Site-saturation Mutagenesis is more Efficient than DNA Shuffling for the Directed Evolution of β-Fucosidase from β-Galactosidase

Parikh, Monal R.; Matsumura, Ichiro

doi:10.1016/j.jmb.2005.07.020

Cited by 90 publications

(40 citation statements)

References 39 publications

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“…Nevertheless, it does not belong to GH1 or GH30, which have ␤-Fuc activity (the CAZy database). It has been reported that E. coli LacZ, which has a GH2 domain, recognizes and catalyzes a reaction with ␤-D-fucosyl moieties weakly (31,32). We showed that truncated MsgA containing only LacZD demonstrated ␤-Fuc activity (Table 4).…”

Section: Discussionmentioning

confidence: 60%

Identification and Characterization of a Novel Secreted Glycosidase with Multiple Glycosidase Activities in Streptococcus intermedius

et al. 2014

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Section: Discussionmentioning

confidence: 60%

Identification and Characterization of a Novel Secreted Glycosidase with Multiple Glycosidase Activities in Streptococcus intermedius

et al. 2014

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“…However, mutations closer to the catalytic core appear to have a greater effect on the selected catalytic activity (30)(31)(32)(33). These residues for mutagenesis may be selected through a random, rational or combined evolutionary approach.…”

Section: Discussionmentioning

confidence: 99%

Conversion of a Cyclodextrin Glucanotransferase into an α-Amylase: Assessment of Directed Evolution Strategies

2007

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Glycoside hydrolase family 13 (GH13) members have evolved to possess various distinct reaction specificities despite the overall structural similarity. In this study we investigated the evolutionary input required to effeciently interchange these specificities and also compared the effectiveness of laboratory evolution techniques applied, i.e., error-prone PCR and saturation mutagenesis. Conversion of our model enzyme, cyclodextrin glucanotransferase (CGTase), into an R-amylase like hydrolytic enzyme by saturation mutagenesis close to the catalytic core yielded a triple mutant (A231V/F260W/F184Q) with the highest hydrolytic rate ever recorded for a CGTase, similar to that of a highly active R-amylase, while cyclodextrin production was virtually abolished. Screening of a much larger, error-prone PCR generated library yielded far less effective mutants. Our results demonstrate that it requires only three mutations to change CGTase reaction specificity into that of another GH13 enzyme. This suggests that GH13 members may have diversified by introduction of a limited number of mutations to the common ancestor, and that interconversion of reaction specificites may prove easier than previously thought.

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“…A combination of saturation mutagenesis (16)(17)(18) for effective diversity generation coupled with highly efficient recombination tools and the use of statistical analysis to maximize screening efficiency was used to improve DvCA. In nine rounds of evolution, over 27,000 DvCA variants were screened at increasingly higher temperatures in the presence of 3.0-4.2 M MDEA to identify enzymes with the desired characteristics.…”

Section: Significancementioning

confidence: 99%

Directed evolution of an ultrastable carbonic anhydrase for highly efficient carbon capture from flue gas

Alvizo

Nguyen²,

Savile

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

161

145

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Carbonic anhydrase (CA) is one of nature's fastest enzymes and can dramatically improve the economics of carbon capture under demanding environments such as coal-fired power plants. The use of CA to accelerate carbon capture is limited by the enzyme's sensitivity to the harsh process conditions. Using directed evolution, the properties of a β-class CA from Desulfovibrio vulgaris were dramatically enhanced. Iterative rounds of library design, library generation, and high-throughput screening identified highly stable CA variants that tolerate temperatures of up to 107°C in the presence of 4.2 M alkaline amine solvent at pH >10.0. This increase in thermostability and alkali tolerance translates to a 4,000,000-fold improvement over the natural enzyme. At pilot scale, the evolved catalyst enhanced the rate of CO 2 absorption 25-fold compared with the noncatalyzed reaction.carbonic anhydrase | directed evolution | carbon capture R eleasing over 9 billion metric tons of CO 2 worldwide each year, coal-fired power plants are the leading anthropogenic source of CO 2 emission. A potential solution to reduce the release of CO 2 into the atmosphere is the implementation of carbon capture and sequestration (CCS) technology on coal-and natural-gas-fired power plants. Currently, one of the most viable postcombustion CCS technology options uses an amine solvent to remove CO 2 from the flue gas (1). Unfortunately, the large amount of energy required to release the CO 2 and regenerate the solvent would significantly increase the cost of electricity generated (2). The ideal solvent capture process uses a solvent such as an aqueous amine with fast CO 2 absorption kinetics and a low heat of desorption to minimize the solvent regeneration energy. However, a high rate of CO 2 absorption kinetics for an amine solvent correlates with a higher temperature of desorption, whereas solvents with low heat of desorption tend to have much slower adsorption kinetics (3, 4).With reaction rates approaching the limits of diffusion, carbonic anhydrase (CA) is one of the fastest enzymes known. The active site of CAs can turnover CO 2 and water to bicarbonate and a proton up to a million times per second, and are used by almost every living organism to maintain pH balance and transport carbon dioxide. It has been shown that CAs can be used to accelerate the capture of CO 2 (5) by serving as a catalyst in alkaline capture solvents with slow absorption kinetics (6, 7). The potential improvements of CAs on CCS process economics stems from several considerations. The faster absorption and desorption kinetics allows for smaller processing equipment with reduced capital and operating costs (6). The improved kinetics also allows the use of lower temperature capture and solvent regeneration process conditions, and decreases energy losses in the capture process. Another benefit is the improved range of solvent candidates with attractive thermochemical stability (e.g., low vapor pressure, low formation of heat stable salts, etc.), but otherwise unacceptable unca...

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Site-saturation Mutagenesis is more Efficient than DNA Shuffling for the Directed Evolution of β-Fucosidase from β-Galactosidase

Cited by 90 publications

References 39 publications

Identification and Characterization of a Novel Secreted Glycosidase with Multiple Glycosidase Activities in Streptococcus intermedius

Identification and Characterization of a Novel Secreted Glycosidase with Multiple Glycosidase Activities in Streptococcus intermedius

Conversion of a Cyclodextrin Glucanotransferase into an α-Amylase: Assessment of Directed Evolution Strategies

Directed evolution of an ultrastable carbonic anhydrase for highly efficient carbon capture from flue gas

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