Purification of cellulase and xylanase enzymes produced from potential microorganisms isolated from compost for industrial application
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Date
2012
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
YSPU
Abstract
The present investigation was carried out to isolate, screen and identify the most efficient cellulolytic and
xylanolytic microorganisms from compost. Enzyme production, optimization, purification, characterization, kinetics,
cellulase/xylanase gene encoding and scale up studies were performed with selected strains to recommend their use for
industries. In total 102 microorganisms including 93 bacteria and 9 fungi were isolated. Among them, three bacteria SH8,
SH0 and BM-1 and two fungal isolates i.e. T2 and W2were screened for cellulase and xylanase enzyme production studies.
The bacterial isolates were identified as B. amyloliquefaciens, B. tequilensis and B. subtilisrespectively by 16S rRNA PCR
technique and registered in NCBI under accession no. JX129360.1, JX129359.1 and JX129361.1 while fungiwere identified
as T. longibracheatum and R. oryzae by ITS 5.8S rRNA technique and being assigned accession no. JX213811.1 and
JX213812.1. in NCBI. Cellulase and xylanase enzymes were optimized through classical approach one factor at a time
(OFAT) as well as response surface methodology (RSM) under submerged fermentation varying medium, pH, temperature,
inoculum size, carbon source, substrate concentration and incubation time and in solid state fermentation using different
pretreatments i.e. pH, temperature, moisture ratioand incubation time. The percent increase in enzyme activity obtained
after optimization of different process parameters was 204.51% for cellulase of B. subtilis BM-1, 222.91% and 210.52% for
xylanase of B. amyloliquefaciensSH8 and B. tequilensisSH0, respectively in SmF. The increase in cellulase and xylanase of
T. longibracheatumT2 was recorded 63.65% and 95.96% while in R. oryzaeW2 it was 107.38% and 125.64%, respectively
under SSF. The purification of hydrolytic enzymes was proceeded following multistep purification technique using ion
exchange chromatography and gel exclusion chromatography and the molecular weight of partially purified xylanase
enzymes of B. amyloliquefaciensSH8, B. tequilensisSH0 and T. longibracheatumT2 were found in the range of 14 kDa to
93.4 kDa, respectively. The partially purified xylanase of both bacterial and fungal origin were further characterized by
studying, the effect of various parameters viz. the effect of pH, temperature, metal ions, substrate specificity, substrate
concentrations and their kinetic parameters were derived. The partially purified thermostable xylanaseof bacterial origin was
active at 90
o
C, pH 6.0, showed high activity on xylan containingsubstrates and depicted cellulase free nature. The K
m
and
V
max
of B. amyloliquefaciens SH8 xylanase for birch wood xylan were 166.67 µmol/mg/min and 5.83 mg/ml, while for B.
tequilensisSH0 were 166.66 µmol/mg/min and 10.99 mg/ml. Similarly, partially purified enzyme of T. longibracheatumT2
was optimally active at 60
o
C, pH 5.0 and showed high activity on xylan containing substrates. The K
m
and V
max
of T.
longibracheatum T2 xylanase for birch wood xylan were 125.0 µmol/mg/min and 1.55 mg/ml. Cellulase and xylanase
abilities of the isolates were targeted by amplification of eglS and Xyn genes. Optimum conditions i.e. inoculum age,
inoculum size, aeration rate and agitation rate were explored at pilot scale for xylanase enzyme by B. amyloliquefaciens
SH8. The inoculum age of 4h, inoculum size @ 10% concentration, 1.0vvm and agitation rate of 200 rpm were found best
for production of xylanase enzyme in 7.5 L bioreactor. Mathematical model based on experimental results for xylanase
production was proposed. The production of xylanasewas found growth associated. The model consists ofa set of ordinary
differential equations taking into account the bacterial growth, substrate utilization and xylanase production with time.
Description
Keywords
Cellulase, Xylanase enzymes, Microorganisms