Improved triglyceride transesterification by circular permuted <i>Candida antarctica</i> lipase B

Yu, Ying; Lutz, Stefan

doi:10.1002/bit.22471

Cited by 24 publications

(11 citation statements)

References 15 publications

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“…CALB is a 33.273 KDa enzyme (Uppenberg et al, 1994 ) that behaves as an esterase in solution with small molecule reactants, but can undergo interfacial activation (classic lipase behavior) with more bulky substrates (Zisis et al, 2015 ). CALB is a versatile biocatalyst that has been used in water and organic solvents for various reactions, including capsaicin hydrolysis, phenolic acid esterification, octyl-β-glucoside synthesis, and alkyl ester production (Ljunger et al, 1994 ; Guyot et al, 1997 ; Anderson et al, 1998 ; Duarte et al, 2000 ; Deng et al, 2005 ; Yu and Lutz, 2010 ).…”

Section: Introductionmentioning

confidence: 99%

How Does Solvation Layer Mobility Affect Protein Structural Dynamics?

Dahanayake

Mitchell-Koch

2018

Front. Mol. Biosci.

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Solvation is critical for protein structural dynamics. Spectroscopic studies have indicated relationships between protein and solvent dynamics, and rates of gas binding to heme proteins in aqueous solution were previously observed to depend inversely on solution viscosity. In this work, the solvent-compatible enzyme Candida antarctica lipase B, which functions in aqueous and organic solvents, was modeled using molecular dynamics simulations. Data was obtained for the enzyme in acetonitrile, cyclohexane, n-butanol, and tert-butanol, in addition to water. Protein dynamics and solvation shell dynamics are characterized regionally: for each α-helix, β-sheet, and loop or connector region. Correlations are seen between solvent mobility and protein flexibility. So, does local viscosity explain the relationship between protein structural dynamics and solvation layer dynamics? Halle and Davidovic presented a cogent analysis of data describing the global hydrodynamics of a protein (tumbling in solution) that fits a model in which the protein's interfacial viscosity is higher than that of bulk water's, due to retarded water dynamics in the hydration layer (measured in NMR τ2 reorientation times). Numerous experiments have shown coupling between protein and solvation layer dynamics in site-specific measurements. Our data provides spatially-resolved characterization of solvent shell dynamics, showing correlations between regional solvation layer dynamics and protein dynamics in both aqueous and organic solvents. Correlations between protein flexibility and inverse solvent viscosity (1/η) are considered across several protein regions and for a rather disparate collection of solvents. It is seen that the correlation is consistently higher when local solvent shell dynamics are considered, rather than bulk viscosity. Protein flexibility is seen to correlate best with either the local interfacial viscosity or the ratio of the mobility of an organic solvent in a regional solvation layer relative to hydration dynamics around the same region. Results provide insight into the function of aqueous proteins, while also suggesting a framework for interpreting and predicting enzyme structural dynamics in non-aqueous solvents, based on the mobility of solvents within the solvation layer. We suggest that Kramers' theory may be used in future work to model protein conformational transitions in different solvents by incorporating local viscosity effects.

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Section: Introductionmentioning

confidence: 99%

How Does Solvation Layer Mobility Affect Protein Structural Dynamics?

Dahanayake

Mitchell-Koch

2018

Front. Mol. Biosci.

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“…CALB is an efficient biocatalyzer in non-aqueous environment. It can be used in a broad range of fields, and possesses excellent catalytic performance than any other lipases in terms of biodiesel production [3], [4], polymer synthesis [5], chiral resolution [6] and pharmaceuticals preparation [7].…”

Section: Introductionmentioning

confidence: 99%

de novo Design and Synthesis of Candida antarctica Lipase B Gene and α-Factor Leads to High-Level Expression in Pichia pastoris

et al. 2013

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Candida antarctica lipase B (CALB) is one of the most widely used and studied enzymes in the world. In order to achieve the high-level expression of CALB in Pichia, we optimized the codons of CALB gene and α-factor by using a de novo design and synthesis strategy. Through comparative analysis of a series of recombinants with different expression components, we found that the methanol-inducible expression recombinant carrying the codon-optimized α-factor and mature CALB gene (pPIC9KαM-CalBM) has the highest lipase production capacity. After fermentation parameters optimization, the lipase activity and protein content of the recombinant pPIC9KαM-CalBM reached 6,100 U/mL and 3.0 g/L, respectively, in a 5-L fermentor. We believe this strategy could be of special interest due to its capacity to improve the expression level of target gene, and the Pichia transformants carrying the codon-optimized gene had great potential for the industrial-scale production of CALB lipase.

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“…More recently, random circular permutation has been successfully applied as the diversity generation step for the directed evolution of Candida antarctica lipase B [7], [8] and Bacillus circulans xylanase Bcx [9]. Circular permutation at select sites in lipase B was found to increase hydrolytic activity up to 175-fold on certain substrates [8], [10]. Such improved variants often have the relocated termini proximal to the active site [7], [9], which may alter activity through local, subtle conformational changes and increased backbone flexibility [11].…”

Section: Introductionmentioning

confidence: 99%

Circular Permutation in the Ω-Loop of TEM-1 β-Lactamase Results in Improved Activity and Altered Substrate Specificity

Guntas¹,

Kanwar²,

Ostermeier

2012

PLoS ONE

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Generating diverse protein libraries that contain improved variants at a sufficiently high frequency is critical for improving the properties of proteins using directed evolution. Many studies have illustrated how random mutagenesis, cassette mutagenesis, DNA shuffling and similar approaches are effective diversity generating methods for directed evolution. Very few studies have explored random circular permutation, the intramolecular relocation of the N- and C-termini of a protein, as a diversity-generating step for directed evolution. We subjected a library of random circular permutations of TEM-1 β-lactamase to selections on increasing concentrations of a variety of β-lactam antibiotics including cefotaxime. We identified two circularly permuted variants that conferred elevated resistance to cefotaxime but decreased resistance to other antibiotics. These variants were circularly permuted in the Ω-loop proximal to the active site. Remarkably, one variant was circularly permuted such that the key catalytic residue Glu166 was located at the N-terminus of the mature protein.

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Improved triglyceride transesterification by circular permuted Candida antarctica lipase B

Cited by 24 publications

References 15 publications

How Does Solvation Layer Mobility Affect Protein Structural Dynamics?

How Does Solvation Layer Mobility Affect Protein Structural Dynamics?

de novo Design and Synthesis of Candida antarctica Lipase B Gene and α-Factor Leads to High-Level Expression in Pichia pastoris

Circular Permutation in the Ω-Loop of TEM-1 β-Lactamase Results in Improved Activity and Altered Substrate Specificity

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