Optimization, Purification and Characterization of b-xylanase by a Novel Thermotolerant Strain of Microbispora siamensis, DMKUA 245T

Abstract

Investigation of factors affecting b-xylanase production by a newly thermotolerant strain of Microbispora siamensis, DMKUA 245T, using a sequence of statistical methods, indicated that the optimized medium was composed of 10 g/L of xylan, 0.7 g/L of casein, 1 g/L of K2HPO4 and 0.15 g/L of MgSO4×7H2O, yielding 44 U/mL of b-xylanase activity in a shaking flask at a temperature of 35 °C for 120 h. This was a 4.4-fold increase compared to when using unoptimized medium. b-Xylanase production of 250 U/mL was obtained in a 3 L stirrer fermenter with an aeration rate of 0.5 vvm, agitation speed of 150 rpm and initial pH of 7.0 for 72 h cultivation. The b-xylanase enzyme from the strain DMKUA 245T was purified, with a specific activity of 219.4 U/mg protein obtained. SDS-PAGE and gel filtration indicated that the molecular weight of the purified b-xylanase as a monomer was estimated to be 65.8 kDa. The optimum pH and temperature for b-xylanase activity were 5.5 and 60 °C, respectively. The activity of Co2+-, K+- and Mg2+-treated purified enzymes was more thermostable than the untreated purified enzyme, particularly the Co2+-treated purified enzyme, which was still stable at temperatures up to 90 °C. The hydrolysis products were a series of short-chain xylooligosaccharides, indicating that the purified enzyme was an endo-b-1, 4-xylanase.