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Glutamine transaminase variant with improved catalytic activity and thermal stability

A glutamine and transaminase technology, applied in the field of transglutaminase variants, can solve the problems of TGase poor stability, high cost, limited application space, etc., and achieve the effect of improving thermal stability and specific enzyme activity

Pending Publication Date: 2022-04-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The poor stability of TGase has always been the main problem limiting its application space
Poor stability makes TGase not easy to store, and the loss is relatively large during transportation and use
In food processing, the addition of TGase is often accompanied by temperature rise to assist food processing. During this period, the loss of TGase will increase the cost.

Method used

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  • Glutamine transaminase variant with improved catalytic activity and thermal stability
  • Glutamine transaminase variant with improved catalytic activity and thermal stability
  • Glutamine transaminase variant with improved catalytic activity and thermal stability

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0076] Preparation of variants

[0077] Transglutaminase variants of the invention can be prepared using any mutagenesis procedure known in the art (eg, site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.).

[0078] Site-directed mutagenesis is a technique for introducing one or more (eg, several) mutations at one or more defined sites in the polynucleotide encoding said parental transglutaminase.

[0079] Site-directed mutagenesis can be accomplished in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. In vitro site-directed mutagenesis can also be performed by cassette mutagenesis, which involves cleavage by restriction enzymes at a site in the plasmid containing the polynucleotide encoding the parental transglutaminase and subsequent insertion of the oligo containing the mutation into Nucleotides are linked in polynucleotides. Typically, the restriction enzym...

Embodiment 1

[0130] The preparation and thermal stability of some transglutaminase variants will be described below in conjunction with one of the specific embodiments.

[0131] The involved Escherichia coli JM109 and Escherichia coli E.coli BL21 (DE3) were purchased from takara-Baori Medical Biotechnology (Beijing) Co., Ltd., and the pET-22b (+) plasmid was purchased from Novagen (the above-mentioned strain E.coli BL21 (DE3) can be purchased and does not need to be preserved for patent procedures), neutral protease is purchased from Beijing Soleibao Technology Co., Ltd. (product number Z8032), Blunting Kination Ligation (BKL) Kit and HS DNA Polymerase was purchased from Biotech Biotechnology (Beijing) Co., Ltd., and Bradford Protein Concentration Assay Kit (detergent-compatible type) was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.

[0132] The media involved are as follows:

[0133] LB liquid medium: yeast powder 5.0g / L, tryptone 10.0g / L, NaCl 10.0g / L, ampicillin 100μg / L. ...

Embodiment 2

[0152] Example 2: Application of transglutaminase variants in meat processing

[0153] Utilize the transglutaminase variant 2 mature enzyme prepared in embodiment 1 to carry out the processing of dried rabbit meat, specifically:

[0154] S1, mince the rabbit meat and dice the chicken to obtain mixed meat;

[0155] S2. After uniformly mixing salt, compound phosphate and water, adding the mixed meat obtained in S1, mixing evenly, sealing with a plastic wrap, and marinating for 10 hours to obtain cured meat;

[0156] S3. Homogenizing the marinated meat to obtain mixed minced meat;

[0157] S4. Add transglutaminase, ovalbumin, ginger powder, etc. to the mixed minced meat obtained in S3, and stir evenly at a temperature of 2° C. to obtain mixed minced meat;

[0158] S5. After sealing the mixed minced meat obtained in S4 with a plastic wrap, place it in a water bath at a temperature of 60° C. for 0.2 h, extrude, dry, and cool naturally to obtain a semi-finished product;

[0159] ...

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Abstract

The invention discloses a glutamine transaminase variant with improved catalytic activity and thermal stability, and belongs to the fields of biology and food. The glutamine transaminase variant provided by the invention is an enzyme with improved thermal stability, and comprises a substitution corresponding to the 287th site of a polypeptide shown as SEQ ID NO.1. The glutamine transaminase variant disclosed by the invention can be applied to the aspects of food treatment, processing, conversion and the like, and can be used for maintaining or improving the quality, consistency, elasticity, moisture or viscosity of food. The glutamine transaminase variant with improved thermal stability is more beneficial to its stable application in harsh industrial environments, such as artificial meat paste processing and meat ball processing.

Description

technical field [0001] The invention relates to a transglutaminase variant with improved catalytic activity and thermal stability, which belongs to the fields of biology and food. Background technique [0002] Transglutaminase (Transglutaminase, hereinafter referred to as TGase, EC 2.3.2.13) derived from Streptomyces mobaraenesis AAT65817 is an industrial enzyme preparation widely used in the food industry. It can catalyze the reaction between the γ-carboxamide group of glutamine residues in proteins and small molecules and the amino groups in acyl acceptors to promote the formation of covalent cross-links. At present, TGase is used as an additive in food, especially during food pretreatment, such as adding TGase to meat, soybean products and dairy products to improve the appearance and taste of food. [0003] The poor stability of TGase has always been the main problem limiting its application space. The poor stability makes TGase not easy to store, and the loss is relati...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54A23C9/12A23C19/032A23C19/06A23G9/36A23L11/00A23L13/40A23L13/70A23L17/00A23L27/60
Inventor 刘松王兴隆杜建辉周景文陈坚堵国成
Owner JIANGNAN UNIV
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