Isolation And Identification Of Cellulase-secreting Probiotic From The Cecum Of Goose And The Enzymy Properties

Cellulose is the most abundant organics on the earth.But the resource is underutilized and even most of them became one of the environmental pollutants.There are a lot of studies on the cellulolytic microorganisms and most of the cellulolytic strains were isolated from different fermented plant residues and soil.Some cellulolytic strains from animal had been isolated,and most of them were isolated from ruminant.To our knowledge,the study of cellulolytic bacterium isolated from the cecum of goose was rare.The goose,as herbivores,had high digestibility to diet fiber.There is a lot of microorganism in goose caecum,so it has been considered that the fermentation function of goose caecum is same as rumen.In order to enhance the utilization of cellulose resources,cellulolytic microorganisms from geese cecum had been isolated and identified.Then the proper culture conditions of cellulase and properties of the cellulase were studied,and the experiment may lay a certain foundation for the research and application of the bacteria.In this study,six 3-month of Yangzhou geese were provided randomly(half of them was male,and the other was female).The goose was killed and the cecum was ligated and taken out.A microorganism secreting cellulase was isolated from the cecum of goose and it showed a high cellulase activity of 1.25 U/m L determined by DNS method.The microorganism was identified as a kind of Bacillus amyloliquefaciens through analysis the gene sequence of 16 S r RNA and named as B.amyloliquefaciens S1.In vitro assays,the proper culture conditions of producing cellulase were studied by single-factor experiment and orthogonal test.The results indicated that the best conditions of producing cellulase were 37 ? of the culture temperature,6.0 of the initial p H,72 h of the incubation time,and 75 ml per 250 ml of the loaded liquid volmne.And the order of the degree of the effection of various factors on the activity of enzyme-producing by B.amyloliquefaciens S1 was: initial p H> incubation time = culture temperature >loaded liquid volmne.Some properties of the cellulase from B.amyloliquefaciens S1 were studied.The results indicated that the optimal p H of the enzymes was 7.0 and the optimal reaction temperature was 60 ?.The enzymes possessed strong resistance against heat and acidity.The cellulase showed a great activity to absorbent cotton,soybean meal and filter paper.This demonstrated that the cellulase acted on extensive fiber substrates.The Fe3+ inhibited cellulase activity slightly,while the Ca2+ K+,Cu2+,Fe2+,Mn2+ and Zn2+ can enhance cellulase activity distinctly.Meanwhile,a gene encoding a kind of cellulase of B.amyloliquefaciens S1 was cloned.The ORF of cellulase gene was 1500 bp in length,which encoded a deduced protein of 499 amino acids amino acids.According to the BLAST comparison and analysis of the sequence with the existing cellulase gene of B.amyloliquefaciens from Genbank,the sequence had 97%-99%homology with other cellulase genes.The gene was consistent with the expected result through the evolution tree analysis by soft MEGA5.1.Then the cellulase gene was linked into prokauryotic expression vector and its prokaryotic expression was also carried out.The cellulase gene encoded a protein of 55 k Da,which was tested through SDS-PAGE and Western blotting.