CN101643712A

CN101643712A - Escherichia coli strain for efficiently converting glutamine to synthesize L-theanine and application thereof

Info

Publication number: CN101643712A
Application number: CN200910035096A
Authority: CN
Inventors: 殷志敏; 沈青红; 吕志祥; 王正才; 傅珒; 张正平
Original assignee: CHANGZHOU AGELUO BIOTECHNOLOGY Co Ltd; Nanjing Normal University
Current assignee: CHANGZHOU AGELUO BIOTECHNOLOGY Co Ltd; Nanjing Normal University
Priority date: 2009-09-15
Filing date: 2009-09-15
Publication date: 2010-02-10
Anticipated expiration: 2029-09-15
Also published as: CN101643712B

Abstract

The invention relates to an Escherichia coli strain for biologically converting to synthesize theanine and an application thereof. The Escherichia coli strain for efficiently converting L- glutamine and ethylamine is Escherichia coli of CCTCC No. M 209166. The strain can prepare theanine synzyme in high yield, can efficiently convert the glutamine and the ethylamine into L-theanine and obtains success in the industrial production process. The converting ratio of the glutamine exceeds 80%.

Description

The coli strain of efficiently converting glutamine to synthesize L-theanine and application thereof

Technical field:

The present invention relates to high-performance bio and transform the coli strain and the application in the preparation of industrialization theanine of producing theanine.

Background technology

The L-theanine is a distinctive seed amino acid in the green tea, the Japan scholar extracted, made with extra care out theanine first from tealeaves the fifties, and determined its chemical structure, after this many scholars have carried out number of research projects in general to it, and the preparation method of theanine mainly contains the training of vegetable cell group, chemosynthesis, microbial fermentation and separates etc. four kinds with ion exchange resin.A large amount of studies show that: theanine has the multiple nutrients nourishing function, calms the nerves and loosens, promotes brain function, hypotensive, antitumor and antifatigue effect.A kind of emerging foodstuff additive of theanine effect have the characteristic of safety non-toxic, have been widely used in medicines and health protection and the foodstuffs industry.

L-theanine (L-theanine) is the main body of tealeaves total free aminoacids, is one of main component that constitutes the tealeaves natural quality, accounts for more than 50% of whole total free aminoacids, and content can reach 1.5%-2% in the spring tea, can be up to 3% in the tender stem.The L-theanine is the material that constitutes the green tea local flavor as a kind of peculiar amino acid in the tealeaves.The L-theanine is synthetic at the tea tree root, transfer to leaf and accumulate in leaf by limb, but one meets solar radiation and will be converted into catechuic acid.Therefore, L-theanine content in the senior green tea of culture underglass is many, and content is less in coarse leaves.Nineteen fifty the wine family more two doctors Lang in that research is beautiful when revealing the bright refreshing flavour composition of tea, isolation identification goes out the L-theanine from young sprout, Japan in 1964 is decided to be foodstuff additive with the L-theanine and uses, its purposes is as the green tea flavour enhancer, but, in food, almost can't use popularization because the L-theanine price of extracting was very expensive at that time.The L-theanine that Japanese sun Chemical Corporation in 1992 succeeds in developing, cheap, purity is high and can large-scale industrialized production, make it can in food, add application as commodity.

Because there is the difficult point of chiral separation in L-theanine (L-theanine) by chemosynthesis, restricted the application of this method in large-scale industrialized production, therefore, utilize the inevitable direction of the large-scale industrialized production of microorganism biological fermentation becoming, but the efficient conversion of screening theanine coli strain is very difficult.

Summary of the invention

The object of the present invention is to provide a plant height to imitate the intestinal bacteria of converting glutamine and ethamine, and the application of this bacterial strain in preparation L-theanine.

The said bacterial strain of the present invention is CCTCC No.M209166 of intestinal bacteria (Escherichia coli) Nan Shi.

The invention also discloses the application of above-mentioned bacterial strains in preparation L-theanine.Specifically: (1) cultivate southern teacher No. to OD600 greater than 2.0, collect thalline, the broken thalline of high pressure, enzyme liquid;

(2) enzyme liquid is joined carry out bio-transformation in glutamine and the ethylamine solution, after fermentation culture, must be rich in the fermented liquid of L-theanine.

Cultivate in the above-mentioned steps (1) and obtain the thalline that enzyme unit alive rises at 12000-23000/.

In the above-mentioned steps (2), glutamine is preferably 0.2-0.8M, ethylamine solution 40-100ml in every liter of reaction solution, and adding enzyme unit alive is the 4000-8000/ liter, invert point is controlled at 20-37 ℃.

The culture condition that the south teacher is No. one: the intestinal bacteria south teacher who screens is inoculated in the LB solid medium No. one, every liter of substratum contains peptone 10g, yeast extract 5g, NaCl 10g, agar 15g, cultivate 12h for 37 ℃, choose single colony inoculation in 10mL LB liquid nutrient medium, every liter of substratum contains peptone 10g, yeast extract 5g, NaCl 10g is settled to 1 liter with deionized water

Adopt this coli strain after shaking table-50 liter fermentor tank-500 liter fermentor tank-10 ton fermentor cultivation, to obtain a large amount of enzymes and live unit at 23000/ liter thalline, in 5 tons of reactors, the 0.6M/L glutamine is carried out bio-transformation, after 25 ℃ of enzyme reactions of 40～48h, obtain the L-theanine of 0.49M/L, transformation efficiency to glutamine surpasses 80%, and this bacterial strain successfully obtains to use in large-scale industrial production.

Beneficial effect of the present invention is embodied in: the ethamine (0.45M) by high density is cultivated screening, it is the coli strain of theanine that a plant height that obtains is imitated the bio-transformation glutamine, this bacterial strain can not only be effectively by utilizing glutamine and ethamine to synthesize the L-theanine, and this bacterial strain can efficiently be converted into the L-theanine to glutamine and ethamine, and its transformation efficiency to glutamine has surpassed 80%; And its characteristic remains unchanged after the number cultivation in generation, and the ability of synthetic theanine is identical with the parental generation of beginning, is the effective production bacterial strain of suitability for industrialized production theanine from now on.

Description of drawings

Fig. 1 is embodiment two bacteria growing curve accompanying drawings

Fig. 2 is embodiment two an enzymes unit alive curve accompanying drawing

Fig. 3 is embodiment three bacteria growing curve accompanying drawings

Fig. 4 embodiment three bacteria growing curve accompanying drawings

Fig. 5 embodiment four bacteria growing curve accompanying drawings

Fig. 6 is embodiment four an enzymes unit alive curve accompanying drawing

Embodiment

Embodiment one bacterial strain screening

Tealeaves set isolate 23 strain coli strains in the soil and be inoculated into respectively in the LB substratum that contains ethamine (0.45M) and carry out incubated overnight.

Cultivate after 24 hours with microscope the coli strain of cultivating is observed, find that the cellular form of 3 strain coli strains wherein is normal and more cell count is arranged, and the cellular form of other 20 strain is irregular shape, and cell count is considerably less.

The glutamine and the 1M second ammonia that add 400mM in the Bacillus coli cells of logarithmic phase are carried out bio-transformation, the further fermentation through 12 hours, most of glutamine will be converted into theanine by the L-theanine synthetase in the coli strain.

The colibacillary fermentation of above 3 strains is transformed the theanine result analyze discovery through HPLC, wherein there are strain intestinal bacteria to have surpassed 80% to the transformation efficiency of glutamine, we are with its called after intestinal bacteria south teacher No., further carrying out the bio-transformation experiment in 1 liter of reaction system finds, the concentration of the synthetic theanine of this bacterial strain is up to 51.2 grams, through the number cultivation in generation, the ability of the synthetic theanine of this bacterial strain remains unchanged.

On July 28th, 2009 southern teacher is delivered Chinese typical culture collection center preservation for No. one, the depositary institution address: life science institute of Wuhan University; Preservation strain classification called after: No. one, intestinal bacteria (Escherichia coli) Nan Shi, deposit number CCTCC No.M 209166.

Embodiment two

(1) the intestinal bacteria south teacher who screens is inoculated in the LB solid medium No. one, every liter of substratum contains peptone 10g, yeast extract 5g, NaCl 10g, agar 15g cultivates 12h for 37 ℃, choose single colony inoculation in 10mL LB liquid nutrient medium, every liter of substratum contains peptone 10g, yeast extract 5g, NaCl 10g, be settled to 1 liter with deionized water, 12h is cultivated in 37 ℃ of concussions, gets 1mL then and is inoculated into 100mL LB liquid nutrient medium, 6～8h, OD are cultivated in 37 ℃ of concussions ₆₀₀Reach 2.0～2.2 (Fig. 1), the OD of enzyme unit alive (Nanjing is built up biotinylation kit and detected) ₅₃₀Reach 12000～14000 (Fig. 2), centrifugal 15min collects thalline, through the high pressure fragmentation, obtains theanine synthetase.

(2) add the theanine synthetase of 4000 units by every 0.6M/L glutamine and 70% (quality) ethylamine solution, after 25 ℃ of enzyme reactions of 40～48h, through HPLC detect find its to the transformation efficiency of glutamine up to 70%.

With 0.2, the 0.8M/L glutamine repeats above-mentioned experiment, the result is basic identical.

Embodiment three

(1) the intestinal bacteria south teacher who screens is inoculated in the LB solid medium No. one, every liter of substratum contains peptone 10g, yeast extract 5g, and NaCl 10g, agar 15g cultivates 12h for 37 ℃, chooses single colony inoculation in the autonomous MB substratum of 10mL, and every liter of substratum contains Na ₂HPO ₄15g, KH ₂PO ₄5g, NaCl 1g, NH ₄Cl2g, glycerine 3ml, peptone 10g; 12h is cultivated in 37 ℃ of concussions, gets 1mL then and is inoculated into the autonomous MB liquid nutrient medium of 100mL, and 6～8h, OD are cultivated in 37 ℃ of concussions ₆₀₀Reach 2.1～2.3 (Fig. 3), the OD of enzyme unit alive (Nanjing is built up biotinylation kit and detected) ₅₃₀Reach 15000～16000 (Fig. 4), centrifugal 15min collects thalline, through the high pressure fragmentation, obtains theanine synthetic gamma-glutamyl transferase

(2) add the theanine synthetase of 5000 units by every 0.6M/L glutamine and ethylamine solution, after 25 ℃ of enzyme reactions of 40～48h, detect through HPLC and to find that its transformation efficiency to glutamine reaches 80%.

Embodiment four

(1) the intestinal bacteria south teacher who screens is inoculated in the LB solid medium No. one, every liter of substratum contains peptone 10g, yeast extract 5g, NaCl 10g, agar 15g cultivates 12h, chooses single colony inoculation in 10mL LB liquid nutrient medium for 37 ℃, every liter of substratum contains peptone 10g, yeast extract 5g, NaCl 10g is settled to 1 liter with deionized water, 12h is cultivated in 37 ℃ of concussions, get 1mL then and be inoculated into 100mL LB liquid nutrient medium, after 4h is cultivated in 37 ℃ of concussions, add 60mL LB liquid nutrient medium, 6～8h, OD are cultivated in 37 ℃ of concussions ₆₀₀Reach 2.8～3.0 (Fig. 5), the OD of enzyme unit alive (Nanjing is built up biotinylation kit and detected) ₅₃₀Reach 18000～22000 (Fig. 6), centrifugal 15min collects thalline, through the high pressure fragmentation, obtains theanine synthetase.

(2) add the theanine synthetase of 6000 units by every 0.6M/L glutamine and ethylamine solution, after the 20-37 ℃ of enzyme reaction of 40～48h, detect through HPLC and to find that its transformation efficiency to glutamine surpasses 80%.

Embodiment five

To after the high pressure fragmentation, get 100 liters of enzyme liquid at 23000/ liter thalline in a large amount of enzymes of acquisition after 10 tons of fermentor cultivation unit alive, join and contain 4 tons of substrates (in the solution of 0.6M/L L-glutaminate and 2M ethamine, after 25 ℃ of enzyme reactions of 48h, obtain the L-theanine of 0.49M/L, the transformation efficiency of glutamine is surpassed 80%.

Laboratory apparatus and material:

Shaking table; Erlenmeyer flask; The microorganism Bechtop; The LB substratum; HPLC; The broken instrument of high pressure; Fermentor tank, reactor.

Claims

1, a kind of efficient conversion L-glutaminate and ethamine produce the colibacillary bacterial strain of L-theanine, are CCTCC No.M 209166 of intestinal bacteria (Escherichia coli) Nan Shi.

2, the application of the synthetic colibacillary bacterial strain of L-theanine of the said efficient conversion of claim 1 in preparation L-theanine.

3, according to the said application of claim 2, it is characterized in that, specifically:

(1) cultivate bacterial strain to OD600 more than 2.0, collect thalline, the broken thalline of high pressure, enzyme liquid;

4, according to the said application of claim 3, it is characterized in that, cultivate in the step (1) and obtain the thalline that enzyme unit alive rises at 12000-23000/.

According to the said application of claim 3, it is characterized in that 5, the glutamine that step (2) is wherein added is 0.2-0.8M, ethylamine solution is the 40-100ml/ liter, adds enzyme and lives unit at the 4000-8000/ liter, and invert point is controlled at 20-37 ℃.