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

Abstract

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.

Keywords: Cassava starch; Corn starch; Ethanol; Gene cloning and expression; Penicillium oxalicum; Raw starch hydrolysis; Raw starch-digesting glucoamylase; Simultaneous saccharification and fermentation.

Fig. 1

SDS-PAGE and native-PAGE analysis of the purified raw starch-digesting enzyme. a SDS-PAGE analysis of the purified raw starch-digesting enzyme. Lane 1 protein molecular weight marker; lane 2 the purified raw starch-digesting enzyme. b Native-PAGE of the purified enzyme. Lane 1 the purified protein stained by Coomassie Brilliant Blue R-250; lane 2 amylase activity visualized by KI/I₂ solution

Fig. 2

HPLC chromatograms of raw cassava starch hydrolysates and SEM analysis of the granules treated by the glucoamylase PoGA15A. A HPLC analysis of the reaction mixture produced by the purified enzyme. a Glucose standard, and products from raw cassava starch hydrolyzed by the enzyme after 0.5 h (b), 2 h (c), 4 h (d), and 8 h (e). B Scanning electron micrographs of raw cassava starch granules digested by the purified enzyme. f An untreated raw cassava starch granule, and raw cassava starch granules treated after enzyme hydrolysis for 0.5 h (g), 2 h (h), 4 h (i), and 8 h (j)

Fig. 3

Effects of pH and temperature on enzymatic activity and the stability of the purified glucoamylase PoGA15A. Data given are mean ± standard deviation from three replicates. The results are from a representative experiment, and similar results were obtained in two other independent experiments. a The effect of pH on enzyme activity. The enzyme activity was assayed in a citrate–phosphate buffer (pH 3.0–7.0) at 37 °C. b The influence of temperature on enzyme activity. The enzyme activity was determined between 30 and 80 °C under optimum pH conditions. c The effect of pH on enzyme stability. The pH stability of PoGA15A was measured by pre-incubating the enzyme in various buffers for 24 h at 4 °C, and the residual enzyme activity was determined using the standard method. d The influence of temperature on enzyme stability. Temperature stability was determined by the standard method after pre-incubating the enzyme at pH 4.5 between 30 and 80 °C for 1 h

Fig. 4

Phylogenetic analysis comparison of PoGA15A with other reported glucoamylases from bacteria and fungi. A phylogenetic tree was generated from the amino acid sequence alignments using Molecular Evolutionary Genetics Analysis (MEGA) software 4.0 and the neighbor-joining method with 1000 bootstrap replicates. All the protein sequences used for the analysis had been functionally identified and their GenBank accession numbers are shown

Fig. 5

Efficient hydrolysis of raw corn flour and raw cassava flour. The experiments were conducted using the rPoGA15A from the recombinant P. pastoris with the addition of commercial α-amylase. Data are mean ± standard deviation from two replicates. The results shown are from a representative experiment, and similar results were obtained in two other independent experiments. The reaction took place in citrate–phosphate buffer (pH 4.5) on a shaker at 180 rpm and 40 °C. The dosage of each enzyme used was 0.05 U/mg raw flour. a Hydrolysis of raw corn flour at 50, 100, and 150 g/L. b Hydrolysis of raw cassava flour at 50, 100, and 150 g/L

Fig. 6

Simultaneous saccharification and fermentation of raw corn flour and raw cassava flour to ethanol. The experiments were carried out using the rPoGA15A from recombinant P. pastoris and commercial α-amylase at a raw flour concentration of 150 g/L. The dosage of each enzyme used was 0.05 U/mg raw flour. The fermentation was conducted at 40 °C. Data are mean ± standard deviation from two replicates. The results shown are from a representative experiment, and similar results were obtained in two other independent experiments. a Simultaneous saccharification and fermentation of raw corn flour to ethanol. b Simultaneous saccharification and fermentation of raw cassava flour to ethanol