The Optimization And Immobilized Study Of The Enzyme Catalymes Catalyzing The Production Of D-hydroxyphenylglycine

Objectives 1 Developed D-hydantoinase with high performance from brand new sources,with a new screening system. 2 Constructed rapid-throughput screening model to obtain higher performance D-hydantoinase and serve as the basis for the optimization of similar enzymes. 3 Researched the immobilization of whole cell on D-hydantoinase-engineered bacterium to obtain the optimal formula and serve as the basis of industrial production of D-hydroxyphenylycine.Methods 1 For obtaining the positive Streptomyces producing D-hydantoinase, a series screening methods were applied. The methods contain: first,regarded Dphydroxypphenylhydantion separated primarily from marine Streptomyces library as the only N element to form the medium; Then, double agar plate method and microporous rapid screening method were used to do the second screening; At last, the final screening was done with the method of molecular biology. Application of D-hydantoinase gene from positive Streptomyces, analysis of D-hydantoinase and the building of engineered bacteria. The determination of enzyme activity and kinetic parameters of D-hydantoinase.Online software Swiss-modle was used for modeling and software Caver Analyst was utilized for structure simulation analysis of catalytic channel. 2 Building D-hydantoinase mutant library by error-prone PCR; Screening mutant enzyme by high-throughput screening model; Determining properties of mutation D-hydantoinase enzyme; 3 Sodium alginate was applied to research the immobilization of whole-cell; To optimize the concentration of sodium alginate, diatomite and glutaraldehyde; Determining the optimum diameter and adding concentration of immobilized pellets; Evaluation of immobilization parameters in batch process; Measurement of the mechanical strength of beads.Results 1 4 positive Streptomyces of D-hydantoinase were screened by series screening,named S1, S14, S29 and S145,and then the kinetic parameters of D-hydantoinase from different bacteria were determined. The enzyme compare energy of D-hydantoinase from S145 bacteria was 9.7 U/mg, the Kcat was 3.2×10-6 /s and Km was 9.5 mmol/L; At last,the homologous model built and analysis of catalytic channel were applied in structure of D-hydantoinase, and the result showed the length of the main catalytic channel, Tunnel₁,was 9.1 ?, and the amino acids residue of bottleneck were histidines of 59 th and 181 st sites, and glutamic acid of 313 rd site, and neck radius was 2.18 ?; The length of the potential catalytic channel, Tunnel₂, was 13.6 ?, and the amino acids residue of bottleneck were the threonine of 62 nd site, asparaginate of 93 rd site and tryptophan of107 th site, and neck radius was 1.52 ?. 2 7 positive mutation bacteria were screened by high-throughput screening model, and the mutations were mainly concentrated in 181 st and 286rd; The enzyme activity of mutation bacterium in 181 st was 11.5 U/mg, and the product tolerance increase in 10 %; The enzyme activity of mutation bacterium in 181 st was 11 U/mg, and the product tolerance increase in 11 %. 3 The optimal formula of immobilized cells: 3.0 % of sodium alginate, 1.5 % of diatomite, 0.05 % of Ca Cl2, 1.0m M of Mn Cl2; The optimum diameter was 2.6 mm, and the adding concentration was30 %; The reused batches of immobilized cells were 12 times, and the production were more than 88 %.Conclusions 1 Developed a new screening strategy of D-hydantoinase to obtain potential enzyme optimization site and served as the theoretical basis to optimize enzyme; 2Obtained higher performance of mutant D-hydantoinase; 3 Obtained a optimized formula to product high-performance immobilized cells and made the enzymatic production Dhydroxyphenylglycine possible.