrate specificity in terms of the specific activity was
established using a 0.5% (w/v) solution of different pectic substrates
viz. polygalacturonic acid and pectin with a 28%, 35%, 65% and
70% degree of esterification (DE). The thermodynamic parameters
(H# , S# , G# , GE#–S and GE#–T ) for the substrate hydrolysis
were calculated by rearranging Eyring’s absolute rate equation as
described earlier [13].
2.6. Thermal stability assay
The thermal deactivation was determined by incubating the
enzyme at 20–70 ◦ C temperature range. Aliquots were withdrawn
at 10 min intervals up to 90 min, cooled on ice for 30 min, and PL
activity was assayed. The residual activity was estimated as [18]:
E 
t
Residual PL activity (%) = 100 (1)
E0
where Et is the activity at time t (min), and E0 is the initial activity
at time t = 0 min.
2.7. Models for PL thermal deactivation kinetics
2.7.1. First-order model
As described by Ortega et al. [18] thermal deactivation of
enzymes can often be explained by a first-order kinetic model.
According to this model an enzyme activity decreases log-linearly
as a function of time as described in the following equation:
E 
t
ln = −Kd t (2)
E0
where Et is the enzyme activity at time t (min), E0 is initial enzyme
activity, t is time of treatments, and Kd is the first-order deactivation
rate constant.
2.7.2. Multi-fraction deactivation model
Multi-fraction first-order deactivation kinetic model according
to Ortega et al. [18] was used to analyze kinetic data. Following the
first-order kinetics, the implicit assumptions were that n fractions
of PL existed and that each fraction was deactivated independently,
i.e.,
N1 → I1 , N1 → I1 , . . . , Ni → Ii (3)
24 V.B. Maisuria, A.S. Nerurkar / Biochemical Engineering Journal 63 (2012) 22–30
where N is the active enzyme and I is the inactive enzyme. The
first-order inactivation rate process is:
dEi
= Ki Ei
dt
After integration
Ei = E0i e−Ki t
Residual activity at time t is the sum of the activities of individual
fractions, i.e.,

n
E= E0i e−Ki t
i=1
where Ki and E0i are the deactivation rate constant and enzyme
activity fraction of the ith fraction. Eq. (6) was fitted to the thermal
deactivation kinetics data for i = 1, 2 and 3, corresponding to first
order, two-fraction first order and three-fraction first order models,
using a non-linear regression routine in a SigmaStat3.5 and vali-
dated with Origin 6.0 software package to obtain deactivation rate
constants and activity fractions.
2.8. Effect of pH or cations on thermal stability
A pH range from 6.0 to 10.0 was selected for determining the
thermal stability of purified PL. The pH of the enzyme solutions
was adjusted using 1 M HCl or 1 M NaOH, followed by a thermal
stability assay according to Gohel and Naseby [19]. The different
salts NaCl (0.1 M), KCl (0.15 M), MgCl2 (5 mM), CaCl2 (1 mM), and
MnCl2 (1 mM) were selected on the basis of observations of activity
enhancement reported by other authors [14,20]. The purified and
dialyzed enzyme solutions was initially incubated with each salt
separately at 4 ◦ C for 10 min, followed by a thermal stability assay
at individual temperature.
2.8.1. Estimation of deactivation rate constant and
thermodynamic parameters for thermal deactivation
Purified PL samples were subjected to temperature between 40
and 70 ◦ C for up to 120 min to observe the thermal stability in pres-
ence of different salts and pH conditions. The residual PL activity
was estimated before and after the incubation period at each tem-
perature under the respective conditions. The deactivation rate of
PL was calculated by first order expression [9]:
dE
= −Kd E
dt
So that,
E 
t
ln = −Kd t
E0
The Kd (deactivation rate constant or first order rate constant)
values were calculated from a plot of time (t) versus ln[Et /E0 ] at a
particular temperature. The half-life of an enzyme is defined as the
time required by the enzyme to lose half of its initial activity, which
is calculated as:
ln 2
t1/2 =
Kd
Thermodynamic parameters were calculated by rearranging the
Eyring absolute rate equation [13]. H* and S* values were esti-
mated from the slope and intercept of a 1/T versus ln[Kd /T] plot,
respectively. So that,
H ∗ = −(slope)R
  K 
S ∗ = R intercept − ln b
h the range of that reported for other bacterial PLs from B. pumilus
where Kb is the Boltzmann’s constant (R/N) = 1.38 × 10−23 J/K,
R the gas constant = 8.314 J/K/mol and h the Planck’s con-
stant = 6.626 × 10−34 J s.
(4) Free energy change were calculated by using the following rela-
tionship
G∗ = H ∗ − T S ∗ (12)
(5)
where T is the absolute temperature (K).
Energy of deactivation was estimated using the Arrhenius equa-
tion:
Kd = Ae(−Ed /RT ) (13)
(6)
So that,
Ed
ln[Kd ] = − + ln A (14)
RT
Energy (Ed ) involved in this deactivation process was calculated
from the slope of a linear plot of 1/T versus ln[Kd ], Ed = −slope × R,
where R (gas constant) = 8.314 J/K/mol. Thermalstability of enzyme
in the presence of different pH and cations was determined by heat-
ing the enzyme solution at 40–70 ◦ C in the presence of the salt or in
specified pH in sealed tubes. The results represented are the mean
values of the experiments conducted in triplicate. Data were statis-
tically analyzed using SigmaStat 3.5, GraphPad Prism 4 and Origin
6.0 software packages.
3. Results and discussion
3.1. Purification and characterization of pectate lyase from Pcc
BR1
The pectate lyase purified from culture supernatant of Pcc BR1
using ion-exchange column chromatography was more than 95%
pure as judged on SDS–PAGE with 34% yield giving 23 fold purifi-
cation. The size of the purified PL was found to be around 40 kDa
as validated by activity staining zymogram for PL (Fig. 1) which
was similar to those reported for E. carotovora, PLI (44 kDa), PLII
(41 kDa) by Lei et al. [11]. PL showed enhanced and optimum activ-
ity at 50 ◦ C and pH 8.5 (data not shown). Sugiura et al. [12] have
also reported similar temperature optima for PL of E. carotovora Er.
The activation energy (Ea ) as determined by Arrhenius model for
PL was 2.02 kJ mol−1 , lower than that reported for PL of B. pumilus
DKS1 (13.37 kJ mol−1 ) [7]. The temperature quotient (Q10 ) for PL of
Pcc BR1 was obtained 1.0 at 20–70 ◦ C, similar to that of PG (1.03) of
same strain reported earlier [13]. It has broad substrate specificity
(7)
as demonstrated by the relative activity (RA) with polygalacturonic
acid (100% RA), 28% DE pectin (92% RA), 35% DE pectin (82% RA),
and 70% DE pectin (80% RA). This unusual substrate preference of
(8) PL toward moderate and high methyl esterified pectin has been
observed earlier for PelB of E. carotovora subsp. carotovora (Ecc)
[10]; and PL3 of E. carotovora subsp. atroseptica (Eca) [21].
3.2. Kinetics and thermodynamic parameters of PL
3.2.1. Michaelis–Menten kinetic parameters
PL activity showed a typical Michaelis–Menten profile with two
(9) different substrates viz. polygalacturonic acid and 70% DE pectin,
in both cases the curves were linear with correlation coefficient
(R2 ) of 0.9945 and 0.9845 respectively. The values of Km and Vmax
were 0.4 g l−1 and 67.57 U ml−1 respectively for polygalacturonic
acid and 0.77 g l−1 and 57.35 U ml−1 respectively for 70% DE pectin.
The values of Km indicate that the PL had a relatively low affinity
for 70% DE pectin than for the polygalacturonic acid. These appar-
(10)
ent values of Km are higher than PLs from Eca (0.27–0.30 g l−1 )
(11) [21] and E. chrysanthemi (Ech) (0.20–0.32 g l−1 ) [22], but within
 V.B. Maisuria, A.S. Nerurkar / Biochemical Engineering Journal 63 (2012) 22–30
Fig. 1. (A) SDS–PAGE analysis and (B) activity staining zymogram of purified PL from Pcc BR1 at different purification steps. Lane M: protein MW markers, 1: purified PL, and
2: dialyzed acetone precipitates.
DKS1 (0.44 g l−1 ) [7] and B. pumilus BK2 (0.24–1.35 g l−1 ) [23]. The
turn over number (Kcat ) and second order rate constant (Kcat /Km )
were 8.7 min−1 and 21.75 respectively with polygalacturonic acid,
while 7 min−1 and 9.1 respectively with 70% DE pectin. Moreover,
the catalytic efficiency (Vmax /Km ) of PL was found to be higher for
polygalacturonic acid (169.0) as compared to 70% DE pectin (70.6).
The catalytic efficiency value provides a practical model for select-
ing the most efficient enzyme for a commercial application process
using a fixed initial substrate concentration [18].
3.2.2. Thermodynamic parameters for substrate hydrolysis
Thermodynamic parameters viz. enthalpy of activation (H# ),
entropy of activation (S# ) and Gibbs free energy of activation
(G# ) measured during enzymatic substrate hydrolysis process
provide a relative idea about the functionality of enzyme toward
specific substrate [13]. The H# , S# and G# of PL for polygalac-
turonic acid hydrolysis were 0.098 kJ mol−1 , −193.73 J mol−1 K and
58.8 kJ mol−1 respectively, while for 70% DE pectin hydrolysis these
values were 0.098 kJ mol−1 , −191.97 J mol−1 K and 58.267 kJ mol−1
respectively. Similar values were observed previously for PG from
Ecc BR1 [13]. The low enthalpy value suggests that the PL is
very efficient for formation of transition state complex between
enzyme–substrate (E–S). The feasibility and extent of the chem-
ical reaction is evaluated by estimating the change in the Gibbs
free energy (G# ) for conversion of substrate to product [13]. G#
value was lower with both substrates indicating that the conversion
of the transition state (E–S) into product is spontaneous, whereas
the low S# value with both substrates suggested that the transi-
tion state complex of PL had less disorder.
The free energy changes for the activation of the substrate
binding (GE#–S ) and free energy changes for the formation of
the activation complex (GE#–T ) of the PL were determined as
−2.31 kJ mol−1 and −7.76 kJ mol−1 respectively for polygalactur-
onic acid; −0.66 kJ mol−1 and −5.563 kJ mol−1 respectively for 70%
DE pectin. This reconfirmed that the PL had a high affinity toward
polygalacturonic acid than 70% DE pectin for hydrolysis which
25
spontaneously forms the product  4:5 unsaturated oligogalac-
turonides.
3.3. Thermalstability kinetics
Thermostable enzymes have substantial potential for many
industrial applications. Some specific applications as in food, paper
and textile industrial processes are carried out at high temper-
ature in order to reduce microbial contamination, maximize the
reaction rate and improve the productivity. Therefore there is
emphasis on the elucidation of thermal deactivation mechanisms
and the development of strategies for enhancing the stability of
thermostable enzyme [18]. Thermalstability is the property of an
enzyme to resist thermal unfolding in the absence of a substrate,
while thermophilicity is the ability of an enzyme to catalyze the
reaction at elevated temperatures in the presence of a substrate
[24].
The inactivation curves of PL of Pcc BR1 depicted in semi-
logarithmic graphs were not linear in the temperature range
50–70 ◦ C (Fig. 2). Evidently, they have multiphase nature there-
fore it is inferred that the thermal inactivation of PL was not
simple first-order process commonly observed for enzyme inac-
tivation but a multi-fraction process. Multi-fraction inactivation
kinetics has been also reported for other pectic enzymes [18]. Nath
et al. [25] have marked that biphasic thermal inactivation is shown
by the enzyme when it consists of two groups of thermostable
and thermolabile subunits or isoenzymes or due to formation of
enzyme aggregates during thermal-inactivation, as observed by
Ortega et al. [18] for Rapidase C80 (commercial preparation of
PG).
Non-linear, multi phase behavior was shown by PL of Pcc BR1,
evident from the inactivation curves at 50 and 60 ◦ C where they
exhibit different slopes at short and long incubation times (Fig. 2).
The multi-fraction first-order model was fitted to inactivation
results obtained for PL from non linear curves at 50 and 60 ◦ C
(Table 1). The high coefficient of determination (0.993 at 50 ◦ C and
0.988 at 60 ◦ C) for the three-fraction first order model and highly
26 V.B. Maisuria, A.S. Nerurkar / Biochemical Engineering Journal 63 (2012) 22–30

o o o o o o
20 C 30 C 40 C 50 C 60 C 70 C

7.36 × 10−6 (2.98 × 10−6 )g

1.56 × 10−3 (4.09 × 10−4 )g
1.24 × 10−1 (1.53 × 10−2 )f
4.5

Three-fraction
4.0

2.74 × 10−2 f

ln [residual PL activity, %]
3.5
26.48

1.268
20.73

0.988
22.00
3.0

2.5

2.0
8.897 × 10−2 (8.86 × 10−3 )f
5.71 × 10−4 (9.48 × 10−5 )f

1.5

1.0
Two-fraction

4.74 × 10−2 f

0.5
48.48

1.267
20.73

0.977

0.0
0 20 40 60 80
–

Time (min)

Fig. 2. Pseudo-first order plot for irreversible thermal inactivation of purified PL

from Pcc BR1. Data presented are average values of n = 3 independent experiments
3.84 × 10−2 (5.58 × 10−3 )f

and error bars represent the standard deviation.

Single-fraction

significant mean square errors at 50 ◦ C (4.46 × 10−3 at P < 0.001)

0.257f

and 60 ◦ C (2.74 × 10−2 at P < 0.001) revealed that the inactivation

69.21

4.146
0.855
60 ◦ C

curve was better explained by the three-fraction first-order model.

–
–

–
–

In both the cases of inactivation curves at 50 and 60 ◦ C, the stan-

dard errors of estimated values of dissociation rate constants (Kd1
and Kd2 ) were higher than that for the two-fraction first-order
7.38 × 10−4 (1.65 × 10−4 )g
2.94 × 10−3 (6.17 × 10−3 )

5.5 × 10−6 (1.2 × 10−6 )g

model. In contrast, as shown in Table 1, in case of three-fraction

first-order model, the standard errors for estimated constants Kd1 ,
Kd2 , and Kd3 were considerably lower than the parameter values
Three-fraction

of inactivation curves at both temperatures 50 and 60 ◦ C. There-

4.46 × 10−3 f
Determination of thermal inactivation using multi fraction first order kinetic models for PL of Pcc BR1.

fore, the three-fraction first-order model was found most suitable

0.0570
66.69
21.61

0.993

than the two-fraction first-order model for explaining the inactiva-

11.7

tion kinetic data of PL. Alternatively, the three-fraction first-order

model was not adequate to explain the inactivation curve of PL
at 70 ◦ C, because PL shows very low i.e. only 1.3% residual activ-
ity after 30 min of treatment at 70 ◦ C (Fig. 2). Similar observations
2.24 × 10−2 (1.35 × 10−3 )g
4.52 × 10−6 (5.7 × 10−5 )

have been reported for PL of B. pumilus DKS1 at temperatures

beyond 70 ◦ C [7] and commercial preparation of PG (Rapidase
C80) at 60 ◦ C [18]. As observed by Ortega et al. [18], at elevated
Two-fraction

temperature, the labile fraction was inactivated rapidly therefore

1.72 × 10−2 f

only rate constants of thermostable fraction could be determined.

Unit for Ei is % PL activity and for Kdi is s−1 where i = 1; 2; 3.
0.971
0.384

Three phases of PL inactivation at higher rate were designated, as

88.3
11.7

phase-I, phase-II and phase-III (Fig. 3). The deactivation energy (Ed )
–

of PL calculated from the Arrhenius plot of thermal inactivation

(Fig. 3) at three phases-I (R2 , 0.97), II (R2 , 0.98) and III (R2 , 0.99)
were 95.94 kJ mol−1 , 97.05 kJ mol−1 and 143.81 kJ mol−1 respec-
2.2 × 10−2 (1.35 × 10−3 )f

Values in parentheses are standard errors.

tively. These values clearly emphasize robustness of inactivation

Predicted residual error sum of square.

model for PL. Furthermore, the effect of temperature on half-life

Statistically significant at P < 0.001
Single-fraction

Statistically significant at P < 0.05.

time of PL has been studied which is summarized in Table 2. The

Temperatures

1.5 × 10−2 f

half-life time (t1/2 ) value is generally assumed as additive [26] and

Coefficient of determination.

therefore values of phase-III are combined t1/2 values of other two

0.971
0.206
50 ◦ C

phases. The t1/2 value of PL in phase-III at 50 ◦ C was 119 folds higher,

97.9
–
–

–
–

whereas at 60 ◦ C the value was 25 fold higher than combined val-

Mean square error.

ues of phase-I and -II. This suggests that the enzyme was more
heat-stable in phase-III (Table 2). The denaturing reactions of con-
Parametersa

formationally intact proteins at high temperatures occur slowly,

suggesting that the conformational stability of the protein struc-
PRESSd
Table 1

MSEb
Kd1 e
Kd2 e
Kd3 e

ture may be important to explain the upper temperature limit for

R2 c
E1
E2
E3

enzyme activity [27].

 V.B. Maisuria, A.S. Nerurkar / Biochemical Engineering Journal 63 (2012) 22–30 27

Table 2
Effect of temperature on half-life time with three-fraction first-order inactivation kinetic model of PL.

Temperature (◦ C) Half life (min)

Phase-I Phase-II Phase-III

20 1.73 × 103 6.93 × 104 2.31 × 107

30 2.24 × 102 1.73 × 104 3.47 × 106
40 1.61 × 102 1.16 × 104 1.39 × 106
50 1.10 × 102 8.66 × 102 1.16 × 105
60 10.44 6.93 × 102 1.73 × 104
70 3.68 2.39 × 102 –

3.4. Thermal inactivation studies of PL
The thermal inactivation studies are crucial to understand-
ing the relationship between structure and function of enzyme.
Enzymes inactivation is a major constraint in the biotechnologi-
cal process development. Understanding the complex process of
enzyme inactivation is helpful in enhancing the feasibility of many
biological processes [28]. There is paucity of information avail-
able concerning transition state parameters of heat inactivation
of PL from soft rot Erwinia. The effect of various combinations
of pH and temperature on inactivation of PL as well as the
effect of different salts on thermal inactivation of PL was evalu-
ated as described in Section 2.8.1. The extent of deactivation is
assessed by the deactivation rate, which is proportional to the
active enzyme concentration and deactivation rate constant (Kd )
[9]. Fig. 3 shows linear inactivation curves in Arrhenius plots for
heat induced inactivation of PL at 20–70 ◦ C temperature range. The
first-order inactivation kinetic model fitted these curves based on
which the deactivation rate constant was calculated for each con-
dition.
3.4.1. Thermal inactivation of PL with different pHs
The deactivation rate constants (Kd ) and half-life time (t1/2 )
were estimated at 40–70 ◦ C temperature range with different pHs
– 6.0, 7.0, 8.0, 9.0, and 10.0 (Fig. 4A and C). pH is one of the most
important factors affecting the tertiary and quaternary structure
of proteins and therfore enzymes. Since the PL exhibits optimum
activity at pH 8.5, the pH range 6–10 was selected for these studies.
The thermalstability of the PL was significantly higher at pH 8.0 at
temperature 50 and 60 ◦ C (Fig. 4A and C). The order of stabiliza-
tion of PL in the various pH conditions at 70 ◦ C was as follows: pH
0 Overall, the cations used in this study were found to increase the
-3
Ln Kd (min )
8.0 > pH 7.0 > pH 9.0 ≥ pH 10.0 > pH 6.0 (Fig. 4A and C). The inacti-
vation of enzyme involves the unfolding of protein structure due
to changes in the balance of electrostatic and hydrogen bonds. This
is governed by pH induced changes of ionization state of ionogenic
groups of proteins [28]. It is envisaged that the thermal unfolding
of the PL in relaxed state at 60 ◦ C and in alkaline pH conditions
due to changes in the balance of the electrostatic and hydrogen
bonds, possibly influence its thermalstability. Also the half-life time
(t1/2 ) of the PL with different pH and temperature combinations,
increased significantly (P < 0.001) toward the alkaline pH (up to pH
8.0) at 40–70 ◦ C. As shown in Fig. 4C, at a elevated temperature
of 70 ◦ C, the t1/2 of the purified PL was significantly higher at pH
8.0 (7.5 min) and lower at pH 6.0 (5.4 min), suggesting that alka-
line pH condition influences the thermal stability of PL to certain
extent.
3.4.2. Effect of cations on the thermal inactivation of PL
Similarly, the deactivation rate constant (Kd ) and half-life time
(t1/2 ) of the PL were calculated at a temperature range of 40–70 ◦ C
in the presence of different cations. The presence of salts ions plays
prominent role on enzyme activity and stability [28]. Influence of
different divalent cations (Ba2+ , Co2+ , Cu2+ , Mg2+ , Mn2+ , Sr2+ and
Zn2+ ) and monovalent cations (Na+ and K+ ) for optimum PL activity
have been extensively investigated [1,7,14,20]. PLs are known to
have an absolute requirement for Ca2+ ions for their production and
activity. Also the Mn2+ ion similar to Ca2+ was reported as a strong
cofactor, that could act as inducer of PL activity [7,29]. This led to
the choice of five different cations (Na+ , K+ , Ca2+ , Mg2+ and Mn2+ )
in chloride form for the study of their effect on thermal inactivation
of PL in the 40–70 ◦ C temperature range.
The PL exhibited reduced deactivation rate and increased half-
life time in presence of cations at each temperatures studied in
comparison to the control PL (without any cations) (Fig. 4B and D).
stability of the PL, especially at temperature 40 and 50 ◦ C. The effec-
tiveness of the various cations during heat induced inactivation
was in the following order: Ca2+ > Mn2+ > Mg2+ > Na+ > K+ , indicat-
ing that the effect of divalent cations on the thermostability of

-6
-1

Phase I the enzyme was higher as compared to monovalent cations. The

stabilization of the PL by salts may have been due to a reduction
-9
in unfavorable electrostatic repulsion leading to reduced unfavor-
able electrostatic free energy. The stability of the PL was found to
-12
Phase II be significantly higher with Ca2+ , with the lowest Kd values being
obtained at and between 40 and 70 ◦ C (Fig. 4B). The t1/2 values of
-15 the enzyme with the different cations were found to be signifi-
Phase III cantly increased particularly with Ca2+ at a temperature range of
-18 40–70 ◦ C (Fig. 4D). These half-life time values of PL were found to
be lower than that for the PG from the same strain Pcc BR1, with
2.9 3.0 3.1 3.2 3.3 3.4 3.5
-1 these cations (except for Ba2+ ) [13].
1000/T (K ) The presence of Ca2+ significantly (P < 0.001) influences ther-
malstability of PL at temperatures 60 and 70 ◦ C. According to
Fig. 3. Arrhenius plot to calculate activation energy ‘Ed ’ for irreversible thermal
inactivation/denaturation of PL using relationship ln Kd = −Ed /RT, where R (gas con- thermalstability data obtained, the stabilizing effect of 1 mM Ca2+
stant) = 8.314 J/K/mol. was optimal where all the enzyme molecules would be present
28 V.B. Maisuria, A.S. Nerurkar / Biochemical Engineering Journal 63 (2012) 22–30

pH-6 pH-7 pH-8 pH-9 pH-10 Control PL NaCl KCl MgCl2 CaCl2 MnCl2

0.18
(A) 0.18
(B)

ab
ab
0.16 0.16

b
a
0.14 0.14

ab
a
a

c
a

a
0.12

b
b
0.12

bc

bc
a
b

b
ab
a
K d (min )

c
b
b
-1

0.10 0.10

d
a

c
a
a

c
0.08 0.08

b
b

0.06 0.06

a
c

d
b
b
0.04 0.04

f
e
cd
c
d
0.02 0.02

0.00 0.00
40 50 60 70 40 50 60 70

18 50

c
(C)
c

16 (D)
40
14

b
12

b
b

d
30
10
t1/2 (min)

b
a

b
a

e
a

8
ac
a

20
a

a
a

c
a

a
a
a

6
a

b
ab
4
10

bc
bc
c
ab
b
a

ac
b
a

a
2

a
a
a
0 0
40 50 60 70 40 50 60 70
o o
Temperature ( C) Temperature ( C)

Fig. 4. Deactivation rate constant (Kd ) and half-life (t1/2 ) of PL in response of different pHs (A and C) and cations (B and D). The dialyzed PL of Pcc BR1 (control PL, without
any salts) was pre-incubated with each salts [NaCl (0.1 M), KCl (0.15 M), MgCl2 (5 mM), CaCl2 (1 mM), and MnCl2 (1 mM)] individually at 4 ◦ C for 10 min, followed by thermal
stability assay. Bars represent average values of n = 3 independent experiments and error bars represent the standard deviation. One way ANOVA with Tukey’s test was used
to determine significant differences (P < 0.001) among enzyme systems at each temperature. Same alphabets above the bars indicate non-significant difference.

as enzyme–Ca2+ complexes which in agreement with inactivation of the number of noncovalent bonds broken and S* the net
model proposed by Violet and Meunier [30] which is as follows: enzyme/solvent disorder change associated with the N → Tn* tran-
K
sition. The number of non-covalent bonds broken during the Tn*
K 2
1
NCa⇔UCa−→ICa (15) state of enzyme is difficult to assess. In the N state, there is
some ambiguity connected with the bond energies and relative
where NCa is native enzyme–Ca2+
complex, UCa is heat unfolded
importance of various non-covalent bonds. Stabilizing factors of
enzyme–Ca2+ complex and ICa is the irreversible inactivated
the native state of enzyme are normally covalent disulfide bonds,
enzyme–Ca2+ complex.
hydrogen bonds, electrostatic forces of ion pairs, van der Waals
The rate-limiting step for the irreversible heat-inactivation of
and hydrophobic interactions [31]. It is assumed that all the other
enzymes is the formation of an unfolded enzyme (U) state at
hydrogen bonds in native confirmation of enzyme i.e. either the
moderate temperatures, describing irreversible inactivation as a
hydrogen bond donor or the hydrophobic bond acceptor, forms a
two-stage reaction [26]:
hydrogen bond with water molecules in addition to the intramolec-
N ⇔ U → I(andN → Ioverall) (16) ular hydrogen bonds. Since the same group should also hydrogen
bond to water molecules in the unfolded state of enzyme, they
where N is the native conformation for PL, U is the heat induced
should no longer contribute significantly to the conformational
unfolded enzyme and I is the irreversible inactivated PL. Results
stability through hydrogen bonding [26].
described in Table 3 and Fig. 5 show thermodynamic parameters
associated with the formation of a transition state (Tn*) according
to Eq. (17): 3.4.3. Enthalpy–entropy compensation during thermal
∗ deactivation
N ↔ Tn ↔ U (17)
In order to understand the behavior of PL in different physiolog-
Thus, H* and S* are the enthalpy and entropy change respec- ical conditions and the complex process of enzyme deactivation,
tively for the N → Tn* reaction. The H* provides a measure in addition to the deactivation kinetics, thermal inactivation
 V.B. Maisuria, A.S. Nerurkar / Biochemical Engineering Journal 63 (2012) 22–30 29

Table 3
Thermal inactivation thermodynamic parameters of PL at temperature range of 40–70 ◦ C.

Enzyme systems H* (kJ mol−1 )† S* (J mol−1 K−1 )† Ed (kJ mol−1 )†

Pectate lyase (PL) 31.2 ± 1.59a

−169.13 ± 4.84 a
33.91 ± 1.59a
PL + 0.1 M NaCl 39.0 ± 2.32b −147.62 ± 6.97b 41.72 ± 2.32b
PL + 0.15 M KCl 35.14 ± 2.07ab −157.6 ± 4.98ab 38.14 ± 1.64ab
PL + 5 mM MgCl2 56.8 ± 1.74c −100.8 ± 2.56c 57.37 ± 3.58c
PL + 1 mM CaCl2 67.88 ± 1.31d −71.0 ± 3.51d 66.74 ± 2.87d
PL + 1 mM MnCl2 61.0 ± 2.01c −90.20 ± 2.79c 62.16 ± 2.01cd
PL + pH 6 12.0 ± 1.46e −226.88 ± 5.45e 15.03 ± 1.76e
PL + pH 7 13.2 ± 2.39ef −225.0 ± 7.55ef 15.91 ± 2.39e
PL + pH 8 16.22 ± 0.52g −218.0 ± 6.41g 19.0 ± 0.52f
PL + pH 9 9.01 ± 1.97e −237.12 ± 6.14e 11.73 ± 1.97e
PL + pH 10 8.6 ± 0.75eh −238.81 ± 2.29eh 11.32 ± 0.75e

Values with same alphabets (a, b, c, d) indicate non-significant difference (P < 0.001).
†
Values represent mean ± standard deviation of n = 3 independent experiments, analyzed by One way ANOVA with Tukey’s test to determine significant differences among
enzyme systems.

thermodynamic parameters (G*, H*, S*, and Ed ) were deter-
mined. The change in enthalpy (H*) and entropy (S*) were
estimated based on the transition state theory. The S* values were
obtained negative for all systems used in this study (Table 3), which
is unique property of biocatalyst processes. The solvent and struc-
tural effects are two important factors which influence the numeric
values of H* and S*. The negative values of S* may be due
to agregation of the partially unfolded enzyme molecules [32]. As
shown in Table 3, the H* and S* values decreased with increase
in alkaline pH conditions (above the pH 8.0) and were significantly
higher at pH 8.0 compared to other pH conditions (Table 3). Prob-
ably at higher pHs, the stable tertiary structure of enzyme gets
compressed, resulting in lower residual activity [9]. In the case
of cations, the enthalpy and entropy values decreased in the fol-
lowing order: Ca2+ > Mn2+ > Mg2+ > Na+ > K+ (Table 3), which can be
attributed to the binding of the Ca2+ ions to the enzyme molecule
providing compactness to the tertiary structure that results in
higher thermostability.
The thermal deactivation energy (Ed ) of PL estimated using
Arrhenius model was significantly higher at pH 8.0 (near to opti-
mum pH 8.5 for the PL activity) and starts decreasing above pH-8.0
(Table 3). Alternatively, the increase in the deactivation energy
in presence of Ca2+ , Mn2+ and Mg2+ suggested that the enzyme
requires more amount of energy to deactivate in presence of diva-
lent cations. However, the effect of monovalent cations (Na+ and
K+ ) on stability of PL was found to be negligible (Table 3).
90 ◦ C and 4.7% at 70 ◦ C, compared to 40 ◦ C, which were considered
as unfavorable reaction condition at higher temperature from the
thermodynamic viewpoint for PL of B. pumilus DKS1.
The kinetic analysis of enzyme inactivation mechanisms is of
prime importance since it allows better control over biocataly-
sis processes. In addition, the information about the resistance of
enzymes to thermal denaturation due to the intrinsic contribution
of the polypeptide chain (i.e. hydrophobic interaction, hydrogen
bonding, and ionic stabilization) is also useful [33]. The broad
nature of the optimum region for the thermostability can be deter-
mined based on the different thermodynamic parameters of the
enzyme with various extrinsic factors. To sum up, these studies
contribute to facilitate better application of the biocatalysts.
It has been mentioned by Solbak et al. [34] that under alka-
line conditions and elevated temperatures (the optimal conditions
for scouring), noncellulosic part of cotton fibers mainly pectic sub-
stances in the form of pectate can rapidly be hydrolyzed by pectate
lyase. The enzyme based bioscouring treatment for cotton fabrics
and degumming of fibers, are generally carried out at temper-
atures above 70 ◦ C and in an alkaline pH, preferably above pH
8.0 [7]. The thermostable PL of Pcc BR1 is a catalytically efficient
enzyme for polygalacturonic acid hydrolysis; also it can act on
40 °C 50 °C 60 °C 70 °C
100

95 cc cc
c cb cb
3.4.4. Free energy changes during PL deactivation b cb
b b
bb b a
The Gibbs free energy (G*) measures the combination of a
ba a a a a
90 a a a a b
changes in heat and entropy that occurs during the spontaneity of a a a
G * (kJ mol )

a a a a
-1

a reaction. The resistance of enzyme to thermal denaturation is due a d

a a d a d d
a a a d a a
to the ‘intrinsic’ contribution of polypeptide chain i.e. hydrophobic a a d a
85 a d a d d
a a a
interactions, hydrogen bonding and ionic stabilization. An increase
in S* implies an increase in the number of protein molecules in a
transition activated state, which in turn gives a lower value of G* 80
[19]. The G* values for the PL deactivation are shown in Fig. 5. It
was observed that with increasing the temperature, the free energy 75
change (G*) increased by 5.7% (control PL), 2.31% (PL with Ca2+ ),
2.94% (PL with Mn2+ ) and 3.31% (PL with Mg2+ ) at 70 ◦ C as compared
to 40 ◦ C. In case of pH range studied, the G* increased in the range 70
) l l
of 7.18–7.92% at 70 ◦ C as compared to 40 ◦ C. Therefore, it reiterated l2
(PL NaC M KC gC CaC
l2 Cl2 H6 H7 H8 H9 10
ase .1M 1 5 M M Mn L+ p L+ p L+ p L+ p + pH
that the reaction conditions were most favorable with Ca2+ , Mn2+ , te
ly . M
+ 0 L+ 0 + 5m + 1m + 1m
M P P P P PL
cta PL P PL PL PL
Mg2+ and alkaline condition (pH 8.0) at elevated temperatures from Pe
the thermodynamic point of view. Possibly the divalent cations Enzyme systems
binding have influence on structural stabilization of PL through
stabilization of native state (decrease in the free energy) and/or Fig. 5. The free energy changes during thermal inactivation of PL in presence of dif-
ferent cations and pH conditions. Bars represent mean ± standard deviation of n = 3
destabilization of transition state (increase in the free energy); as
independent experiments, analyzed by One way ANOVA with Tukey’s test to deter-
observed by other authors for peroxidase stability induced by heme mine significant differences (P < 0.001) among enzyme systems at each temperature.
binding [31]. Basu et al. [7] reported that G* increased by 7% at Same alphabets above bars indicate non-significant difference.
30 V.B. Maisuria, A.S. Nerurkar / Biochemical Engineering Journal 63 (2012) 22–30
methylated pectins under the alkaline conditions. Based on its
kinetic and thermodynamic parameters, PL of Pcc BR1 can be
expected to be versatile and efficient for industrial application.
Therefore this enzyme could be utilized efficiently for agro-
industrial pectic waste treatment, as well as industrial degumming
treatment of plant fibers such as cotton, jute and ramie. Work on
this aspect is under progress in our laboratory. The feasibility of
obtaining a PL with novel industrial potential is higher from a new
microbial strain not previously reported for this purpose as in this
case should not be ignored.
4. Conclusions
The present studies explore the important biochemical proper-
ties of the PL produced by Pcc BR1 for the first time. It demonstrated
activity on polygalacturonic acid as well as 70% methylated pectin.
Its nonlinear inactivation curves at 50 and 60 ◦ C fitted to the
three-fraction first-order model. Based on the thermodynamic
parameters of thermal inactivation of PL, it was found that alkaline
pH 8.0 and Ca2+ ions favor the thermal stability at 60 and 70 ◦ C. The
thermoactive PL of Pcc BR1 has an alkaline pH optimum (pH 8.5),
low activation energy and broad substrate specificity, which sug-
gests that this strain could be utilized effectively as a novel source
of PL for the pectic waste management and degumming of plant
fibers such as cotton, jute and ramie.
Acknowledgement
Author MVB acknowledges the University Grants Commission
(New Delhi, India) for a meritorious student research fellowship
(UGC sanction no. F.4-1/2006/XI plans/BSR).
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