Isolation And Identification Of Geobacillus Thermodenitrificans And Characterization Of Its Producing Enzyme

Feed enzymes are used in feed industry widely owing to green, security andeconomy. Thermostability is essential for feed enzyme to apply because hightemperature is used during the feed processing. There are many thermophilicmicroorganisms living in various natural or artificial environments with hightemperature in the earth. They are one of the important sources of thermostableenzyme. The purpose of the present work was to isolate thermophiles from hot springsand compost samples in Guangxi and analyse the existence or not of eight feedenzymes and the producing enzyme characterization of an isolated strain ofGeobacillus thermodenitrificans. Main contents and results of this work were asfollows:1. Eleven thermophilic microorganisms were isolated from samples describedpreviously, and identified as six Bacillus licheniformis strains, two Bacillus coagulansstrains, Geobacillus thermodenitrificans, Bacillus smithii and Geobacillusstearothermophilus by16S rDNA analysis. And eight kinds of feed enzymesincluding protease, esterase, amylase, ?-galactosidase, ?-glucanase, xylanase,CMCase and ?-galactosidase were detected for the isolated strains by the method ofsingle carbon source or chromogenic reaction. The results showed all the strains hadrich enzyme systems. Among them, Bacillus licheniformis and Bacillus smithiisynthetized more kinds of these enzymes than others, seven and six kinds respectively,Geobacillus thermodenitrificans and Geobacillus stearothermophilus synthetized fivekinds, and Bacillus coagulans only synthetized four kinds.2. A new bacterial strain producing extracellular ?-galactosidase was finallyidentified as Geobacillus thermodenitrificans and named YWX5based on thephylogenetic tree, and the characterization of the ?-galactosidase was investigated.The results showed that the enzyme had great thermal and pH stability. The enzymeshowed optimal activity at temperature of65?and pH7.0. There was only less than5%loss of the enzyme activity after120min incubation at50-65?, and about83%ofthe initial activity was detected after120min at70?. Specifically, The enzymeretained about36%activity after10min at75?. Meanwhile, it retained more than80%activity after120min at the pH3.0-10.0. Enzyme activity was strongly inhibitedby the presence of Ag+, sodium dodecylsulfate (SDS), Cu2+and lactose, and there wasno obvious effect with the presence of ethylenediaminetetraacetic acid (EDTA), other metal ions and sugars tested in this work. Moreover, the ?-galactosidase wastolerant towards the stomach and intestine conditions in vitro, and it retained57.09%and84.41%activity respectively after incubation120min.3. It was investigated that the effect of medium (including carbon sources,nitrogen sources and initial pH), culture temperature and time on Geobacillusthermodenitrificans YWX5producing ?-galactosidase. The results showed raffinoseis an effective inducer for the strain producing the enzyme, but there was no efficientto supplement moderate raffinose into soybean meal medium with concentrationoptimized; the most effective nitrogen source was KNO3with concentration at0.5%,and enzyme producing was promoted by supplementation with0.5%yeast extract asextra nitrogen source; it was also enhanced by supplementation with0.5%NaCl,0.1%K2HPO4and something to hold pH. In addition, The optimal culture temperatureand initial pH of the medium for the strain producting ?-galactosidase were60?and7.0-8.0, respectively. And the activity of the ?-galactosidase reached0.306U/mLwithincubation47h in the final medium?...