Screening And Identification Of Alginate Lyase Producing Bacteria And Its Heterologous Expression

As the main structural component of brown algae,alginate is the most abundant polysaccharide from brown algae.It has been widely used in fertilizer,feed,health products,medicine,and other fields because of its safe and non-toxic properties,such as promoting plant seed germination,inhibiting pathogenic bacteria,lowering blood pressure,anti-oxidation.Alginate lyase is often used to prepare alginate oligosaccharides by microbial methods.However,the alginate lyase produced by wild bacteria has poor stability,low enzyme activity,low degradation efficiency,so it can’t be industrialized.Based on the present situation,this paper starts from the strain screening,separation,identification of a strain producing alginate lyase.Then this paper focused on producing directed alginate oligosaccharides,the synergistic action of cellulase,degradation of kelp,constructing engineering bacterium by molecular biology techniques,whole genome sequencing technology to obtain integrated enzyme information.The specific content of this study is as follows:（1）Pseudoalteromonas sp.Alg6B producing extracellular alginate lyase was screened from the sea of Rongcheng,Shandong Province.This strain grew rapidly and its enzyme activity reached 33.7 U·m L^-1.Final hydrolysates identified by TLC and LC-MS were monosaccharide,disaccharide,tetrasccharide,hexaose.（2）Alg6B can be directly added to the kelp degradation medium to degrade kelp.After 24h fermentation,the kelp degradation rate is 58%.Moreover,Alg6B could be degraded by solid cellulase（source of Tricderma viflora）.Adding 0.4%（0.2 g/50 m L）solid cellulase to Alg6B,the degradation rate of kelp reached 97%.At that time,all the lumps of kelp are degraded and integrated into the kelp degradation solution,with only a few particles.The degradation solution of kelp has high nutrient content and is suitable for the preparation of biological fertilizer.（3）Using gene mining techniques and bioinformatics analysis,a template strain Pseudoalteromonas distincta strain ATCC 700518 was found in the NCBI database.5 genes Alg-1,Alg-2,Alg-3,Alg-4,Alg-5 were successfully amplified by designing primers.Finally,only p ET-28a-Alg-2（E.coli BL21）was soluble expressed,and the produced alginate lyase was an intracellular enzyme with the activity of 14.5 U·m L^-1.（4）Alg6B genome was de novo sequenced and the basic information of the Alg6B genome was obtained.Gene annotation showed that there were 6 genes encoding alginate lyase in Alg6B,which were gene1992,gene2243,gene2250,gene2251,gene2804,gene2944.Gene1992corresponds to Alg-1,gene2804 corresponds to Alg-2,gene2251 corresponds to Alg-3,gene2250 corresponds to Alg-4,and gene2243 corresponds to Alg-5.