Efficient hydrolysis of raw starch and ethanol fermentation: a novel raw starch-digesting glucoamylase from Penicillium oxalicum.

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 α-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 α-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.

Highly Efficient Synthesis of Fructooligosaccharides by Extracellular Fructooligosaccharide-Producing Enzymes and Immobilized Cells of Aspergillus aculeatus M105 and Purification and Biochemical Characterization of a Fructosyltransferase from the Fungus.

In this work, Aspergillus aculeatus M105 was obtained to produce high extracellular fructooligosaccharide-producing enzyme activity. The maximum yields of fructooligosaccharides produced by its extracellular enzymes and immobilized cells were 67.54 and 65.47% (w/w), respectively. A fructosyltransferase (FTase), AaFT32A, was purified from M105. The optimal pH and temperature of AaFT32A were pH 5.0-6.0 and 65 °C, respectively. The Km, Vmax, and kcat values for the transfructosylating activity of AaFT32A were 2267 mM, 1347 µmol/min/mg protein, and 1550.2 s(-1), respectively, and those values for the hydrolytic activity of AaFT32A were 6.10 mM, 32.44 µmol/min/mg protein, and 37.3 s(-1), respectively. The sequence of AaFT32A deduced from the cloned gene shared 99.4% identity with a FTase from Aspergillus japonicus CB05 and a fructofuranosidase from Aspergillus niger and 96.5% identity with a FTase (Aspacl_37092) from A. aculeatus ATCC 16872. The fungal strain and its FTase may have potential applications in the prebiotics industry.

Production of raw cassava starch-degrading enzyme by Penicillium and its use in conversion of raw cassava flour to ethanol.

A newly isolated strain Penicillium sp. GXU20 produced a raw starch-degrading enzyme which showed optimum activity towards raw cassava starch at pH 4.5 and 50 °C. Maximum raw cassava starch-degrading enzyme (RCSDE) activity of 20 U/ml was achieved when GXU20 was cultivated under optimized conditions using wheat bran (3.0% w/v) and soybean meal (2.5% w/v) as carbon and nitrogen sources at pH 5.0 and 28 °C. This represented about a sixfold increment as compared with the activity obtained under basal conditions. Starch hydrolysis degree of 95% of raw cassava flour (150 g/l) was achieved after 72 h of digestion by crude RCSDE (30 U/g flour). Ethanol yield reached 53.3 g/l with fermentation efficiency of 92% after 48 h of simultaneous saccharification and fermentation of raw cassava flour at 150 g/l using the RCSDE (30 U/g flour), carried out at pH 4.0 and 40 °C. This strain and its RCSDE have potential applications in processing of raw cassava starch to ethanol.