Study On The Strain Screening, Gene Cloing And Expression Of β-mannanase

Hemicelluloses are a kind of regenerate energy sources which second only to cellulose and account 1/4~1/3 of the plant dry weight. Its decomposition means a lot to the energy sources and environment problem in the future. The enzymatic reaction in hemicelluloses decomposition and the enzyme system screening of hemicelluloses hydrolysis have been the focal point of research. Twoβ-mannanase overproducing strains were isolated from soil by enriching and isolating. The strain 2 with enzyme activity of 271.85 U/mL was 5.39 times higher than strain 1. The strain 1 was identified as Pseudomonas sp. and the strain 2 was identified as bacillus subtilis by morphological and physiological characteristics analysis and 16S rDNA BLAST.The optimal conditions for producingβ-mannanase by the two strains were determined and the compositions of fermentation medium of Pseudomonas sp. were: 1 g/l00 mL maizena, 2 g/100mL konjak powder, pH6.0 and the optimal temperature is 32℃, under which the activity ofβ-mannanase was up to 185.37 U/mL,3.68 times as before. The strain which rapidly producedβ-mannanase showed the highestβ-mannanase activity after culturing for 12 h. The study of the enzymatic character of theβ-mannanase revealed that it was the acidicβ-mannanase, and the optimal condition of the enzyme was pH4.0 and 60℃, respectively. The activity ofβ-mannanase was stable at pH4.0～6.0 and 40～55℃。The compositions of fermentation medium of bacillus subtilis were: 1g/l00 mL Yeast, 2 g/100mL konjak powder, pH6.0 and the optimal temperature is 32℃, under which the activity ofβ-mannanase was up to 1231.41 U/mL which was higher values as far as now,4.53 times as before.The strain which rapidly producedβ-mannanase showed the highestβ-mannanase activity after culturing for 9 h, shorter than other common strains obviously. The study of the enzymatic character of theβ-mannanase revealed that it was the acidicβ-mannanase, and the optimal condition of the enzyme was pH5.0 and 35℃, respectively. The activity ofβ-mannanase was stable at pH4.0～8.0 and 35～65℃.And the strain has a high industrial value.In this study we designed the specific primers and successfully amplified the full-length sequence of the man based on the separation characteristics of man and combined with published man sequences of bacillus subtilis. The man had been cloned into the prokaryotic expression vector pET-32a, creating the recombinant plasmid pET-32a-man, which was then transformed into BL21 (DE3). After it was induced with1.0 mM IPTG for 6h, the recombinant protein showed the highest expression. SDS-PAGE analysis showed that the man was expressed in the strain BL21 (DE3) with the molecular mass of approximately 60 kDa.The activity ofβ-mannanase was not detected by the methord of DNS from the recombinant bacteria and the recombinant protein analysis showed that the protein existed mainly in the form of inclusion bodies.