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Thermal stability improved lipase mutant constructed through orthogenesis

A technology of thermal stability and directed evolution, 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: 2012-09-05
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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  • Thermal stability improved lipase mutant constructed through orthogenesis
  • Thermal stability improved lipase mutant constructed through orthogenesis
  • Thermal stability improved lipase mutant constructed through orthogenesis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Construction of a Pichia pastoris library expressing lipase mutants using the error-prone PCR method

[0052] 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:

[0053]

[0054] Wherein, the sequences of the upstream primer F and the downstream primer R are:

[0055] F: 5'-TCAAGATCCCTAGGGTTCCTGTTGGTCATAAAGGTTC-3';

[0056] R: 5'-AATTCCAGTGCGGCCGCTTACAAACAGCTTCCTTCG-3'.

[0057] PCR amplification conditions: 94°C for 3 min; 94°C for 1 min, 59°C for 1 min, 72°C for 2 min, 30 cycles; 72°C for 10 min.

[0058] After the error-prone PCR amplification product is purified by DNA purification kit, restriction endonuclease Avr II and not I respectively digest the error-prone PCR amplification product and the plasmid pPIC9K, connect to obtain the expression plasmid containing the mutant gene, and transform it into E.coli JM109 Compet...

Embodiment 2

[0060] Example 2, Construction of a Pichia pastoris library expressing lipase mutants using the DNA Shuffling method

[0061] 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:

[0062] extract easy The genome of the Pichia pastoris library expressing the lipase mutant constructed by the wrong PCR method was digested with DNase I for 30 min, and the digested product was extracted with phenol-chloroform to remove the protein and dissolved in 30 μL sterile water. Using the genome as a template, proceed as follows:

[0063] Step 1: PCR reaction system:

[0064] Taq (2.5U) 0.5 μL 5×buffer (Mg 2+ plus) 10 μL Genome 0.5 μL dNTP (25mmol / L) 4 μL dd H 2 o 34.5 μL

[0065] PCR amplification conditions: 94°C for 3 min; 10 cycles of 94°C for 1 min, 59°C for 1 min, and 72°C for 2 min; 72°C for 10 min.

[0066] Step 2: Add...

Embodiment 3

[0068] Embodiment 3, the screening of lipase mutant with high enzymatic activity

[0069] 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.

[0070] 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 min, cool to room temperature, and pour Fast- The blue RR dye was about 15 mL, and within 2 minutes, the black-brown colony of the single clone was darker than that of the control colony, which was the target strain of the primary screening.

[0071] 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 min. Insert the strain obtained from the primary screening into it, and use the Pichia pastoris expressing the lipas...

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Abstract

The invention relates to a thermal stability improved lipase mutant constructed through orthogenesis, belonging to the technical field of enzymatic genetic engineering. The invention discloses a thermal stability improved lipase mutant obtained through a molecular biological technology by using a Rhizopuschinensis CCTCC M201021 lipase as a parent. In amino acid sequences of each mutant, the related amino acid mutant is one or more 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 represented by a half-life period of t50 at 65 DEG C, and the thermal stability of the mutants are improved compared with the parent of Rhizopuschinensis lipase. The invention also discloses a DNA (Deoxyribonucleic Acid) sequence, an expression vector and a host cell for encoding the lipase mutant.

Description

technical field [0001] The invention relates to a lipase mutant with improved thermostability constructed through directed evolution, 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. Most Rhizopus lipases have high 1,3-position selectivity, so they are often used in oil processing. However, oil processing usually needs to be carried out at higher temperatures, and Rhizop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12N15/55C12N15/81C12N1/19C12R1/84
Inventor 喻晓蔚徐岩王睿
Owner 金湖县农副产品营销协会
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