Cloning And Molecular Engineering Of Alkaline ?-Amylase

Amylase is the first enzyme produced industrially and used most frequently,which account for about 30%of total global enzyme market.Alkaline alpha amylase is one kind of amylaes that have the highest catalytic activity and stability at the alkaline conditons,which have been widely applied in desizing processes and detergents industries.However,some deficiencies of commercially used alkaline amylases,like low catalytic efficiency,high alkali-instability and thermal instability,significantly limited the range of its applications.It is urgent to discover new alkaline alpha amylases to expand gene databases,and improve their properties by genetic engineering to meet the demands of industrial application.In the present study,starting from discovering new alkaline alpha amylases,we successfully cloned a novel alkaline alpha amylase,and analyzed its enzymatic characteristics.Based on the homology modeled structure,domain engineering was conducted to improve its thermal stability,catalytic efficiency and production.The main contents and results are as follows:1.Gene discovery.A high alkaline amylase producing strain was isolated from a paddy field by Horikoshi medium.The 16S rDNA sequence analysis shows that this isolate share 100%identity with facultative alkaliphilic Bacillus pseudofirmus OF4,and named as Bacillus pseudofirmus 703.The amylase gene Amy703 was then cloned by PCR,and its amino acid sequence analysis suggests that Amy703 contains seven conserved sequence regions of alpha amylase family enzymes.Cluster analysis shows that Amy703 is a novel alkaline alpha amylase belonging to a new clade of GH13.2.Enzymatic characteristics.The gene Amy703 was heterologous expressed in E.coli,and enzyme assays of recombinant Amy703 were performed.The results show that Amy703 is a metalloenzyme with maximal activity at 40 ? and pH 9.0.Amy703 was stable below 40?,above 95%of activity was retained after 20 min incubation at 35 ?,and it displays good alkali-stability,retained about 80%of its activity between 8.0 and 10.5 after incubation at 4? for 12 h.Its activity was significantly enchanced by 2-and 2.48-fold at the presence of 10 mM Ca2+ and Mg2+,respectively,while EDTA almost abolished its activity.Amy703 has a highest affinity for soluble starch with Km=3.92 mg/ml and Vmax=17.664 nmol/min/mg.In addition,site-directed mutagenesis was conducted and verified its catalytic triad.3.Homology modeling and molecular engineering.Based on the protein backbone theory of De novo computational design of enzymes,although the backbone of a enzyme changed arbitrarily,a active enzyme couble still be obtained as long as the conformation of active centre was maintained.We speculated that a mutant with improved enzymatic characteristics,like thermostability and catalytic efficiency,couble be achieved by domain engineering by the fact that most of extant proteins are modular structures,namely many proteins consist of two or more independently stable and functional structural domains.Homology modeling of Amy703 was conducted by MOE program,and the modeled structure shows that Amy703 consists of four structural domains and the N-terminal domain is a unique domain of Amy703.As a validation of the above hypothesis,we truncated the N-terminal domian of Amy703(N-Amy703),the C-terminal domain(C-Amy703)and both N-and C-terminal domian((N+C)-Amy703)respectively.4.Enzymatic assays of mutants.After purification of these mutants,enzymatic assays show that the specific activity(U/mg)of N-Amy703 was 170.2,which was about 40-fold of Amy703.While the activity of C-Amy703 and(N+C)-Amy703 were both lower than Amy703.So N-Amy703 were chosed for fucther analysis.The results show that the optimum temperature of N-Amy703 increased from 40 ? to 45?,and the optimum pH shifted from 9.0 to 9.5.The thermal stability of N-Amy703 was also enhanced,the T1/2 at 50? improved by 3-4 fold.N-Amy703 hydrolyzed soluble starch with Km of 11.88 mg/ml,and Vmax of 659.21?mol/min/mg.The catalytic constant(Kcat)of N-Amy703 was 50181.98 min-1,which was 25.8-fold that of Amy703.The catalytic efficiency(Kcat/Km)of N-Amy703 was 4224.07 L/(g·min),which was 8.52 times of Amy703.Amino acids analysis of the N-terminal domain shows that it contains a conserved domain family E_set,a subset of these members were recently identified as members of the CBM48(Carbohydrate Binding Module 48)family.Substrate binding assay shows that N-Amy703 lose the ability to bind amylose,means N-terminal domain may be involved in binding insoluble substrate,and it may be a CBM.