Gene Mining And Expression Of Alginate Lyase Based On Genome-wide Analysis

Alginate lyase is an important tool enzyme for the development and utilization of marine polysaccharide resources,which can specifically degrade the marine polysaccharides of alginate into unsaturated alginate oligosaccharides at the non-reducing terminal（AOS）.AOS is widely used in medicine,chemical energy,food and other fields by further derivation or modification.At present,several microorganisms harboring alginate lyase have been screened from nature,however,the enzyme yield is low and the stability is not high.In addition,most of these enzymes exhibit the substrate preference for polymannuronic acid fragment（poly M）,the ability to degrade rigid polyguluronic acid（poly G）fragment is limited,which restricts the application of the alginate lyase.Therefore,further exploration of alginate lyases with superior properites and improvement of the catalytic performances are important approaches to expand the application.In our preliminary research,a strain of Isoptericola halotolerans CGMCC 5336 with high yield of alginate lyase was obtained.This lyase possessed the characteristics of poly G preference and high enzymatic activity.In this present study,the genome-wide sequencing and analysis of I.halotolerans CGMCC 5336 were carried out,the encoding gene of alginate lyase was excavated and heterologous expressed,the catalytic activity of the recombinant enzyme was improved by combination modification and optimization,the enzymatic properties were characterized.The main research contents include:（1）The whole genome of I.halotolerans CGMCC 5336 was sequenced,and the metabolic characteristics of the strain were analyzed by gene function annotation.The genes related to halophilic characteristics in I.halotolerans CGMCC 5336 were analyzed by functional annotation.The metabolic pathway of I.halotolerans CGMCC 5336 using alginate was predicted.At the same time,three alginate lyase genes were excavated that were alg912、alg913、alg1075.（2）The gene of alg913 encoding alginate lyase was heterologously expressed.In E.coli system,various expression plasmids and hosts were investigated and screened.By comparison and optimization,the engineering strain of E.coli Rosetta-p ET-32a（+）-alg913 was successfully constructed.The extracellular expressed alginate lyase of Alg913 was obtained with the enzymatic activity of 530.2 U·m L^-1.（3）Alg913 was modified by combination strategies.Through site-directed mutagenesis,the mutant of S221F with 84.65%increase in enzymatic activity was obtained compared with the initial enzyme.Structural simulation analysis showed that the increase in enzyme activity might be related to the improvement of the binding ability of enzyme to substrate by the change in catalytic pocket shape.Furthermore,the enzyme with the activity of 1822.9 U·m L^-1was obtained through the replacement and combination of promoters,RBS and signal peptides,of which the enzymatic activity was 2.4-fold higher than the initial value,which was denoted as Alg913-CM.（4）Fermentation optimization and characterization of enzymatic properties of Alg913-CM were performed.The medium composition,culture and induction conditions were optimized.The purified Alg913-CM was obtained by nickel ion affinity chromatography.The protein concentration was 0.39 mg·m L^-1 and the specific enzymatic activity was 3846.9U·mg^-1.The optimal temperature and p H of the recombinant enzyme Alg913-CM were 50°C and 7.5,respectively.Alg913-CM could specifically degrade sodium alginate,poly M and poly G,with a preference for poly G.The K_m and V_max were 1.78 mg·m L^-1 and 2.44 mg·m L^-1?min^-1,respectively.With sodium alginate as substrate,AOS mainly composed of disaccharide,trisaccharide and tetrasaccharide was obtained with the highest yield of 46.7%.