Clone And Expression Of High Yield Recombinant β-mannanase In Pichia Pastoris

In this thesis, the β-mannanase genes of Bacillus sp.QYW-1was cloned and wasexpressed efficiently in the vector, then the large target protein was acquired, whichavoid the control of metabolic modulation mechanism to β-mannanase genes. The stablerecombinant was obtained and enzyme quantity and enzyme activity was improvedsignificantly, lay a foundation for the future industrialized production of theβ-mannanase.This study aimed to identify a mannanase-producing strain isolated from konjacsoil. The mannanase-producing strain were grown on selective plates (LB;0.5%konjacglucomannan;0.1%Congo Red;2.0%agar).The strain was identified according to themorphological,physiological,biochemical characteristics and sequencing analysis of the16S rRNA fragment,and the strain was identified as Bacillus sp, named Bacillussp.QYW-1（GenBank Accession Number JX524224）.Using degenerate polymerase chain reaction a new β-mannanase gene, denoted asmanW （GenBank Accession Number JX869490）, was obtained from Bacillussp.QYW-1genome. An open reading frame (ORF) of1084bp encoded a protein of360amino acids including a putative25-residue signal peptide and belonging to Glycosylhydrolase family26.The expressed enzyme had a molecular mass of approximately40kD determined by SDS-PAGE. A codon optimized β-mannanase gene from Bacillussp.QYW-1cleaved by EcoR I and Not I was inserted into the expression vectorpPIC9K,downstream of an α-factor signal peptide sequence, and transformed intoEscherichia coli DH5α. The resulting plasmid linearized with Sac I was transformedinto Pichia pastoris GS115.After extensive screening, the recombinant strain MANW1that expresses thesecretory protein at high level was obtained. Fermentation conditions and enzymaticcharacteristics were studied preliminarily. And after subculture for fifteen generation,the plasmid wasn’t loss, indicating that the genetically engineered Pichia pastoris hadgenetic stability.Optimum pH and temperature for the recombinant mannanase was6.5and37°C, respectively. The recombinant enzyme was stable at pH5.0～7.4andmaintained over40％of original activity after incubation at70℃for 20min.β-mannanase activity was assayed using3,5-dinitrosalicylic acid (DNS) method.The activity of the recombinant mannanase reached1200U/mL.