High-level Expression Of Novel Chitosanase And Preparation Of Chitooligosaccharides

Chitooligosaccharides have abundant biological activities and multiple applications in food,agriculture,medicine,cosmetics and other fields.The main preparation technology of chitooligosaccharides is the enzymatic hydrolysis of chitosan,but the exploitation of chitosanase with high enzyme activity,good stability and low cost is insufficient,which had affected the development of chitooligosaccharides industry.The purpose of this study is to obtain chitosanase with high enzyme activity and good stability by gene cloning and expression technology,and to study the preparation process of chitooligosaccharides.Firstly,a novel chitosanase gene with a full length of 843 bp was cloned from Bacillus amyloliquefaciens ECU08.It was successfully expressed in Escherichia.coli with a molecular weight of 31 kDa.The specific activity of the electrophoresis-grade pure enzyme obtained by Ni-IDA affinity chromatography one-step purification was 1031.2 U/mg.The optimum temperature and pH were 50? and 6.0,respectively.The study of hydrolytic properties showed that this enzyme was an endo-type chitosanase,its smallest substrate was chitotetraose.The final products of chitosan hydrolyzed by this enzyme were chitobiose and chitotriose,and no glucosamine was produced.Chitooligosaccharides which own different degree of polymerization in the range of DP 2-10 could be obtained by controlling the amount of enzyme and reaction time in the hydrolysis of chitosan.Secondly,the above mentioned chitosanase gene without signal peptide was efficiently expressed in Pichia pastoris GS115.Recombinant chitosanase was expressed efficiently by high-density fermentation in batch-fed culture.The enzyme activity of fermentation supernatant reached 4314.0 U/mL at pH 5.5 and 50?.The protein content and the cell wet weight were 4.2 g/L and 355 g/L,respectively.The optimum temperature and pH of recombinant chitosanase were 55? and 6.5,respectively,which showed good stability.The specific activity of pure enzyme purified by Q-Sepharose Fast Flow strong anion exchange column was 2380.5 U/mg at the optimal reaction condition,the recovery was 64.9%,and the purification multiple was 1.03.Finally,the effects of pH,temperature,substrate concentration and reaction time on the preparation of chitooligosaccharides were studied.Preparation process of chitooligosaccharides was then determined and scale-up experiment was also carried out.The optimal reaction condition for the preparation of chitooligosaccharides with DP 2-3 was obtained:pH 5.0,45?,3%substrate and reaction time 24 h.And the final products were chitobiose and chitotriose,a small amount of acetylated chitotriose was also detected.The scale-up experiment of 30 L reaction kettle showed that the yield of production and reaction were 75.8%and 85.1%,respectively.Material balance and economic accounting showed that the process had high profit,and was suitable for industrial production.In this study,a novel chitosanase with high stability and specificity was obtained,the degree of polymerization of products was definite.The recombinant proteins were prepared by high-density fermentation of Pichia pastoris expression system and the high-efficiency enzymatic preparation of chitooligosaccharides with chitobiose and chitotriose as main components was implemented.