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A kind of preparation method of partially inactivated chymosin

A technology of rennet and preparation method, which is applied in the field of protein refolding, can solve the problems of inactivity, etc., and achieve the effect of simple operation, high enzyme activity and obvious refolding effect

Active Publication Date: 2020-04-17
SHANDONG POLYTECHNIC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the expression system itself or the treatment of Guo Chen in the experiment, these proteins mostly exist in an inactive form and need to be refolded

Method used

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  • A kind of preparation method of partially inactivated chymosin
  • A kind of preparation method of partially inactivated chymosin
  • A kind of preparation method of partially inactivated chymosin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The chymosin solution containing 0, 0.50, 0.75, 1.00, 1.25 and 1.5mmol / L ectoine was placed at 69°C for 0, 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, respectively. 9min, 10min, obtain partially inactivated chymosin; figure 1 It is the time-course curve of the change of chymosin activity under different concentrations of ectoine. Depend on figure 1 It can be seen that after adding ectoine, the thermal stability of chymosin in each group is reduced to varying degrees. In each group containing ectoine, when ectoine is 0.75mmol / L, the heat treatment time for enzyme activity can be measured At the longest time, 9% of the relative activity remained after 7 minutes of treatment; for other concentrations, the relative activity dropped to about 10% at 4-6 minutes.

Embodiment 2

[0040] The chymosin solution containing 0.75mmol / L ectoine was placed at 61, 63, 65, 67, 68, 69 and 70°C for heat inactivation treatment for different periods of time to obtain partially inactivated chymosin; said figure 2 It is the time-course curve of the change of chymosin activity under the same ectoine concentration.

[0041] Refolding experiment

[0042] 1. Effect of heat treatment time on refolding of chymosin

Embodiment 3

[0044] The partially inactivated rennet in Example 1 was rapidly cooled to 4° C. and kept in a refrigerator for static storage. Samples were taken every 6 hours from 0 to 42 hours, and the water bath was preheated to 35°C to measure the change of residual rennet activity. image 3 ,Depend on image 3It can be seen that with the prolongation of renaturation time, the activity of chymosin increased. The residual enzyme activity of the non-renaturation control group was 61.5% after incubation for 1 minute; after 42 hours of renaturation, the residual enzyme activity rose to 83.3%. In addition, the heat treatment time at which rennet activity could be detected was also significantly prolonged. In the control group, the enzyme activity could not be detected after 6 minutes of heat treatment. After 12 minutes of heat treatment, 8.8% of the remaining enzyme activity could be detected in the experimental group after renaturation for 36 hours and 42 hours.

[0045] 2. Effect of refol...

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Abstract

The invention belongs to the technical field of protein renaturation and especially relates to a preparation method for partially inactivated chymosin. The preparation method comprises the following steps: (1) placing a chymosin solution containing ectoine under the temperature at 61-70 DEG C and treating for 1-20min, thereby acquiring the partially inactivated chymosin, and (2) quickly cooling the partially inactivated chymosin prepared in the step (1) to 4 DEG C, standing by and refrigerating for 0-42h, thereby acquiring the chymosin after renaturation. The method is different from the treating method for high concentration urea, and the like; a heat inactivation method is adopted for preparing the partially inactivated chymosin; the operation is simple and easy, the repeatability is high and the state of the partially inactivated chymosin is more approximate to the heat inactivation state of enzyme; the inactivated chymosin prepared according to the method has the characteristics of obvious renaturation effect, high enzyme activity after renaturation, and the like.

Description

technical field [0001] The invention belongs to the technical field of protein refolding, in particular to a preparation method of partially inactivated chymosin. Background technique [0002] Rennet is an essential enzyme preparation for making cheese and casein, and it can hydrolyze Phe of κ-casein in milk 105 -Met 106 Peptide bonds make the milk coagulate, and after a series of processing, it is made into cheese. In addition, rennet also has a clinical effect in treating dyspepsia in children. Like other enzyme preparations, rennet may be denatured and inactivated by high heat, freeze-thaw, radiation, organic solvents, etc. during production and application, resulting in loss of enzyme activity. Cheese processing has strict requirements on the amount of rennet added, and as a pharmaceutical preparation, it is also required that rennet can maintain high stability. Methods such as immobilization, chemical modification, and protein engineering can all improve the thermal...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/50
CPCC12N9/50C12Y304/23004
Inventor 武彬
Owner SHANDONG POLYTECHNIC
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