Study On Screening Of Pine Needle Degradation Bacteria And Its Enzymatic Characteristics

There are many different kinds of pine resources in our country.Pine needle is a kind of sustainableuseful natural renewable resource.It is rational utilization of discarded pine needles for our environment.Wecan use pine needle resources for soil conditioner and improve soil diversity at the same time.Therefore pineneedles resources development and utilization on soil ecological environment plays a particularlyimportant role.Use the humus comeing from DA Xingan－Ling of Heilongjiang Province as sample of theoriginal bacteria，use the powder of pine needle as the only carbon source witch is use to domesticatethe untreatedc original sample,use CMCase enzyme as target ofseed selection,after separation andpurification,finely obtain5strains witch have high cellulase activity of bacterial.To select large circleof hydrolysis fast growing strains of2,4,8,15and18, respectively we use congo red differential mediumstaining.The hydrolysis circle of38bacteria grow fastest of five strains.CMC enzyme activity and FPAactivity of No.15strain bacteria are both highest.Molecular identification: Application kit DNAextraction,PCR amplification,by the Beijing Genomics sequencing,sequencing the the BLAST genepool and then we determine five strains bacteria to belong to Bacillus Bacillus,Bacillus cereus,Bacillusand short rod Bacillusfour categories.At the same time,we do the identification of molecular biologyand physiological and biochemical indicators identified for five strains.Optimization of fermentation conditions: mix four strains of two and three and four kinds of thebacteria in same inoculum size,and determination of CMC enzyme activity,FPA enzyme activity,andmicrocrystalline cellulose enzyme activity.The highest combination is2’4’8,so we select2’4’8mixedstrains for further experiments.From the initial pH,temperature,nitrogen source and external carbonsource to the optimization of fermentation conditions.External carbon source for sucrose and pineneedles powder mass ratio of2:1,plus the nitrogen source is urea,the initial pH value of7.55,adding asurfactant is polyethylene glycol,mixed strain2’4’8maximum of enzyme production.The mixed strainsbdh combination inoculum size of5%(V: V),to develop the first72hours of the enzyme activityreached the highest value.CMC enzyme activity is70.9IU.FPA enzyme activity is39.19IU.Beta－glucosidase activity is6.36IU.Microcrystalline cellulose enzyme activity is59.94IU.The hemicellulaseactivity is165.22IU． Enzymatic Characteristics: the2’4’8combination of mixed strains secrete cellulase maintained at atemperature of45°C to65°C to reach its higher enzyme activity,stability,cellulase and hemicellulasedynamic optimum temperature are50°C;Fe2+activated for cellulase and hemicellulase enzyme activity，part of the metal ions,significantly inhibited the enzyme activity of cellulase and hemicellulase．Stabilitywhen the pH value of a range between5.5to6.5will maintain a high enzyme activity，where incellulase activity of the optimum pH value of7.0,while the the hemicellulase activity optimum pH valueof6.0.Meanwhile,the cellulase can still maintain more than70%of the enzyme activity.