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PRODUCTION, PURIFICATION AND CHARACTERIZATION OF CELLULASE FREE XYLANASE FROM Cellulosimicrobium cellulans IN SOLID STATE FERMENTATION OF APPLE POMACE AND ITS APPLICATION IN PULP BIOBLEACHING

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Title PRODUCTION, PURIFICATION AND CHARACTERIZATION OF CELLULASE FREE XYLANASE FROM Cellulosimicrobium cellulans IN SOLID STATE FERMENTATION OF APPLE POMACE AND ITS APPLICATION IN PULP BIOBLEACHING
 
Creator WALIA, ABHISHEK
 
Contributor SHRIKOT, C.K.
 
Subject vermicomposting, soybeans, yields, sowing, zinc, iron, planting, crops, farmyard manure, root nodulation
APPLE POMACE
 
Description ABSTRACT
Alkalophilic Cellulosimicrobium cellulans CKMX1 isolated from mushroom compost is first report on
actinomycete from Cellulomonas genera to produce cellulase-free xylanase, which is an important industrial
enzyme used in the pulp and paper industry. Strain CKMX1 was characterized by metabolic fingerprinting,
whole-cell fatty acids methyl ester analysis and 16Sr DNA and found to be Cellulosimicrobium cellulans
CKMX1. Cultural conditions and process parameters i.e. type of medium, particle size of carbon source,
incubation period, temperature, initial pH, inoculum size, yeast extract, NH4NO3, urea, peptone, CMC, MgSO4
and CaCO3 were optimized using one factor at a time approach and xylanase activity was increased to 570.0 U/g
DBP. CMCase, avicelase, FPase and -glucosidase activities were not detected, highlighting the novelty of the
xylanase enzyme produced by CKMX1. Further optimization of enzyme production was carried out using
central composite design following response surface methodology with eight independent variables which
resulted in very high levels of xylanase (1027.65 U/g DBP). The optimization resulted in 3.1-fold increase of
xylanase production, compared with the lowest xylanase production of 331.50 U/g DBP. The enzyme was
purified by gel permeation and anion exchange chromatography and had a molecular mass of 29 kDa. Xylanase
activity was maximum at pH 8.0 and 60ºC. The enzyme was somewhat thermostable, retaining 50% of the
original activity after incubation at 50ºC for 30 min. The xylanase had Km and Vmax values of 26.4 mM and 2,000
μmol/min/mg protein, respectively. All metal ions except HgCl2, CoCl2 as well as CdCl2 were well tolerated and
did not adversely affect xylanase activity. The deduced internal amino acid sequence of C. cellulans CKMX1
xylanase by MALDI-TOF MS resembled the sequence of acetyl xylan esterase of the Thermotoga sp. EMP
(Accession. no. WP_008192031). Molecular cloning was done by using pGEM-T easy vector and sequence of
xylanase gene of C. cellulans CKMX1 showed maximum homology (98%) with xylanase gene of
Cellulosimicrobium sp. (Accession no. FJ859907.1). The hydrolytic products of the insoluble oat spelt xylan
incubated with xylanase were identified by xylose standards using HPLC. Xylanase effectively hydrolyzed
xylan and the hydrolysis by xylanase produced mainly xylose as the main product after 5 min incubation time.
Cellulase-free xylanase from C. cellulans CKMX1 under C-EP-D sequence has been shown to bring about a
12.5% reduction of chlorine, decrease of 0.8 kappa points (40%) and gain in brightness was 1.42 % ISO points
in 0.5% enzyme treated pulp as compared to control where no enzyme pre-treatment was given, when
enzymatically prebleached pulp was charged with 7.4% of total chlorine. From the present studies it is clear that
C. cellulans CKMX1 xylanase is having the characteristic suited for an industrial enzyme (xylanases that are
active and stable at elevated temperatures and alkaline pH
 
Date 2016-05-31T14:31:07Z
2016-05-31T14:31:07Z
2013
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/66489
 
Language en
 
Format application/pdf