Improving hydrolases for organic synthesis

doi:10.1016/S1367-5931(98)80043-4

Current Opinion in Chemical Biology

Volume 2, Issue 1, February 1998, Pages 121-126

https://doi.org/10.1016/S1367-5931(98)80043-4 Get rights and content

Abstract

Improving hydrolases by site-directed mutagenesis continues to be important, but an alternative method — directed evolution — also gains favor. Directed evolution combines random mutagenesis with screening or selection for the desired property. Directed evolution is especially useful for cases like solvent tolerance or thermostability where current theories are inadequate to predict which structural changes will give improvement. Researches have also recently made significant progress on several practical problems: how to maintain the high activity of proteases and lipases in nonpolar organic solvents, how to resolve amines, and how to efficiently recycle the unwanted enantiomer in kinetic resolutions. Besides the lipases and proteases, researchers are also developing new hydrolases, notably dehalogenases and epoxide hydrolases.

References (66)

KK Kim et al.
The crystal structure of a triacylglycerol lipase from Pseudomonas cepacia reveals a highly open conformation in the absence of a bound inhibitor
Structure
(1997)
JD Schrag et al.
The open conformation of a Pseudomonas lipase
Structure
(1997)
D Lang et al.
Crystal structure of a bacterial lipase from Chromobacterium viscosum ATCC 6918 refined at 1.6 Å resolution
J Mol Biol
(1996)
HC Holzwarth et al.
Computer-aided modelling of stereoselective triglyceride hydrolysis catalyzed by Rhizopus oryzae lipase
J Mol Catal B Enzym
(1997)
YF Wang et al.
An efficient synthesis of chiral amino acid and peptide alkylamides via CLEC-subtilisin catalyzed-coupling and in situ resolution
Tetrahedron Lett
(1996)
D Rotticci et al.
Enantiomerically enriched bifunctional sec-alcohols prepared by Candida antarctica lipase B catalysis. Evidence of non-steric interactions
Tetrahedron Asymm
(1996)
PP Wangikar et al.
Structure and function of subtilisin BPN′ solubilized in organic solvents
J Am Chem Soc
(1997)
JL Schmitke et al.
The mechanistic dissection of the plunge in enzymatic activity upon transition from water to anhydrous solvents
J Am Chem Soc
(1996)
Balkenhohl F, Hauer B, Ladner W, Pressler U, Rettenmaier H, Adam G: A method to increase the activity of hydrolytic...
PM Dinh et al.
Catalytic racemisation of alcohols: application of enzymatic resolution reactions
Tetrahedron Lett
(1996)

L Ember

Detoxifying nerve agents

Chem Eng News

(1997, Sept 15)

K Faber et al.

Microbial epoxide hydrolase

Acta Chem Scand

(1996)

M Kohno et al.

The crystal structure of lipase II from Rhizopus niveus at 2.2 Å resolution

J Biochem

(1996)

M Holmquist et al.

A structural basis for enantioselective inhibition of Candida rugosa lipase by long chain alcohols

Protein Sci

(1996)

RR Klein et al.

Altered acyl chain length specificity of Rhizopus delemar lipase through mutagenesis and molecular modeling

Lipids

(1997)

R Sakowicz et al.

PArtial reversal of the substrate stereospecificity of an L-lactate dehydrogenase by site directed mutagenesis

J Am Chem Soc

(1995)

H Smidt et al.

Preparation of optically-pure chiral amines by lipase-catalyzed enantioselective hydrolysis of N-actyl amines

Biotechnol Tech

(1996)

RJ Kazlauskas et al.

A structure-based rationalization of the enantiopreference of subtilisin toward secondary alcohols and isosteric primary amines

J Mol Catal B Enzym

(1997)

MD Ballinger et al.

Furilisin — a variant of subtilisin BPN′ engineered for cleaving tribasic substrates

Biochemistry

(1996)

P Berglund et al.

Chemical modification of cysteine mutants of subtilisin Bacillus lentus can create better catalysts than the wild-type enzyme

J Am Chem Soc

(1997)

RD Kidd et al.

A weak calcium binding site in subtilisin BPN′ has a dramatic effect on protein stability

J Am Chem Soc

(1996)

L You et al.

Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide

Protein Eng

(1996)

JC Moore et al.

Directed evolution of a p-nitrobenzyl esterase for aqueous—organic solvents

Nat Biotechnol

(1996)

A Sattler et al.

Thermostable variants of subtilisin selected by temperature-gradient gel electrophoresis

Electrophoresis

(1996)

A Shinkai et al.

Substitutions of Ser for Asn-163 and Pro for Leu-264 are important for stabilization of lipase from Pseudomonas aeruginosa

J Biochem

(1996)

S Yamaguchi et al.

The consequences of engineering an extra disulfide bond in the Penicillium camembertii mono- and diglyceride specific lipase

Protein Eng

(1996)

MT Reetz et al.

Creation of enantioselective biocatalysts for organic chemistry by in vitro evolution

Angew Chem Int Ed Engl

(1997)

UT Bornscheuer et al.

Directed evolution of an esterase for the stereoselective resolution of a key intermediate in the synthesis of epothilones

Biotech Bioeng

(1998)

H Zhao et al.

Combinatorial protein design: strategies for screening protein libraries

Curr Opin Struct Biol

(1997)

LE Janes et al.

Quick E. A fast spectrophotometric method to measure the enantioselectivity of hydrolases

J Org Chem

(1997)

AM Klibanov

Why are enzymes less active in organic solvents than in water?

Trends Biotechnol

(1997)

JC Lee et al.

The stabilization of proteins by sucrose

J Biol Chem

(1981)

SJ Prestrelski et al.

Separation of freezing- and drying-induced denaturation of lyophilized proteins using stress-specific inhibitors

Arch Biochem Biophys

(1993)

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Herein we report a new approach to enantiomerically enriched acetates using a lipase-catalyzed hydrolysis in non-aqueous media by alkaline carbonate salts. The use of sodium carbonate in the enzymatic hydrolysis with Candida antarctica lipase B (CAL-B) of racemic acetates shows a large enhancement of the reactivity and selectivity of this lipase. The role of the carbonate salts, the amount and the nature of the alkaline earth metal on the efficiency of this new pathway are investigated. The enzymatic kinetic resolution of acetates 1a–9a, by enzymatic-carbonate hydrolysis under mild conditions is described. In all cases, the resulting alcohols and remaining acetates were obtained in high ee values (up to >99%) while the selectivities reached E >500.
A molecular modelling approach to rationalize the stereochemical outcome of the Burkholderia cepacia lipase-catalyzed transesterification of aromatic primary alcohols with vinyl esters with different chain lengths in chloroform
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The Burkholderia cepacia lipase-catalyzed transesterification of 2-methyl-3-phenyl-1-propanol with vinyl esters proceeds with high enantioselectivity independently of the acyl chain length and the low enantioselectivity of the same reaction with 2-phenyl-1-propanol is not affected by chain length of the vinyl esters. A molecular modelling approach has been developed in order to rationalize the enzymatic results.
Lipase action on some non-triglyceride substrates
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Approaches for improving methodologies of kinetic resolution of enantiomers as well as of regioselective protection of functional groups of complex chiral molecules involving lipase-catalytic reactions are highlighted. Decyclization of hemiacetals by lipase as well as exclusive pathways of lipase-catalyzed derivatization of prostanoids are brought out. Lipase-triggered cascade-reactions are noticed.
Structure and mechanism of the epoxide hydrolase from Agrobacterium radiobacter AD1
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The X-ray structure of the epoxide hydrolase from Agrobacterium radiobacter AD1 has been determined by isomorphous replacement at 2.1 Å resolution. The enzyme shows a two-domain structure, with the core having the α/β hydrolase fold topology which provides the scaffolding for the catalytic triad residues, Asp 107, Asp 246, and His 275. Based on biochemical and structural data, a mechanism is proposed to illuminate the peculiar chemical strategy to activate harmful epoxide substrates for hydrolysis and detoxification. The structure suggests Tyr 152/Tyr 215 as the residues involved in substrate binding, stabilization of the transition state, and possibly protonation of the epoxide oxygen. Site-directed mutagenesis studies confirmed the structural results and, in addition, showed a significant increase in enantioselectivity for styrene oxide, and substituted variants thereof, for a Tyr 215→Phe mutant. Since Tyr 215 is conserved among all epoxide hydrolase sequences, an activating role of this residue is suggested for other epoxide hydrolases as well, and mutation of this residue might also result in mutant enzymes with improved enantioselectivity in other cases.
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The x-ray structure of the lipase fromPseudomonas aeruginosa PAO1 has been determined at 2.54 Å resolution. It is the first structure of a member of homology family I.1 of bacterial lipases. The structure shows a variant of the α/β hydrolase fold, with Ser⁸², Asp²²⁹, and His²⁵¹ as the catalytic triad residues. Compared with the “canonical” α/β hydrolase fold, the first two β-strands and one α-helix (αE) are not present. The absence of helix αE allows the formation of a stabilizing intramolecular disulfide bridge. The loop containing His²⁵¹ is stabilized by an octahedrally coordinated calcium ion. On top of the active site a lid subdomain is in an open conformation, making the catalytic cleft accessible from the solvent region. A triacylglycerol analogue is covalently bound to Ser⁸² in the active site, demonstrating the position of the oxyanion hole and of the three pockets that accommodate thesn-1, sn-2, and sn-3 fatty acid chains. The inhibited enzyme can be thought to mimic the structure of the tetrahedral intermediate that occurs during the acylation step of the reaction. Analysis of the binding mode of the inhibitor suggests that the size of the acyl pocket and the size and interactions of the sn-2 binding pocket are the predominant determinants of the regio- and enantio-preference of the enzyme.

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Improving hydrolases for organic synthesis

Abstract

Structure

Structure

J Mol Biol

J Mol Catal B Enzym

Tetrahedron Lett

Tetrahedron Asymm

J Am Chem Soc

J Am Chem Soc

Tetrahedron Lett

Chem Eng News

Acta Chem Scand

The crystal structure of lipase II from Rhizopus niveus at 2.2 Å resolution

J Biochem

A structural basis for enantioselective inhibition of Candida rugosa lipase by long chain alcohols

Protein Sci

Altered acyl chain length specificity of Rhizopus delemar lipase through mutagenesis and molecular modeling

Lipids

PArtial reversal of the substrate stereospecificity of an L-lactate dehydrogenase by site directed mutagenesis

J Am Chem Soc

Preparation of optically-pure chiral amines by lipase-catalyzed enantioselective hydrolysis of N-actyl amines

Biotechnol Tech

A structure-based rationalization of the enantiopreference of subtilisin toward secondary alcohols and isosteric primary amines

J Mol Catal B Enzym

Furilisin — a variant of subtilisin BPN′ engineered for cleaving tribasic substrates

Biochemistry

Chemical modification of cysteine mutants of subtilisin Bacillus lentus can create better catalysts than the wild-type enzyme

J Am Chem Soc

A weak calcium binding site in subtilisin BPN′ has a dramatic effect on protein stability

J Am Chem Soc

Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide

Protein Eng

Directed evolution of a p-nitrobenzyl esterase for aqueous—organic solvents

Nat Biotechnol

Thermostable variants of subtilisin selected by temperature-gradient gel electrophoresis

Electrophoresis

Substitutions of Ser for Asn-163 and Pro for Leu-264 are important for stabilization of lipase from Pseudomonas aeruginosa

J Biochem

The consequences of engineering an extra disulfide bond in the Penicillium camembertii mono- and diglyceride specific lipase

Protein Eng

Creation of enantioselective biocatalysts for organic chemistry by in vitro evolution

Angew Chem Int Ed Engl

Directed evolution of an esterase for the stereoselective resolution of a key intermediate in the synthesis of epothilones

Biotech Bioeng

Combinatorial protein design: strategies for screening protein libraries

Curr Opin Struct Biol

Quick E. A fast spectrophotometric method to measure the enantioselectivity of hydrolases

J Org Chem

Why are enzymes less active in organic solvents than in water?

Trends Biotechnol

The stabilization of proteins by sucrose

J Biol Chem

Separation of freezing- and drying-induced denaturation of lyophilized proteins using stress-specific inhibitors

Arch Biochem Biophys