Food-grade nattokinase expression bacterium

A technology of nattokinase and expressing bacteria, applied in the directions of enzymes, peptidases, bacteria, etc., can solve the problem of no recombinant Bacillus natto, etc., and achieve the effect of high purity

Active Publication Date: 2016-11-09
WUHAN KANGFUDE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] So far, there have been no reports of using a food-grad

Method used

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  • Food-grade nattokinase expression bacterium

Examples

Experimental program
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example 1

[0060] Construction of example 1 thermosensitive plasmid pKGFP194ts

[0061] Using the pE194 plasmid as a template, the thermosensitive replicon 194ts fragment of the vector was amplified with primers F1 and R1.

[0062] The PCR system is: 10×PCR Buffer 5μL, 2mM dNTP 5μL, 25mM MgSO 4 5 μL, 1.5 μL each of 10 μM primer F / R, 0.5 μL template DNA, 1 μL KOD-Plus-Neo, dH 2 O 32.5 μL.

[0063] The PCR reaction conditions are as follows: 94°C; 30 cycles (98°C for 10s, 58°C for 30s, 68°C for 1.5min); 68°C for 5min, 4°C for heat preservation; the PCR system and reaction conditions in subsequent experiments are configured and set according to the above conditions. The annealing temperature and extension time are adjusted according to the actual situation, and other parameters remain unchanged.

[0064] Refer to the method in CN201410430501 patent literature to construct pWEBK15 plasmid.

[0065] The pWEBK15 plasmid construction method is as follows:

[0066] Extract pET-28a(+) vector...

example 2

[0072] Construction of example 2 temperature-sensitive knockout (in) plasmids

[0073] Genomic DNA of Bacillus was extracted by CTAB method (cetyltrimethylammonium bromide) as a template for gene amplification (Acta Microbiology, 2006, 46(1):7-12.).

[0074] Genomic DNA of Bacillus licheniformis CICC10266 was used as a template, the upstream homology arm apr-U of the apr gene was amplified with apr-UF / R primers, and the downstream homology arm apr- d.

[0075] Using Bacillus natto CICC20132 genomic DNA as a template, using aprN-F2 / R2 as a primer to amplify the nattokinase gene aprN gene fragment 1; using the mixture of apr-U fragment, apr-D fragment and aprN fragment 1 as a template, and using apr-UF / apr-DR is the overlapping fragment aprUD-aprN of primer amplification three fragments; figure 2 shown.

[0076] The pKGFP194ts plasmid was used as a template, and the vector fragment was amplified with primers F4 and R1. The amplified product of the vector fragment was digeste...

example 3

[0079] Example 3 Nattokinase gene seamless knock-in into Bacillus licheniformis

[0080] (1) Single-copy strain construction of nattokinase gene

[0081] Plasmid pKGFP194-aprUD-aprN was extracted and electrotransformed into Bacillus licheniformis CICC10266 strain. The preparation of competent cells and electrotransformation methods refer to the following literature: J Microbiol Meth.1999,34(3):183-191.

[0082] The transformed cells were spread on 20μg / ml karamycin LB solid resistance plate and cultured at 30°C for 16h, and the positive line clones were verified by PCR, and the positive line clones were inoculated into LB liquid medium and cultured at 44°C, every 8-12h Transfer 0.2mL of bacterial liquid to 20mL of fresh LB medium for subculture, take samples from the second subculture, streak on the resistance plate, and use the outer primers apr-UF2 and apr- DR2 and the primers in the gene knockout vector to be knocked out were used to perform colony PCR amplification on a s...

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Abstract

The invention discloses a food-grade nattokinase expression bacterium. The recombinant bacterium is bacillus licheniformis which is transformed so that the genome contains at least one copy nattokinase gene. Preferably, in the genome of the expression bacterium, the negative regulatory factor gene hrcA is inactivated. The production strain provided by the invention accords with the requirements of a food-grade expression system, multiple copy nattokinase genes are tracelessly integrated into the genome of the bacillus licheniformis to be expressed, the expression quantity is obviously improved compared with that in the prior art, through the inactivation of the negative regulatory factor gene hrcA, the enzyme activity and expression quantity are further improved, the enzyme activity achieves 1350FU/mL, and the protein content achieves 1.45g/L. The nattokinase prepared by the recombinant bacterium is high in purity, can be processed into a health product or can be taken as an active ingredient of a medicine for treating cerebral infarction.

Description

technical field [0001] The invention relates to the technical field of microbial genetic engineering, in particular to a food-grade nattokinase expressing bacterium, wherein the expressing bacterium is a recombinant bacillus licheniformis. Background technique [0002] Japanese scholar Hiroyuki Sumi first discovered in 1987 in the traditional Japanese food natto and extracted the active substance with the function of dissolving thrombus—Nattokinase (Nattokinase, NK) (Cellular and Molecular Life Sciences, 1987, 43 (10): 1110 -1111.). Nattokinase is a protease with strong thrombolytic function produced by Bacillus subtilis var.natto, and it is a subtilisin. The enzyme is not only easy to extract and purify, low in cost, good in thrombolytic effect, quick in action, long in efficacy time, good in safety, without any side effects; Tissue plasminogen activator has unique advantages, it can directly hydrolyze fibrin, especially cross-linked fibrin, into small peptides and amino ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75A61K38/48A61P9/10A23L29/00A23L33/00C12R1/10
CPCA61K38/00C12N9/54C12N15/75C12N2800/101C12Y304/21062
Inventor 汪小锋汪卫叶聪张濛刘艳红马飞
Owner WUHAN KANGFUDE BIOTECH CO LTD
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