The Research On Diversity And Functional-enzymes Of Deep-sea Microbes

The ocean is the largest biological inhabiting system on earth which occupies about 71%of the earth's surface.It contains huge microbial resources because of its complex and changeable environmental characteristics.Especially in the deep-sea area,microorganisms can tolerate extreme conditions such as high-salinity,high-pressure,low-temperature?or local high-temperature?,oligosaccharide and low-light,and then they have formed varieties of enzyme systems.Therefore,the development of abundant enzyme gene resources in deep-sea microbes is one of the effective ways to realize high value utilization of marine biological resources.Among them,pullulanase?EC3.2.1.1/41?is a kind of starch debranching enzyme widely existed in marine microbes,which can hydrolyze the?-1,6-glycoside bond of amylopectin and make it into amylose.This characteristic determines its wide applications in the industries related to starch processing.However,the pullulanase used in our country are almost entirely dependent on imports,which has raised the production cost of the related enterprises and restricted the rapid development of the related industries in our country because of its high prices.Hence,we look for high yield pullulanase-producing strain and develop its pullulanase gene resources from deep-sea area in order to provide the experimental basis for the commercial production of pullulanase in our country in the future.In this paper,taking“isolation and identification of culturable microbes—screening of functional enzymes—cloning and expression of the pullulanase gene"as the clue,the research made on diversity and functional-enzymes of deep-sea microbes from the following aspects:?1?Taking the sea-water with different depths in the Northwest Pacific and the middle India Ocean as the research object,1 225 strains of bacteria were isolated and identified,and these culturable bacteria were analyzed statistically later.The results showed that these bacteria belonged to four groups,including Proteobacteria,Actinomycetes,Bacteroidetes and Firmicutes,especially widely distributed in the?-proteobacteria and?-proteobacteria.?2?The 1 225 isolates were screened for the 7 functional-enzymes activities?including amylase,cellulase,protease,lipase,chitoamylase,alginate lyase and?-carrageenase?,and then their potential of functional-enzyme activities were preliminarily evaluated.The results indicated that there were 220 strains with amylase activities,231 strains with protease activities,only 16 strains with lipase activities,302 srains with chitoamylase activities,297 strains with alginate lyase activities,289strains with?-carrageenase activities,and no strain with cellulase activity.?3?The 220 strains with amylase activities were screened again for pullulanase activities,and one wild strain named Alteromonas sp.Y-389 was selected by initial screening with transparent circle method in solid medium and rescreening with DNS reagent method in liquid medium,which can produce high yield of extracellular-pullulanase activity.Subsequently,the optimum design of culture medium and culture conditions for its fermentation were made in order to explore the maximum enzyme-producing ability of this wild strain.By single factor analysis,the results showed that the pullulanase-producing activity of Alteromonas sp.Y-389reached 7.69 U/m L,which proved that the wild strains have strong ability to produce extracellular-pullulanase and has great value for follow-up research.?4?Using the method of molecular biology,the gene encoding pullulanase pulA of the wild strain Alteromonas sp.Y-389 were cloned,sequenced and analyzed by bioinformatics analysis.The results indicated that the pullulanase gene pulA full-length ORF is 4347 bp,encoding a protein PulA with 1449 amino acid.The protein PulA has closer relationship with multiple pullulanases from Alteromonas sp.through phylogenetic analysis.The molecular weight of the protein PulA is 158.68kDa,and the theoretical isoelectric point is 4.15,which belongs to the PulA superfamily by means of further bioinformatics analysis.?5?With the plasmid pET-28a?+?as the carrier,inserted gene pul A into the carrier and then constructed the gene engineering bacteria.Under the induction of IPTG,the target pullulanase PulA was successfully expressed in the cytoplasm of engineered strains.Then,the purification and characterization of the pullulanase Pul A were studied.The results showed that the specific activity of the purified pullulanase PulA reached 54.53 U/mg.The optimal pH and temperature for the pullulanase was 6.0and 35 ^oC,respectively.Mn²⁺had an obvious activation effect on enzyme activity,while Cu²⁺,Zn²⁺,Fe²⁺had significant inhibitory effects.It belongs to the?pullulanase,and its Km and Vmax were 14.97 g/L and 0.36 g/?L·min?respectively.This result built a good foundation for the industrial production of pullulanase in the future.