Status of protein engineering for biocatalysts: how to design an industrially useful biocatalyst

Bommarius, Andreas S.; Blum, Janna K.; Abrahamson, Michael J.

doi:10.1016/j.cbpa.2010.11.011

Cited by 200 publications

(103 citation statements)

References 42 publications

Supporting

Mentioning

101

Contrasting

Unclassified

Order By: Relevance

“…The structure-guided consensus approach refers to the introduction of specific mutations to the protein sequence predicted by utilizing sequence-based alignment, phylogenetic analysis, structural information, and bioinformatics (39,61). About 1,000 colonies of the consensus library were initially screened for improved activity following incubation for 30 min in 50% methanol.…”

Section: Resultsmentioning

confidence: 99%

“…The high functionality of the consensus library can be attributed to the choice of specific point mutations and the application of eliminating criteria to reduce destructive mutations. It was previously shown that the datadriven consensus approach is useful in creating mutant libraries of high functionality, which enables the screening effort to be minimized (39,42,47,61). Low functionality of a random mutagenesis library may be a result of a high mutation rate and multiple alterations that can lead to destabilization and unfolding of the enzyme (36,66).…”

Section: Figmentioning

confidence: 99%

See 1 more Smart Citation

Protein Engineering by Random Mutagenesis and Structure-Guided Consensus of Geobacillus stearothermophilus Lipase T6 for Enhanced Stability in Methanol

Dror

Shemesh

Dayan

et al. 2014

Appl Environ Microbiol

116

107

View full text Add to dashboard Cite

The abilities of enzymes to catalyze reactions in nonnatural environments of organic solvents have opened new opportunities for enzyme-based industrial processes. However, the main drawback of such processes is that most enzymes have a limited stability in polar organic solvents. In this study, we employed protein engineering methods to generate a lipase for enhanced stability in methanol, which is important for biodiesel production. Two protein engineering approaches, random mutagenesis (error-prone PCR) and structure-guided consensus, were applied in parallel on an unexplored lipase gene from Geobacillus stearothermophilus T6. A high-throughput colorimetric screening assay was used to evaluate lipase activity after an incubation period in high methanol concentrations. Both protein engineering approaches were successful in producing variants with elevated half-life values in 70% methanol. The best variant of the random mutagenesis library, Q185L, exhibited 23-fold-improved stability, yet its methanolysis activity was decreased by one-half compared to the wild type. The best variant from the consensus library, H86Y/ A269T, exhibited 66-fold-improved stability in methanol along with elevated thermostability (؉4.3°C) and a 2-fold-higher fatty acid methyl ester yield from soybean oil. Based on in silico modeling, we suggest that the Q185L substitution facilitates a closed lid conformation that limits access for both the methanol and substrate excess into the active site. The enhanced stability of H86Y/A269T was a result of formation of new hydrogen bonds. These improved characteristics make this variant a potential biocatalyst for biodiesel production.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

Protein Engineering by Random Mutagenesis and Structure-Guided Consensus of Geobacillus stearothermophilus Lipase T6 for Enhanced Stability in Methanol

Dror

Shemesh

Dayan

et al. 2014

Appl Environ Microbiol

116

107

View full text Add to dashboard Cite

show abstract

“…In contrast to our previous single-site iterative saturation mutagenesis, we created mutant libraries based on the CAST principle. CAST was designed to exploit the synergistic geometric effects of saturating small por- tions of a given protein's active site (36). It was applied successfully in broadening substrate specificity (14) or altering stereoand/or regio-selectivity (28,37,38).…”

Section: Discussionmentioning

confidence: 99%

Engineering a Monolignol 4-O-Methyltransferase with High Selectivity for the Condensed Lignin Precursor Coniferyl Alcohol

Cai

Bhuiya²,

Shanklin

et al. 2015

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background: Guaiacyl lignin dominates the polymer's condensation, therefore, negatively affecting plant cell wall's digestibility. Results: A promiscuous O-methyltransferase was incrementally evolved to constrain its substrate specificity to guaiacyl lignin precursors. Conclusion:The enzyme with nine amino acid substitutions is functionally specialized for selectively methylating the condensed lignin precursors. Significance: The obtained enzyme is a candidate for specifically altering lignin structure to improve plant cell wall digestibility.

show abstract

“…Several comprehensive reviews discussed the discovery and engineering tools developed to obtain naturally evolved or tailor-made enzymes with enhanced properties for chemical synthesis [29,30,40]. In the case of microbial strains isolated from natural environment, enzymes could be discovered through protein purification, insertional mutation, chromosome walking, shotgun library, etc.…”

Section: Discovery and Characterization Of Opbe Reductasesmentioning

confidence: 99%

“…Various enzyme discovery and protein engineering tools have been proposed and proved, which would greatly shorten the developing period and increase the availability of robust enzymes. Several literatures on the biocatalytic asymmetric reduction for pharmaceuticals, protein engineering strategies for robust enzymes in chemical synthesis have been reviewed [28][29][30][31][32]. Also, various OPBE reductases have been successfully identified from natural samples or genome databases.…”

Section: Introductionmentioning

confidence: 99%

Bioreductive preparation of ACE inhibitors precursor (R)-2-hydroxy-4-phenylbutanoate esters: Recent advances and future perspectives

2015

Bioresour. Bioprocess.

View full text Add to dashboard Cite

Optically active (R)-2-hydroxy-4-phenylbutanoate esters ((R)-HPBE) are key precursors for the production of angiotension-converting enzyme (ACE) inhibitors, which are important prescriptive drugs for preventing the formation of angiotensin II and lowering the blood pressure. The biocatalytic asymmetric reduction of ethyl 2-oxo-4-phenylbutanoate (OPBE) to (R)-HPBE with carbonyl reductases has several advantageous attributes, including high enantioselectivity, mild reaction condition, high catalytic efficiency, and environmental benignity. An increasing number of OPBE reductases have been discovered owing to the drastic achievements in genomics, screening and evolution technologies, and process engineering. The potential of (R)-HPBE production process has also been intensively evaluated. This review covers recent progress on the bioreductive preparation of (R)-HPBE, especially on various screening approaches for the identification of OPBE reductases and their characterization.

show abstract

Status of protein engineering for biocatalysts: how to design an industrially useful biocatalyst

Cited by 200 publications

References 42 publications

Protein Engineering by Random Mutagenesis and Structure-Guided Consensus of Geobacillus stearothermophilus Lipase T6 for Enhanced Stability in Methanol

Protein Engineering by Random Mutagenesis and Structure-Guided Consensus of Geobacillus stearothermophilus Lipase T6 for Enhanced Stability in Methanol

Engineering a Monolignol 4-O-Methyltransferase with High Selectivity for the Condensed Lignin Precursor Coniferyl Alcohol

Bioreductive preparation of ACE inhibitors precursor (R)-2-hydroxy-4-phenylbutanoate esters: Recent advances and future perspectives

Contact Info

Product

Resources

About