Protein engineering for development of new hydrolytic biocatalysts

Widersten, Mikael

doi:10.1016/j.cbpa.2014.03.015

Cited by 47 publications

(16 citation statements)

References 41 publications

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“…Lipases A and B from Candida antarctica are two biocatalysts that have found widespread applications in organic transformations, and both enzymes work very well in a range of organic solvents 22. The enzyme Candida antarctica lipase A (CalA) has been employed to a lesser extent compared to Candida antarctica lipase B (CalB).…”

Section: The Amino Acid (Aa) Residues Included In the Cala Librarymentioning

confidence: 99%

Combinatorial Library Based Engineering of Candida antarctica Lipase A for Enantioselective Transacylation of sec‐Alcohols in Organic Solvent

2015

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Am ethod for determining lipase enantioselectivity in the transacylation of sec-alcohols in organic solvent was developed. The method was applied to am odel library of Candida antarctica lipase A( CalA) variants for improved enantioselectivity (E values) in the kinetic resolution of 1-phenylethanol in isooctane.Afocused combinatorial gene library simultaneously targeting seven positions in the enzyme active site was designed. Enzyme variants were immobilized on nickel-coated 96-well microtiter plates through ah istidine tag (His 6 -tag), screened for transacylation of 1-phenylethanol in isooctane,and analyzed by GC.The highest enantioselectivity was shown by the double mutant Y93L/L367I. This enzyme variant gave an Evalue of 100 (R), whichi sadramatic improvement on the wild-type CalA (E = 3). This variant also showed high to excellent enantioselectivity for other secondary alcohols tested.

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Section: The Amino Acid (Aa) Residues Included In the Cala Librarymentioning

confidence: 99%

Combinatorial Library Based Engineering of Candida antarctica Lipase A for Enantioselective Transacylation of sec‐Alcohols in Organic Solvent

2015

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“…In recent decades, numerous enzymes have demonstrated the promiscuous activity for a large scope of reactions, which suggests the universe of enzyme promiscuous activities available in nature is tremendous. New advanced approaches have been developed to help us identify promiscuous activity, providing conditions for synthetic biology to construct novel biocatalysts via protein engineering …”

Section: Introductionmentioning

confidence: 99%

Probing the Mechanism of CAL‐B‐Catalyzed aza‐Michael Addition of Aniline Compounds with Acrylates Using Mutation and Molecular Docking Simulations

Yang

et al. 2019

ChemistrySelect

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CALÀ B (Lipase B from Candida antarctica) catalyzed aza-Michael addition of a set of aniline compounds with acrylates under mild conditions was described. A systematic study allowed to determine the appropriate solvent, enzyme loading, reaction temperature and time. In order to speculate and verify its mechanism, the wild-type and three mutants (S105 A, H224 A and I189 A) of CALÀ B were expressed. Some control experiments demonstrated the active site was responsible for the enzymatic process, in which Ser105 and His224 played a crucial role. Besides, the mutation of Ile189 also affected its activity a lot. Based on these results, a docking experiment was performed to speculate the mechanism: the oxyanion hole (Thr40 and Gln106) of the active site activated the acrylates and stabilized the transition states. The Ile189 residues, as an important part of active cavity, could form a strong hydrophobic interaction with substrates. And the Ser105 and His224 residues were responsible for proton transfer during the catalytic process. This would help to understand the promiscuity of CALÀ B, and provide ideas to design novel enzyme to improve the efficiency of its promiscuity.[a] B.

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“…Protein engineering by rational design requires structural information, and the precise regions for mutagenesis are identified usually by computational tools or prior knowledge (17,(22)(23)(24). One recently developed concept for enzyme stabilization in organic solvents is the modification of residues buried in tunnels within the protein structure.…”

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confidence: 99%

Filling the Void: Introducing Aromatic Interactions into Solvent Tunnels To Enhance Lipase Stability in Methanol

Gihaz

Kanteev

Pazy

et al. 2018

Appl Environ Microbiol

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Enhanced stability in organic solvents is a desirable feature for enzymes implemented under industrial conditions. Lipases potential as biocatalysts is mainly limited by denaturation in polar alcohols. In this study we focused on selected solvent tunnels in lipase from T6 to improve its stability in methanol during biodiesel synthesis. Using rational mutagenesis, bulky aromatic residues were incorporated in order to occupy solvent channels and induce aromatic interactions leading to a better inner core packing. Each solvent tunnel was systematically analyzed with respect to its chemical and structural characteristics. Selected residues were replaced with Phe, Tyr or Trp. Overall, 16 mutants were generated and screened in 60% methanol, from which 3 variants showed elevated stability up to 81-fold compared with wild-type. All stabilizing mutations were found in the longest tunnel detected in the "closed-lid" x-ray structure. Combining the Phe substitutions created double mutant A187F/L360F with an increase in T of +7°C in methanol and a 3-fold increase in biodiesel synthesis yield from waste chicken oil. Kinetic analysis with -nitrophenyl laurate revealed that all mutants displayed lower hydrolysis rates ( ), though mostly their stability properties determined the transesterification capability. Seven crystal structures of different variants were solved disclosing new π-π or CH/π intramolecular interactions emphasizing the significance of aromatic interactions to improved solvent stability. This rational approach could be implemented in other enzymes for stabilization in organic solvents. Enzymatic synthesis in organic solvents holds increasing industrial opportunities in many fields, however, one major obstacle is the limited stability of biocatalysts in such a denaturing environment. Aromatic interactions play a major role in protein folding and stability and we were inspired by this to re-design enzyme voids. Rational protein engineering of solvent tunnels of lipase from is presented here, offering a promising approach to introduce new aromatic interactions within the enzyme core. We discovered that longer tunnels leading from the surface to the enzyme active site were more beneficial targets for mutagenesis for improving lipase stability in methanol during biodiesel biosynthesis. Structural analysis of the variants confirmed the generation of new interactions involving aromatic residues. This work provides insights into stability-driven enzyme design by targeting solvent channels void.

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Protein engineering for development of new hydrolytic biocatalysts

Cited by 47 publications

References 41 publications

Combinatorial Library Based Engineering of Candida antarctica Lipase A for Enantioselective Transacylation of sec‐Alcohols in Organic Solvent

Combinatorial Library Based Engineering of Candida antarctica Lipase A for Enantioselective Transacylation of sec‐Alcohols in Organic Solvent

Probing the Mechanism of CAL‐B‐Catalyzed aza‐Michael Addition of Aniline Compounds with Acrylates Using Mutation and Molecular Docking Simulations

Filling the Void: Introducing Aromatic Interactions into Solvent Tunnels To Enhance Lipase Stability in Methanol

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