Lipase mutants with enhanced thermal stability

A thermal stability and mutant technology, applied in the field of genetic engineering of enzymes, can solve problems such as poor stability, and achieve the effect of improving thermal stability

Active Publication Date: 2011-02-16
金湖县农副产品营销协会
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the contrary, the stability is poor

Method used

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  • Lipase mutants with enhanced thermal stability
  • Lipase mutants with enhanced thermal stability
  • Lipase mutants with enhanced thermal stability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1. Construction of a Pichia pastoris library expressing lipase mutants using the error-prone PCR method

[0051] Nucleotide mutations were introduced into the lipase gene proRCL of Rhizopus sinensis in vitro by error-prone PCR. The reaction conditions for error-prone PCR are as follows:

[0052] dCTP (25mmol / L) 2μL

[0053] dTTP (25mmol / L) 2μL

[0054] dGTP (10mmol / L) 1μL

[0055] dATP (10mmol / L) 1μL

[0056] F (20pmol / μL) 1μL

[0057] R (20pmol / μL) 1μL

[0058] Mg 2+ (25mM) 14μL

[0059] mn 2+ (5mM) 1.5μL

[0060] pPIC9K-proRCL

[0061] (Plasmid carrying the Rhizopus sinensis lipase gene proRCL) 0.5 μL

[0062] Taq DNA polymerase (2.5U) 1 μL

[0063] 10×PCR buffer (without MgCl 2 ) 5μL

[0064] wxya 2 O 20 μL

[0065] Wherein, the sequences of the upstream primer F and the downstream primer R are:

[0066] F: 5'-TCAAGATCCCTAGGGTTCCTGTTGGTCATAAAGGTTC-3';

[0067] R: 5'-AATTCCAGTGCGGCCGCTTACAAACAGCTTCCTTCG-3'.

[0068] PCR amplification conditi...

Embodiment 2

[0071] Example 2, Construction of a Pichia pastoris library expressing lipase mutants using the DNA Shuffling method

[0072] The mutation sites in the lipase mutants constructed by error-prone PCR were randomly combined by DNA shuffling method. The conditions for DNA shuffling are as follows:

[0073] The genome of the Pichia pastoris library expressing lipase mutants constructed by the error-prone PCR method was extracted, digested with DNase I for 30 min, and the digested product was extracted with phenol-chloroform to remove protein and dissolved in 30 μL sterile water. Using the genome as a template, proceed as follows:

[0074] Step 1: PCR reaction system:

[0075] Taq (2.5U) 0.5μL

[0076] 5×buffer(Mg 2+ plus) 10μL

[0077] Genome 0.5 μL

[0078] dNTP (25mmol / L) 4μL

[0079] dd H 2 O 34.5 μL

[0080] PCR amplification conditions: 94°C for 3min; 94°C for 1min, 59°C for 1min, 72°C for 2min, 10 cycles; 72°C for 10min.

[0081] Step 2: Add 1 μL each of primers F a...

Embodiment 3

[0083] Embodiment 3, the screening of lipase mutant with high enzymatic activity

[0084] Copy the Pichia library stored on the MD plate to the MM plate, culture at 30°C for 2 days, and use the Pichia expressing the lipase of the parent Rhizopus sinensis as the control bacteria.

[0085] Plate initial screening: Add 200 μL of methanol to the cover of the MM plate every 12 hours to induce the expression of recombinant lipase. After 2-3 days of induction, heat the plate at 65°C for 60 minutes, cool to room temperature, and pour Fast-blue RR staining evenly on the plate. Inject about 15mL, and within 2 minutes, the dark brown colony of the monoclonal is darker than the control colony, which is the strain of primary screening purpose.

[0086] 96-well plate screening method: add 300 μL of BMGY medium to a 1.8 mL / well (flat bottom) 96-well plate, and sterilize at 121° C. for 20 minutes. Insert the strain obtained from the primary screening into it, and use the Pichia pastoris expr...

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Abstract

The invention relates to lipase mutants with enhanced thermal stability, which belongs to the technical field of enzyme gene engineering. Rhizopus chinensis CCTCC No.M201021 lipase used as the parent is processed by molecular biotechnology to obtain the lipase mutants with enhanced thermal stability. In the amino acid sequences of the mutants, the related amino acid mutation(s) is(are) one or a plurality of Met101Thr, Glu107Gly, Ala129Ser, Ser151Asn, Cys160Leu, Lys161Arg, Pro168Leu, Pro168His, Leu180His, Asp182Tyr, Thr183Ala, Thr218Ser, Lys219Asp, Ala230Phe, Ser234Phe, Val261Gly, His317Pro, Val329Ala, Glu363Arg, Asn366Asp and Ser373Cys. The mutants are expressed by half-life period t50 at 65 DEG C. The thermal stability of the mutants is enhanced as compared with the parent Rhizopus chinensis lipase. The invention also discloses a DNA sequence, an expression carrier and a host cell for coding the lipase mutants.

Description

technical field [0001] The invention relates to a lipase mutant with improved thermostability, in particular to a lipase mutant with improved thermostability obtained by using molecular biology techniques, and belongs to the technical field of enzyme genetic engineering. Background technique [0002] Lipase (EC 3.1.1.3) can not only catalyze the hydrolysis of oil, but also catalyze ester synthesis, transesterification, acid hydrolysis and other reactions in the non-aqueous phase. It is widely used in chemistry, food, pharmaceutical and detergent or bioenergy in industry. Microorganisms are an important source of lipase, and Rhizopus is an important producer of microbial lipase. Today, more than 30 Rhizopus lipases have been commercially produced. Rhizopus lipases mostly have high 1,3-position selectivity, so they are often used in oil processing. However, oil processing usually needs to be carried out at a relatively high temperature, and Rhizopus lipase belongs to a medi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12N9/20
Inventor 喻晓蔚徐岩王睿
Owner 金湖县农副产品营销协会
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