Cloning And Expression Of Genes Encoding Polysaccharide Hydrolases Of Bacillus Sp. HS81 From A Tropical Anthill

Microorganisms are producers of many hydrolytic enzymes,among which polysaccharide hydrolases are main players in degradading biomass-related wastes.Polysaccharide hydrolases are thought to be potential in bioconversion and utilization of biomass wastes as renewal resources.It has been shown that the polysaccharide hydrolases from bacteria and fungi vary largely in enzymatic type and activity.Compared to fungi hydrolases,bacterial ones are more diverse and adapted to wider range of pH and temperature.And bacteria are generally fast growing which require less expensive fermentation equipment,and thus lower the cost of industrial production.In this study,a Bacillus strain HS81 was isolated from soil samples of an anthill in tropical forest,Badam Island,Indonesia.It was shown to have multiple activities of degradading sodium carboxymethyl-cellulose,xylan,mannan and lichenin concurrently.According to the phenotypic characteristics and 16S rRNA gene sequence analysis,this strain was shown to be a member of Bacillu subtilis subsp.spizizenii.The strain was shown to have the highest rate of growth and enzymatic activity among the strains tested,including Bss6,a reference strain of Bacillus subtilis subsp.spizizenii.However,there is no report of the functional analysis and properties of polysaccharide hydrolases from this subspecies.In this study,it is aimed to explore the unique hydrolases from this special isolate from a tropical anthill,by means of gene cloning,expression,functional confirmation and enzymatic analysis.Since there was no genomic data available when the study started,protein sequences were selected at Bacillus genus or species level and analyzed to design the conserved and degenerate primers for PCR amplification.Isolation,identification and cloning of four target genes putatively encoding ?-1,4-glucanase CelB1n,?-1,4-mannosidase manBln,?-1 3-1,4-glucanase gluBn and ?-1 4-xylosidase XylBln,were conducted by PCR-based cloning and subcloning.The high fidelity PCR systems were applied to minimize the error.Individual gene amplicon produced by fidelity PCR was subsequently expressed in E.coli Transetta(DE3)through the bacteria expression vector pET26b(+).Plate experiments were done verifying that strains expressing the individual hydrolase gene clearly displayed larger hydrolytic zone compared to that by crude enzymatic preparation from Bss6.Functional and enzymatic analysis of the expressed genes using DNS assay showed that the above-mentioned four hydrolases all possessed superior specific activities,ranging from 2409.67 to 104132.55 U/mg.The optimum temperatures of these enzymes ranged from 55? to 80?,and the enzymes were stable at 50-60?.The optimal pH range was 5.0-6.0,and they were stable at pH 5.0-10.0 and maintained over 80%relative activity after being treated with pH buffers for 1 hour.In addition,the engineered enzymes had good resistance to most of the tested metal ions and chemical reagents.Among the expressed hydrolases,the activity of ?-1,4-mannosidase was the most stable.Not only its optimal temperature was 80?,but also it maintained respectively 92.7%and 78.6%relative activity,after being incubated at 80?and 90 ? for 90 min.Moreover,it remained 81.3%residual activity after being incubated at 100? for 60 min,indicating that P-1,4-mannosidase manB1n was an extremely thermophilic enzyme,being stable at 100?.Sequence analysis indicated that genes encoding the four polysaccharide hydrolases of HS81 were quite different from the known counterparts from other members within genus Bacillus even species B.sbtilis,implying the structural diversity of putative enzymes encoded.The diversity in gene sequences may reflect the uniqueness of tropical anthill as a special environmental niche.In addition,adoption of pET26b(+)as an efficient expression system and Ni-NTA protein purification procedure may all make contribution to the high activity of transgenic hydrolases.Together,our results demonstrated that the four engineered hydrolytic enzymes from HS81 were thermostable and alkali tolerant,having potential in bio-treatment of the wastes produced by industries including agriculture,forestry,the detergent and textile and the pulp bleaching.