Enhanced thermostability and tolerance of high substrate concentration of an esterase by directed evolution

Kim, Ji-Heui; Choi, Gi-Sub; Kim, Seungbum; Kim, Won‐Ho; Lee, Jin Young; Ryu, Yeon‐Woo; Kim, Geun-Joong

doi:10.1016/j.molcatb.2003.11.010

Cited by 39 publications

(13 citation statements)

References 26 publications

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“…Thus, we sought to use directed evolution to improve its thermostability. Directed evolution has become a powerful tool for improving enzyme function and has been successfully used to improve the thermal stability of many different enzymes (9,15,19,27). Here we report the directed evolution of a highly thermostable PTDH mutant for NAD(P)H regeneration.…”

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Directed Evolution of a Thermostable Phosphite Dehydrogenase for NAD(P)H Regeneration

Johannes

Woodyer²,

Zhao

2005

Appl Environ Microbiol

142

106

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NAD(P)H-dependent oxidoreductases are valuable tools for synthesis of chiral compounds. The expense of the cofactors, however, requires in situ cofactor regeneration for preparative applications. We have attempted to develop an enzymatic system based on phosphite dehydrogenase (PTDH) from Pseudomonas stutzeri to regenerate the reduced nicotinamide cofactors NADH and NADPH. Here we report the use of directed evolution to address one of the main limitations with the wild-type PTDH enzyme, its low stability. After three rounds of random mutagenesis and high-throughput screening, 12 thermostabilizing amino acid substitutions were identified. These 12 mutations were combined by site-directed mutagenesis, resulting in a mutant whose T 50 is 20°C higher and half-life of thermal inactivation at 45°C is >7,000-fold greater than that of the parent PTDH. The engineered PTDH has a half-life at 50°C that is 2.4-fold greater than the Candida boidinii formate dehydrogenase, an enzyme widely used for NADH regeneration. In addition, its catalytic efficiency is slightly higher than that of the parent PTDH. Various mechanisms of thermostabilization were identified using molecular modeling. The improved stability and effectiveness of the final mutant were shown using the industrially important bioconversion of trimethylpyruvate to L-tert-leucine. The engineered PTDH will be useful in NAD(P)H regeneration for industrial biocatalysis.The potential of enzyme-catalyzed transformations in the pharmaceutical and fine-chemical industries has long been considered to have great promise (7,10,23). Despite the development of rapid and efficient methods for creating designer biocatalysts, inherent limitations in the chemistry that these enzymes perform still persist. The majority of biocatalysts used in industry are hydrolytic in action (ϳ65%) and perform rather simple chemistry (4). More complicated enzymatic reactions often require one or more costly cofactors, which, when added in stoichiometric quantities, make the process not economically feasible. Oxidoreductases, for example, catalyze a myriad of regio-, chemo-, and stereospecific reactions on a variety of functional groups, but often require either NADH or NADPH as a cofactor (8, 11).Various in situ regeneration methods have been developed to allow the use of catalytic quantities of NAD(P) ϩ and NAD(P)H (20). The most successful and widely used enzymatic NADH regeneration system is based on the Candida boidinii formate dehydrogenase (FDH) (1). We have attempted to develop an NADH regeneration process using the enzyme phosphite dehydrogenase (PTDH) from Pseudomonas stutzeri (2). This enzyme catalyzes the nearly irreversible oxidation of phosphite to phosphate with the concomitant reduction of NAD ϩ to NADH. The kinetic and practical advantages of using PTDH for NADH regeneration have been recently reported (22).In addition to NADH regeneration, the enzyme PTDH also has considerable potential in regenerating NADPH. The cost of NADPH is significantly higher than that of NADH, and no widel...

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confidence: 99%

Directed Evolution of a Thermostable Phosphite Dehydrogenase for NAD(P)H Regeneration

Johannes

Woodyer²,

Zhao

2005

Appl Environ Microbiol

142

106

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“…Combining rational engineering principles with an evolutionary approach has proven particularly powerful: small focused libraries are synthesised, guided by structure-function information, and then screened/ selected for properties of interest 10 . Such strategies have been used to alter properties such as stereospecificity, expression level 11 and Michaelis constants (K M ) 12 and to overcome functional constraints, such as inhibition by substrates and/or products leading to improved reaction yields 13 .…”

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Hacking nature: genetic tools for reprograming enzymes

2017

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Enzymes have many modern industrial applications, from biomass decomposition in the production of biofuels to highly stereospecific biotransformations in pharmaceutical manufacture. The capacity to find or engineer enzymes with activities pertinent to specific applications has been essential for the growth of a multibillion dollar enzyme industry. Over the course of the past 50-60 years our capacity to address this issue has become increasingly sophisticated, supported by innumerable advances, from early discoveries such as the co-linearity of DNA and protein sequence 1 to modern computational technologies for enzyme design. The design of enzyme function is an exciting nexus of fundamental biochemical understanding and applied engineering. Herein, we will cover some of the methods used in discovery and design, including some 'next generation' tools.Traditionally, enzymes with useful biochemical properties have been sourced from nature, tapping into the natural diversity generated by evolution. Where known physiological functions are useful in an industrial setting, it is relatively simple to match an enzyme to an application (e.g. amylase-mediated glucose production from starch). Where novel functions are required, enrichment culturing of microbes can be used: for example, the recent isolation of bacteria capable of using nylon intermediates as a nitrogen source with potential utility in nylon manufacture (Figure 1) 3 . Non-culture based methods can also be applied to enzyme discovery, for example by exploiting the explosion of genetic information that followed the 'omics' revolution. Driven by technological advances in DNA sequencing, and computational power, this has provided an enormous resource, accessible by bioinformatic analysis, and leading to the discovery of enzymes with industrial applications, such as novel imine reductases for asymmetric organic synthesis 4 .Methods have also been developed to move beyond the repertoire of enzymes currently known in nature. One of the most prevalent approaches has been to use atomic information about structure and function to rationally redesign enzymes, often to expand or alter substrate range, change stereospecificity or alter physical however, the catalytic properties of such synthetic and semisynthetic enzymes can be improved by direction evolution and related methodologies 21,22 .In the approaches considered above, enzyme engineers have been content to explore the chemical space provided by the 20 canonical

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“…The eluted fragments were cloned into the BamHI site of pD3-P2 and transformed into E. coli XL1-Blue. The transformed cells were then cultured at 37°C in LB agar medium for 20 h. To screen for clones with esterase activity, activity staining was conducted using an overlaid soft agar (0.8%) containing Į-naphthyl acetate (45 ȝg/ml) and fast blue RR (45 ȝg/ml) (Kim et al, 2004). These clones were further assayed for final selection under oxidation-sensitive open conditions with cell lysate.…”

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confidence: 99%

“…The putative active site and catalytic triad were indicated by the box and underlines, respectively. was rapidly (<10 min) visualized as a deep brown color (Kim et al, 2004).…”

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Screening and identification of a novel esterase EstPE from a metagenomic DNA library

2011

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Esterases represent a large family of hydrolases with broad substrate specificity and functional sequence space. Although many attempts to screen new esterases have been conducted, there have been few reports conducted to discriminate unique enzymes from typical ones based on novel structure and function. In this study, we discovered an esterase and a novel family through a successive assay of whole cells and crude lysates (oxidative open condition). The screened putative esterases from the metagenomic DNA of salted shrimp consisted of 753 bp encoding 27 kDa of polypeptide, namely PE esterase. Sequence analyses revealed that an identical gene was reported from whole genome sequencing of Stenotrophomonas maltophilia K279a. However, its biochemical and phylogenetic characteristics have not yet been evaluated. PE esterase was overexpressed only by the MBP fusion state in E. coli and was easily purified using an affinity column. This enzyme showed a typical spectrum of substrate specificity and possessed the consensus motifs, Ser-Asp-His and GXSXG, which are essential for most esterase/lipase superfamilies. Interestingly, the entire organization of the ORF and consensus sequence around the active site were distinct from the related enzymes, and its structure could be affected by a reducing agent, DTT.

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Enhanced thermostability and tolerance of high substrate concentration of an esterase by directed evolution

Cited by 39 publications

References 26 publications

Directed Evolution of a Thermostable Phosphite Dehydrogenase for NAD(P)H Regeneration

Directed Evolution of a Thermostable Phosphite Dehydrogenase for NAD(P)H Regeneration

Hacking nature: genetic tools for reprograming enzymes

Screening and identification of a novel esterase EstPE from a metagenomic DNA library

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