Enhancement of the thermostability of a recombinant β-agarase, AgaB, from Zobellia galactanivorans by random mutagenesis

Jang, Min-Kyung; Lee, Seung Woo; Lee, Dong‐Geun; Kim, Nam‐Young; Yu, Ki Hwan; Jang, Hye Ji; Kim, Suhkman; Kim, Andre; Lee, Sang-Hyeon

doi:10.1007/s10529-010-0237-5

Cited by 28 publications

(22 citation statements)

References 15 publications

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“…Jang et al created a mutant β-agarase from Zobellia galactanivorans that had enhanced thermostability by random mutagenesis. The melting temperature (Tm) was increased by 5.2 °C over that of the wild-type enzyme (54.6 °C) [31]. Yu et al enhanced the thermostability of a Rhizopus chinensis lipase by two rounds of error-prone polymerase chain reaction (PCR) and two rounds of DNA shuffling.…”

Section: Introductionmentioning

confidence: 99%

Directed evolution and secretory expression of a pyrethroid-hydrolyzing esterase with enhanced catalytic activity and thermostability

et al. 2017

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BackgroundPyrethroids are potentially harmful to human health and ecosystems. It is necessary to develop some efficient strategies to degrade pyrethroid residues. Biodegradation is generally considered as a safe, efficient, and inexpensive way to eliminate environmental contaminants. To date, although several pyrethroid-hydrolyzing esterases have been cloned, there has been no report about a pyrethroid hydrolase with high hydrolytic activity, good stability, and high productivity, indispensable enzymatic properties in practical biodegradation. Almost all pyrethroid hydrolases are intracellular enzymes, which require complex extraction protocols and present issues in terms of easy inactivation and low production.ResultsIn this study, random mutagenesis was performed on one pyrethroid-hydrolyzing esterase, Sys410, to enhance its activity and thermostability. Two beneficial mutations, A171V and D256N, were obtained by random mutagenesis and gave rise to the mutant M2. The mutant displayed ~1.5-fold improvement in the kcat/Km value and 2.46-fold higher catalytic activity. The optimal temperature was 10 °C higher than that of the wild-type enzyme (55 °C). The half-life at 40–65 °C was 3.3–310 times longer. It was surprising that M2 has a half-life of 12 h at 70 °C while Sys410 was completely inactivated at 70 °C. In addition, the desired gene was extracellularly expressed in a Pichia pastoris host system. The soluble expression level reached up to 689.7 mg/L. Remarkably, the enzyme could efficiently degrade various pyrethroids at moderate temperature for 15 min, exceeding a hydrolysis rate of 98%, which is the highest value ever reported.ConclusionsThis is the first report about random mutagenesis and secretory expression of pyrethroid-hydrolyzing esterase with high-level productivity and purity in P. pastoris. Broad substrate specificity, enhanced activity and thermostability make M2 an ideal candidate for the biodegradation of pyrethroid residues.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0698-5) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Directed evolution and secretory expression of a pyrethroid-hydrolyzing esterase with enhanced catalytic activity and thermostability

et al. 2017

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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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“…However, mainly due to their low efficiency in agarose degradation, natural agarases are normally unsatisfactory in industrial preparation of agarose oligosaccharides. To improve the enzyme properties, two wild-type agarases were modified by random mutations via error-prone PCR or DNA shuffling (Jang et al, 2010;Shi et al, 2008). Although these two studies have succeeded in increasing the thermostabilities of agarases, the enzyme activities and other properties have been poorly improved (Lee et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

Deletion of a non-catalytic region increases the enzymatic activity of a β-agarase from Flammeovirga sp. MY04

Han

Liu

et al. 2015

J. Ocean Univ. China

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A Glycoside hydrolase (GH) typically contains one catalytic module and varied non-catalytic regions (NCRs). However, effects of the NCRs to the catalytic modules remain mostly unclear except the carbohydrate-binding modules (CBMs). AgaG4 is a GH16 endo-β-agarase of the agarolytic marine bacterium Flammeovirga sp. MY04. The enzyme consists of an extra sugar-binding peptide within the catalytic module, with no predictable CBMs but function-unknown sequences in the NCR, which is a new characteristic of agarase sequences. In this study, we deleted the NCR sequence, a 140-amino acid peptide at the C-terminus and expressed the truncated gene, agaG4-T140, in Escherichia coli. After purification and refolding, the truncated agarase rAgaG4-T140 retained the same catalytic temperature and pH value as rAgaG4. Using combined fluorescent labeling, HPLC and MS/MS techniques, we identified the end-products of agarose degradation by rAgaG4-T140 as neoagarotetraose and neoagarohexaose, with a final molar ratio of 1.53:1 and a conversion ratio of approximately 70%, which were similar to those of rAgaG4. However, the truncated agarase rAgaG4-T140 markedly decreased in protein solubility by 15 times and increased in enzymatic activities by 35 times. The oligosaccharide production of rAgaG4-T140 was approximately 25 times the weight of that produced by equimolar rAgaG4. This study provides some insights into the influences of NCR on the biochemical characteristics of agarase AgaG4 and implies some new strategies to improve the properties of a GH enzyme.

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Enhancement of the thermostability of a recombinant β-agarase, AgaB, from Zobellia galactanivorans by random mutagenesis

Cited by 28 publications

References 15 publications

Directed evolution and secretory expression of a pyrethroid-hydrolyzing esterase with enhanced catalytic activity and thermostability

Directed evolution and secretory expression of a pyrethroid-hydrolyzing esterase with enhanced catalytic activity and thermostability

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

Deletion of a non-catalytic region increases the enzymatic activity of a β-agarase from Flammeovirga sp. MY04

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