Optimization Of Conditions For Production Of β-mannanase And Its Enzymatic Properties

By using konjak powder as the only carbon source, using the methods ofenriching culture, isolation and selective plate culture,13mannanase producing strainswere isolated by observing the clear circles on selective plates from32soil samples,which were gathered from nature of grape yards and konjak soils. Five strains of highenzyme activity were got which showed bigger clear circles. Then one strain was gotafter the second svreening by shaking culture and rhe research of stability aboutenzyme-production. The strain shows better stability of enzyme activity and higherenzyme activity was named QYW-1. It was identified as Bacillus subtilis by itsphenotype, morphological characteristics, the traditional physiological andbiochemical characteristics, together with the result of16s rDNA sequence.Comparetive16s rDNA sequence analyses revealed that QYW-1was most related tothe strain Bacillus subtilis（Identities＞99%）.The strain QYW-1has been registered inGenbank under the accession number of JX524224.Based on single factor experiments which were used to optimize the medium andcultural conditions, the most important of11factors which affected enzymy productionselection with Plackett-Burman design was used. PB design was established todetermine which factors in the fermentation medium mainly influence the enzymeproduction (konjak powder, pepdone, MgSO4). Further optimization of enzymeproduction was carried out using Box-Behnken design of experiments, and its resultwas the optimum medium ingredients forβ-Mannanase production in a250mL flaskwere as follows: konjak powder26g/L, peptone10g/L, MgSO43.8g/L, NaCl10g/L,KCl6g/L, NaNO36g/L, K2HPO43g/L, initial pH nature. The30mL cultivation wascarried out at37℃、180r/min for15~16h. Under the RSM-optimized conditions, theβ-mannanase were improved from115.62U/ml to233.86U/ml.The crudeβ-Mannanase of the strain Bacillus sp. QYW-1were investigated. Thethe optimal pH for the enzyme reaction was6.5and optimal temperature was55℃, itwas stable between pH4.5~9.0and35~60℃. The enzyme activity was partlystimulated by Ca2+、Mg2+and inhibited by Ag+、Hg2+、EDTA.We use the crude enzymewhich was producted by the strain Bacillus sp.QYW-1to catalyze the waste brewer’syeast cellwall to product manno-oligosaccharides. The hydrolysis conditions of wastebrewer’s yeast cellwall were optimized according to the single factor. Under the conditions of β-mannanase70U/g, hydrolytic temperature50℃and hydrolytictime7h, the hydrolysate was mainly oligo-saccharides with little trace ofmono-saccharides analyzed by thin-layer chromatography. It can enlarge the source ofmanno-oligosaccharides, increase the use of waste brewer’s yeast in use, reduceenvironmmenal pollution coming from the beer-production. The research is providedwith important significance and a bright future for the beer-ptoduction.