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Expression of a secretory ?-glucosidase II from Apis cerana indica in Pichia pastoris and its characterization.

ABSTRACT: BACKGROUND: ?-glucosidase (HBGase) plays a key role in hydrolyzing ?-glucosidic linkages. In Apis mellifera, three isoforms of HBGase (I, II and III) have been reported, which differ in their nucleotide composition, encoding amino acid sequences and enzyme kinetics. Recombinant (r)HBGase II from A. cerana indica (rAciHBGase II) was focused upon here due to the fact it is a native and economic honeybee species in Thailand. The data is compared to the two other isoforms, AciHBGase I and III from the same bee species and to the three isoforms (HBGase I, II and III) in different bee species where available. RESULTS: The highest transcript expression level of AciHBGase II was found in larvae and pupae, with lower levels in the eggs of A. cerana indica but it was not found in foragers. The full-length AciHBGase II cDNA, and the predicted amino acid sequence it encodes were 1,740 bp and 579 residues, respectively. The cDNA sequence was 90% identical to that from the HBGase II from the closely related A. cerana japonica (GenBank accession # NM_FJ752630.1). The full length cDNA was directionally cloned into the pPICZ?A expression vector in frame with a (His)(6) encoding C terminal tag using EcoRI and KpnI compatible ends, and transformed into Pichia pastoris. Maximal expression of the rAciHBGase II-(His)(6) protein was induced by 0.5% (v/v) methanol for 96 h and secreted into the culture media. The partially purified enzyme was found to have optimal ?-glucosidase activity at pH 3.5 and 45°C, with > 80% activity between pH 3.5-5.0 and 40-55°C, and was stabile (> 80% activity) at pH 4-8 and at < 25-65°C. The optimal substrate was sucrose. CONCLUSIONS: Like in A. mellifera, there are three isoforms of AciHBGase (I, II and III) that differ in their transcript expression pattern, nucleotide sequences and optimal enzyme conditions and kinetics.

SUBMITTER: Kaewmuangmoon J

PROVIDER: S-EPMC3599193 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Expression of a secretory α-glucosidase II from Apis cerana indica in Pichia pastoris and its characterization.

Kaewmuangmoon Jirattikarn J Kilaso Manlika M Leartsakulpanich Ubolsree U Kimura Kiyoshi K Kimura Atsuo A Chanchao Chanpen C

BMC biotechnology 20130218

<h4>Background</h4>α-glucosidase (HBGase) plays a key role in hydrolyzing α-glucosidic linkages. In Apis mellifera, three isoforms of HBGase (I, II and III) have been reported, which differ in their nucleotide composition, encoding amino acid sequences and enzyme kinetics. Recombinant (r)HBGase II from A. cerana indica (rAciHBGase II) was focused upon here due to the fact it is a native and economic honeybee species in Thailand. The data is compared to the two other isoforms, AciHBGase I and III ...[more]

PMID: 23419073

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Project description:The methylotrophic yeast Pichia pastoris has been utilized for heterologous protein expression for over 30 years. Because P. pastoris secretes few of its own proteins, the exported recombinant protein is the major polypeptide in the extracellular medium, making purification relatively easy. Unfortunately, some recombinant proteins intended for secretion are retained within the cell. A mutant strain isolated in our laboratory, containing a disruption of the BGS13 gene, displayed elevated levels of secretion for a variety of reporter proteins. The Bgs13 peptide (Bgs13p) is similar to the Saccharomyces cerevisiae protein kinase C 1 protein (Pkc1p), but its specific mode of action is currently unclear. To illuminate differences in the secretion mechanism between the wild-type (wt) strain and the bgs13 strain, we determined that the disrupted bgs13 gene expressed a truncated protein that had reduced protein kinase C activity and a different location in the cell, compared to the wt protein. Because the Pkc1p of baker's yeast plays a significant role in cell wall integrity, we investigated the sensitivity of the mutant strain's cell wall to growth antagonists and extraction by dithiothreitol, determining that the bgs13 strain cell wall suffered from inherent structural problems although its porosity was normal. A proteomic investigation of the bgs13 strain secretome and cell wall-extracted peptides demonstrated that, compared to its wt parent, the bgs13 strain also displayed increased release of an array of normally secreted, endogenous proteins, as well as endoplasmic reticulum-resident chaperone proteins, suggesting that Bgs13p helps regulate the unfolded protein response and protein sorting on a global scale.IMPORTANCE The yeast Pichia pastoris is used as a host system for the expression of recombinant proteins. Many of these products, including antibodies, vaccine antigens, and therapeutic proteins such as insulin, are currently on the market or in late stages of development. However, one major weakness is that sometimes these proteins are not secreted from the yeast cell efficiently, which impedes and raises the cost of purification of these vital proteins. Our laboratory has isolated a mutant strain of Pichia pastoris that shows enhanced secretion of many proteins. The mutant produces a modified version of Bgs13p. Our goal is to understand how the change in the Bgs13p function leads to improved secretion. Once the Bgs13p mechanism is illuminated, we should be able to apply this understanding to engineer new P. pastoris strains that efficiently produce and secrete life-saving recombinant proteins, providing medical and economic benefits.

Project description:BACKGROUND: Recently, the increased demand of energy has strongly stimulated the research on the conversion of lignocellulosic biomass into reducing sugars for the subsequent production, and ?-glucosidases have been the focus because of their important roles in a variety fundamental biological processes and the synthesis of useful ?-glucosides. Although the ?-glucosidases of different sources have been investigated, the amount of ?-glucosidases are insufficient for effective conversion of cellulose. The goal of this work was to search for new resources of ?-glucosidases, which was thermostable and with high catalytic efficiency. RESULTS: In this study, a thermostable native ?-glucosidase (nBgl3), which is secreted by the lignocellulose-decomposing fungus Aspergillus fumigatus Z5, was purified to electrophoretic homogeneity. Internal sequences of nBgl3 were obtained by LC-MS/MS, and its encoding gene, bgl3, was cloned based on the peptide sequences obtained from the LC-MS/MS results. bgl3 contains an open reading frame (ORF) of 2622 bp and encodes a protein with a predicted molecular weight of 91.47 kDa; amino acid sequence analysis of the deduced protein indicated that nBgl3 is a member of the glycoside hydrolase family 3. A recombinant ?-glucosidase (rBgl3) was obtained by the functional expression of bgl3 in Pichia pastoris X33. Several biochemical properties of purified nBgl3 and rBgl3 were determined - both enzymes showed optimal activity at pH 6.0 and 60°C, and they were stable for a pH range of 4-7 and a temperature range of 50 to 70°C. Of the substrates tested, nBgl3 and rBgl3 displayed the highest activity toward 4-Nitrophenyl-?-D-glucopyranoside (pNPG), with specific activities of 103.5 ± 7.1 and 101.7 ± 5.2 U mg-1, respectively. However, these enzymes were inactive toward carboxymethyl cellulose, lactose and xylan. CONCLUSIONS: An native ?-glucosidase nBgl3 was purified to electrophoretic homogeneity from the crude extract of A. fumigatus Z5. The gene bgl3 was cloned based on the internal sequences of nBgl3 obtained from the LC-MS/MS results, and the gene bgl3 was expressed in Pichia pastoris X33. The results of various biochemical properties of two enzymes including specific activity, pH stability, thermostability, and kinetic properties (Km and Vmax) indicated that they had no significant differences.

Dataset Information

Expression of a secretory ?-glucosidase II from Apis cerana indica in Pichia pastoris and its characterization.

Publications

Expression of a secretory α-glucosidase II from Apis cerana indica in Pichia pastoris and its characterization.

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