Analysis Of Bacterial Community And The Screening And Identification Of Enzyme-producing Bacteria In Intestine Of Antheraea Pernyi

Antherea pernyi, belonged to Lepidoptera,Saturniidae,is a kind of biological model insect with high economic value.It distributes in more than10provinces and areas in China,mainly in Shandong, Liaoning, Henan and other north areas of China. It has been confirmed that intestinal microflora play an important role on nutrition, immunization of host insect and so on.There are a lot of studies on intestinal bacteria of different insects. Reling on combination of molecular technique and pure culture method, microbial diversity of gut of silkworm has also been a lot of research and few studies on other wild silkworm,such as A. pernyi.Due to the difference of intestinal environment, intestinal microbial of different insects have physiological variance.The variance also provides more resources to be developing microbial resources. The bacterial community and enzyme-producing bacteria in intestine of A. pernyi larvae were isolated and identified to develop microecological agents for increasing leaf-silk conversation rate and disease resistance.Bacterias were isolated from intestine of fifth instars A. pernyi larvae reared on oak leaves by isolated cultures NA, LB and Sabouraud.12intestinal bacterias were identified according to physiological and biochemical characteristics and phylogenetic analysis based on16S rDNA sequences. Cellulase, protease, lipase-producing strains were screened on selective mediums,and the D/d value of inhibition zone and enzyme activity were determinated for comparing the ability of enzyme production of strains.The intestinal bacterias isolated from A. pernyi larvae belong to Bacillus, Staphylococcus and Enterobacter. Among them, Bacillus is the main bacteria and the main enzyme-producing bacteria which could produce cellulase and protease, maximum D/d and enzyme activity,1.5and5.0,100U/ml and24U/ml respectively Staphylococcus could produce protease weakly, about10U/ml Enterobacter couldn’t produce enzyme. Intestinal bacteria community of A. pernyi were similar to that of Bombyx mori,which could be developed as microecological agent in sericulture for the enzyme-producing strains exhibiting high activity.Cellulase can’t be synthesized in epithelial cells of silkworm gut,intestinal bacteria using cellulose may play an important role on digestion and utilization of feed.Cellulose has important significant of practical application in feed, environment and other industries. A cellulase producing strain B. subtilis was screened from the A. pernyi gut, enzyme activitiy118.5U/ml in50℃and pH7.0.The endoglucanase (endo-β-glucannase) gene of B. subtilis was obtained by PCR amplification. The length of ORF is1.5kb, encoding499amino acids, molecular weight of enzyme protein55Kd, isoelectric point7.49, expressed in E. coli BL21. By subculture, extracellular enzyme activity can reach126.8U/ml. A. pernyi alkaline gut provides a new environment for studying bacterial cellulase, also provides theoretical basis for studying A. pernyi gut bacteria and the mechanism of action of cellulase.