A Variant of<i>Orpinomyces joyonii</i>1,3-1,4-β-Glucanase with Increased Thermal Stability Obtained by Random Mutagenesis and Screening

Kim, Suk-Am; Cheng, K.-J.; Liu, Jinhao

doi:10.1271/bbb.66.171

Cited by 6 publications

(3 citation statements)

References 7 publications

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“…In the last few decades, directed evolution has become a very important method to improve enzyme properties. Many research studies have been concerned with improving thermostability, acid tolerance or the catalytic efficiency of β‐1,3‐1,4‐glucanases by directed evolution . For instance, by using this technology, the T m of β‐1,3‐1,4‐glucanases from Orpinomyces joyonii increased 14 °C, the T m of β‐1,3‐1,4‐glucanases from Bacillus amyloliquefaciens increased 5.5 °C, the optimal pH of β‐1,3‐1,4‐glucanases from Paecilomyces thermophila J18 decreased by 2 …”

Section: Introductionmentioning

confidence: 99%

“…Many research studies have been concerned with improving thermostability, acid tolerance or the catalytic efficiency of -1,3-1,4-glucanases by directed evolution. 14 -21 For instance, by using this technology, the T m of -1,3-1,4-glucanases from Orpinomyces joyonii increased 14 ∘ C, 14 the T m of -1,3-1,4-glucanases from Bacillus amyloliquefaciens increased 5.5 ∘ C, 19 the optimal pH of -1,3-1,4-glucanases from Paecilomyces thermophila J18 decreased by 2. 21 In previous work in our laboratory, a -1,3-1,4-glucanase gene from Bacillus altitudinis YC-9 was isolated, characterized and expressed in Escherichia coli.…”

Section: Introductionmentioning

confidence: 99%

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Engineering of a thermostable β‐1,3‐1,4‐glucanase from Bacillus altitudinisYC‐9 to improve its catalytic efficiency

Mao

Gao

et al. 2015

J Sci Food Agric

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The three-dimensional structure predicted by SWISS-MODEL of the mutated enzyme Glu-3060 showed that the substitution of three amino acids had an effect on the catalytic activity, stability and optimal pH of the enzyme, through changing the charge properties or electron density, forming secondary keys, the acidity of the amino acids and the side chain group. The sum effects of all the factors were increased activity of the mutated enzyme and decreased optimal pH, while the same thermostability was maintained, thereby increasing the suitability of the enzyme for industrial use.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Engineering of a thermostable β‐1,3‐1,4‐glucanase from Bacillus altitudinisYC‐9 to improve its catalytic efficiency

Mao

Gao

et al. 2015

J Sci Food Agric

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“…The fusion enzyme in this study retained more than 50% of activity at 80°C for 30 min (Fig. 4) which is one of the highest levels of thermostability among the reported b-1,3-1,4-glucanases (Chen et al 1997;Kim et al 2002;Teng et al 2006;Yang et al 2008;Qiao et al 2009;Luo et al 2010). Improved thermal stability has been attributed to protein folding and the formation of disulfide bonds (Han et al 2009).…”

Section: Discussionmentioning

confidence: 61%

Construction and characterization of a fusion β-1,3-1,4-glucanase to improve hydrolytic activity and thermostability

Sun

Wang

et al. 2011

Biotechnol Lett

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A new fusion gene (Bgl-licMB), encoding β-1,3-1,4-glucanase both from Bacillus amyloliquefaciens (Bgl) and Clostridium thermocellum (licMB), was constructed via end-to-end fusion and expressed in Escherichia coli to improve hydrolytic activity and thermostability of β-1,3-1,4-glucanase. The results of enzymatic properties showed that the catalytic efficiency (K(cat)/K(m)) of the fusion enzyme for oat β-glucan was 2.7 and 20-fold higher than that of the parental Bgl and licMB, respectively, and that the fusion enzyme can retain more than 50% of activity following incubation at 80°C for 30 min, whereas the residual activities of Bgl and licMB were both less than 30%. These properties make this particular β-1,3-1,4-glucanase a good candidate for application in brewing and animal-feed industries.

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Improvement of enzyme activity of β-1,3-1,4-glucanase from Paenibacillus sp. X4 by error-prone PCR and structural insights of mutated residues

Baek

Lee

et al. 2017

Appl Microbiol Biotechnol

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β-1,3-1,4-Glucanase (BGlc8H) from Paenibacillus sp. X4 was mutated by error-prone PCR or truncated using termination primers to improve its enzyme properties. The crystal structure of BGlc8H was determined at a resolution of 1.8 Å to study the possible roles of mutated residues and truncated regions of the enzyme. In mutation experiments, three clones of EP 2-6, 2-10, and 5-28 were finally selected that exhibited higher specific activities than the wild type when measured using their crude extracts. Enzyme variants of BG, BG, and BG were mutated at two, two, and six amino acid residues, respectively. These enzymes were purified homogeneously by Hi-Trap Q and CHT-II chromatography. Specific activity of BG was 2.11-fold higher than that of wild-type BG, whereas those of BG and BG were 0.93- and 1.19-fold that of the wild type, respectively. The optimum pH values and temperatures of the variants were nearly the same as those of BG (pH 5.0 and 40 °C, respectively). However, the half-life of the enzyme activity and catalytic efficiency (k /K) of BG were 1.92- and 2.12-fold greater than those of BG at 40 °C, respectively. The catalytic efficiency of BG increased to 3.09-fold that of BG at 60 °C. These increases in the thermostability and catalytic efficiency of BG might be useful for the hydrolysis of β-glucans to produce fermentable sugars. Of the six mutated residues of BG, five residues were present in mature BGlc8H protein, and two of them were located in the core scaffold of BGlc8H and the remaining three residues were in the substrate-binding pocket forming loop regions. In truncation experiments, three forms of C-terminal truncated BGlc8H were made, which comprised 360, 286, and 215 amino acid residues instead of the 409 residues of the wild type. No enzyme activity was observed for these truncated enzymes, suggesting the complete scaffold of the α/α-double-barrel structure is essential for enzyme activity.

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A Variant ofOrpinomyces joyonii1,3-1,4-β-Glucanase with Increased Thermal Stability Obtained by Random Mutagenesis and Screening

Cited by 6 publications

References 7 publications

Engineering of a thermostable β‐1,3‐1,4‐glucanase from Bacillus altitudinisYC‐9 to improve its catalytic efficiency

Engineering of a thermostable β‐1,3‐1,4‐glucanase from Bacillus altitudinisYC‐9 to improve its catalytic efficiency

Construction and characterization of a fusion β-1,3-1,4-glucanase to improve hydrolytic activity and thermostability

Improvement of enzyme activity of β-1,3-1,4-glucanase from Paenibacillus sp. X4 by error-prone PCR and structural insights of mutated residues

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