Optimization Of Alginate Lyase-producing Strains And Study On Alginate Oligosaccharides

China's seaweed culture area is vast,brown algae resources are abundant,and the annual output of alginate has been ranked first in the world.However,alginate has a large molecular weight,high degree of polymerization,high viscosity,and poor absorption and utilization,which limits its application range.Its degradation product,alginate oligosaccharide,has many biological activities such as antihypertensive,hypoglycemic,immunomodulatory and anti-tumor in medicine,and its development and utilization has attracted more and more attention from researchers in various countries.Based on the versatility of alginate oligosaccharides,the degradation of kelp polysaccharides and the screening of kelp polysaccharide-degrading strains or degrading enzymes are crucial.In this paper,the microbial diversity of high-efficiency kelp degrading flora and its algin degrading enzyme system were studied,and a strain Vibrio sp.B4 with high yield of algin lyase was screened,and its fermentation conditions were optimized and enzymatic properties were studied.Furthermore,alginate oligosaccharides was prepared by degrading sodium alginate with the strain Vibrio sp.B4,and its degradation products were analyzed,providing preliminary data for the research and development of marine oligosaccharide innovative drugs.In order to investigate systematically the diversity and brown algal polysaccharide degrading enzymes of Laminaria japonica degrading microbes,the metagenome of these microbes was sequenced with massively parallel sequencing technology in this study.A total of 36,552,330 reads were obtained and 105,145 genes were modeled.The results showed that the microbes in phylum Firmicutes accounted for 75.41%among the L.japonica digesting microbes.The dominant bacteria were from genera Lachnoclostridium,Bacillus,Paenibacillus,Aneurinibacillus and Brevibacillus.In total,4,526 genes were found to encode enzymes in L.japonica degrading microbes.The number of glycoside hydrolase?GH?genes was the largest?1,520?,which was followed by that of carbohydrate binding module?CBM?genes?764?,carbohydrate esterase?CE?genes?706?and glycosyl transferase?GT?genes?693?.The polysaccharide lyase?PL?genes?107?and their auxiliary protein?AP?genes?126?were the two least.A large portion of GH genes belonged to GH109,GH13,GH18 and GH43 families.In addition,a small number of cellulosome component protein genes were found.We also found abundant alginate lyase,cellulase,pectinase and amylase producing bacteria,which provided basic data for further technological developments of highly efficient artificial degradation of L.japonica and brown algal polysaccharide exploitation.In order to screen the alginate lyase-producing strain and promote its commercial application,7 strains producing alginate lyase were screened from kelp,conch and abalone viscera using sodium alginate as the sole carbon source screening medium.Among them,strain B4 from abalone viscera has the highest enzyme activity,so B4 was selected for the following experiments.The strain was identified as Vibrio sp.B4according to morphological observation and phylogenetic analysis.Vibrio sp.B4 is milky yellow on the screening plate,round and slightly convex,opaque,smooth surface,neat edges,Gram-negative,nearly spherical or elliptical,flagellate,atypical vibrio.Single factor experiments showed the optimum carbon source of its fermentation medium was 10 g/L sodium alginate and the optimum nitrogen source was 10 g/L?NH₄?₂SO₄.The optimum fermentation conditions were temperature 30?,initial pH 6.0,inoculation 1%.On the basis of single factor experiment,3 significant factors affecting enzyme activity were screened out by Plackett-Burman?PB?design:?NH₄?₂SO₄,pH and inoculation amount.The optimization by a response surface methodology revealed that the optimal fermentation medium of B4 was comprised of sodium alginate 10 g/L,?NH₄?₂SO₄ 10.91 g/L,NaCl 10 g/L,KH₂PO₄ 1 g/L,MgSO₄?7H₂O 0.5 g/L,CaCl₂ 0.2 g/L,FeSO₄?7H₂O 0.02 g/L.The optimal fermentation conditions were temperature 30?,inoculation amount 1.1%,initial pH 6.07,speed 150 rpm.The alginate lyase activity was19.09 U/mL under the optimized condition,which was 3.67 times higher than that under basic medium and culture conditions.The enzyme activity of Vibrio sp.B4 in this work has been greatly improved by optimization,and its enzyme production time is short,which can provide reference for further research and application of alginate lyase.The crude enzyme solution was separated and purified by anion exchange chromatography and molecular exclusion chromatography,and a single band of pure enzyme was obtained by SDS-PAGE electrophoresis.Then the enzymatic properties of AL-B4 enzyme were preliminarily analyzed.It was found that the optimum temperature of alginate lyase AL-B4 was 40°C,the optimum pH was 8.0,and the enzyme was stable at pH 6.0-8.0 and below 35°C.Al³⁺,Zn²⁺,SDS and EDTA have strong inhibitory effects on enzyme activity,Cu²⁺make AL-B4 enzyme almost inactive;Na⁺,Co²⁺and Fe³⁺promote the enzyme activity,and Mn²⁺has a strong promoting effect on AL-B4,while K⁺Ca²⁺,Mg²⁺,and Ba²⁺have little effect on the enzyme activity;sodium chloride can greatly increase the activity of the enzyme.The enzyme activity is the largest in the concentration of sodium chloride 0.75 mol/L-1.5 mol/L.The obtained crude enzyme solution was used to hydrolyze sodium alginate solution,and the impurities such as macromolecular protein were removed by ultrafiltration.After freeze drying,alginate oligosaccharides was obtained.It was analyzed by TIC and HPLC and HPLC-MS.TLC identified that the alginate oligosaccharides obtained by enzymolysis was mainly composed of six components,and further analyzed by HPLC and LC-MS,the results were monosaccharide to hexasaccharide,of which tetrasaccharide had the highest content.The research on degradation products provides an important research foundation for the commercial application of algin lyase and a good enzyme source for the enzymatic preparation of alginate oligosaccharides.