Analysis On Active-site Architectures Of Degrading Enzymes For Main Seaweed Polysaccharides And Their Degradation Patterns

With their rich content and industrial output value,algae can create hundreds of millions of dollars' production every year.Brown algae and red algae,whose cell walls consist of agar,carrageenan and algin,are gradually cultivated by artificial breeding.With the development of high-throughput sequencing technology,large amounts of genome of marine bacteria have been obtained and indicate that enzymes secreted by marine bacteria play a vital role in microbial catabolism pathways.However,the mechanism of biomass which can be efficiently converted has not been figured out especially in molecule level.Countless microbes associated with seaweed-degrading CAZymes secrete GH or PL to involve in the metabolic process.Analyzing the GH or PL in molecular level will provide a new method for ocean carbon cycle research.Based on the analysis above,this study aims at screening natural sample from sea or land to obtain species resources and genetic resources of seaweed-degrading CAZymes.At the same time,the efficient degradation enzyme can be heterologously expressed and the properties can be characterized.Structural bioinformatics methods were used to explore the active-site architecture and substrate-binding patterns of three types of seaweed-degrading CAZyme families.Therefore,this study intends to mutant the enzymes with rational transformation in molecular level,and speculates the function of key residues in the active-site architecture to exploit enzymes with high application value.Besides,the novel species are identified by using polyphasic taxonomic method.1.Screening bacterial strains from natural samples and identifying two novel strains.In the research on the microbial community diversity,two novel strains,N39^T and XZ17^T,were isolated from lake water sample and sea cucumber culture pond,respectively.Morphological,physiological,biochemical.molecular phylogenetic analysis and chemotaxonomic characteristics indicated that strain N39C and XZ17^T are considered to represent two novel species within the genus Kordiimonas and Nocardioides,therefore Kordiimonas sediminis sp.nov.and Nocardioides gilvus sp.nov.are recommended as their name respectively.2.Structural bioinformatics methods were used to explore the active-site architecture and substrate-binding patterns of three types of seaweed-degrading CAZymes families.Seaweed-degrading CAZymes play a key role in degrading algal polysaccharides which are major carbon source in the marine environment.Structural bioinformatics methods were used to explore the active-site architecture and substrate-binding patterns of three types of seaweed-degrading CAZymes families.Apart from aromatic residues,a higher frequency of alkaline residues His and Arg was found in the active-site architecture than in the whole enzyme.Conserved residues displayed a higher frequency in-2 and +3 subsites which are common recognition sites for each family,and conserved residues at other subsites showed a scattered distribution in active-site.This study explores preferential substrate-recognition patterns from various perspectives,leading to an improved understanding of the molecular basis of substrate-binding specificity and promiscuity.The results explain seaweed-degrading CAZymes are capable of digesting complex algal polysaccharides with a synergistic action,providing a potential method for the rational design of enzymes.3.Purification,characterization and cloning of alginate lyase Aly V4.The alginate lyase gene alyV4 in Vibrio sp.QY104 was cloned and overexpressed in E.coli BL21?DE3?.The characters of AlyV4 were determined after the purification and ultrafiltration of protein.The enzyme activity was optimal when assays were conducted at 40? and when the pH was 7.6.AlyV4 showed an obvious preference for alginate in the substrate specificity test.The evolutionary relationships showed that AlyV4 belongs to PL7-5 family.The specific activity was 5686.28 U/mg,k_m and k_cat were 1216 mg/mL and 6274.03 S^-1,respectively.4.The function analysis of key residues in the active-site architecture of alginate lyase AlyV4.The site-directed mutagenesis of AlyV4 was performed by mutating conserved residues or key residues in Lid-loop in the active-site architecture.The enzyme activity experiments indicated that the mutants almost lost more than 90%activity compared with the wild type.Therefore,the catalytic kinetic constant like k_m and k_cat cannot be measured.Fluorescence spectrometry method can be used to measure the binding constant of the mutant protein.The result showed that most of the mutant proteins lost less 45%binding energy than the wild type.The contradictory relation between enzyme activity and the binding energy showed that the enzyme catalysis process is not only consisted of the substrate binding,catalysis and product release,but also subdivided into some intermediate states.After substrate binding,some key residues in the active-site architecture play an important role in assisting catalysis.For example,the residues in the-1 site provide the ability to maintain the unique twisted conformation to ensure the catalyst,like the transform from boat to chair conformation.Using FACE to characterize product of the wild and mutant protein,the result showed that the oligose degradation ability of S76A/S76R and Y81A/Y81N decreased compared with the wild protein.The data presented here provided new insight into this important step of enzyme catalysis and therefore an increased understanding of how enzyme degrades seaweed polysaccharides.