Genomic Analysis And Enzymatic Characterizations Of ?-amylase Producing Strain Flavobacterium Sp.HSL13

Alpha-amylase,namely 1,4-?-D-glucanohydrolase,is an endoglucosidase,which can hydrolyze the?-1,4-glycosidic bond inside starch,and the hydrolyzed product is glucose?dextrin and oligosaccharides.Alpha-amylase is the most common type of amylase,which has a wide range of applications in industry,such as food processing,feed,washing,textile,papermaking and so on.Due to the extremely wide application of amylase in industry,its demand is also increasing,mining amylase genes with different characteristics for industrial production has become a hot topic now.In order to obtain more amylase genes with different characteristics,researchers will screen amylase genes from different environments,such as oceans,hot springs,and icebergs.In this study,14 amylase-producing strains were isolated from the mangroves in Bailu Park,Sanya,Hainan.Among them,the strain HSL13 has the strongest starch hydrolysis ability.At the same time,its physiological and biochemical characteristics analysis,molecular biology identification and genome analysis revealed that this strain contains a new type?-amylase gene amy2,which realized the heterologous expression of this gene in P.pastoris,and preliminary analysis of its related enzymatic properties.The main results of this study are as follows:(1)According to the amylase activity of the strain and the size of the transparent circle,a strain HSL13 with the strongest ability to hydrolyze starch was screened out.It produces yellow pigment,after Gram staining,it becomes a short red rod shape under an optical microscope,it was a Gram-negative bacteria.Biolog analysis showed that the bacteria can use dextrin,D-maltose,L-alanine,L-arginine and other sugars and amino acids as carbon sources for growth and metabolism.It was more sensitive to fusidic acid,guanidine hydrochloride and other chemical reagents.Its genetic relationship was closer to F.johnsoniae,and it was first established as Flavobacterium sp.HSL13.(2)Through the sequencing and analysis of the whole genome of Flavobacterium sp.HSL13,the total length of the genome of this strain was 5,483,844 bp,the GC content was33.7%,and there were a total of 4674 protein-coding genes and 124 non-coding RNAs.According to the general database annotation analysis,it is known that the bacteria contained amino acid metabolism,sugar metabolism,lipid metabolism and some other basic metabolism related proteins;according to the proprietary database annotation analysis,it is known that it was rich in glycoside hydrolase and contained alpha-amylase encoding gene,thus verified that this strain has the function of hydrolyzing starch.(3)The new?-amylase gene amy2 derived from Flavobacterium sp.HSL13 was transferred into P.pastoris and successfully achieved extracellular expression.The optimum reaction temperature for amylase amy2 was 40?,and the optimal reaction p H was 5.0,it belongs to low-temperature acid amylase,it could maintain more than 60%activity after incubating at p H 5.0?11.0 for 1 h;the metal ions Co²⁺and Ca²⁺can promote the enzyme activity;the specific enzyme activity under the optimal conditions was 81U/mg;According to the determination of substrate specificity,it has hydrolysis effect on soluble starch,corn starch and other starch substrates,and can be used in various starch hydrolysis processes.