Screening And Protein Engineering Of Stereoselective Amidase And Its Application In The Synthesis Of L-Phosphinothricin

Amidases,also named amidohydrolases,is a hydrolase capable of breaking non-peptidic amide bonds.Amidase exhibited high activity,strict enantioselectivity and broad range of substrate specificity.Most of the studies focused on the utilization of amidases in the synthesis of enantiomerically pure agrochemicals,pharmaceuticals and chiral amino acids.L-phosphinothricin?L-PPT?is a naturally occurring L-type phosphorus-amino acid found in Streptomyces hygroscopicus.It is also a broad-spectrum,contact-killing,and biocide herbicide that is harmless to the environment.Compared with the traditional synthesis of L-PPT via chemical methods,biosynthesis of L-PPT by amidase would be an alternative method because of its strict stereoselectivity,mild reaction conditions,high conversion rate,subsequent separation and purification operations,and environmental friendliness.In this study,stereoselective amidases were obtained by screening from amidase-producing microorganisms which were screened from soil samples and database mining as well,then secretory expressed in B.subtiis.The catalytic activities of amidase were tested.The catalytic property of Bm-Ami among them was improved by directed evolution,and applied into the synthesis of L-PPT.The dissertation mainly includes the following contents:1.A novel and simple high-throughput method for the detection of phosphinothricin produced by biosynthesis approach was developed.By using this method,ten amidase-producing strains were screened from soil,of which two strains Kluyvera cryocrescens ZJB-17005 and Leclercia adecarboxylata ZJB-17008 showed higher activity and stereoselectivity.By using genome hunting and database mining technology,35 amidases were obtained and an amidase library was constructed.Finally,three amidases,Bm-Ami,Kc-Ami and La-Ami that exhibited high activity and stereoselectivity were selected as target enzymes for further study.2.A highly active secretory expression system in Bacillus subtilis was constructed for enhancing the heterogenous secretory expression efficiency of amidase?Bm-Ami?.A secretory expression system pBSHdd2-20 containing a dual-promoter P_amyE-cdd and a signal peptide Pac was constructed via promoters and signal peptides screening.The system showing an extracellular Bm-Ami activity of135.58 U mL^-1 during shake-flask cultivation,which was 3.58-fold greater than that of the original plasmid pBSH1.Finally,extracellular amidase of Kc-Ami and La-Ami was 3.02-fold and 3.12-fold greater than that with corresponding parent secretory expression systems.3.Bm-Ami,Kc-Ami and La-Ami were purified and biochemically characterized.SDS-PAGE analysis of the purified Bm-Ami,Kc-Ami and La-Ami indicated that the enzymes were heterodimer composed of two subunits with molecular weights of about 26 and 61 kDa,24 and 64 kDa,26 and 62 kDa respectivity.All of them were Ntn-hydrolase family amidases,containing N-terminal Ser?1 and corresponding amino acids.The optimum pH of Bm-Ami,Kc-Ami and La-Ami were 8.5.The optimum temperature of Bm-Ami,Kc-Ami and La-Ami were 55?C,50?C,55?C.The half-lives of Bm-Ami,Kc-Ami and La-Ami at 50?C were 19.8 h,59.1 h,47.6 h.Bm-Ami with the maximum catalytic efficiency,it had 2.24-fold and 1.72-fold than that of La-Ami and Kc-Ami respectivity.Furthermore,Bm-Ami,Kc-Ami and La-Ami exhibited capacities for the biosynthesis of chiral amino acids.4.Structural modeling and active pocket prediction were applied to hunt hotspots.Mut-R?381F/Y?382F as the optimal mutant were obtained with 204%activity of the wild-type of Bm-Ami by directed evolution.Furthermore,Bm-Ami and the mutants were expressed and purified to determine the kinetic parameters.The results indicated the catalytic efficiency(k_cat/K_m)of mutants all improved than that of the wild type of Bm-Ami,the catalytic efficiency of mut-R?381F/Y?382F showd 2.84-fold than the wild type of Bm-Ami.5.The amidase mut-R?381F/Y?382F was used for catalytic preparation of L-phosphinothricinwiththe rac-4-?hydroxy?methyl?phosphoryl?-2-?2-phenylacetamido?butanoic acid?rac-S?as subtrate.The optimum pH,temperature and amidase loading of the reaction were determined as 9.0,55?C and 15?g mL^-1(753.3 U L^-1).Under the optimal reaction conditions,49.2%conversion,99.9%e.e.value and 584.52 g L^-1 d^-1 space-time yield was obtained at 500 mM substrate loading after 2 h reaction.The refined L-PPT was obtained by vacuum distillation,organic solvent extraction,different solubility of PPT and rac-S in different acidity-alkalinity,adsorption of cation-exchange resin and crystallization with a purity of 99%and a total yield of 90%.