Identification Of A Bacterium Strain ALW1 Degrading Laminaria Japonica And Characterization Of Its β-glucosidase MGlu1A

The research on the microorganism degrading kelp(Laminaria japonica)and its polysaccharide is of great significance for the value-added utilization of kelp and its application in the biofuel industry.In our early study,Microbulbifer sp.ALW1 was isolated from the rotten kelp.In this study,the strain ALW1 was identified.The evolution and structural characteristics ofβ-glucosidase were studied based on bioinformatics.The molecular cloning technology was used to clone,express and purify β-glucosidase,and to identify the function of β-glucosidase.This study not only lays a good foundation for the application of strain ALW1.but also lays a foundation for the enzyme’s application,and the relationship between the structure and function of β-glucosidase.In this study,strain ALW1 was identified by chemical taxonomy.DNA-DNA molecular hybridization and physiological characteristics.The results showed that strain ALW1 was Gram-stain-negative,rod-shaped or oval-shaped,non-spore-forming,and non-motile.Colonies were circular with unregular edge.wet surface,opaque and yellow in colour.The best growth conditions of strain ALW1 were 30℃,pH 6.0-7.0 and 2.0%(w/v)NaCl.The 16S rRNA gene sequence comparisons indicated that strain ALW1 was most closely related to Microbulbifer rhizosphaerae Cs16bT(98.33%similarity).The predominant respiratory quinone of ALW1 was ubiquinone-8(Q-8).Its major fatty acids were C1 8:1ω7c/C18:1ω6c.iso-C15:0,iso-C17:1ω9c/C16:0 10-methyl,and C16:1ω7c/C16:1 ω6c.Its major cell-wall sugars were ribose and galactose.The DNA G+C content of strain ALW1 was 58.2 mol%.Its DNA-DNA relatedness values of strain ALW1 with type strains of Mlicrobulbifer gwangyangensis GY2T,Microbulbifer aggregans CCB-MM1T,Microbulbifer maritimus TF-17T,Microbulbifer okinawensis ABABA23T and Microbulbifer rhizosphaerae Cs16bT were 28.9%,43.3%,41.2%,35.4%and 45.6%,respectively.In conclusion,strain ALW1 was a novel species of the genus Microbulbifer,we named it as Microbulbifer amoykelpensis ALW1.The physicochemical properties,phylogenetic tree and structure of β-glucosidase MGlulA were analyzed by using the online and offline tools of bioinformatics.The gene of β-glucosidase MGlulA was 1419 bp encoding 472 amino acids.Bioinformatics analysis showed that the coding product of β-glucosidase MGlu1A gene belonged to glycoside hydrolase GH1 family,which has 79%sequence similarity with GH1 family β-glucosidase from Microbulbifer mangrovi.The N-terminal 24-467 amino acids of MGlulA constitute the catalytic domain of enzymes belonging to GH1 family.SDS-PAGE analysis showed that the molecular weight of MGlulA was about 63 kDa.The enzymatic properties of β-glucosidase MGlulA were studied with 4-nitrophenylβ-D-glucopyranoside(pNPG)as substrate.The results showed that the optimal reaction temperature and pH of β-glucosidase MGlulA were 40℃ and 4.5.Na+,K+,Mg2+,Ba2+promoted the activity of β-glucosidase MGlulA.Zn2+,Cu2+,Fe3+inhibited the activity of MGlulA.In the presence of Dithiothreitol(DTT)and β-Mercaptoethanol(β-ME),there was no significant inhibition of enzyme activity.Sodium dodecyl sulfate(SDS)almost completely inhibited the activity of β-glucosidase MGlulA,and the recombinant enzyme had excellent resistance to Ethylene diamine tetraacetic acid(EDTA),Tween 20 and Tween 80.The relative activities of recombinant β-glucosidase MGlulA were 175%in 800 mmol/L glucose,127%in 1000 mmol/L xylose,and 123%in 600 mmol/L galactose.With pNPG as the substrate,Km and Vmax of β-glucosidase MGlulA were 2.71 mmol/L and 4.52 U/mg,respectively.When the enzyme amount was 3.56 U MGlulA/0.05 g cellobiose in the experiment of cellobiose degradation,the reaction became stable after 4 h,and the yield of glucose was about 99%.The results of molecular docking showed that the binding energy of pNPG and 4-nitrophenylβ-D-Galactopyranoside(pNPGala)to β-glucosidase MGlulA were-8.2 kJ/moL and-8.0 kJ/moL,respectively.The number of hydrogen bonds formed between pNPG and MGlulA was more than that of pNPGala.These indicated that the interaction between pNPG and MGlulA was larger,which was conducive to the catalytic reaction.