Cloning, expression and characterization of a thermotolerant endoglucanase from Syncephalastrum racemosum (BCC18080) in Pichia pastoris

Wonganu, Benjamaporn; Pootanakit, Kusol; Boonyapakron, Katewadee; Champreda, Verawat; Tanapongpipat, Sutipa; Eurwilaichitr, Lily

doi:10.1016/j.pep.2007.10.022

Cited by 46 publications

(12 citation statements)

References 23 publications

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“…Brown rot fungi are considered important microbes that maintain soil fertility, and G. trabeum is widely reported brown rot wood-decaying fungus. Many endoglucanses can be separated from their signal peptides for protein expression [32]. It was believed that G. trabeum relies on the Fenton degrading system, but the present study, as well as the Wndings by Cohen et al [5], demonstrated that G. trabeum also has the ability to express cellulolytic enzymes.…”

Section: Discussionmentioning

confidence: 41%

Characteristics of bifunctional acidic endoglucanase (Cel5B) from Gloeophyllum trabeum

Kim

Lee

Patel

et al. 2012

Journal of Industrial Microbiology and Biotechnology

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The endoglucanase (Cel5B) from the filamentous fungus Gloeophyllum trabeum was cloned and expressed without a signal peptide, and alanine residue 22 converted to glutamine in Pichia pastoris GS115. The DNA sequence of Cel5B had an open reading frame of 1,077 bp, encoding a protein of 359 amino acid residues with a molecular weight of 47 kDa. On the basis of sequence similarity, Cel5B displayed active site residues at Glu-175 and Glu-287. Both residues lost full hydrolytic activity when replaced with alanine through point mutation. The purified recombinant Cel5B showed very high specific activity, about 80- to 1,000-fold and 13- to 70-fold in comparison with other endoglucanases and cellobiohydrolase, on carboxymethylcellulose and filter paper, respectively, at pH 3.5 and 55°C. Cel5B displayed bifunctional characteristics under acidic conditions. The kinetic properties of the enzyme determined using a Lineweaver-Burk plot indicated that Cel5B is a catalytically efficient cellulolytic enzyme. These results suggest that Cel5B has high bifunctional endo- and exoglucanase activity under acidic conditions and is a good candidate for bioconversion of lignocellulose.

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Section: Discussionmentioning

confidence: 41%

Characteristics of bifunctional acidic endoglucanase (Cel5B) from Gloeophyllum trabeum

Kim

Lee

Patel

et al. 2012

Journal of Industrial Microbiology and Biotechnology

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“…3B), suggesting that the recombinant Cel5A was not N-linked glycosylated. It is likely that the recombinant Cel5A was heavy O-linked glycosylated, just as the reported endoglucanase in Syncephalastrum racemosum [32]. Effects of temperature and pH on enzyme activity.…”

Section: Characterization Of the Two Endoglucanases Molecular Masses mentioning

confidence: 82%

Molecular cloning and characterization of two major endoglucanases from Penicillium decumbens

Wei

Qin²,

Qu³

2010

Journal of Microbiology and Biotechnology

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Two major endoglucanase genes (cel7B and cel5A) were cloned from Penicillium decumbens 114-2 using the method of modified thermal asymmetric interlaced polymerase chain reaction (TAIL−PCR). The result of Southern blotting suggested that P. decumbens has a single copy of the cel5A gene and a single copy of the cel7B gene in its chromosomal DNA. The expression levels of cel5A and cel7B were determined by means of real-time quantitative PCR, suggesting that the two genes were coordinately expressed, and repressed by glucose and induced by cellulose. Both endoglucanase genes were expressed in Saccharomyces cerevisiae and the recombinant proteins were purified. The recombinant Cel7B and Cel5A were both optimally active at 60 o C and pH 4.0. The recombinant Cel7B showed more than 8-fold, 30fold, and 5-fold higher enzyme activities toward carboxymethyl cellulose, barley β-glucan, and PASC, respectively, in comparison with that of Cel5A. However, their activities toward pNPC and Avicel showed minor differences. The results suggested that Cel7B is a strict endoglucanase, whereas Cel5A showed processivity because of its relative higher ability to hydrolyze the crystal cellulose.

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“…This result demonstrated that the α-factor secretion signal sequence adopted from the yeast host could enhance the secretion for recombinant protein. Furthermore, although the signal sequence of Syncephalastrum racemosum (BCC18080) endoglucanase was recognized and properly processed by P. pastoris, the endoglucanase was truncated; a recombinant vector carrying the α-factor secretion signal sequence could secrete the full-length endoglucanase protein [37]. In our study, we constructed two vectors for improving recombinant endoglucanase secretion and expression.…”

Section: Discussionmentioning

confidence: 99%

Cloning and Heterologous Expression of a Novel Endoglucanase Gene egVIII from Trichoderma viride in Saccharomyces cerevisiae

Huang

Yang

Liu

et al. 2009

Appl Biochem Biotechnol

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Endoglucanase is a major cellulolytic enzyme produced by the fungus Trichoderma viride. The 1,317 bp cDNA of endoglucanase gene egVIII was cloned from T. viride AS3.3711, encoding a 438 amino acid protein with a calculated molecular mass of 46.86 kDa and isoelectric point of 4.32. Sequence analysis suggested that EGVIII belonged to the glycosyl hydrolase family 5. The N-terminal region of EGVIII contains a signal peptide sequence of 19 amino acid residues, indicating that it is an extracellular enzyme. Transcription of the egVIII gene in T. viride AS3.3711 can be induced by carboxymethyl cellulose sodium (CMC-Na), sucrose, microcrystalline cellulose, and corn stalk, and inhibited by glucose and fructose. The alpha-mating factor signal can effectively enhance the secretion of the recombinant EGVIII in Saccharomyces cerevisiae, as demonstrated by the enzymatic activity of recombinant yeast IpYEMalpha-xegVIII in the supernatant, which was 0.86 times higher than that of the IpYES2-egVIII. Recombinant endoglucanase EGVIII showed optimal activity at a temperature of 60 degrees C and pH of 6.0. It was stable when incubated from 35 degrees C to 70 degrees C for 1 h. The enzymatic activity of recombinant EGVIII was stable at a pH 3.0 to 7.5 at 50 degrees C and reached the highest level at 0.174U when activated by 75 mM of Zn(2+). The Michaelis-Menten constant (Km) and Kcat values for CMC-Na and cellotriose hydrolysis were 3.82 mg/ml, 9.56 s(-1) and 1.75 mg/ml, 7.08 s(-1), respectively. Transgenic yeast strain IpYEMalpha-xegVIII might be useful for renewable fuels industries.

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Cloning, expression and characterization of a thermotolerant endoglucanase from Syncephalastrum racemosum (BCC18080) in Pichia pastoris

Cited by 46 publications

References 23 publications

Characteristics of bifunctional acidic endoglucanase (Cel5B) from Gloeophyllum trabeum

Characteristics of bifunctional acidic endoglucanase (Cel5B) from Gloeophyllum trabeum

Molecular cloning and characterization of two major endoglucanases from Penicillium decumbens

Cloning and Heterologous Expression of a Novel Endoglucanase Gene egVIII from Trichoderma viride in Saccharomyces cerevisiae

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