Raw-starch-digesting α-amylase (Amyl III) was purified to an electrophoretically pure state from the extract of a koji culture of Aspergillus awamori KT-11 using wheat bran in the medium. The purified Amyl III digested not only soluble starch but also raw corn starch. The major products from the raw starch using Amyl III were maltotriose and maltose, although a small amount of glucose was produced. Amyl III acted on all raw starch granules that it has been tested on. However, it was considered that the action mode of the Amyl III on starch granules was different from that of glucoamylase judging from the observation of granules under a scanning electron microscope before and after enzyme reaction, and also from the reaction products. Glucoamylase (GA I) was also isolated and it was purified to an electrophoretically pure state from the extract. It was found that the electron micrographic features of the granules after treatment with the enzymes were quite different. A synergistic effect of Amyl III and GA I was observed for the digestion of raw starch granules.
Trichoderma reesei is known to be one of the organisms capable for producing various types of cellulase in high concentrations. Among these cellulases, the highest catalytic efficiency of endoglucanases II (EGII, EC 3.2.1.4) are considered important for industrial application. The characterization of the EGII is necessary since it is widely used in high-temperature reactions in the industries. In this study, the recombinant EGII protein was expressed in Pichia pastoris and it has a molecular mass of approximately 52 kDa. Recombinant EGII was purified using Ni-NTA affinity chromatography and characterized by SDS-PAGE and western blot analyses. The enzyme activity of recombinant EGII was measured using the Nelson Somogyi method to determine its optimum pH and temperature. The result showed that the maximum EGII expression was achieved after 72 h of culture incubation. The crude enzyme has optimum activity at pH 5.0, resulting in 16.3 U/mL and 14.6 U/mL activity at 40 °C and 50 °C, respectively. While the purified enzyme gave the specific activity of 115.7 U/mg under the optimum condition. Finally, our study demonstrated that recombinant EGII could retain the endoglucanase activity for 89% and 80% at 40 °C and 50 °C, respectively.
Complementary DNAs encoding α-amylases (Amyl I, Amyl III) and glucoamylase (GA I) were cloned from Aspergillus awamori KT-11 and their nucleotide sequences were determined. The sequence of Amyl III that was a raw starch digesting α-amylase was found to consist of a 1,902 bp open reading frame encoding 634 amino acids. The signal peptide of the enzyme was composed of 21 amino acids. On the other hand, the sequence of Amyl I, which cannot act on raw starch, consisted of a 1,500 bp ORF encoding 499 amino acids. The signal peptide of the enzyme was composed of 21 amino acids. The sequence of GA I consisted of a 1,920 bp ORF that encoded 639 amino acids. The signal peptide was composed of 24 amino acids. The amino acid sequence of Amyl III from the N-terminus to the amino acid number 499 showed 63.3% homology with Amyl I. However, the amino acid sequence from the amino acid number 501 to C-terminus, including the rawstarch-affinity site and the TS region rich in threonine and serine, showed 66.9% homology with GA I.
A serious global energy crisis is thought to be originated from the imbalance rapid consumption and the non-renewable nature of the fossil fuels. A potential, yet promising route for diminising this problem might involve rapid conversion of organic waste and biomass into fuels as an alternative. Oil-palm empty fruit bunch (EFB) is the waste from the oil palm plantation which abundant amount of lignocellulosic EFB biomass. EFB biomass was used as raw material of the second generation of bioethanol production. EFB was converted into ethanol through enzymatic hydrolysis and fermentation simultaneously. Cellulose waste was then turned into glucose by enzymatic saccharification and finally fermented into ethanol. The experiment of 20 liter broth resulted in ethanol concentration of about 7.93% (w/w). Conversion of cellulose into glucose was about 60.02%, and conversion of glucose into ethanol was about 88.44%. Following distillation, ethanol of 1970 mL was obtained at a concentration of 63% (v/v).Keywords: EFB, saccharification, fermentation, glucose, ethanol ABSTRAKAdanya krisis energi minyak bumi secara global disebabkan oleh ketimpangan antara konsumsi dan produksi minyak bumi. Guna mengimbangi ketimpangan tersebut, maka dilakukan konversi limbah organik dan biomassa menjadi bahan bakar secara tepat dan cepat. Tandan Kosong Sawit (TKS) merupakan limbah dari perkebunan sawit yang melimpah jumlahnya. Penelitian etanol generasi kedua berbahan baku biomassa lignoselulosa dilakukan melalui proses sakarifikasi selulosa menjadi glukosa secara enzimatis dan fermentasi glukosa menjadi etanol. Berdasarkan hasil yang diperoleh dari 20 liter hidrolisat didapat konsentrasi etanol sebesar 7,93% (b/b). Hasil konversi selulosa menjadi glukosa sebesar 60,02%, sedangkan konversi glukosa menjadi etanol sebesar 88,44%. Setelah dilakukan distilasi didapatkan etanol sebanyak 1970 mL dengan konsentrasi 63% (v/v).Kata kunci: TKS, sakarifikasi, fermentasi, glukosa, etanol
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