Gene Cloning And Expression Of Amylase From Microorganisms Isolated From Various Environments

Amylases are a group of enzymes that catalyze the hydrolysis of starch and glycogen.Amylases are extensively applicated in food industries such as baking,brewing,fruit juices and starch syrups.In addition,they are also applied in paper,textile,detergent and pharmacy industries.In this study,amylase producing microorganisms were isolated from various environments and a amylase gene was cloned,expressed and characterized.The details of the results were listed as follows:A total of 38 strains which had amylase-producing capabilities were isolated from 11 environmental samples such as hot spring in Tengchong,Dabusu Lake and Mangroves.These strains were identified by morphological characteristics and 16S rDNA sequence analysising and results suggested that the strains were mainly belong to the genus such as Bacillus,Vibrio,Marinobacter,Arthrobacter and Photobacterium.Strain WQJ-1 and WQJ-60 were identified as Geobacillus and Alicyclobacillus,respectively.The pH and temperature optimum of the amylase from strain WQJ-1 were 6 and 75?,respectively.The pH and temperature optimum of the amylase from strain WQJ-60 were 3.5 and 80?,respectively.Amylase-producing medium and fermentation condition were optimized for the strain WQJ-60,and the optimal conditions were as follow:maltose 1.0%(w/v),peanut cake powder 1.5%(w/v),NaCl 0.5%(w/v),K2HPO4 50 mM,Tween-80 0.06%(w/v)with initial pH of 3.0.The fermentation should maintain at 60? and 200 r/min with 4%(v/v)inoculation in 50 mL/250 mL liquid volumn for 42 h.Under these conditions,amylase activity increased by 12 times higher from 0.4 U/mL to 4.8 U/mL.The optimal reaction pH and temperature of amylase was 3.5 and 80?,respectively.The amylase had a pH tolerance range from pH3.0 to 5.0.Mn2+,Cu2+ and ?-Mercaptoethanol activated its activity while Hg2+,SDS,Pb2+ and Fe2+ inhibited it.Starch soluble was the optimum substrate for the amylase.Degenerate primers were used to amplify 14 amylase gene fragments from the DNA of 38 strains.Sequence analysis showed that these genes had identities from 70%to 100%with known amylase in GenBank Database.Based on the sequences of a-amylase gene fragment obtained,a full-length a-amylase gene named amyWQJ was cloned directly from DNA of the strain WQJ-1.It encoded 537 amino acids and one termination codon,1-22 amino acids of the whole amino acids were predicted to be the signal peptide.The gene amyWQJ was transformed into Escherichia coli BL21(DE3)and expressed,The recombinant rAmyWQJ was purified by Ni-affinity chromatography and characterized.The optimal reaction pH and temperature of enzyme was 6 and 70?,respectively.The enzyme had a wide pH tolerance range from pH5.0 to 10.0.The stability of the recombinant enzyme was enhanced in the presence of Ca2+.Co2+,Na+ and P-Mercaptoethanol activated amylase activity while Hg2+,EDTA and SDS inhibited it obviously.Starch cassava was the optimal substrate for the recombinant rAmyWQJ,followed by the starch soluble,starch corn and starch potato.By using starch soluble as substrate,the Vmax and Km were 2197.80 ?mol/(min-mg)and 6.62 mg/mL,respectively.The TLC experiments showed that the main hydrolysates to starch soluble were oligosaccharides,which were predicted as dextrin.Amylase-producing microorganisms were isolated from various environments and a amylase gene was cloned,expressed and characterized in this study.These results may pave the way for mining new amylase gene and the application of amylase in industry in the future.