Purification, Characterization And Preliminary Gene Cloning Of Glucanase From Amylomyces.rouxii HN0512

In this paper,we used species Amylomyces.rouxii HN0512 isolated from bagasse (Hainan) as our material.This strain had high cellulase producing ability proved by the congored dying method on the culture plates combined with measuring the enzyme activity of liquid culture filtrates by DNS method.By measuring activity of cellulase growing in medium with different level of CMC-Na,Soy Peptone.Wheat Bran.it(orthogonal experiment L₉(3⁴)) found that the enzyme activity of liquid culture filtrates could reach 4.07 IU/mg.An cellulase was purified from Amylomyces.rouxii HN0512 by means of ammonium sulfate fractional precipitation,Hiprep^TM desalting(26×10) and HiTrap^TM Q FF.The Molecular weight was 18.7KD determinded by SDS-PAGE.The specific activity of purified endoglucanase was 140 IU/mg,and increased 66.0-fold and coefficient of recovery was 26.7%.The results of TLC revealed that this enzyme belongs to endoglucanase and named CMCase.The optimal reaction temperature of the purified endoglucanase was 60℃;the optimal pH was 5.0;It can keep half of its activity even at 70℃and was stable between pH 4.0-6.5.Experiments manifested that Km=12mg/ml, Vm=0.5878μM/min.The acticity of CMCase was enhancd by Fe³⁺,Zn²⁺.K⁺,Ba²⁺,Fe²⁺ and inhibited by Urea,Co²⁺,Mg²⁺,Cu²⁺,Na⁺.Preliminary study was carried out on the cloning of the CMCase.As Mucor mycelium containing a great quantity of polysaccharide substances,it could easily generate nucleic acid polysaccharide complex in the RNA extracted process by TRIZOL. So the RNA we got by this method is poor,it can not satisfy the following experiments. This article explored in this regard,by the improved methods we got more ideal RNA. According to Genbank homologous sequences,we designed degenerate primers in order to amplify the cDNA fragment from Amylomyces.rouxii HN0512 by RT-PCR.Although no reslults had been got,it provides some reference for the gene cloning.In this work.a kind of Mucor HN0512 with endoglucanase-producing ability was obtained;the endoglucanase was purified to a single band and its characterization was analyzed.This enzyme can be widely used in feed industy,fermentation industry,food elaboration,et al.