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Purification and Enzymatic Properties of a Difunctional Glycoside Hydrolase from Aspergillus oryzae HML366.

ABSTRACT: In the study, an extracellular enzyme HML CBH1 was purified from the fermentation solution of Aspergillus oryzae HML366, and characterized by biological and molecular analysis. Following the culturing of A. oryzae HML366 under the optimized conditions for enzyme production, an enzyme named HML CBH1 with a molecular weight of 48 kDa was purified using 3000 Da cellulose ultrafiltration column and anion exchange chromatography. The specific activity of the purified enzyme was 9.65 U/mg, and the optimum temperature and pH for the enzyme were 50 and 5.0 °C, respectively. The enzyme was stable at temperatures below 60 °C and pH ranging from 3.0 to 10.0. The partial amino acid sequence of HML CBH1 was analyzed by time-of-flight mass spectrometry, and Mascot and Blast analysis showed that the HML CBH1 sequence was identical to the protein gi:22138643, belonging to the glycoside hydrolase family 7, and had exoglucanase and endoglucanase activity.

SUBMITTER: Qin Y

PROVIDER: S-EPMC7539257 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Purification and Enzymatic Properties of a Difunctional Glycoside Hydrolase from Aspergillus oryzae HML366.

Qin Yongling Y Fu Yue Y Li Qiqian Q Luo Fengfeng F He Haiyan H

Indian journal of microbiology 20200606 4

In the study, an extracellular enzyme HML CBH1 was purified from the fermentation solution of Aspergillus oryzae HML366, and characterized by biological and molecular analysis. Following the culturing of A. oryzae HML366 under the optimized conditions for enzyme production, an enzyme named HML CBH1 with a molecular weight of 48 kDa was purified using 3000 Da cellulose ultrafiltration column and anion exchange chromatography. The specific activity of the purified enzyme was 9.65 U/m ...[more]

PMID: 33087997

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Project description:Aspergillus oryzae spores, when sprinkled onto steamed rice and allowed to propagate, are referred to as rice "koji." Agmatine, a natural polyamine derived from arginine through the action of arginine decarboxylase (ADC), is abundantly produced by solid state-cultivated rice koji of A. oryzae RIB40 under low pH conditions, despite the apparent absence of ADC orthologs in its genome. Mass spectrometry imaging revealed that agmatine was accumulated inside rice koji at low pH conditions, where arginine was distributed. ADC activity was predominantly observed in substrate mycelia and minimally in aerial mycelia. Natural ADC was isolated from solid state-cultivated A. oryzae rice koji containing substrate mycelia, using ammonium sulfate fractionation, ion exchange, and gel-filtration chromatography. The purified protein was subjected to sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE), and the detected peptide band was digested for identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The gene AO090102000327 of strain RIB40 was identified, previously annotated as phosphatidylserine decarboxylase (PSD), and encoded a 483-amino acid peptide. Recombinant protein encoded by AO090102000327 was expressed in Escherichia coli cells cultivated at 20°C, resulting in the detection of 49 kDa and 5 kDa peptides. The protein exhibited pyruvoyl-dependent decarboxylase activity, favoring arginine over ornithine and showing no activity with phosphatidylserine. The gene was designated Ao-adc1. Ao-ADC1 expression in rice koji at pH 4-6 was confirmed through western blotting using the anti-Ao-ADC1 serum. These findings indicate that Ao-adc1 encodes arginine decarboxylase involved in agmatine production.IMPORTANCEGene AO090102000327 in A. oryzae RIB40, previously annotated as a PSD, falls into a distinct clade when examining the phylogenetic distribution of PSDs. Contrary to the initial PSD annotation, our analysis indicates that the protein encoded by AO090102000327 is expressed in the substrate mycelia area of solid state-cultivated A. oryzae rice koji and functions as an arginine decarboxylase (ADC). The clade to which Ao-ADC1 belongs includes three other Ao-ADC1 paralogs (AO090103000445, AO090701000800, and AO090701000802) that presumably encode ADC rather than PSDs. Regarding PSD, AO090012000733 and AO090005001124 were speculated to be nonmitochondrial and mitochondrial PSDs in A. oryzae RIB40, respectively.

Dataset Information

Purification and Enzymatic Properties of a Difunctional Glycoside Hydrolase from Aspergillus oryzae HML366.

Publications

Purification and Enzymatic Properties of a Difunctional Glycoside Hydrolase from <i>Aspergillus oryzae</i> HML366.

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