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Recombination methyl nourishment bacillus and application thereof

A technology of methyl nutrition and recombinant bacteria, applied in the direction of bacteria, transferase, fermentation, etc., can solve the problems of high cost, low output, low efficiency, etc., and achieve the effect of low cost, high profit, and broad application prospects

Inactive Publication Date: 2008-04-23
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Domestically, protein hydrolysis is mainly used to produce L-serine, but its output is low, efficiency is low, and cost is high. The method used more abroad is to use microbial fermentation to produce L-serine

Method used

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  • Recombination methyl nourishment bacillus and application thereof
  • Recombination methyl nourishment bacillus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Preparation of Recombinant Methylotrophic Bacillus MB202

[0039] 1. Cloning of glyA gene

[0040] 1. Screening of the recipient bacterium-Methylobacterium sp. MB200 CGMCC No 1526 of the three-parent hybrid

[0041] Collect 0.5g of mud sample from a biogas digester in Long'an County, Guangxi, add it to an Erlenmeyer flask containing 100ml medium III liquid culture medium, culture in a shaker at 32°C for 3 days, transfer the cultured bacteria liquid to 1% inoculum In 100ml medium II liquid medium, continue to culture at 32°C on a shaker for 3 days, take 100μl of the above bacterial liquid and spread it on the medium II solid medium, and cultivate for 3 days at 32°C. When the bacteria grow out, use an inoculating loop to pick the sheet. The colonies were inoculated on the medium II solid medium, and the single colonies that grew were numbered respectively. The strain numbered MB200 was identified as Methylobacterium sp., and its preservation number was CGMCC No 152...

Embodiment 2

[0059] Example 2. Detection of the ability of Methylobacterium sp. MB202 CCTCC № M207126 to produce serine hydroxymethyltransferase and L-serine

[0060] 1. Methylobacterium sp. MB202 CCTCC № M207126 produces serine hydroxymethyltransferase

[0061] In order to determine the relationship between the SHMT enzyme activity of the strain and the growth period, the strains MB200 and MB202 were respectively inoculated into medium II liquid medium. After 12 hours of cultivation at 32°C, the OD of the bacterial solution was measured every 3 hours 600 Value, and collect 0.6mg of wet bacteria (2×10 6 -2×10 7 CFU) Determination of enzyme activity.

[0062] The method for determining enzyme activity is: 1ml enzyme reaction system containing 50mM phosphate buffer (pH8.5), 0.05mmol / ml DL-3-phenylserine, 0.05umol / ml pyridoxal phosphate, 0.03% CTAB and 0.6 mg of wet bacteria (2×10 6 -2×10 7 CFU), the reaction temperature is 45°C, and the enzyme activity unit is an enzyme activity unit that produc...

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Abstract

The present invention discloses one kind of recombinant methylotrophic bacillus and its application. The recombinant methylotrophic bacillus MB202 is obtained through inserting glyA into the polyclonal site of pLAFR3 to obtain recombinant vector pLAFRg, and the subsequent three parent crossbreeding to introduce pLAFRg into methylotrophic bacillus MB200 to obtain the recombinant methylotrophic bacillus MB202. Through shaking table culture of MB202 at 32 deg.c in 50mM Tris-HCl culture medium with glycine in 10 mg / ml, methanol in 50 mg / ml, and biomass of 10<8>-10<9> CFU / ml, in pH 8.5 for 48 hr, L-serine yield may reach 11.5mg / ml; and through fermentation culture of MB202 at 32 deg.c for 12 hr, the activity of serine hydroxymethlase may reach 115 U. The recombinant methylotrophic bacillus MB202 has broad application foreground in producing L-serine and treating methanol waste water.

Description

Technical field [0001] The invention relates to a recombinant methylotrophic bacterium and its application. Background technique [0002] L-serine is a sugar-producing amino acid and is one of the 20 basic amino acids that make up protein. Although L-serine is a non-essential amino acid, it has many important physiological functions and functions: L-serine is the precursor for the synthesis of purine, thymine, and choline; after phosphorylation, the hydroxyl group of L-serine can derive important functions. Physiological functions of phosphoserine, phosphoserine can relieve fatigue and restore physical strength; L-serine has the effect of stabilizing the pH value of eye drops, and there is no irritation after eye drops; L-serine is an important natural moisturizing factor (NMF) One is the main role of skin stratum corneum to keep moisture, a key additive in high-end cosmetics; at the same time, serine derivatives also have excellent medicinal and biological activities, such as α-...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N9/10C12P13/06
Inventor 武波申佩弘欧倩
Owner GUANGXI UNIV
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