Genetic Improvement Of Bacillus Licheniformis And Application In The Expression Of Starch Debranching Enzyme

Debranching enzyme is an important emzyme,and it is indispensable for complete hydrolysis of starch.So far,there are two kinds of debranching enzymes that can hydrolyze a-1,6 glycosidic bond in starch molecules efficiently and specificlly:pullulanase and isoamylase.Previous study established a relatively perfect secretory system in Bacillus licheniformis and it is an excellent workhorse for production of a-amylase and ?-amylase.Therefore,we studyed heterologous expression of pullulanase and isoamylase using B.licheniformis as host strains.The main contents are as follows:Based on the expression system B.licheniformis Z902??Aamy?,mutant strain Z113 was constructed with deletion of an alkaline protease gene.The extracellular protease activity was 21%compared to original strain Z902,that means the secretion of protease was decreased significantly.At the same time,the lack of alkaline protease gene had little effect on the normal growth of the strains.The expression of pullulanase and isoamylase in B.licheniformis:1)The plasmid with pullulanase gene pulB was electroporated into B.licheniformis Z902 and Z113.From the result of fermentation at shake-flask level,we can not detect enzyme activity in recombinant bacterium Z902/pulB,while the enzyme activity in recombinant bacterium Z113/pulB was 41 U/mL.The activity was almost 2 times higher than previous data of our lab.The expression level of pullulanase has been further improved,which indicates that it is necessary to remove the alkaline protease gene.2)Isoamylase gene iso was amplified by PCR,and cloned into expression vector pHY-WZX and chemically transformed into Bacillus subtilis 1A717.The recombinant plasmid obtained was subsequently electroporated into B.licheniformis Z902 and Z113.At the shake-flask level,the activity of isoamylase in the recombinant bacterium Z902/ISO reached 330 U/mL,and Z113/ISO reached its maximum of 425 U/mL,suggesting its heterologous overexpression.By-investigating the biochemical properties of the enzyme,its optimal reaction conditions were determined to be 50-55 1C and pH 6.5-9.0.K⁺,Ca²⁺ and Mg²⁺ could enhance the enzyme activity,while other tested metal ions or chemicals strongly inhibited its activity.The order of the substrate utilization was amylopectin>glycogen>glutinous rice starch>pullulan.As analyzed by HPLC,the two kinds of debranching enzyme can significantly improve the production rate of maltose,78.61%and 76.35%respectively.But the recombinant isoamylase was better than pullulanase in preparing high-purity maltose syrup.Furthermore,we studied integration expression of pullulanase in host Z902,due to the recombinant strains with plasmids exist its genetic instability.On the basis of the alkaline protease gene deletion mutant box Aapr,pullulanase expression cassette was inserted into the mutation box.The recombinant plasmid pT2-apr::pulB obtained was subsequently electroporated into B.licheniformis Z902.Homologous recombination was induced by culture at high temperature,and the pulB gene was site-specific integrated into the genome.At shake-flask level,the pullulanase activity was 15 U/mL in the recombinant bacterium and genetic stability was 100%.