Lipase mutant and application thereof in decontamination

A lipase and mutant technology, applied in the field of enzyme engineering, can solve the problems of poor lipase stability, loss of enzyme activity, loss of activity, etc., and achieve the effect of improving pH stability

Active Publication Date: 2020-03-24
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] However, most applications of lipase have certain requirements on the stability of lipase. For example, in the process of drug synthesis, higher temperatures are often used, which has higher requirements on the thermal stability of lipase. Need to use strong alkali in leather processing and papermaking process, this has higher requirement to the pH stability of lipase under alkaline condition, and existing most lipase stability is all relatively poor, for example, derives from The lipase RAL of Rhizopus arrhizus has an optimum temperature of only 35°C. After heat treatment at 50°C for 10 minutes, only 40% of the enzyme activity remains, and after 40 minutes of treatment, the enzyme activity will be completely lost. It will lose more than 70% of its activity when the pH is greater than 9.5, which undoubtedly greatly limits the further development of lipase in the above applications

Method used

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  • Lipase mutant and application thereof in decontamination
  • Lipase mutant and application thereof in decontamination
  • Lipase mutant and application thereof in decontamination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1: Expression of wild-type lipase

[0083] Specific steps are as follows:

[0084] The gene encoding the lipase whose amino acid sequence is shown in SEQ ID NO.1 is amplified from Rhizopus chinonsis; the obtained gene proRCL is connected to the pPIC9K vector by Not I and EcoR I double digestion to obtain the recombinant Plasmid pPIC9K-proRCL; Transform the recombinant plasmid pPIC9K-proRCL into Escherichia coli JM109 to obtain E. L Kanamycin), cultured at 37°C for 10 h, screened positive clones and confirmed by sequencing, obtained the correct recombinant plasmid pPIC9K-proRCL; linearized the recombinant plasmid pPIC9K-proRCL with Sal I, and electrotransformed into Pichia In the yeast GS115, the Pichia GS115 / pPIC9K-proRCL was obtained; the Pichia GS115 / pPIC9K-proRCL was spread on the MD plate (containing 100mg / L kanamycin), and cultured at 37°C for 10h Afterwards, the genome was extracted, and positive clones were identified by PCR to obtain positive transform...

Embodiment 2

[0088] Example 2: Preparation and expression of lipase mutants

[0089] Specific steps are as follows:

[0090] Using the whole plasmid PCR technology, the recombinant plasmid pPIC9K-proRCL obtained in Example 1 was used as a template for site-directed mutation to obtain mutants S151N, S235A, K171R, S343Y, G275A, Q332F, D310V, L180H, F316C\G340C, S178C\Q238C, F316C\G340C\S178C\Q238C、S235A\S343Y\Q332F\D310V、S151N\K171R\G275A\L180H、S235A\S343Y\Q332F\D310V\S151N\K171R\G275A\L180H、S235A\S343Y\Q332F\D310V\S151N\ K171R\G275A\L180H\F316C\G340C\S178C\Q238C, D159R and T319V;

[0091] Wherein, the mutation primer is:

[0092] S151N:

[0093] S151N-Fm: 5'-CTGCTTACTGTCGT AAC GTCGTTCCAGGTACC-3' (SEQ ID NO. 4);

[0094] S151N-Rm: 5'-GGTACCTGGAACGAC GTT ACGACAGTAAGCAG-3' (SEQ ID NO. 5);

[0095] S235A:

[0096] S235A-Fm: 5'-GTTTCCTTTCC GCA TACAACCAAGTTGTCAAAG-3' (SEQ ID NO. 6);

[0097] S235A-Rm: 5'-CTTTGACAACTTGGTTGTA TGC GGAAAGGAAAC-3' (SEQ ID NO. 7);

[0098] K171R:

[00...

Embodiment 3

[0136] Embodiment 3: Research on the enzymatic properties of different lipases

[0137] Specific steps are as follows:

[0138] 1. The optimum temperature (T opt )

[0139] The wild type obtained in Example 1 and the L1, L2, L3, L4, For the lipase activity of L5, L6, L7, L8, L9, L10, L11, L12, L13, L14, L15, L16 and L17, the highest enzyme activity is 100%, and the other enzyme activities are compared with it to calculate the relative enzyme activity live to investigate the optimum action temperature of the enzyme (see the test results in Figure 3-4 ).

[0140] Depend on Figure 3-4 It can be seen that the optimum temperature of the wild type is 40°C; the optimum temperature of L1, L2, L3, L4, L5, L6, L8, L9, L10, L16 and L17 is also 40°C, which is no different from that of the wild type. The optimum temperature of L7 increased to 45°C; the optimum temperature of L13 increased to 43°C; the optimum temperature of L11 and L12 increased to 45°C; the optimum temperature of ...

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Abstract

The invention discloses a lipase mutant and its application in decontamination, and belongs to the technical field of enzyme engineering. The lipase mutant of the invention has good thermal stability,good pH stability and good washing performance. Therefore, the lipase mutant of the invention has very important applications in decontamination, oil processing, dairy processing, pasta processing, meat processing, drug synthesis, diesel oil synthesis, polymer synthesis, chiral compound synthesis, leather production, detergent preparation, papermaking and the like.

Description

technical field [0001] The invention relates to a lipase mutant and its application in decontamination, belonging to the technical field of enzyme engineering. Background technique [0002] Lipase (Lipase, EC 3.1.1.3, glyceride hydrolase) is a general term for a class of enzymes that hydrolyze fat. In some reaction systems, lipase can catalyze hydrolysis, alcoholysis, transesterification, hydrogenolysis, and indirect redox transesterification. In addition, lipase also has the ability to hydrolyze racemic mixtures and synthesize esters and synthesize peptide bonds. Therefore, lipase has important applications in many fields, such as: [0003] 1. Lipase can be used in oil processing, and the lipase hydrolysis of oil can be carried out at normal temperature and pressure, so it will not denature biological substances such as highly unsaturated fatty acids and tocopherol in oil; [0004] 2. Lipase can be used in dairy processing. Applying lipase to hydrolyze milk fat in dairy p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12N15/55C11D3/386C11D7/42C11D11/00
CPCC11D3/38627C12N9/20C12N15/815C12Y301/01003
Inventor 王睿喻晓蔚徐岩
Owner JIANGNAN UNIV
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