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Candida antarctica lipase B mutant as well as transformation method and application thereof

A technology of Candida Antarctica and mutants, applied in the field of bioengineering, can solve problems such as hindering industrial application, poor enantioselectivity, and prolonging the production cycle, and achieve high-efficiency enantioselectivity and high enantioselectivity , The effect of reducing the production cycle

Active Publication Date: 2017-11-24
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In our previous research, CALB was used to catalyze the alcoholysis of 3-substituted glutaric anhydride to prepare (R)-3-substituted glutaric acid monoester, but the enantioselectivity was poor, and the wild CALB was S-selective. Transform it, fine-tune the active pocket, and obtain a mutant EF5, at 5°C, its ee R is 98.5%, and decreases with increasing temperature
However, low temperature (5°C) greatly prolongs the production cycle, increases the cost, and hinders its industrial application.

Method used

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  • Candida antarctica lipase B mutant as well as transformation method and application thereof
  • Candida antarctica lipase B mutant as well as transformation method and application thereof
  • Candida antarctica lipase B mutant as well as transformation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Selection and screening of mutation sites

[0041] The active pocket of CALB is mainly composed of two parts: the acyl-binding pocket and the alcohol-binding pocket. The catalytic triad Asp187-His224-Ser105 is located in between. The acyl binding pocket is mainly composed of A141, L144, V149, D134, T138, and Q157, the alcohol binding pocket is mainly composed of T42, K47, W104, L278, A281, and A282, and A281, A282, and I285 are oriented toward the alcohol moiety of the substrate, limiting the size of the channel.

[0042] Selection of sites: Acyl pockets (D134, A148, V149, I189, V190, and Q157), alcohol pockets (T42, T43, W104, A281, and A282), A281 and A282 were taken into account because they are located at the channel; additionally , the proximity of D223 and T186 to the catalytic histidine, aspartic acid may have an effect on the conformation of the active pocket, taking into account, as figure 1 shown.

Embodiment 2

[0043] Example 2: Experimental verification of the effect of amino acid residues in simulated screening on enantioselectivity

[0044] For the amino acid residues D134, A148, V149, I189, V190, Q157, T42, T43, W104, A281, A282, D223 and T186 in the simulation screening, the enantioselectivity was verified by constructing a mutation library and high-throughput screening Impact. Combine the above mutation sites into library 1 (A148 / V149), library 2 (I189 / V190), library 3 (Q157), library 4 (T42 / T43), library 5 (W104), library 6 (A281 / A282) , library 7 (D223), library 8 (T186), library 9 (D134), construct the above nine mutant libraries, and through high-throughput screening, screen all mutants in all mutant libraries to verify enantiomeric selection Sexual changes. After screening nearly 7,000 mutants, according to the screening results, only D223V, A281S and D223V / A281S had significant changes in enantioselectivity, and A282S, W104A, and Q157N were selected for experimental ver...

Embodiment 3

[0045] Embodiment 3: the construction of mutant

[0046] Six mutants D223V, A281S, A282S, W104A, Q157N and D223V / A281S were successfully constructed by site-directed mutagenesis. First, use the plasmid connected with the parental EF5 gene on the T vector as a template, respectively with SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO .10, SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14 are primers, utilize high-fidelity enzyme PrimerStar to amplify, utilize restriction endonuclease XhoI and XbaI double enzyme digestion (37 °C, 3h), the mutant gene was recovered. The obtained mutant gene was ligated with the PGAPZαA expression vector recovered by the same endonuclease digestion at 16°C overnight, and the ligated product was transformed into JM109 competent cells for amplification. The recombinant plasmid was extracted, linearized with endonuclease AVrII (37°C, 3h), recovered from the column, electrotransformed into GS115 compet...

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Abstract

The invention discloses a Candida antarctica lipase B mutant as well as a transformation method and application thereof, belonging to the technical field of bioengineering. According to the transformation method, a key locus capable of decreasing dynamics of the conformation of a pocket is found by virtue of conformational dynamics engineering of a combined pocket of CALB through molecular dynamics simulation, and an optimal mutant is designed, so that the limitation that previous enzyme EF5 only presents high enantio-selectivity at a relatively low temperature is overcome, and the optimal mutant is experimentally verified. Finally, the obtained optimal mutant D223V / A281S is used for catalyzing and preparing (R)-3-substituted glutaric acid alkyl monoester compounds, an eeR value of the optimal mutant can reach above 99% under an industrially acceptable temperature (20-55 DEG C), the yield is more than 80.0%, the space time yield can reach 107.54g.L<-1>d<-1>, the cost caused by a previous low-temperature condition is greatly lowered, the production period is shortened, and foundation is laid for the industrialization of the (R)-3-substituted glutaric acid alkyl monoester compounds.

Description

technical field [0001] The invention relates to a mutant of Candida antarctica lipase B, a transformation method and an application thereof, and belongs to the technical field of bioengineering. Background technique [0002] Rosuvastatin Calcium, also known as Keding, belongs to the third generation of statin drugs, which can effectively reduce the concentration of low-density cholesterol, reduce the content of triglyceride and apoprotein B, and at the same time increase high-density cholesterol. The drug is mainly obtained by condensing a chiral side chain (phosphonium salt) with a pyrimidine core through a ylide reaction. In the synthetic route of rosuvastatin calcium, the reaction steps that can be replaced by biological enzymatic methods include: the synthesis of side chain phosphonium salt ; side chain carbonyl reduction; side chain terminal ester hydrolysis. (3R)-TBDMSO Methyl glutarate is an important precursor for the assembly of phosphonate side chains, and its abs...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12N15/63C12P9/00A61K38/46A61P3/06
CPCA61K38/00C12N9/20C12P9/00A61P3/06C12P7/62C12Y301/01003
Inventor 刘立明杨彬
Owner JIANGNAN UNIV
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