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Application of a Protease Spectrum Electrophoresis Detection Method in Rapid Identification of Bacterial Extracellular Proteases

A detection method and protease technology, applied in the field of protease species identification, can solve problems such as unintuitive results, and achieve the effect of eliminating cumbersome steps

Active Publication Date: 2017-03-29
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the pure enzyme is used in this method, the type of protease can be judged intuitively, but if it is a crude enzyme extract, the result is not intuitive, that is because the crude enzyme liquid contains a variety of proteases. Roughly judge which types of enzymes may be contained in the crude enzyme solution, and it is not intuitive to see which one or several proteases are inhibited

Method used

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  • Application of a Protease Spectrum Electrophoresis Detection Method in Rapid Identification of Bacterial Extracellular Proteases
  • Application of a Protease Spectrum Electrophoresis Detection Method in Rapid Identification of Bacterial Extracellular Proteases
  • Application of a Protease Spectrum Electrophoresis Detection Method in Rapid Identification of Bacterial Extracellular Proteases

Examples

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Embodiment 1

[0042]Example 1: Using the method of the present invention to detect the extracellular protease of Pseudoalteromonas sp. SJN2. Pseudoalteromonas sp. SJN2 is a strain isolated from sea sand in the intertidal zone of Haikou Holiday Beach in our laboratory.

[0043] Proceed as follows:

[0044] (1) Streak-inoculate Pseudoalteromonas on solid medium, activate and culture at 18°C ​​for 48h, then inoculate in 5ml liquid LB medium, shake and culture at 18°C ​​and 200rpm for 24h, then press 5% (v / v) inoculum was inoculated into 100ml of fermentation medium, cultured with shaking at 18°C ​​and 200rpm for 4 days, and centrifuged at 10000rpm to obtain a fermentation broth;

[0045] The above-mentioned solid medium components are as follows, all in parts by weight:

[0046] 1 part of peptone, 0.5 parts of yeast powder, 1.5 parts of agar, 100 parts of distilled water, pH 7.5-8.0;

[0047] The components of LB medium are as follows, all in parts by weight:

[0048] 1 part of peptone, 0....

Embodiment 2

[0055] Example 2: The steps of detecting the extracellular protease of marine bacteria (Bacillus sp.SQN6-1) by the method of the present invention are the same as in Example 1, and the fermentation and cultivation time is 72 hours. Marine Bacillus sp.SQN6-1 is a strain isolated from Haikou Holiday Beach seawater in our laboratory. Step is with embodiment 1. Protease bands such as figure 2 shown. OP specifically binds divalent metal ions and can inhibit the activity of metalloproteases. PMSF is a serine protease inhibitor, so it can be obtained from figure 2 It is clearly seen in the figure that 3 of them are serine protease bands, because the active bands disappear after adding PMSF, and 1 is a metalloprotease, and the degradation band disappears on the active electrophoresis map containing OP, indicating that it is strongly inhibited by OP , loss of enzymatic activity.

Embodiment 3

[0056] Embodiment 3: The operation steps of using the method of the present invention to detect trypsin Trypsin are the same as in Example 1, except that the commercial enzyme preparation purchased by Shanghai Sangong, trypsin Trypsin, is used. The purpose of this experiment is to establish an experimental method. Protease is a typical serine protease, which can be inhibited by PMSF inhibitor, but inhibitor OP has no effect on its enzyme activity. Trypsin bands such as image 3 shown. Trypsin is a typical serine protease. When the substrate solution is added with PMSF, its active bands completely disappear after incubation, while the degradation bands on the active electrophoresis spectrum containing OP do not change.

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Abstract

The invention discloses application of a protease graph electrophoresis detection method to rapid identification of extracellaluar protease variety of bacteria. The application is implemented by the following steps: a, preparing SDS (sodium dodecyl sulfate) polyacrylamide gel containing no a protease substrate; b, evenly mixing a protease sample with a loading buffer solution containing no beta-mercaptoethanol and then loading the sample without heating in boiling water bath; c, carrying out electrophoresis; d, washing the gel; e, soaking the gel in a substrate solution containing different protease inhibitors; and f, reacting and developing. The protease graph electrophoresis detection method is capable of determining the natures of different proteases and obviously shortened in time in contrast with a traditional method; complex steps of separating and purifying the proteases are omitted; the method is good in repeatability, high in sensitivity and accurate in active stripe measurement, thus being highly advantageous for subsequent protease identification and purposeful purification operation; the method can be applied to detection of various types of proteases and has wide use space.

Description

technical field [0001] The invention belongs to the technical field of identification of protease species, and relates to an application method for rapidly identifying protease species by utilizing the activity electrophoresis detection technology of protease spectrum and the reaction of protease inhibitors. Background technique [0002] Proteases (proteinases), also known as peptidases, are a class of enzymes that can hydrolyze proteins and polypeptides. They are widely found in animals, plants and microorganisms, and perform many different physiological functions. The MEROPS database records information about proteases and peptide inhibitors of proteases. According to the amino acids involved in catalysis in the catalytic center, proteases in the database can be divided into six categories: aspartic proteases, cysteine ​​proteases, gluten proteases, metalloproteases, serine proteases, threonine proteases and proteases of unknown catalytic type. Protease is one of the most...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/447
Inventor 何海伦刘丹武翠玲杨兴昊吴日帮黄嘉封黄雅雯
Owner CENT SOUTH UNIV
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