Directed Evolution of α-Aspartyl Dipeptidase from Salmonella typhimurium

Kong, Xiangduo; Liu, Yiming; Gou, Xin; Zhu, Shizhen; Zhang, Hongying; Wang, Xiaoping; Zhang, Jin

doi:10.1006/bbrc.2001.5937

Cited by 7 publications

(3 citation statements)

References 13 publications

(9 reference statements)

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“…Unlike protein engineering by rational design, directed molecular evolution does not require any knowledge a priori of protein structure or structure-function relationships. It is well-known that screening efficiency was the hinge of directed evolution (34). In our present research, a simple and rapid twostep screening method was constructed, which was sensitive enough to ensure that even a small enhancement to achieve the desired result could be observed.…”

Section: Discussionmentioning

confidence: 96%

Directed Evolution and Characterization of a Novel d-Pantonohydrolase from Fusarium moniliforme

Liu

Sun

Leng

2006

J. Agric. Food Chem.

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D-Pantonohydrolase has attracted increasing attention as a biocatalyst for stereospecific production of D-pantoic acid. The Fusarium moniliforme D-pantonohydrolase was selected for directed evolution through error-prone Polymerase Chain Reaction (PCR) combined with DNA shuffling for improved activity and pH stability using a convenient two-step high-throughput screening method based on the product formation and pH indicator. After three sequential error-prone PCRs and two rounds of DNA shuffling followed by screening, about 60 positive mutants were produced and a best mutant, Mut H-1287, with improved activity and pH stability was obtained. As compared to wild-type D-pantonohydrolase, Mut H-1287 showed a 10.5-fold higher specific activity; moreover, it could retain 85% of its original activity after incubation under low pH. Gene analysis indicated that the Mut H-1287 had D63H, K118Q, and V241I substitutions. The wild-type and evolved D-pantonohydrolase (Mut H-1287) was purified in three steps. The activities and characteristics of purified wild-type and evolved D-pantonohydrolase were also studied and compared.

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Section: Discussionmentioning

confidence: 96%

Directed Evolution and Characterization of a Novel d-Pantonohydrolase from Fusarium moniliforme

Liu

Sun

Leng

2006

J. Agric. Food Chem.

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“…Thermostability, for example, could be conferred by any one of the at least three different genetic elements. Because of the importance of proteolytic enzymes, directed evolution of proteases and peptidases remains one of the most actively pursued research areas (10,12,34,100,160,210,211,285,297,304,(327)(328)(329)349).…”

Section: Directed Evolution Of Biochemical Catalystsmentioning

confidence: 99%

“…In another example, the trehalose-6-phosphate synthase/phosphatase operon was evolved to achieve greater trehalose production in E. coli (159,160). In E. coli, trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase are encoded by the otsBA operon.…”

Section: Directed Evolution Of Metabolic Pathwaysmentioning

confidence: 99%

Laboratory-Directed Protein Evolution

Yuan

Kurek

English

et al. 2005

Microbiol Mol Biol Rev

176

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Systematic approaches to directed evolution of proteins have been documented since the 1970s. The ability to recruit new protein functions arises from the considerable substrate ambiguity of many proteins. The substrate ambiguity of a protein can be interpreted as the evolutionary potential that allows a protein to acquire new specificities through mutation or to regain function via mutations that differ from the original protein sequence. All organisms have evolutionarily exploited this substrate ambiguity. When exploited in a laboratory under controlled mutagenesis and selection, it enables a protein to “evolve” in desired directions. One of the most effective strategies in directed protein evolution is to gradually accumulate mutations, either sequentially or by recombination, while applying selective pressure. This is typically achieved by the generation of libraries of mutants followed by efficient screening of these libraries for targeted functions and subsequent repetition of the process using improved mutants from the previous screening. Here we review some of the successful strategies in creating protein diversity and the more recent progress in directed protein evolution in a wide range of scientific disciplines and its impacts in chemical, pharmaceutical, and agricultural sciences

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Dipeptidase E

Miller¹

2013

Handbook of Proteolytic Enzymes

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Directed Evolution of α-Aspartyl Dipeptidase from Salmonella typhimurium

Cited by 7 publications

References 13 publications

Directed Evolution and Characterization of a Novel d-Pantonohydrolase from Fusarium moniliforme

Directed Evolution and Characterization of a Novel d-Pantonohydrolase from Fusarium moniliforme

Laboratory-Directed Protein Evolution

Dipeptidase E

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