Isolation Of Cellulose-digesting Thermophilic Bacteria And Cloning Of The Correlative Gene

In total,60 strains of thermophilic bacteria were isolated from samples of three hot springs in Yongtai County,Fuzhou of China.To facilitate the identification of thermophilic strains,SDS-PAGE of whole-cell proteins and 16S rDNA sequences analysis of these strains were performed to identify them.The results showed that there existed 8 differen strains which fell into eight species of five genera as follow, Geobacillus stearothermophilus,Geobacillus kaustophilus,Geobacillus thermocatenulatus,Geobacillus thermoleovorans,Anoxybacillus kualawohkensis,Thermus sp.,Meiothermus sp.,Aranicola sp..Thermophiles and their enzymes are important resources in industry and biotechnology.Nowadays,with the gradual energy shortage,the study of cellulase has been very important to ultilize cellulose——the renewable source which has large potential.Two cxtracellular cellulase-produced thermophilic bacterial strains,named as DT-M7 and TC-S8 were isolated.They were identified as species of Alicyclobacillus and Geobacillus.Fermentation conditions of the extracellular cellulase secreted by Geobacillus sp.TC-S8 were researched.The result shows that,in the condition that initial pH of culture medium was 6.5,culture-broth-quantum was about 20%,inoculum was 2%, speed of shake-culture was 120 r/min and culture time was 36 hours,the total CMCase activity secretied by strain TC-S8 reached its peak.The optimum condition for the cellulase secreted by strain TC-S8 was 60-70℃and pH 7.0.HPLC was used to analyse what the digest product was.The result showed that the digest product was mainly glucose and few cellobiose.Becauseβ-glucosidase hydrolyze cellobiose to produce glucose finally,so the result showed that strain TC-S8 not only secreted endoglucanase and exoglucanase but alsoβ-glucosidase.These types of cellulase make up of the compound system of cellulase.The result of cloningβ-glucosidase gene GL showed that the length of sequence in nucleotides was 1415nt,and the max ORF encoded 470 amino acid.This sequence shared 90%similarity withβ-glucosidase gene of Geobacillus kaustophilus HTA426 and 89%similarity withβ-glucosidasc gcne of Geobacillus thermodenitrificans NG80-2.Based on phylogenetic trees,β-glucosidase gene of strain TC-S8,β-glucosidase gene of Geobacillus kaustophilus HTA426 andβ-glucosidase gene of Geobacillus thermodenitrificans NG80-2 are on the same branch.Cloning ofβ-glucosidase gene of strain TC-S8 founded the base for constructing the engineering strain with high-yield cellulase.