Identification Of Raw Starch Digesting Enzymes Produced By Rhizopus Microsporus Var. Chinensis CICIM F0088

A newly isolated active producer of raw-starch-digesting amyloytic enzymes, CICIM F0088, was isolated from the strains library including 365 fungi able to digest starch, and was identified by morphological characteristics and molecular phylogenetic analyses. This fungus was kept in the Culture and Information Center of Industrial Microorganism of China University (http://cicim-cu.jiangnan.edu.cn) at JiangNan University as Rhizopus microsporus var. chinensis CICIM F0088. The amount of enzymes production and their compositions were different between solid-state fermentation (SSF) and submerged fermentation (SmF).For the first time, comparative research of amyloytic enzymes produced in SSF and SmF was carried out in fungus Rhizopus microsporus. Two glucaomylases named as GA A and GA B, and oneα-amylase named as AA from SSF were purified to homogeneity through ammonium sulfate precipitation, aqueous two-phase systems, Toyopearl DEAE-650M chromatography, prep cell and Toyopearl HW-55F gel filtration chromatography. The mocular weights of three proteins were 53, 52, and 55 kDa, respectively. In addition, two amyloytic enzymes from SmF were also purified to homogeneity by the procedures of ammonium sulfate precipitation, Toyopearl DEAE-650M chromatography, prep cell, separately named as GA C and GA D. The mocular weights of two proteins were 53, and 52 kDa, respectively.Glucoamylases produced from SSF were much more thermostable and pH tolerate than enzymes produced from SmF. Raw-starch-digesting abilities were found in all the four glucoamylases except theα-amylase.Based on the analysis of conserved amino acid sequences from Rhizopus sp., and the codon preference of Rhizopus microsporus, DNA sequence of gluE was amplified, and then the cDNA sequence of gluE was amplified by RT-PCR. Results showed a 2763 bp gluE gene including 4 introns was cloned. The deduced unprocessed glucoamylase from CICIM F0088 was 579 amino acids long and shows 80% identity to the Rhizopus glucoamylase. It was the first time of glucoamylase gene cloning from Rhizopus microsporus.GA E cDNA fragment encoding mature peptide was inserted into the plasmid pET-28a(+) and expressed in E. coli BL21. The purified recombinant enzyme displayed raw starch digesting ability.