β淀粉酶的异源表达与酶学性质轻工技术与工程发酵专业论文.docxVIP

AB AB STRACT 13-Amylase，one of crucial enzymes involved in starch—bioprocessing．catalyzes hydrolyzation of starch to yield food manufacturing important disaccharide mahose．has important application value in medicine，food processing，fermentation，textile and other industries．In this research，we expect to study the hctcrologous expression of barlev 13-amylase and the properties of recombinant B-amylase with molecular cloning technique， and to explore the feasibility of．the heterologous preparation．The main results are as follows： The feasibility of the heterologous expression of barley B-amylase in Bacillus was explored．Then recombinant plasmid pUB—BBA was successfully transformed into BacilluJ subtilis and Bacillus licheniformis，the gene of barley B-amylase was correctly expressed． The barley B-amylase coding gene sequences bba was cloned by PCR and then a barlev B。amylase overexpressed recombinant Pichia pasloris strain GS．BBA was developed followed by the expressing plasmid pPIC．BBA constructed and screeing by MD medium， Starch transparent circle medium and G4 1 8-YPD．Strain GS．BBA secretorily produced about 1 80 U／ml of fl-amylase induetively in 0．5％(v／v)Methanol for 1 20 hour in shakin2 flask fermentation． The biochemical properties of the recombinant D-amylase was examined．It performed the maximum activity under the condition ofpH 5．0 and 55℃with the good stability under the temperature no higher than 55。C and pH among 3．0．10．0．the recombinant enzyme activity was obviously inhibited stimulated by Ca2+and M92+．The recombinant enzyme hydrolyzed the starch to form maltose its majority product．In the sanle conditions，the conversion rate of recombinant enzyme hydrolyzed the substxate to form maltose reached 58．53％．With the aid of pullulanase， it hydrolyzed the starch to form maltose with efficiency up to 78．28％，which is similar to the 13-amylase extracted from plants． Key words：D-Amylase，Heterologous expression，Enzymatic properties 万方数据 目 目 录 1前言 · 1 1．1 B．淀粉酶的来源