Directed evolution of Tau class glutathione transferases reveals a site that regulates catalytic efficiency and masks co-operativity

2016 ◽  
Vol 473 (5) ◽  
pp. 559-570 ◽  
Author(s):  
Irine Axarli ◽  
Abdi W. Muleta ◽  
Dimitrios Vlachakis ◽  
Sophia Kossida ◽  
Georgia Kotzia ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Catalytic Efficiency ◽  
Glutathione Transferases ◽  
Dna Shuffling ◽  
A Site ◽  
Metabolism Of Xenobiotics

GSTs (glutathione transferases) are enzymes involved in the metabolism of xenobiotics. A GST created through DNA shuffling showed allosteric kinetics and enhanced detoxifying potential towards the herbicide fluorodifen. Its structure was determined. New engineered GSTs could be useful in biotechnology as efficient bioscavengers.

Nucleotide Exchange and Excision Technology DNA Shuffling and Directed Evolution

2010 ◽  
pp. 333-344 ◽  
Author(s):  
Janina Speck ◽  
Sabine C. Stebel ◽  
Katja M. Arndt ◽  
Kristian M. Müller
Keyword(s):  
Directed Evolution ◽  
Dna Shuffling ◽  
Nucleotide Exchange

scholarly journals Directed evolution improves the catalytic efficiency of TEV protease

2019 ◽  
Vol 17 (2) ◽  
pp. 167-174 ◽  
Author(s):  
Mateo I. Sanchez ◽  
Alice Y. Ting
Keyword(s):  
Directed Evolution ◽  
Catalytic Efficiency ◽  
Tev Protease

Improved catalytic efficiency of Endo-β-1,4-glucanase from Bacillus subtilis BME-15 by directed evolution

2009 ◽  
Vol 82 (4) ◽  
pp. 671-679 ◽  
Author(s):  
Ling Lin ◽  
Xin Meng ◽  
Pengfu Liu ◽  
Yuzhi Hong ◽  
Gaobing Wu ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Directed Evolution ◽  
Catalytic Efficiency

Multi-substrate–activity space and quasi-species in enzyme evolution: Ohno's dilemma, promiscuity and functional orthogonality

2009 ◽  
Vol 37 (4) ◽  
pp. 740-744 ◽  
Author(s):  
Bengt Mannervik ◽  
Arna Runarsdottir ◽  
Sanela Kurtovic
Keyword(s):  
Multivariate Analysis ◽  
Directed Evolution ◽  
Enzyme Evolution ◽  
Activity Space ◽  
Glutathione Transferases ◽  
Multidimensional Space ◽  
Alternative Substrates ◽  
Catalytic Activities ◽  
Evolutionary Trajectories

A functional enzyme displays activity with at least one substrate and can be represented by a vector in substrate–activity space. Many enzymes, including GSTs (glutathione transferases), are promiscuous in the sense that they act on alternative substrates, and the corresponding vectors operate in multidimensional space. The direction of the vector is governed by the relative activities of the diverse substrates. Stochastic mutations of already existing enzymes generate populations of variants, and clusters of functionally similar mutants can serve as parents for subsequent generations of enzymes. The proper evolving unit is a functional quasi-species, which may not be identical with the ‘best’ variant in its generation. The manifestation of the quasi-species is dependent on the substrate matrix used to explore catalytic activities. Multivariate analysis is an approach to identifying quasi-species and to investigate evolutionary trajectories in the directed evolution of enzymes for novel functions.

A semi-rational design strategy of directed evolution combined with chemical synthesis of DNA sequences

2007 ◽  
Vol 388 (12) ◽  
pp. 1291-1300 ◽  
Author(s):  
Ai-Sheng Xiong ◽  
Ri-He Peng ◽  
Jing Zhuang ◽  
Jin-Ge Liu ◽  
Feng Gao ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Dna Sequences ◽  
Active Sites ◽  
Rational Design ◽  
Specific Activity ◽  
Dna Shuffling ◽  
Gene Encoding ◽  
Library Construction ◽  
Key Sites

Abstract Directed evolution in vitro is a powerful molecular tool for the creation of new biological phenotypes. It is unclear whether it is more efficient to mutate an enzyme randomly or to mutate just the active sites or key sites. In this study, the strategy of a semi-rational design of directed evolution combined with whole sequence and sites was developed. The 1553 bp gene encoding the thermostable β-galactosidase of Pyrococcus woesei was chemically synthesized and optimized for G+C content and mRNA secondary structures. The synthesized gene product was used as a template or as a wild-type control. On the basis of the first round of DNA shuffling, library construction and screening, one mutant of YH6754 was isolated with higher activity. Eight potential key sites were deduced from the sequence of the shuffled gene, and 16 degenerate oligonucleotides were designed according to those eight amino acids. Two variants of YG6765 and YG8252 were screened in the second part of DNA shuffling, library construction and screening. For comparison, one mutant of YH8757 was screened through the same routine rounds of directed evolution with YH6754 as template. The purified β-galactosidase from YH8757 exhibited a lower specific activity at 25°C than those purified from mutated YG6755 and YG8252.

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