CN107988109B

CN107988109B - flavobacterium mutant strain and application thereof

Info

Publication number: CN107988109B
Application number: CN201711390815.5A
Authority: CN
Inventors: 王莹; 寇玲赟; 于梅
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2017-12-21
Filing date: 2017-12-21
Publication date: 2019-12-10
Anticipated expiration: 2037-12-21
Also published as: CN107988109A

Abstract

The invention provides a flavobacterium mutant strain, the preservation number of which is CGMCC No. 14855. The strain protected by the invention is obtained by carrying out long-term fermentation and screening on the existing flavobacterium which produces fucosan sulfatase. In large-scale fermentation, the capability of the strain for producing the fucosan sulfatase by fermentation is obviously improved, and the capability of producing the fucosan sulfatase can reach 310U/ml at most.

Description

Flavobacterium mutant strain and application thereof

Technical Field

The invention belongs to the technical field of beneficial microorganism mutation screening, and particularly relates to a flavobacterium mutant strain and application thereof.

Background

Fucoidan (Fucoidan), also known as Fucoidan, is a high molecular weight heteropolysaccharide with sulfate groups, and exists in marine brown algae and some spiny skin animals, in recent years, Fucoidan has become a new pet in scientific research due to its excellent biological activities, including anti-tumor, anticoagulant, antibacterial, antiviral, antioxidant, anti-inflammatory, immunoregulation, antithrombotic, anticomplementary, anti-liver disease, blood lipid lowering, antiallergic, anti-gastric ulcer, etc., and is a main focus in the research and development fields of marine drugs and functional foods at present.

Researches show that the polysaccharide has a very complex chemical structure, mainly comprises L-fucose and sulfuric acid groups, and also contains a small amount of galactose, mannose, uronic acid, glucose, rhamnose, xylose, hexosamine, arabinose and the like, and the chemical structure of the polysaccharide can be obviously changed along with different source species, harvesting seasons, geographical positions and extraction methods. Meanwhile, the polysaccharide has large molecular weight and high viscosity, and is easy to generate antigenicity and toxic and side effects, and the application of the polysaccharide is severely limited by the problems. The most effective solution to the above problem is to convert high molecular weight polysaccharides into components of moderate molecular weight and relatively stable structure by a precise degradation technique, thereby clearing obstacles for the application of the polysaccharides. Further studies have shown that the biological activity of fucoidan is molecular weight dependent, i.e. exhibits the highest activity against a certain activity only within a specific molecular weight range, and from this point of view, it is also necessary to degrade the polysaccharide using "precision degradation techniques".

At present, the methods for degrading fucoidan sulfate mainly include chemical degradation, physical degradation and enzymatic degradation. The chemical degradation method is non-specific degradation, the reaction condition is harsh, the process is difficult to control, and the product conversion rate and the functionality are low. Although the physical degradation method is simple in operation and good in controllability, the degradation efficiency is generally low, and a product in a certain molecular weight range is difficult to obtain. The enzymolysis method can specifically cut off the fucoidan chain under mild conditions, the reaction process is easy to control, oligosaccharide products in the required molecular weight range are easy to obtain, the obtained products have high content and strong functionality, and the enzymatic degradation of the polysaccharide is also the most ideal method recognized in academia.

Although enzymatic methods show great advantages in Fucoidan degradation, no commercial enzyme preparations are currently available worldwide, which greatly hinders the use of Fucoidan sulfate. The main reason for this situation is that the enzyme-producing activity of the existing enzyme-producing microorganisms is generally low and cannot meet the requirement of large-scale production. Therefore, the screening of high-activity microorganisms at the present stage is an important way for solving the application problem of the fucoidan sulfate.

Disclosure of Invention

The invention aims to provide a flavobacterium mutant strain and application thereof, wherein the mutant strain can effectively improve the yield of fucosan sulfatase in large-scale fermentation, thereby making up the defects of the prior art.

The Flavobacterium mutant strain is a Flavobacterium (Flavobacterium sp.) RC2-3mut strain, which is preserved in the China general microbiological culture Collection center of Microbiol, China institute of Microbiol culture Collection, Zhongkoyao institute of Michidaceae, North West Lu province, Tokyo, 11 months and 03 days in 2017, and the preservation number is CGMCC No. 14855.

The screened bacteria are used for producing fucosan sulfatase;

The viable bacteria of the flavobacteriaceae RC2-3mut are used for preparing bacteria liquid; the prepared bacterial liquid can be used for extracting intracellular enzymes so as to degrade fucosan sulfate.

the strain protected by the invention is obtained by carrying out long-term fermentation and screening on the existing flavobacterium which produces fucosan sulfatase. In large-scale fermentation, the capability of the strain for producing fucosan sulfatase is obviously improved, and the enzyme production capability can reach 310U/ml at most.

Detailed Description

The applicant prepared fucosan sulfatase by fermenting using flavobacterium (flavobacterium sp.) RC2-3 strain with the collection number of CGMCC No.6932, but found that the fermentation enzyme production capacity of the flavobacterium (flavobacterium sp.) RC2-3 strain is obviously reduced and the large-scale production potential is not realized when a large-scale fermentation experiment is carried out. Therefore, the applicant carries out ultraviolet induced mutation from Flavobacteraceae sp.RC2-3 strain to finally obtain the target strain RC2-3mut strain of the invention, the optimal growth temperature of the mutant strain is increased from 25 ℃ to 30-35 ℃, and the maximum fucosan sulfatase producing capability of the mutant strain can reach 310U/ml in large-scale fermentation.

The present invention will be described in detail with reference to examples.

Example 1 mutagenesis screening of strains

The method comprises the steps of inoculating flavobacterium RC2-3 with the preservation number of CGMCC No.6932 into a liquid culture medium (Fucoidan0.2%, peptone 1%, ammonium nitrate 5mg/mL, prepared by membrane-coated seawater and natural pH), culturing for 12h at 25 ℃ and 180rpm to obtain a seed culture solution, inoculating the seed culture solution into a fresh liquid culture medium according to the inoculation amount of 5%, and culturing for 48h at 25 ℃ and 150rpm to obtain the flavobacterium solution.

And centrifugally collecting thalli, washing twice by using sterile normal saline, adding the sterile normal saline to enable the number of bacteria to reach 10 ⁶ orders of magnitude, oscillating to obtain cell suspension, carrying out ultraviolet mutagenesis, wherein the ultraviolet mutagenesis irradiation time is 10-45s respectively, the time gradient interval is 5s, coating a selective plate with the bacterial suspension which is not subjected to ultraviolet irradiation treatment, culturing for 24h in a dark place, and calculating the lethality as a control group.

Wherein the selective medium plate has the following composition: 0.2% of Fucoidan, 0.2% of peptone, 5mg/mL of ammonium nitrate and 2% of agar, and is prepared by using membrane seawater;

The results show that the lethality of the bacteria is gradually increased along with the extension of the ultraviolet irradiation time, and the lethality is 90% at 40 s. When the time reaches 45s, the lethality rate approaches 100%, so the invention selects the irradiation time of 40s for ultraviolet mutagenesis.

Treating the bacterial suspension by taking the irradiation time with the lethality reaching 90 percent as a mutagenic dose and coating the bacterial suspension on a selective culture medium plate to obtain a purified strain of the mutant bacteria;

Respectively inoculating the slant strains to 50mL of liquid culture medium (Fucoidan 0.2%, peptone 1%, ammonium nitrate 5mg/mL, prepared by using membrane-coated seawater and natural pH), shake-culturing at 35 ℃ and 180rpm for 12h to serve as seed liquid, inoculating the seed liquid to a fermentation culture medium (Fucoidan 0.5%, beef extract 1%, prepared by using membrane-coated seawater and natural pH) according to the inoculation amount of 10%, fermenting in a 5L fermentation tank (filled with 3.5L), culturing at 35 ℃ and 150rpm for 72h, and measuring the Fucoidan content by using a methine blue method. The mutant strains were selected based on the Fucoidan content, and the strain RC2-3mut of the present invention was finally obtained.

The method for measuring the Fucoidan content by the methine blue method comprises the following steps:

Adding 50 mu L of sample to be detected into 9mL of distilled water, uniformly mixing, adding 1mL of 0.41mmol/L methylene blue solution, uniformly mixing, and measuring the absorbance at 559 nm. The calculation was performed using a Fucoidan standard (Sigma) as a standard curve.

Example 2: detection of fermentation enzyme production activity of RC2-3mut strain

the strain RC2-3mut was inoculated into a liquid medium (Fucoidan 0.2%, peptone 1%, ammonium nitrate 5mg/mL, prepared with membrane-coated seawater, pH natural), shake-cultured at 35 ℃ and 180rpm for 12 hours as a seed solution, the seed solution was inoculated into a fermentation medium (Fucoidan 0.5%, beef extract 1%, prepared with membrane-coated seawater, pH natural) in an amount of 10% inoculum size, fermented in a 5L fermentor (3.5L charge), and cultured at 35 ℃ and 150rpm for 72 hours. Sampling 1mL at 24h under sterile condition, inactivating enzyme at 80 deg.C for 15min, centrifuging at 10000rpm for 10min, and collecting supernatant. The change in molecular weight of the polysaccharide was detected by high performance gel exclusion chromatography (conditions of chromatography: Aglient1100 HPLC; TSK-gel G3000PW X1 (30cm X7.5 mm) column; column temperature 40 deg.C; 0.2M NaCl as mobile phase; flow rate 0.5 mL/min; differential detector). The results show that after 0h, 24h, 48h and 72h of culture, the Fucoidan contents with the retention time of 13.6min are respectively 100%, 63%, 20% and 15%.

The enzyme activity detection method comprises the following steps: the intracellular enzyme was mixed with 0.2% Fucoidan (dissolved in 20mM Tris-HCl at pH 8.0) in equal volume, and shake-cultured at 25 ℃ for 2 hours at 120rpm in a shaker, and the reducing sugar content was measured by Somogyi-Nelson method. 1 enzyme activity unit is defined as the amount of enzyme required to produce 1 nanomole of reducing sugar per hour at 25 ℃ and pH 8.0. Through determination, the intracellular enzyme activity of the bacterial liquid produced after the strain RC2-3mut is cultured for 72h reaches 310U/mL. The screened strain has better enzyme production performance in large-scale fermentation.

The experimental results show that the enzyme-producing bacteria screened by the invention have better fermentation enzyme-producing capability and can effectively degrade fucosan sulfate with high molecular weight.

Claims

1. A flavobacterium strain (Flavobacterium sp.) RC2-3mut is characterized in that the preservation number of the flavobacterium is CGMCC No. 14855.

2. Use of flavobacterium according to claim 1 for the production of fucoidan sulfatase.

3. A method for producing fucosan sulfatase, wherein the method comprises fermenting the flavobacterium of claim 1 to produce fucosan sulfatase.

4. A method for producing fucosan oligosaccharide, wherein the method is produced by fermentation using the Flavobacterium of claim 1.