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Cloning of a gene encoding raw-starch-digesting amylase from a Cytophaga sp. and its expression in Escherichia coli.

ABSTRACT: A raw-starch-digesting amylase (RSDA) gene from a Cytophaga sp. was cloned and sequenced. The predicted protein product contained 519 amino acids and had high amino acid identity to alpha-amylases from three Bacillus species. Only one of the Bacillus alpha-amylases has raw-starch-digesting capability, however. The RSDA, expressed in Escherichia coli, had properties similar to those of the enzyme purified from the Cytophaga sp.

SUBMITTER: Jeang CL

PROVIDER: S-EPMC126781 | biostudies-literature | 2002 Jul

REPOSITORIES: biostudies-literature

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Cloning of a gene encoding raw-starch-digesting amylase from a Cytophaga sp. and its expression in Escherichia coli.

Jeang Chii-Ling CL Chen Li-Shien LS Chen Ming-Yu MY Shiau Rong-Jen RJ

Applied and environmental microbiology 20020701 7

A raw-starch-digesting amylase (RSDA) gene from a Cytophaga sp. was cloned and sequenced. The predicted protein product contained 519 amino acids and had high amino acid identity to alpha-amylases from three Bacillus species. Only one of the Bacillus alpha-amylases has raw-starch-digesting capability, however. The RSDA, expressed in Escherichia coli, had properties similar to those of the enzyme purified from the Cytophaga sp. ...[more]

PMID: 12089056

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Project description:BACKGROUND:Starch is a very abundant and renewable carbohydrate and is an important feedstock for industrial applications. The conventional starch liquefaction and saccharification processes are energy-intensive, complicated, and not environmentally friendly. Raw starch-digesting glucoamylases are capable of directly hydrolyzing raw starch to glucose at low temperatures, which significantly simplifies processing and reduces the cost of producing starch-based products. RESULTS:A novel raw starch-digesting glucoamylase PoGA15A with high enzymatic activity was purified from Penicillium oxalicum GXU20 and biochemically characterized. The PoGA15A enzyme had a molecular weight of 75.4 kDa, and was most active at pH 4.5 and 65 °C. The enzyme showed remarkably broad pH stability (pH 2.0-10.5) and substrate specificity, and was able to degrade various types of raw starches at 40 °C. Its adsorption ability for different raw starches was consistent with its degrading capacities for the corresponding substrate. The cDNA encoding the enzyme was cloned and heterologously expressed in Pichia pastoris. The recombinant enzyme could quickly and efficiently hydrolyze different concentrations of raw corn and cassava flours (50, 100, and 150 g/L) with the addition of ?-amylase at 40 °C. Furthermore, when used in the simultaneous saccharification and fermentation of 150 g/L raw flours to ethanol with the addition of ?-amylase, the ethanol yield reached 61.0 g/L with a high fermentation efficiency of 95.1 % after 48 h when raw corn flour was used as the substrate. An ethanol yield of 57.0 g/L and 93.5 % of fermentation efficiency were achieved with raw cassava flour after 36 h. In addition, the starch-binding domain deletion analysis revealed that SBD plays a very important role in raw starch hydrolysis by the enzyme PoGA15A. CONCLUSIONS:A novel raw starch-digesting glucoamylase from P. oxalicum, with high enzymatic activity, was biochemically, molecularly, and genetically identified. Its efficient hydrolysis of raw starches and its high efficiency during the direct conversion of raw corn and cassava flours via simultaneous saccharification and fermentation to ethanol suggests that the enzyme has a number of potential applications in industrial starch processing and starch-based ethanol production.

Project description:The alpha-amylase from Bacillus sp. no. 195 (BAA) consists of two domains: one is the catalytic domain similar to alpha-amylases from animals and Streptomyces in the N-terminal region; the other is the functionally unknown domain composed of an approx. 90-residue direct repeat in the C-terminal region. The gene coding for BAA was expressed in Streptomyces lividans TK24. Three active forms of the gene products were found. The pH and thermal profiles of BAAs, and their catalytic activities for p-nitrophenyl maltopentaoside and soluble starch, showed almost the same behaviours. The largest, 69 kDa, form (BAA-alpha) was of the same molecular mass as that of the mature protein estimated from the nucleotide sequence, and had raw-starch-binding and -degrading abilities. The second largest, 60 kDa, form (BAA-beta), whose molecular mass was the same as that of the natural enzyme from Bacillus sp. no. 195, was generated by proteolytic processing between the two repeat sequences in the C-terminal region, and had lower activities for raw starch binding and degrading than those of BAA-alpha. The smallest, 50 kDa, form (BAA-gamma) contained only the N-terminal catalytic domain as a result of removal of the C-terminal repeat sequence, which led to loss of binding and degradation of insoluble starches. Thus the starch adsorption capacity and raw-starch-degrading activity of BAAs depends on the existence of the repeat sequence in the C-terminal region. BAA-alpha was specifically adsorbed on starch or dextran (alpha-1,4 or alpha-1,6 glucan), and specifically desorbed with maltose or beta-cyclodextrin. These observations indicated that the repeat sequence of the enzyme was functional in the starch-binding domain (SBD). We propose the designation of the homologues to the SBD of glucoamylase from Aspergillus niger as family I SBDs, the homologues to that of glucoamylase from Rhizopus oryzae as family II, and the homologues of this repeat sequence of BAA as family III.

Dataset Information

Cloning of a gene encoding raw-starch-digesting amylase from a Cytophaga sp. and its expression in Escherichia coli.

Publications

Cloning of a gene encoding raw-starch-digesting amylase from a Cytophaga sp. and its expression in Escherichia coli.

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