Engineering of LadA for enhanced hexadecane oxidation using random- and site-directed mutagenesis

Dong, Yanpeng; Jiang, Yan; Du, Huiqian; Chen, Miao; Ma, Ting; Feng, Lu

doi:10.1007/s00253-012-4035-y

Cited by 23 publications

(21 citation statements)

References 45 publications

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“…A recent study by Dong et al (2012) reported the use of random- and site-directed mutagenesis to enhance the activity of LadA. Three mutants, A102D, L320V, and F146C/N376I from random-mutagenesis, together with six more mutants, A102E, L320A, F146Q/N376I, F146E/N376I, F146R/N376I, and F146N/N376I from site-directed mutagenesis, were obtained and the hydroxylation activity of purified mutants on hexadecane was found to be between 2- and 3.4-fold higher than that of the wild-type LadA, with the activity of F146N/N376I being the highest.…”

Section: Diversity Of Alkane Hydroxylasesmentioning

confidence: 96%

Structural insights into diversity and n-alkane biodegradation mechanisms of alkane hydroxylases

Ji¹,

Mao²,

Wang³

et al. 2013

Front. Microbiol.

175

113

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Environmental microbes utilize four degradation pathways for the oxidation of n-alkanes. Although the enzymes degrading n-alkanes in different microbes may vary, enzymes functioning in the first step in the aerobic degradation of alkanes all belong to the alkane hydroxylases. Alkane hydroxylases are a class of enzymes that insert oxygen atoms derived from molecular oxygen into different sites of the alkane terminus (or termini) depending on the type of enzymes. In this review, we summarize the different types of alkane hydroxylases, their degrading steps, and compare typical enzymes from various classes with regard to their three-dimensional structures, in order to provide insights into how the enzymes mediate their different roles in the degradation of n-alkanes and what determines their different substrate ranges. Through the above analyzes, the degrading mechanisms of enzymes can be elucidated and molecular biological methods can be utilized to expand their catalytic roles in the petrochemical industry or in bioremediation of oil-contaminated environments.

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Section: Diversity Of Alkane Hydroxylasesmentioning

confidence: 96%

Structural insights into diversity and n-alkane biodegradation mechanisms of alkane hydroxylases

Ji¹,

Mao²,

Wang³

et al. 2013

Front. Microbiol.

175

113

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“…Directed evolution has emerged as a ubiquitous technique to enhance the stability, activity, and selectivity of enzymes [28]. Notably, it has not relied on a priori structural and mechanistic information about the proteins [29], more suitable for modifying novel enzymes. Over the past decade, a large number of molecular biological methods for gene mutagenesis has been developed [29].…”

Section: Introductionmentioning

confidence: 99%

“…Notably, it has not relied on a priori structural and mechanistic information about the proteins [29], more suitable for modifying novel enzymes. Over the past decade, a large number of molecular biological methods for gene mutagenesis has been developed [29]. Random mutagenesis, as a powerful tool, has been advocated for modifying various enzyme functions [28, 30].…”

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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“…By screening 7,500 clones of a random mutagenesis library, 3 mutants with 2-fold improved activity were found. The improved variants were further subjected to site specific saturation mutagenesis which resulted in a mutant performing 3.4-fold better than the wild type [67]. The data-driven approach on the other hand, focuses on the mutagenesis of targeted positions with high potential to influence the desired property along with the use of restricted codon degeneracy for the creation of relatively small libraries.…”

Section: Discussionmentioning

confidence: 99%

Engineering Non-Heme Mono- And Dioxygenases for Biocatalysis

Dror

Fishman

2012

Computational and Structural Biotechnology Journal

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Oxygenases are ubiquitous enzymes that catalyze the introduction of one or two oxygen atoms to unreactive chemical compounds. They require reduction equivalents from NADH or NADPH and comprise metal ions, metal ion complexes, or coenzymes in their active site. Thus, for industrial purposes, oxygenases are most commonly employed using whole cell catalysis, to alleviate the need for co-factor regeneration. Biotechnological applications include bioremediation, chiral synthesis, biosensors, fine chemicals, biofuels, pharmaceuticals, food ingredients and polymers. Controlling activity and selectivity of oxygenases is therefore of great importance and of growing interest to the scientific community. This review focuses on protein engineering of non-heme monooxygenases and dioxygenases for generating improved or novel functionalities. Rational mutagenesis based on x-ray structures and sequence alignment, as well as random methods such as directed evolution, have been utilized. It is concluded that knowledge-based protein engineering accompanied with targeted libraries, is most efficient for the design and tuning of biocatalysts towards novel substrates and enhanced catalytic activity while minimizing the screening efforts.

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Engineering of LadA for enhanced hexadecane oxidation using random- and site-directed mutagenesis

Cited by 23 publications

References 45 publications

Structural insights into diversity and n-alkane biodegradation mechanisms of alkane hydroxylases

Structural insights into diversity and n-alkane biodegradation mechanisms of alkane hydroxylases

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

Engineering Non-Heme Mono- And Dioxygenases for Biocatalysis

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