Breeding Of Alginate Lyase-Producing Microorganisms

Algin was a water-soluble acidic polisaccharides between cell walls and intercellular of brown algae.Alginate lyase degraded algin by beta elimination reaction,it is important in algae oligose preparation and genetic engineering and biomass energy development of seaweed.In this study,28 strains producing alginate lyase had been screened;26 alginate lyase gene fragments and 4 full-length genes had been cloned;optimization of enzyme production,gene cloning expression and recombinant enzymology properties were conducted.specific research results are as follows:(1)28 alginate lyase producing microorganisms were screened and purified from laminaria,asparagus,abalone farms,etc.By morphological observation and molecular identification,these strains were identified as Pseudoalteromonas sp.,vibrio sp.,streptomyces sp.,psychrobacter sp.and so on.Results showed that the optimum pH for alginate lyase crude enzyme activity were generally between 7.0?8.0,the optimum reaction temperature was generally between 30??50?.(2)Based on the results of fermentation experiments about Pseudoalteromonas sp.BYS-2,the optimal liquid medium were composed as follow:sodium alginate 1g/L,glucose 0.5g/L,yeast extract 10g/L,soybean cake powder 3g/L,(NH4)2HPO4 3g/L,pH 8.0.The optimal fermentation conditions include inoculation 2%,medium volume 50ml/250ml,incubate temperature 20? and shaking speed 200r/min.After fermenting about 24h under this condition,alginate lyase activity could reach 5.29 U/mL which was 2.37 folds higher than the ordinary condition(1.57 U/mL).(3)26 alginate lyase gene fragments and 4 full-length genes(alg390,alg738,alg752,alg1089)were obtained from Pseudoalteromonas sp.by degenerate PCR.The gene alg738 had a total length of 2217 bp.It encoded a protein of 950 amino acids,and 1-25 amino acids were predicted to form signal peptide.The theoretical molecular weight of mature protein was 79.512kDa,and the isoelectric point was 6.28.(4)Gene alg 738 was successfully expressed in Ecoli BL21.Characteristic study on the recombinant enzyme showed that:the optimal enzyme reaction pH was 8.0,the optimum reaction temperature was 45?,and the enzyme was stable under 43? and pH6.0?9.0;the enzymatic activity could be stimulated by 5mmol/L concentration of Na+,Mg2+,Fe2+,Mn2+,and inhibited by Ni2+,Co2+,Cu2+,Hg2+,Zn2+,EDTA,SDS;The Km,Vmax for the Alg738 on algin were 1.81 mg/ml and 0.51?mol/(mg·min).Substrate specificity analysis showed that the recombinant enzyme had a function preference polyM to polyG.In summary,we screened alginate lyase producing microorganisms from various environments and carry out enzyme production optimization.Then several alginate lyase genes were cloned.We prepared enzyme by means of gene cloning and expression.All above aimed to find effective alginate lyase genes and lay the foundation for development and application of alginate lyase.