Improving the thermostability of Geobacillus stearothermophilus xylanase XT6 by directed evolution and site-directed mutagenesis

Zhang, Zhigang; Yi, Zhuolin; Pei, Xiaokun; Wu, Zhong‐Liu

doi:10.1016/j.biortech.2010.07.060

Cited by 97 publications

(39 citation statements)

References 35 publications

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“…6A). Hence, restoring this consensus mutation has been crucial to improve heterologous AAO expression in yeast while enhancing thermostability, which is in excellent agreement with previous reports on ancestral library design by introducing consensus/ancestor mutations to improve the heterologous expression and thermostability of different enzymes (46)(47)(48). Indeed, the discovery of a consensus mutation by focused evolution, rather than using the consensus method based on sequence alignment or an inference phylogenetic analysis for ancestor mutations, highlights the potential of random domain mutagenesis approaches to reveal beneficial consensus/ancestor mutations.…”

Section: Resultssupporting

confidence: 86%

Focused Directed Evolution of Aryl-Alcohol Oxidase in Saccharomyces cerevisiae by Using Chimeric Signal Peptides

Viña‐Gonzalez

González-Pérez

Ferreira

et al. 2015

Appl Environ Microbiol

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c Aryl-alcohol oxidase (AAO) is an extracellular flavoprotein that supplies ligninolytic peroxidases with H 2 O 2 during natural wood decay. With a broad substrate specificity and highly stereoselective reaction mechanism, AAO is an attractive candidate for studies into organic synthesis and synthetic biology, and yet the lack of suitable heterologous expression systems has precluded its engineering by directed evolution. In this study, the native signal sequence of AAO from Pleurotus eryngii was replaced by those of the mating ␣-factor and the K 1 killer toxin, as well as different chimeras of both prepro-leaders in order to drive secretion in Saccharomyces cerevisiae. The secretion of these AAO constructs increased in the following order: prepro␣-AAO > pre␣proK-AAO > preKpro␣-AAO > preproK-AAO. The chimeric pre␣proK-AAO was subjected to focused-directed evolution with the aid of a dual screening assay based on the Fenton reaction. Random mutagenesis and DNA recombination was concentrated on two protein segments (Met[␣1]-Val109 and Phe392-Gln566), and an array of improved variants was identified, among which the FX7 mutant (harboring the H91N mutation) showed a dramatic 96-fold improvement in total activity with secretion levels of 2 mg/liter. Analysis of the N-terminal sequence of the FX7 variant confirmed the correct processing of the pre␣proK hybrid peptide by the KEX2 protease. FX7 showed higher stability in terms of pH and temperature, whereas the pH activity profiles and the kinetic parameters were maintained. The Asn91 lies in the flavin attachment loop motif, and it is a highly conserved residue in all members of the GMC superfamily, except for P. eryngii and P. pulmonarius AAO. The in vitro involution of the enzyme by restoring the consensus ancestor Asn91 promoted AAO expression and stability.A ryl-alcohol oxidase (AAO; EC 1.1.3.7) is a flavoenzyme of the GMC (glucose-methanol-choline) oxidoreductase superfamily, the members of which share a N-terminal FAD-binding domain containing the canonical ADP-binding motif. Secreted by several white-rot fungi, this monomeric flavoprotein plays an essential role in natural lignin degradation (1). Accordingly, AAO oxidizes lignin-derived compounds and aromatic fungal metabolites, releasing H 2 O 2 that is required by ligninolytic peroxidases to attack the plant cell wall (2). Moreover, the H 2 O 2 produced by AAO is an efficient vehicle to generate highly reactive hydroxyl radicals through the Fenton reaction (Fe 2ϩ ϩ H 2 O 2 ¡ OH˙ϩ OH Ϫ ϩ Fe 3ϩ ), such that OH˙can act as a diffusible electron carrier to depolymerize plant polymers. AAO oxidizes a variety of aromatic benzyl (and some aliphatic polyunsaturated) alcohols to the corresponding aldehydes. In addition, AAO participates in the oxidation of aromatic aldehydes to the corresponding acids and also has activity on furfural derivatives (3).The past few years have witnessed an intense effort to discern the basis and mechanism of action underlying AAO catalysis (3-10). The AAO catalytic cycle involves dehydroge...

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Section: Resultssupporting

confidence: 86%

Focused Directed Evolution of Aryl-Alcohol Oxidase in Saccharomyces cerevisiae by Using Chimeric Signal Peptides

Viña‐Gonzalez

González-Pérez

Ferreira

et al. 2015

Appl Environ Microbiol

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“…Random mutagenesis techniques such as errorprone PCR and DNA shuffling can be easily applied for the improvement of enzyme thermostability (8,9,12,28) because these techniques do not require detailed structural information or accurate predictions at the substituted residues (14).…”

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confidence: 99%

Improvement in the Thermostability of d -Psicose 3-Epimerase from Agrobacterium tumefaciens by Random and Site-Directed Mutagenesis

Choi

Yeom

et al. 2011

Appl Environ Microbiol

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The S213C, I33L, and I33L S213C variants of D-psicose 3-epimerase from Agrobacterium tumefaciens, which were obtained by random and site-directed mutagenesis, displayed increases of 2.5, 5, and 7.5°C in the temperature for maximal enzyme activity, increases of 3.3-, 7.2-, and 29.9-fold in the half-life at 50°C, and increases of 3.1, 4.3, and 7.6°C in apparent melting temperature, respectively, compared with the wild-type enzyme. Molecular modeling suggests that the improvement in thermostability in these variants may have resulted from increased putative hydrogen bonds and formation of new aromatic stacking interactions. The immobilized wild-type enzyme with and without borate maintained activity for 8 days at a conversion yield of 70% (350 g/liter psicose) and for 16 days at a conversion yield of 30% (150 g/liter psicose), respectively. After 8 or 16 days, the enzyme activity gradually decreased, and the conversion yields with and without borate were reduced to 22 and 9.6%, respectively, at 30 days. In contrast, the activities of the immobilized I33L S213C variant with and without borate did not decrease during the operation time of 30 days. These results suggest that the I33L S213C variant may be useful as an industrial producer of D-psicose.D-Psicose (D-allulose), a carbon-3 epimer of D-fructose, is present in small quantities as a nonfermentable constituent of cane molasses (1), as a sugar moiety of the nucleoside antibiotic psicofuranine (4), and as a free sugar in wheat (21) and Itea plants (5). This rare sugar provides suitable sweetness, smooth texture, favorable mouthfeel, and long-time storage stability in food products (23). Psicose is considered to be a potential reduced energy sweetener because the sugar suppresses hepatic lipogenic enzyme activity (18) and does not contribute to calorie production (19).Biological production of psicose from fructose has been studied using D-psicose 3-epimerase from Agrobacterium tumefaciens (10, 13, 16) and D-tagatose 3-epimerases from Pseudomonas cichorii (7,24) and Rhodobacter sphaeroides (26). D-Psicose 3-epimerase from A. tumefaciens has been more effective than the D-tagatose 3-epimerases for the production of psicose. However, D-psicose 3-epimerase is inefficient for the industrial production of psicose because of its short half-life of 63 min at 50°C (10). Thus, improvement in the thermostability of A. tumefaciens D-psicose 3-epimerase is essential for the industrial production of psicose.Random mutagenesis and rational protein design are typical tools for improving enzyme thermostability in the protein engineering field. Random mutagenesis techniques such as errorprone PCR and DNA shuffling can be easily applied for the improvement of enzyme thermostability (8, 9, 12, 28) because these techniques do not require detailed structural information or accurate predictions at the substituted residues (14).In the present study, thermostable variants of D-psicose 3-epimerase from A. tumefaciens were obtained by random and site-directed mutagenesis. The temperature fo...

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“…The mean and standard deviation of the absorbance values from 96 samples were 1.74 and 0.18, respectively (Fig. 3), making the coefficient of variance (CV) of the screening to be 10.3% (CV = [standard deviation/(mean) × 100%]), which indicates lower variability of the high-throughput screening systems [25,26]. The expected false positives exhibiting higher than 1.5 times the mean value would be close to zero in a typical library of 10 4 -10 5 colonies according to statistical calculations [26].…”

Section: Application Of Cnpf In a High-throughput Mannermentioning

confidence: 99%

A practical high-throughput screening system for feruloyl esterases: Substrate design and evaluation

Zhang¹,

Ma²,

Pei³

et al. 2012

Journal of Molecular Catalysis B: Enzymatic

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Improving the thermostability of Geobacillus stearothermophilus xylanase XT6 by directed evolution and site-directed mutagenesis

Cited by 97 publications

References 35 publications

Focused Directed Evolution of Aryl-Alcohol Oxidase in Saccharomyces cerevisiae by Using Chimeric Signal Peptides

Focused Directed Evolution of Aryl-Alcohol Oxidase in Saccharomyces cerevisiae by Using Chimeric Signal Peptides

Improvement in the Thermostability of d -Psicose 3-Epimerase from Agrobacterium tumefaciens by Random and Site-Directed Mutagenesis

A practical high-throughput screening system for feruloyl esterases: Substrate design and evaluation

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