Novel holomonas and method for producing tetrahydropyrimidine by novel holomonas

A Halomonas, tetrahydropyrimidine technology, applied in the field of microorganisms, can solve problems such as high requirements for equipment and operation, unsuitable for large-scale industrial production, and corrosion of fermentation equipment.

Active Publication Date: 2013-12-18
ZHEJIANG HISUN PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the bacterial milking method can obtain relatively high ectoine production through multiple hypotonic shocks, but the production of ectoine decreases with the increase in the number of shocks. In addition, this method has high requirements for equipment and operations. thus limiting its application
There is a large difference between the NaCl concentration for the optimum growth of moderate halophilic bacteria and the NaCl concentration for the optimal synthesis of ectoine. They grow well at lower salt concentrations and can obtain higher biomass. The amount of ectoine is less; growth is inhibited at higher salt concentrations, but the amount of ectoine accumulated per unit of bacteria is larger
The reported high-yield ectoine fermentation production process requires a high NaCl concentration to stimulate the bacteria to accumulate more products, and a high-concentration NaCl solution severely corrodes the fermentation equipment and is not suitable for large-scale industrial production. At the same time, the high-salt fermentation waste liquid has caused great pressure on the environment

Method used

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  • Novel holomonas and method for producing tetrahydropyrimidine by novel holomonas
  • Novel holomonas and method for producing tetrahydropyrimidine by novel holomonas
  • Novel holomonas and method for producing tetrahydropyrimidine by novel holomonas

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Obtaining Halomonas sp. HS-2255 (CGMCC NO.6248)

[0028] Take a 1g sample of sea mud from the coast of Taizhou, Zhejiang, and serially dilute it with sterile water, spread it on an LB plate containing different concentrations of NaCl (30-100g / L), incubate at 30°C, pick a single colony after the colony grows, and continue to separate Purify and screen until pure bacteria are obtained, cultivate them in a fermentation medium with a lower NaCl concentration (not exceeding 100g / L, optimally 75g / L), and detect the ectoine production of the strains by high-pressure liquid chromatography. Select the mutant strains obtained through mutagenesis screening against high-dose metabolic end products and structural analogs, carry out shake flask screening, measure and evaluate the ability and genetic stability of ectoine production, and obtain an excellent strain with significantly improved yield, The mutant strain for mutagenesis screening and resistance screening, and the...

Embodiment 2

[0029] Example 2: 16S rDNA amplification, sequencing and sequence alignment of Halomonas sp. HS-2255 (CGMCC NO.6248).

[0030] Collect the fresh bacteria of the strain to be tested, extract the total DNA template by the Pitcher method improved by the solution bacterial enzyme method, and use the universal primers (27F and 1495R) to amplify the 16S rDNA gene. After the PCR product is detected and purified, it is directly sequenced. Sequencing was performed by Shanghai Sangon Bioengineering Technology Co., Ltd.

[0031] The primer sequences are as follows:

[0032] a) 27F:5'-GAGAGTTTGATCCTGGCTCAG-3'(E.coli 7-27)

[0033]b) 1495R: 5'-CTACGGCTACCTTGTTACGA-3' (E.coli 1495-1514)

[0034] The 50μl PCR reaction system is as follows:

[0035] wxya 2 o

35μl

25 μM Primer 27F

1μl

25 μM Primer 1495R

1μl

10×Poly.Buffer

5μl

2.5mM dNTPs

4μl

25mM MgCl 2

3μl

DNA template

0.5ul

Polymerase*

0.5μl

...

Embodiment 3

[0038] Example 3: Culture method of Halomonas sp. HS-2255 (CGMCC NO.6248)

[0039] Insert the strain of the present invention into the seed medium, the composition of the seed medium is: fish peptone 20g / L, glucose 20g / L, yeast extract 20g / L, NaCl 40g / L, pH7.5, and the culture temperature is 28-35°C , the shaker speed is 180-250r / min, the culture time is 24-48h, until the seed solution OD 600 The absorption value is 0.1-1.0 (with water as a reference, the seed liquid is diluted 30 times).

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Abstract

The invention discloses novel halomonas sp. HS-2255 which can be used for producing tetrahydropyrimidine, and a mutant strain thereof. The preservation number of the novel halomonas sp. HS-2255 is CGMCC (China General Microbiological Culture Collection Center) No. 6248. The invention further discloses a culture method of the strain and a method for producing the tetrahydropyrimidine by fermentation. The strain has a higher tetrahydropyrimidine yield in a culture medium with a carbon source and a nitrogen source which are assimilable, and lower NaCl content, and the content of a byproduct hydroxyl tetrahydropyrimidine is lower, and therefore, the novel halomonas sp. HS-2255 can be used for industrial fermentation production of tetrahydropyrimidine, and has good application prospect.

Description

Technical field: [0001] The invention relates to the field of microorganisms, in particular to the application of a new halomonas and its mutant strains in the fermentative production of ectoine. Background technique: [0002] Halophilic bacteria belong to a kind of extremophile microorganisms. According to their tolerance to NaCl, they can be divided into mild halophilic bacteria (the optimum NaCl concentration is 10-30g / L), moderate halophilic bacteria (the optimum NaCl concentration is 50 -100g / L) and extreme halophilic bacteria (the optimum NaCl concentration is 130-150g / L). In order to maintain the osmotic pressure balance inside and outside the cells, some substances will be accumulated in the cells of halophilic bacteria to resist the external hypertonic environment. Such substances mainly include amino acids and their derivatives, polyols, sugars and ectoine, among which Tetrahydropyrimidine has important application value. [0003] Tetrahydropyrimidine (1,4,5,6-te...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P17/12C12R1/01
Inventor 朱皖宜邱海潇罗红瑜李振武许江丽张宝新韩双林丹丹彭飞宇陈华郑玲辉王玲萍陶正利
Owner ZHEJIANG HISUN PHARMA CO LTD
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