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Production method, purification method and applications of protein glutaminase (PG)

A glutaminase and protein technology, which is applied in the field of screening protein glutaminase producing bacteria, can solve the problems of poor substrate specificity, few strains, low enzyme activity, etc.

Active Publication Date: 2017-11-07
EAST CHINA NORMAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are certain shortcomings: excessive use of transglutaminase will cause protein aggregation; protease can hydrolyze the protein as a whole, the substrate specificity is poor, and it is easy to produce bitter taste; peptide glutaminase is sensitive to high molecular weight polypeptides and Protein does not function and deamidation is not high
The purpose of the present invention is to solve the problem of few existing protein-producing glutaminase strains and low enzyme activity, to provide a protein-producing glutaminase that can be safely and efficiently applied to food, and improves protein solubility, foamability, emulsification and stability. New strain of glutaminase

Method used

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  • Production method, purification method and applications of protein glutaminase (PG)
  • Production method, purification method and applications of protein glutaminase (PG)
  • Production method, purification method and applications of protein glutaminase (PG)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Screening and isolation of Chryseobacterium proteolyticum (Chryseobacterium proteolyticum) YF810 bacterial strain

[0057] The YF810 strain was obtained by screening the enrichment medium, which was prepared by mixing 54mL solution A, 6mL 1% CBZ, 60μL mother solution I, 60μL mother solution II, 60μL mother solution III and 120μL mother solution IV.

[0058] Among them, the formula of liquid A is: 5g / L glucose, 0.2g / L KH 2 PO 4 , 0.2g / L MgSO 4 ·7H 2 O, 0.01% NaCl

[0059] The formula of mother liquor Ⅰ is: 20g / L CaCl 2

[0060] The formula of mother liquor Ⅱ is: 2g / L FeSO 4 ·7H 2 O, 5g / L MnSO 4 4H 2 o

[0061] The formula of mother liquor Ⅲ is: 5g / L NaWO 4 4H 2 O, 5g / L NaMO 4 2H 2 o

[0062] The formula of mother liquor IV is: 50g / L CuSO 4 ·5H 2 o

[0063] The screening steps are:

[0064] The first step is to weigh 10g of soil sample, add 90mL of sterile water, put a small amount of glass beads into an Erlenmeyer flask, and vibrate on a shaker...

Embodiment 2

[0070] Example 2 Identification of Chryseobacterium proteolyticum (Chryseobacterium proteolyticum) YF810 strain

[0071] 1. Morphological identification

[0072] (1) Observe the shape of the colony

[0073] After the suspected strains were cultured on LB plates, the colonies were round, with a diameter of 2mm-4mm, neat edges, golden or orange, opaque, and the surface of the colonies was moist, smooth and shiny, such as figure 1 shown.

[0074] (2) Microscope observation

[0075] After Gram staining and spore staining, observation under an ordinary light microscope showed that the strain was rod-shaped, immobile, non-spore-free, and Gram-negative, such as figure 2 shown.

[0076] Observed under the scanning electron microscope, the bacteria are smooth without flagella, immobile, 0.2μm-0.4μm wide, 0.8μm-2.2μm long, such as image 3 shown.

[0077] 2. Physiological and biochemical identification

[0078] Physiological and biochemical identification of the Chryseobacterium...

Embodiment 3

[0090] Example 3 DNA Sequence Determination and Amino Acid Sequence Prediction of Encoding Protein Glutaminase

[0091] 1. Determination of the full-length DNA sequence of the coding protein glutaminase and its amino acid sequence prediction

[0092] Extract the total genomic DNA of the YF810 bacterial strain, and use this as a template to amplify the full-length DNA sequence of the protein glutaminase produced by the bacterial strain. The primer sequence is:

[0093] Seq ID No.4, F: 5'-CCAACCAACTTAACAAAAACTCACCATTAAAC-3'

[0094] Seq ID No.5, R: 5'-GGAACCCGAACTACCGGAGCAGGATG-3'

[0095] After the PCR product was detected by 1% agarose gel electrophoresis, it was recovered by tapping the gel and sent to a commercial company for sequencing. The sequencing work was completed by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0096] The DNA sequence measured above, namely Seq ID No.10; was translated with the software Clone Manager to obtain the corresponding ami...

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Abstract

The invention relates to preparation and applications of a bacterium for generating protein glutaminase (PG) and a fermentation product of the bacterium. The bacterium is a chryseobacterium proteolyticum YF810 strain, and the preservation number of the strain is CGMCC No. 10532. The strain is obtained from soil through enrichment and sieving, is safe and nontoxic, and can produce protein glutaminase after fermentation. Moreover, the activity of produced protein glutaminase is high. After preparation and purification, the purity of protein glutaminase is high, and the protein glutaminase is used in the industrial food processing field, can obviously improve the solubility, foaming property, emulsification property and stability of protein, and has a wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of food microorganism screening and application, and more specifically relates to the screening of protein glutaminase-producing bacteria and the preparation, purification and application of protein glutaminase, a fermentation product. Background technique [0002] Natural proteins contain more glutamine and asparagine residues, which are cross-linked with other amino acids in the form of hydrogen bonds, resulting in a decrease in the solubility of the protein, which in turn affects the technological properties of the protein, such as emulsification, foaming, Gel properties, etc. (Fennema OR. Food Chemistry [Z]. Beijing: China Light Industry Press, 2003316-317). This limits the many applications of protein in food, beverage, health products, medicine and other fields. Deamidation is an important way to solve this problem. Studies have shown that through deamidation, the amide group in the protein is convert...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/80C12N15/55C12N15/11A23L2/38A21D2/26C12R1/01
CPCA21D2/267A23L2/38C12N9/80C12Y305/01044A23V2002/00C12R2001/01C12N1/205A23V2250/5488
Inventor 黄静严文娟曲瑞丹常忠义高红亮康立叶坚鲁伟
Owner EAST CHINA NORMAL UNIV
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