Screening And Analysis Of Cellulose Degrading Microbial Strains From Intestinal Tract Of Protaetia Brevitarsis Larva

There are abundant microorganisms related to cellulose degradation in the intestines of insects feeding on plant roots and weeds,such as the larvae of Protaetia brevitarsis,which have great potential for screening cellulose degrading bacteria and high-quality cellulase.In this research,90 strains of bacteria and 5 strains of fungi were isolated from the intestinal tract of the Protaetia brevitarsis larva.A strain h9 with endoglucanase（CMC）activity up to 0.19 U/mL was identified as Cellomonas through 16S rDNA analysis,and named Cellulomonas sp.h9.After analysis of the whole genome of h9,20 genes related to cellulose degradation were found,including 6 endoβ-1,4-glucanase genes,12β-glucosidase genes and 2 exoβ-1,4-glucanase genes.One endoβ-1,4-glucanase gene and fourβ-glucosidase genes namely Cen137 and Bgl381,Bgl263,Bgl211 and Bgl204 were cloned and expressed.The enzymatic properties of recombinant Cen137 and Bgl381 were further characterized.The length of Cen137 is 1036 bp and the GC content is 72.0%.It encodes an endo-β-1,4-glucanase with 321 amino acids carrying signal peptide sequence at the N terminus.Cen137 was heterologously expressed in E.coli.And the specific activity of recombinant Cen137 is 11.86 U/mg.Additionally,the optimum reaction temperature is 37℃,and the optimum pH is 6.5.The relative enzyme activity can maintained above 65%between pH 4.0 and 9.0.And the remaining enzyme activity is above 90%after being treated in pH 4.0-12.0 buffer,indicating that the enzyme has strong acid and alkaline resistance.After treated at 30℃,40℃and 50℃,the remaining activity of the enzyme was still about 85%.It was obviously activated by Mn²⁺,and Co²⁺and mercaptoethanol can moderately improve its activity.While its activity was significantly decreased by Cu²⁺and SDS,and Pb³⁺has certain inhibition.The length of Bgl381 is 1446 bp and the GC content is 76.0%.It encodes aβ-glucosidase with 481amino acids.Bgl381 was heterologously expressed in E.coli.And the specific activity of recombinant Bgl381 is 3.84 U/mg.In addition,the optimum reaction temperature is 37℃and the optimum pH is 6.0.And the relative activity of the enzyme is above 70%in the range of pH 7.0-10.0,which indicates that the enzyme has strong alkali resistance.Cr³⁺and SDS significantly inhibited the enzymatic activity of Bgl381.Na⁺,Mg²⁺,Ca²⁺,Cu²⁺,Mn²⁺,Pb³⁺,Zn²⁺,and Co²⁺inhibited its activity to a certain extent.While mercaptoethanol,Fe³⁺and EDTA have a certain activation effect on the activity of Bgl381.This is the first time to study the cellulase gene from the bacteria in the larva of scarab.It helps not only understood the mechanism of cellulose degradation by scarab but also enrich the gene bank of cellulase and provide the cellulase with potential application.