Cloning of a gene encoding thermostable glucoamylase from Chaetomium thermophilum and its expression in Pichia pastoris

Abstract

Aims: Chaetomium thermophilum is a soil‐borne thermophilic fungus whose molecular biology is poorly understood. Only a few genes have been cloned from the Chaetomium genus. This study attempted to clone, to sequence and to express a thermostable glucoamylase gene of C. thermophilum. Methods and Results: First strand cDNA was prepared from total RNA isolated from C. thermophilum and the glucoamylase gene amplified by using PCR. Degenerate primers based on the N‐terminal sequences of the purified glucoamylase according to our previous works and a cDNA fragment encoding the glucoamylase gene was obtained through RT‐PCR. Using RACE‐PCR, full‐length cDNA of glucoamylase gene was cloned from C. thermophilum. The full‐length cDNA of the glucoamylase was 2016 bp and contained a 1797‐bp open reading frame encoding a protein glucoamylase precursor of 599 amino acid residues. The amino‐acid sequence from 31 to 45 corresponded to the N‐terminal sequence of the purified protein. The first 30 amino acids were presumed to be a signal peptide. The alignment results of the putative amino acid sequence showed the catalytic domain of the glucoamylase was high homology with the catalytic domains of the other glucoamylases. The C. thermophilum glucoamylase gene was expressed in Pichia pastoris, and the glucoamylase was secreted into the culture medium by the yeast in a functionally active form. The recombinant glucoamylase purified was a glycoprotein with a size of about 66 kDa, and exhibited optimum catalytic activity at pH 4·5–5·0 and 65°C. The enzyme was stable at 60°C, the enzyme activity kept 80% after 60 min incubation at 70°C. The half‐life was 40 and 10 min under incubation at 80 and 90°C respectively. Conclusions: A new thermostable glucoamylase gene of C. thermophilum was cloned, sequenced, overexpressed successfully in P. pastoris. Significance and Impact of the Study: Because of its thermostability and overexpression, this glucoamylase enzyme offers an interesting potential in saccharification steps in both starch enzymatic conversion and in alcohol production.

A Thermostable Glucoamylase from Bispora sp. MEY-1 with Stability over a Broad pH Range and Significant Starch Hydrolysis Capacity

“…Compared with its counterparts, the most important distinguished property of GLA15 is its excellent thermostability at 70°C. It retained 98% initial activity after incubation at 70°C for 1 h. By contrast, the commercial A. niger glucoamylase only retained 10% activity after incubation at 70°C for 30 min [5]; the T. emersonii glucoamylase only retained stable at 65°C [15]; the glucoamylase from C. thermophilum lost 20% activity after incubation at 70°C for 1 h [17]; and another A. niger glucoamylase with temperature optimum at 75°C was only stable at 40°C [30]. Even though the pH optimum of recombinant GLA15 was 4.5, it retained stable over a broader pH range (2.2–11.0) than glucoamylases from A. niger and Aspergillus niveus (pH 4.0–9.5) [32] and endophytic fungus EF6 (pH 4.0–7.0) [33].…”

“…Thermophilic fungi represent an ideal source for thermostable glucoamylases with high yield. So far several thermophilic glucoamylases have been identified in Talaromyces [15], Thermomyces [16], and Chaetomium [17]. These glucoamylases usually show extremely high temperature optima and thermostability, but the glucose yield from starch degradation is quite low [18].…”

Cloning, expression, and characterization of serine protease from thermophilic fungus Thermoascus aurantiacus var. levisporus

“…The molecular weight variation of the protease expressed in P. pastoris might be a result of N-linked glycosylation. The possible explanation is that in many enzymes, glycosylation is involved in protein rigid structure formation that increases the thermostability (Chen et al, 2007).…”

“…Electrocompetent P. pastoris GS115 cells were prepared as described by Chen et al (2007). A total of 10 g of XbalI-linearized recombinant plasmid pPIC9K/Tapro was transformed into P. pastoris strain GS115 by electroporation using an Eppendorf Electroporator 2510.…”

Advanced Strategies for Improving the Production of Industrial Enzymes in Heterologous Host Systems

“…With the development of genetic engineering, a lot of studies have been carried out to improve the production of industrial enzymes from the molecular level. Genes of some enzymes, such as glucoamylase, amylase, lipase, cellulase and phytase, etc., have been cloned and expressed efficiently in heterologous host systems, such as Saccharomyces cerevisiae; Aspergillus niger and Pichia pastoris [1][2][3][4][5][6][7][8][9][10][11]. In this paper, advanced strategies for improving the production of industrial enzymes were reviewed from the following aspects: the introduction of the strong promoter, increasing the copy number of genes, changing the signal peptide sequence and designing the codon bias ( Figure 1).…”