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Lipase mutant with improved heat stability as well as preparation method and application thereof

A technology of thermal stability and mutant, applied in the field of molecular biology, can solve the problems of poor thermal stability, poor stability, low catalytic ability, etc., and achieve the effect of improving thermal stability and thermal stability.

Active Publication Date: 2019-03-15
浙江容锐科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, although there are also many lipases that have unique advantages in some aspects, it is difficult to achieve large-scale applications due to their low catalytic ability or poor stability. For example, Rhizopus oryzae lipase ROL (Rhizopus oryzae lipase) has good 1, 3-position specificity, preferentially catalyze medium and long chain fatty acids, although they have good selectivity, their use is greatly limited due to poor thermal stability

Method used

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  • Lipase mutant with improved heat stability as well as preparation method and application thereof
  • Lipase mutant with improved heat stability as well as preparation method and application thereof
  • Lipase mutant with improved heat stability as well as preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0051] 1. Selection of hotspot amino acids

[0052] Multiple sequence alignment was performed using the online analysis website ClustalW (http: / / www.genome.jp / tools-bin / clustalw). Lipase RML from Rhizomucor miehei and lipase TLL from Thermomyces lanuginosa have better heat resistance than lipase ROL from Rhizopus oryzae, And it has high homology with ROL, the sequence homology is 53.4% ​​and 29.7%, respectively. Therefore, these two lipases were selected for multiple sequence alignment with ROL to identify potential sites that could enhance the thermostability of ROL.

[0053] Each sequence was uploaded in FASTA format and the results were uploaded to Espript 3.0 (http: / / espript.ibcp.fr / ESPript / cgi-bin / ESPript.cgi) and plotted for a more intuitive alignment.

[0054] The result is as figure 1 As shown, amino acid residues marked with asterisks indicate potential sites selected based on multiple sequence alignment results that may affect the thermal stability of ROL. Among ...

Embodiment 2

[0074] 1. Design of disulfide bonds

[0075]Upload the crystal structure of ROL (PDB ID: 1LGY) to Disulfide by Design 2 (DbD2, http: / / cptweb.cpt.wayne.edu / DbD2 / index.php), and perform calculations. After the initial analysis of the protein structure, the prediction of the protein structure begins, analyzing the potential disulfide bond positions. The results showed that 32 pairs of amino acid residues may form disulfide bonds after mutation to cysteine ​​(Table 2). Score and classify predictions based on energy, bad contacts, thermal mobility, sequence separation, etc. According to the scoring results, the 9 pairs of amino acids with the highest score (>90) were selected for mutation.

[0076] Table 2 Disulfide bond design results

[0077]

[0078]

[0079] Cysteine ​​was introduced at the specific site of lipase ROL by whole-plasmid PCR. PCR primers are shown in Table 3.

[0080] Table 3 Mutation primer design table based on disulfide bond design results

[0081] ...

Embodiment 3

[0092] Enhanced thermostability through combinatorial mutations of hot spots and disulfide bonds

[0093] It is possible to obtain mutants with further improved thermostability by combining mutations at the amino acid sites that affect the thermostability of the enzyme. Based on the results obtained above, we performed combined mutations on ROL, and the mutation sites were V209L, D262G and E190C and E238C. Mutant V209L-D262G still retains about 76% of its initial activity after being incubated at 55°C for 30 minutes;

[0094] Since the mutant E190C / E238C showed improved thermostability at 65°C, the thermostability test of the combined mutations was also performed at 65°C. Such as Figure 7 As shown, compared with the mutant E190C / E238C, all the combined mutants showed enhanced thermal stability, among which, the triple-point mutant V209L-E190C-E238C still retained about 42% of the initial activity; D262G-E190C-E238C retained about 45% of the initial activity after incubatio...

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Abstract

The invention discloses a lipase mutant with improved heat stability as well as a preparation method and application thereof, and belongs to the technical field of biology. The amino acid sequence ofthe lipase mutant is as shown in SEQ ID NO.1 or SEQ ID NO.3 or SEQ ID NO.5 or SEQ ID NO.7 or SEQ ID NO.9 or SEQ ID NO.11 or SEQ ID NO.13. Through multiple sequence alignment and the disulfide bond predicting result, rhizopus oryzaelipase ROL is subjected to heat stability transformation, so that the heat stability of the rhizopus oryzaelipase ROL is greatly improved; meanwhile, by virtue of a computer simulating technology, the heat stability improving principle is explained in molecular level. The heat stability of the lipase mutant provided by the invention is significantly improved; and through combination with the high Sn-1,3 selectivity of the lipase mutant, the lipase mutant has industrialized application value.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a lipase mutant and its preparation method and application. Background technique [0002] Lipase, triacylglycerol acyl hydrolase, catalyzes the hydrolysis of natural substrate oils to produce fatty acids, glycerol and mono- or di-glycerides. Because lipase can catalyze a series of reactions, it occupies an important position in the field of biocatalysis. So far, a variety of lipases have realized their value in industrial production, including lipase RML, CALB and so on. However, although there are also many lipases that have unique advantages in some aspects, it is difficult to achieve large-scale applications due to their low catalytic ability or poor stability. For example, Rhizopus oryzae lipase ROL (Rhizopus oryzae lipase) has good 1, 3-Position specificity, preferentially catalyzing medium and long-chain fatty acids, although they have good selectivity, their us...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12N15/55C12N15/81C12P7/64
CPCC12N9/20C12N15/815C12P7/6454C12P7/649C12Y301/01003Y02E50/10
Inventor 杨立荣吴绵斌赵炯烽林建平
Owner 浙江容锐科技有限公司
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