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Rennin mutant with improved enzyme activity and thermal stability

A rennet and mutant technology, applied in the field of enzyme engineering, can solve the problems of decreased rennet activity, undiscovered research on bacterial-derived rennet site-directed mutation, etc., to reduce thermal stability, improve commercial value, and process Concise and clear effect

Active Publication Date: 2017-05-31
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chitpinityol et al. (1998) used site-directed mutagenesis to change threonine to aspartic acid at position 77, resulting in a decrease in the activity of the mutant compared with the recombinant wild-type chymosin; so far, for calf chymosin, etc. There are many studies on animal chymosin and fungal chymosin such as Mucor micromyces, but no research on site-directed mutation of bacterial chymosin has been found in China

Method used

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  • Rennin mutant with improved enzyme activity and thermal stability
  • Rennin mutant with improved enzyme activity and thermal stability
  • Rennin mutant with improved enzyme activity and thermal stability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] (1) Determine the site-directed mutation site of Bacillus amyloliquefaciens-derived chymosin gene

[0035] Simulate the tertiary structure of Mucor pumila chymosin gene by computer, and compare it with the tertiary structure and nucleic acid sequence of bovine chymosin, select three key sites for site-directed mutation, and select respectively those that may be related to hydrolysis or heat stability Sex-related loci 75, 81 and 222.

[0036] The 75th position was originally leucine, the 81st position was originally valine, and the 222nd position was originally methionine. The present invention pre-mutates the 75th position into glutamine, the 81st position into aspartic acid, and the 222nd position Point mutations to threonine were performed by a PCR-mediated one-step method.

[0037] Primers were designed for the selected mutation sites by DNAMAN, as shown in Table 1.

[0038] Table 1 Primer sequences used for site-directed mutagenesis

[0039]

[0040] (2) PCR-m...

Embodiment 2

[0049] (1) Expression of chymosin mutants in Pichia pastoris

[0050] For the expression steps of the chymosin mutant in Pichia pastoris, refer to the relevant operation steps in Example 1 of the patent application with application number 201611157248.4.

[0051] The recombinant vectors pPIC9K-cMCE-Leu75Gln, pPIC9K-cMCE-Val81Asp, pPIC9K-cMCE-Met222Thr, pPIC9K-cMCE-Leu75Gln-Val81Asp, pPIC9K-cMCE-Leu75Gln-Met222Thrp, and PIC9K-cMCEVal81Asphr were linearized with acIThrase , 2500V, 5ms electrotransformation of Pichia pastoris GS115 competent cells onto MM and MD plates successively, and His + and Mut + Type transformant P-cMCE.

[0052] Select transformants for colony PCR verification, perform agarose gel electrophoresis on the PCR products, select transformants in BMGY medium, and culture them at 30°C / 300rpm until OD600=2~6; collect the cells, resuspend the cells with BMMY, Make OD600=5.0 or so, add anhydrous methanol to the medium to a final concentration of 0.5% (v / v, 5mL m...

Embodiment 3

[0057] (1) Comparison of chymosin activity, protease activity and C / P value properties

[0058] The C / P value refers to the ratio of rennet activity to protease activity per unit mass of pure enzyme powder. It can effectively and accurately characterize the milk-clotting ability of rennet.

[0059] Mutant plasmids Leu75Gln, Val81Asp, Met222Thr, Leu75Gln-Val81Asp, Leu75Gln-Met222Thr, Val81Asp-Met222Thr were transformed into Pichia pastoris GS115, 30 transformants were randomly selected, and the inoculation amount was 0.5%. The starting strain P-cMCE without mutation was used as a control After BMGY culture and methanol induction, the pH is 6.5-6.7, the fermentation temperature is 30°C, and the fermentation is carried out in BMMY medium for 96 hours. The supernatant is collected by centrifugation, and the rennet activity and proteolytic activity of the crude enzyme liquid are measured, and the C / P ratio is calculated. The results are shown in Table 2.

[0060] It can be seen f...

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Abstract

The invention discloses a rennin mutant with improved enzyme activity and thermal stability, and belongs to the technical field of enzyme engineering. Glutamine replaces leucine at the 75th position of rennin, threonine replaces methionine at the 222nd position, the amino acids at the positions are subjected to amphimutation to construct the rennin mutant, the mutant is subjected to heterologous expression in pichia pastoris GS115, and expressed crude enzyme is subjected to rennin activation, protease activation and heat resistance detection. The rennin activity of the crude enzyme of double mutants Leu75Gln-Met222Thr reaches 28.0 SU / mL, the C / P value reaches 11.7, the rennin activity and the C / P value are higher than the rennin activity and the C / P value of an original strain P-cMCE by 155.6% and 195.0%. The thermal stability of the rennin mutant after the rennin mutant is maintained at the constant temperature of 45 DEG C for half an hour is reduced by 13.6%.

Description

technical field [0001] The invention relates to a chymosin mutant with improved enzyme activity and thermal stability, belonging to the technical field of enzyme engineering. Background technique [0002] Rennet is an aspartic protease first discovered in the stomach of unweaned calves, which can specifically cleave the peptide bond between Phe105-Met106 of κ-casein in milk, destroying casein micelles and making Milk coagulation, rennet's milk coagulation ability and proteolysis ability make it a key enzyme in cheese production to form texture and special flavor, and is widely used in the production of cheese and yogurt. [0003] Enhancing the milk-clotting ability of chymosin, reducing the ability to hydrolyze protein and reducing heat resistance have always been the hot direction of protein transformation of rennet. Chitpinityol et al. (1998) used site-directed mutagenesis to change threonine to aspartic acid at position 77, resulting in a decrease in the activity of the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64C12N1/19C12N15/81C12R1/84
CPCC12N9/6483C12Y304/23004
Inventor 丁重阳张琦艾连中王琼杭锋苑畅石贵阳
Owner JIANGNAN UNIV
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