Molecular Modification And Characterization Of Alginate Lyase

Alginate is one of the main components of the most abundant plant seaweed in the ocean.Alginate can be converted into alginate oligosaccharides（AOS）by chemical,physical,and biological enzyme methods,but only AOS produced by biological enzyme methods have the best biological activity.Therefore,the development of efficient alginate lyase to produce AOS has research significance.In this study,point mutations were made on the alginate lyase gene Alg2944 from Pseudoalteromonas sp.Alg6B through sequence alignment and structural analysis to obtain mutants with increased enzyme activity and thermal stability.The mechanism of enzyme activity and thermal stability improvement was explained by structure and property analysis of positive mutants and wild types through molecular docking and molecular dynamics simulation.The optimal mutant was successfully secreted and expressed in Bacillus subtilis through signal peptide screening.The main results of the work are as follows:（1）The conditions for inducing enzyme production in the recombinant strain E.coli BL21（DE3）-p ColdⅡ-Alg2944 were optimized through shake flask fermentation.The results showed that the optimal pre-culture time before induction was around 5 hours;the optimal final concentration of IPTG in the culture medium was 1.0 mmol·L^-1;the optimal calcium compound was CaCl₂,and the optimal CaCl₂ concentration was 10 mmol·L^-1;the induction time was 12hours.（2）The screening of mutants and characterization of their enzymatic properties were conducted.Site-directed mutagenesis of the alginate lyase Alg2944 was performed through Through sequence alignment and structural analysis,and 5 mutants with higher enzyme activity,N145D,A195F,A195I,A195L,and F246Y,were screened.The purified enzyme activities of the mutants were 8559.2 U·mg^-1,12938.3 U·mg^-1,9753.3 U·mg^-1,12854.2 U·mg^-1,and 8291.7U·mg^-1,respectively,which were increased by 21.9%,84.2%,38.9%,83.0%,and 18.1%compared to the wild type.The optimal reaction temperature for the wild type alginate lyase Alg2944 and mutants N145D,A195F,and F246Y was 40℃,while the optimal reaction temperature for mutants A195L and A195I was 45℃.Mutants A195L and A195I also showed significantly better thermal stability than the wild type at 35℃and 40℃.The optimal p H was8.0,and the p H stability was strong between p H 7.0～8.0.Ca²⁺,Mg²⁺,and Ba²⁺had a significant activating effect on the enzymatic activity of alginate lyase Alg2944,while Ag⁺,Zn²⁺,and Co²⁺had a significant inhibitory effect on the activity of Alg2944.The addition of the metal ion chelating agent EDTA significantly inhibited the activity of alginate lyase Alg2944.（3）Molecular dynamics simulation analysis showed that the Root Mean Square Fluctuation（RMSF）of mutant A195L near the mutation site was much lower than that of wild-type alginate lyase Alg2944,explaining the reason why A195L was more thermally stable than the wild-type.Binding free energy calculations showed that A195L(-59.9 kcal·mol^-1)was higher than wild-type Alg2944(-33.7 kcal·mol^-1),explaining why A195L had a lower K_m(2.137mg·m L^-1)than wild-type Alg2944(4.122 mg·m L^-1).Solvent Accessible Surface Area（SASA）calculations showed that A195L（180.34 nm²）was lower than wild-type Alg2944（193.92 nm²）,indicating that Leu195 provided a hydrophobic pocket for the catalytic center entrance.In conclusion,molecular dynamics analysis of A195L and wild-type Alg2944 demonstrated that rigidity and hydrophobicity in the entrance loop region of the catalytic pocket are important for its catalytic activity.（4）The mutant A195L was expressed in Bacillus subtilis 168,and the enzymatic product were analyzed.Through signal peptide screening,it was found that the optimal signal peptide for secretion expression of the enzyme in B.subtilis 168 was Npr B.The fermentation supernatant had an enzyme activity of 1237.4 U·m L^-1 after shaking flask fermentation for 24hours at 37℃.During the optimization of the enzymatic conditions,it was found that the optimal substrate concentration was 10 mg·m L^-1,and the optimal enzyme addition amount in a500μL reaction system was 240 U.Using thin layer chromatography（TLC）to analyze the enzymatic hydrolysis products.The main products were monosaccharides,disaccharides and trisaccharides,and the disaccharides are the final products.