Increased Rigidity of Domain Structures Enhances the Stability of a Mutant Enzyme Created by Directed Evolution†

Biochemistry ◽  
2003 ◽  
Vol 42 (49) ◽  
pp. 14469-14475 ◽  
Author(s):  
Jun Hoseki ◽  
Akihiro Okamoto ◽  
Naoki Takada ◽  
Atsushi Suenaga ◽  
Noriyuki Futatsugi ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Mutant Enzyme ◽  
Domain Structures ◽  
The Stability

scholarly journals Directed Evolution and Immobilization of a Novel Lipase LIP 906

2020 ◽  
Author(s):  
Shuang Dai ◽  
He Li
Keyword(s):  
Enzyme Activity ◽  
Directed Evolution ◽  
Early Stage ◽  
Temperature Stability ◽  
Mutant Enzyme ◽  
Random Mutation ◽  
Optimum Reaction ◽  
Pcr Technology ◽  
Solid Foundation ◽  
The Stability

Abstract Background: The object of this experimental study is a new lipase screened from metagenomic libraries in the early stage of the laboratory and named it LIP 906. In order to improve the stability of the enzyme and develop and apply it as soon as possible, experiments use directed evolution and immobilization.Results: A random mutation library was constructed by error-prone PCR technology, and finally a mutant lipase LIP 5-D with improved enzyme activity was screened out and then immobilized. Compared with the wild-type lipase LIP 906, the enzyme activity of the mutant enzyme LIP 5-D increased 4 times; the optimum reaction temperature was increased by 4 °C by mutation and 3 ℃ by immobilization; and the optimum reaction pH is changed from 7.8 to 7.5; temperature stability and pH stability has been improved. The mutant enzyme LIP 5-D can maintain a relative enzyme activity of about 70% at a temperature below 65 °C for 2 hours, and can also maintain a relative enzyme activity of about 60% at different pH 3 -10.Conclusions: Error-prone PCR and immobilization improved the catalytic activity and stability of the enzyme, and promoted its development and application in many industries. The research on the properties and modification of the new lipase LIP906 provides a solid foundation for my next innovative research in application and environmental protection.

scholarly journals Directed evolution and immobilization of new lipase Lip 906

2021 ◽  
Vol 233 ◽  
pp. 02030
Author(s):  
Shuang Dai ◽  
Shan Liu ◽  
Yun Jeonyun ◽  
Xiong Xiao ◽  
Zujun Deng ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Metagenomic Library ◽  
Temperature Stability ◽  
Relative Activity ◽  
Mutant Enzyme ◽  
Wild Type ◽  
Random Mutation ◽  
Optimal Reaction Temperature ◽  
The Stability ◽  
Optimal Reaction

In this experimental study, a new lipase named Lip 906 was screened out from a metagenomic library in the laboratory. To improve the stability of the enzyme and develop and apply it as soon as possible, we adopted directed evolution and immobilization methods. A random mutation library was constructed by error-prone PCR and finally, a mutant lipase Lip 5-D with increased enzyme activity was screened out and immobilized. The activity of the mutant enzyme Lip 5-D was improved by 4 times compared with the wild-type lipase Lip 906. The optimal reaction temperature rose by 4 °C, and by 3 °C after immobilization. The optimal reaction pH increased from 7.8 to 7.5. Both temperature stability and pH stability were improved. The mutant enzyme Lip 5-D can maintain about 70% of the relative activity after incubation at 65 °C for 2 h, and it can keep 60% at pH 3-10. Error-prone PCR and immobilization improve the catalytic activity and stability of the enzyme, and promote its development and application in many industries.

scholarly journals Engineering Improves Enzymatic Synthesis of L-Tryptophan by Tryptophan Synthase from Escherichia coli

2020 ◽  
Vol 8 (4) ◽  
pp. 519
Author(s):  
Lisheng Xu ◽  
Fangkai Han ◽  
Zeng Dong ◽  
Zhaojun Wei
Keyword(s):  
Thermal Stability ◽  
Amino Acid ◽  
Directed Evolution ◽  
Amino Acid Residue ◽  
Rational Design ◽  
Molecular Engineering ◽  
Mutant Enzyme ◽  
Tryptophan Synthase ◽  
Mutant Library ◽  
Thermal Stability Of

To improve the thermostability of tryptophan synthase, the molecular modification of tryptophan synthase was carried out by rational molecular engineering. First, B-FITTER software was used to analyze the temperature factor (B-factor) of each amino acid residue in the crystal structure of tryptophan synthase. A key amino acid residue, G395, which adversely affected the thermal stability of the enzyme, was identified, and then, a mutant library was constructed by site-specific saturation mutation. A mutant (G395S) enzyme with significantly improved thermal stability was screened from the saturated mutant library. Error-prone PCR was used to conduct a directed evolution of the mutant enzyme (G395S). Compared with the parent, the mutant enzyme (G395S /A191T) had a Km of 0.21 mM and a catalytic efficiency kcat/Km of 5.38 mM−1∙s−1, which was 4.8 times higher than that of the wild-type strain. The conditions for L-tryptophan synthesis by the mutated enzyme were a L-serine concentration of 50 mmol/L, a reaction temperature of 40 °C, pH of 8, a reaction time of 12 h, and an L-tryptophan yield of 81%. The thermal stability of the enzyme can be improved by using an appropriate rational design strategy to modify the correct site. The catalytic activity of tryptophan synthase was increased by directed evolution.

Directed Evolution of a Formate Dehydrogenase for Increased Tolerance to Ionic Liquids Reveals a New Site for Increasing the Stability

ChemBioChem ◽  
2014 ◽  
Vol 15 (18) ◽  
pp. 2710-2718 ◽  
Author(s):  
Julie L. L. Carter ◽  
Mourad Bekhouche ◽  
Alexandre Noiriel ◽  
Loïc J. Blum ◽  
Bastien Doumèche
Keyword(s):  
Ionic Liquids ◽  
Directed Evolution ◽  
Formate Dehydrogenase ◽  
The Stability

scholarly journals Accommodation of large plastic strains and defect accumulation in nanocrystalline Ni grains

2007 ◽  
Vol 22 (8) ◽  
pp. 2241-2253 ◽  
Author(s):  
X.L. Wu ◽  
E. Ma
Keyword(s):  
Deformation Behavior ◽  
Boundary Migration ◽  
Temperature Deformation ◽  
Plastic Strains ◽  
Domain Structures ◽  
Transmission Electron ◽  
Defect Accumulation ◽  
Pile Up ◽  
Slip Planes ◽  
The Stability

A transmission electron microscopy (TEM) study has been carried out to uncover how dislocations and twins accommodate large plastic strains and accumulate in very small nanocrystalline Ni grains during low-temperature deformation. We illustrate dislocation patterns that suggest preferential deformation and nonuniform defect storage inside the nanocrystalline grain. Dislocations are present in individual and dipole configurations. Most dislocations are of the 60° type and pile up on (111) slip planes. Various deformation responses, in the forms of dislocations and twinning, may simultaneously occur inside a nanocrystalline grain. Evidence for twin boundary migration has been obtained. The rearrangement and organization of dislocations, sometimes interacting with the twins, lead to the formation of subgrain boundaries, subdividing the nanograin into mosaic domain structures. The observation of strain (deformation)-induced refinement contrasts with the recently reported stress-assisted grain growth in nanocrystalline metals and has implications for understanding the stability and deformation behavior of these highly nonequilibrium materials.

Improvement of the stability and activity of the BPO-A1 haloperoxidase from Streptomyces aureofaciens by directed evolution

2014 ◽  
Vol 192 ◽  
pp. 248-254 ◽  
Author(s):  
Ryosuke Yamada ◽  
Tatsutoshi Higo ◽  
Chisa Yoshikawa ◽  
Hideyasu China ◽  
Hiroyasu Ogino
Keyword(s):  
Directed Evolution ◽  
Streptomyces Aureofaciens ◽  
The Stability

scholarly journals Directed Evolution of Xylose Isomerase for Improved Xylose Catabolism and Fermentation in the Yeast Saccharomyces cerevisiae

2012 ◽  
Vol 78 (16) ◽  
pp. 5708-5716 ◽  
Author(s):  
Sun-Mi Lee ◽  
Taylor Jellison ◽  
Hal S. Alper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ethanol Production ◽  
Directed Evolution ◽  
Ethanol Yield ◽  
Xylose Isomerase ◽  
Mutant Enzyme ◽  
Xylose Utilization ◽  
Xylose Consumption ◽  
Content Type ◽  
Consumption Rates

ABSTRACTThe heterologous expression of a highly functional xylose isomerase pathway inSaccharomyces cerevisiaewould have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways inS. cerevisiaesuffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of thePiromycessp. xylose isomerase (encoded byxylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing agre3knockout andtal1andXKS1overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.

scholarly journals Directed evolution  improves the enzymatic synthesis of L-5-hydroxytryptophan by an  engineered tryptophan synthase

2021 ◽  
Author(s):  
Xu Lisheng ◽  
Tingting Li ◽  
Ziyue Huo ◽  
Qiong Chen ◽  
Qiuxia Xia ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Enzymatic Synthesis ◽  
High Throughput Screening ◽  
Screening Method ◽  
Mutant Enzyme ◽  
Tryptophan Synthase ◽  
Effective Strategy ◽  
Reaction Conditions ◽  
Important Amino Acid

Abstract L-5-Hydroxytryptophan is an important amino acid that is widely used in food and medicine. In this study, L-5-hydroxytryptophan was synthesized by a modified tryptophan synthase. A direct evolution strategy was applied to engineer tryptophan synthase from Escherichia coli to improve the efficiency of L-5-hydroxytryptophan synthesis. Tryptophan synthase was modified by error-prone PCR. A high activity mutant enzyme (V231A/K382G) was obtained by a high-throughput screening method. The activity of mutant enzyme (V231A/K382G) is 3.79 times higher than that of its parent, and kcat/Km of the mutant enzyme (V231A/K382G) was 4.36 mM− 1∙s− 1. The mutant enzyme (V231A/K382G) reaction conditions for the production of L-5-hydroxytryptophan were 100 mmol/L L-serine at pH 8.5 and 35°C for 15 h, reaching a yield of L-5-hydroxytryptophan of 86.7%. Directed evolution is an effective strategy to increase the activity of tryptophan synthase.

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