Characterization Of Novel Alginate Lyase And Its Thermostability Modification

Alginate oligosaccharides(AOS)are oligosaccharides produced from alginate,which have a variety of physiological activities,such as regulating human immunity,anti-oxidation,antitumor,and promoting plant growth.Alginate lyase(Aly)is an important biological catalyst to produce alginate oligosaccharides.Alginate lyases come from a wide range of sources,and most of the reported alginate lyases generally suffer from poor thermostability,which could not meet the requirement of industrial application.In this study,four kinds of alginate lyases from different microbial sources were screened using computer-aided gene mining techniques,and their enzymatic properties were identified respectively.Then an alginate lyase with good stability from Paenibacillus sp.YN15 was selected for mutation through the rational design of thermostability.The main research is as follows:(1)Targeted mining of novel alginate lyase enzymes.Using the tool Enzyme Miner,24 potential alginate lyase protein sequences were screened.In this step,highly active and stable alginate lyases were used as the probes,and the reported conserved catalytic sites were used as the filtering benchmarks,respectively.Through preliminary experiments,four alginate lyases with certain catalytic activity were obtained and characterized from different microorganisms:Microbulbifer salipaludis Aly(named Misa-aly),Reinekea thalattae(named Reth-aly),Paenibacillus ehimensis NBRC 15659 Aly(named Paeh-aly),Paenibacillus sp.YN15 Aly(named Payn-aly),and to study their enzymatic properties.(2)Enzymatic properties of the new enzymes: The results showed that the optimum temperatures of Misa-aly,Reth-aly,Paeh-aly,and Payn-aly were 45 ℃,35 ℃,55℃,and 55 ℃,respectively,and the optimum p H was 8.5,7.5,7.5 and 8.0,respectively,and the optimum Na Cl concentrations were 100 mmol/L,600 mmol/L,50 mmol/L,and 50 mmol/L.By contrast,Paehaly and Payn-aly derived from the polysaccharide lyase 31 family(PL-31)displayed higher thermostability,and the residual enzyme activity of them at 55℃ for 5 h incubation was 61.0%and 47.1%,respectively.In terms of substrate specificity,Misa-aly,Paeh-aly,and Payn-aly showed preferred activity for the poly M region,while Reth-aly displayed higher activity for the poly G region.Under the optimum reaction conditions,the specific activities on the substrate sodium alginate of Misa-aly,Reth-aly,Paeh-aly,and Payn-aly were 53.0 U/mg,765.7 U/mg,125.7 U/mg,and 90.3 U/mg,respectively.The oligosaccharide products obtained from the reactions of the four novel alginate lyases were analyzed by TLC and LC-MS.It was found that all four alginate lyases were endocytic lyases,and oligosaccharides with polymerization degree of 2～4 could be obtained from the decomposed products,and Payn-aly could also produce monosaccharides.(3)Rational design modification for thermostability improvement.Using Payn-aly as the basic template,based on the prediction of protein folding free energy change and sequence comparison analysis,the potential mutation sites were selected.Through preliminary screening,three positive mutations were obtained,including K48 P,Y106G,and S190 G.Among them,K48 P displayed the best thermostability improvement.Compared with the wild-type Payn-aly,the catalytic activity changed very little,while its optimum temperature was increased by 10℃,and the residual enzyme activity at 65℃ for 30 min incubation was increased by 44.2%.From the structural analysis,the proline introduced in the position of helices would generally increases the structure stability.The increase in stability brought about by Y106 G and S190 G were because these two residues were remote from the ionizable residues and catalytic residues on the β-folded.In general,the introduction of glycine strictly followed evolutionary conservation and made the whole structure more stable.