Mining Of Cold-active Chitin Deacetylase From Polar Marine Bacteria And Its Enzymatic Property Analysis

Chitin is a straight-chain polysaccharide linked by?-N-acetyl-D-glucosamine monomer connected by a?-1,4 glycosidic bond.The N-acetyldegree of linear polysaccharide is still chitin when the degree of N-acetylation is more than 50%.If the degree of N-acetylation is reduced to less than 50%,it can be derived into chitosan.Chitin deacetylase is an important enzyme that catalyzes the conversion of chitin to chitosan.Chitin deacetylation catalyzed?-N-acetyl-D-glucosamine deacetylation to convert chitin to chitosan.Screening a new type of cold-active and high activity chitin deacetylase is beneficial to solve the problems of high energy consumption and environmental pollution in industrial preparation of chitosan.The strategy has important scientific significance and application value.This paper takes the microorganisms in the polar marine environment as the research object.A strain with chitin degradation activity was isolated from the shallow sea surface sediments of the Great Wall Station,Antarctica.The chitin deacetylase gene was obtained by sequencing the whole genome of the strain.Then,the synthesis,prokaryotic expression,optimization of expression conditions,purification of recombinant enzyme and enzymatic properties of chitin deacetylase gene were studied.Genomic analysis also sho wed that the strain belonged to Pseudomonas and was named Pseudomonas sp.GWSMS-1.Genomic characterization analysis showed that the genome size of the strain was about 4.61 Mb and the proportion of GC was about 59%.The genome contains a total of 4599 genes,of which 3049 have clear functional annotations.Genome annotation analysis showed that 111 genes related to carbohydrate metabolism were 33glycosyltransferase,24 glycosylesterase,23 glycoside hydrolase,8 carbohydrate binding domain and 2 polysaccharide lyase.In this paper,the expression of recombinant chitin deacetylase was optimized as follows:induction temperature 20 ^oC,inoculum size 1.5%,inducer concentration?IPTG?0.1 mM,induction time 16 h.The specific activity of the recombinant enzyme was 150.50 U/mg and the purification folds were 27.12 times.The enzyme yield of recombinant was 53.27%.The analysis of the enzymatic properties of the recombinant enzyme showed that the optimum catalytic temperature was 15 ^oC.When the catalytic temperature is in the range of 10 ^oC to 20 ^oC,the catalytic activity is still more than80%.When the catalytic temperature is greater than or equal to 50 ^oC,the catalytic activity of the recombinant enzyme is less than 20%,indicating that the recombinant enzyme has the characteristic of low temperature enzyme.The enzyme activity was greatly lost when the recombinant enzyme was incubated at 35 ^oC for 1 hour.The enzyme activity was lost when the temperature was more than 45 ^oC.The optimum catalytic activity of the recombinant enzyme was 7.0 for pH,but 98.46%of the enzyme activity could be preserved when pH was 8.0.The results showed that the catalytic efficiency of the recombinant enzyme was the highest under the condition of partial alkalinity.It can promote the catalysis of the recombinant enzyme for K⁺,Na⁺,Zn²⁺,Mg²⁺,Ni²⁺,EDTA and DTT.It has different inhibitory effects on the catalysis of the recombinant enzyme for Li⁺,NH⁴⁺,Ca²⁺,Mn²⁺,Cu²⁺,Fe²⁺,Fe³⁺,Co²⁺,SDS and TritonX-100.When the concentration of sodium chloride is 0.1 M,the recombinant enzyme has the highest catalytic activity,indicating that the catalysis of the recombinant enzyme requires certain salt concentration conditions.When the salt concentration was higher than 0.5 M,the catalytic activity of the recombinant enzyme was inhibited.Even at low temperature?4 ^oC?for 1 hour,too high salt concentration?1 M?still reduced the recombinant enzyme activity to less than 50%.The results showed that the high salt environment had a great inhibitory effect on the enzyme activity.The enzyme activity was significantly inhibited by acetonitrile and methanol at the concentration of 20%or more.Secondly,30%or more ethanol and acetone had significant inhibitory effect on enzyme activity.Finally,50%or more of DMSO had a significant inhibitory effect on enzyme activity.The results showed that the recombinant enzyme had relatively high tolerance to DMSO,acetone and ethanol.In this paper,a well-characterized chitin deacetylase gene was obtained from the genome of Antarctic marine cold fungu.Recombinases are obtained by gene synthesis and heterologous expression.Analysis of enzymatic properties indicated that the recombinase was a typical low temperature enzyme.This study can lay a foundation for the screening and analysis of low-temperature chitin deacetylase from polar microbial sources and its application to lay the foundation for the enzyme-producing strain and its novel low-temperature enzyme gene resources.