Production, purification and characterization of cellulase free xylanase from Cellulosimicrobium cellulans in solid state fermentation of apple pomace and its application in pulp biobleaching
Loading...
Date
2013
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
YSPU
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).
Description
Keywords
enzymes, productivity, bacteria, fungi, polysaccharides, apples, fermentation, acidity, poultry equipment, concentrates