CN109913386A

CN109913386A - One plant height produces tea tree endophyte CsE7 and its application of theanine

Info

Publication number: CN109913386A
Application number: CN201910208436.2A
Authority: CN
Inventors: 孙俊; 常慢慢; 宛晓春; 张照亮; 张正竹
Original assignee: Anhui Agricultural University AHAU
Current assignee: Anhui Agricultural University AHAU
Priority date: 2018-12-17
Filing date: 2019-03-19
Publication date: 2019-06-21
Anticipated expiration: 2039-03-19
Also published as: CN109913386B; CN109402028A

Abstract

The invention discloses tea tree endophyte and its applications that a plant height produces theanine, the tea tree endophyte classification naming is Luo Dannuo Bacteriaceae gamboge Bacillus (Rhodanobacteraceae Luteibacter sp.) CsE7, it is preserved in China typical culture collection center, deposit number is CCTCC NO:M 2018816, and preservation address is Wuhan University, Wuhan City, Hubei China province.The present invention produces the bacterial strain of L-thiamine, for the production theanine endophyte Luo Dannuo Bacteriaceae gamboge Bacillus CsE7 separated from tea tree for the first time.Bacterial strain CsE7 can synthesize L-thiamine on the LB culture medium for being added to glutamine and ethamine, and the theanine of synthesis can rapid secretion to extracellular, secernment efficiency is up to 90% or more, the present invention prepares L-thiamine using glutamine and ethamine as substrate using the CsE7 bacterial strain, operating method is simple, theanine high production efficiency has good industrial applications prospect.

Description

One plant height produces tea tree endophyte CsE7 and its application of theanine

Technical field

The invention belongs to the preparation technical fields of theanine, and in particular to the tea tree endophyte of plant height production theanine CsE7 and its application.

Background technique

Theanine (L-Theanine) is N- ethyl-gamma-glutamyl amine, accounts for 70% or more of total amino acid content in tealeaves, It is distinctive nonprotein free amino acid in tea tree, is also to confer to the main matter of tealeaves flavour, content is directly determined Determine tea leaf quality.Theanine, which has, to be loosened neural, antitumor, antidepression, protection blood vessel, blood pressure lowering, inhibits fat and improve machine The medicine effect such as body immunity function, further, it is also possible to be applied to field of food as functional food additives.Therefore, tea ammonia Acid is increasingly favored by people, and international market is also very big to its demand.

Currently, there are mainly three types of the methods of production theanine.One is extract from tealeaves.Theanine in Tea content compared with It is low, and raw material sources are limited, cause that the production cost is very high.Second method is chemical synthesis.This method not only results in poison Property substance residual, and the theanine produced is the mixture of D- theanine and L-thiamine isomers, and it is complicated to split process for refining. The third method is to utilize microorganism and its enzyme fermentation production theanine.That reports at present, which can synthesize the microorganism of theanine, includes Escherichia coli, bacillus subtilis etc., but these are all nature free microorganisms, are not isolated out of tea tree body.

Summary of the invention

Goal of the invention: being directed to the deficiencies in the prior art, and the object of the present invention is to provide a plant heights to produce theanine Tea tree endophyte satisfies the use demand for the endophyte separated from tea tree for the first time.It is a further object of the present invention to provide one kind The tea tree endophyte of above-mentioned high yield theanine is producing the application in L-thiamine.

Technical solution: in order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention are as follows:

One plant height produces the tea tree endophyte of theanine, and classification naming is Luo Dannuo Bacteriaceae gamboge Bacillus (Rhodanobacteraceae Luteibacter sp.) CsE7, preservation and China typical culture collection center, preservation are compiled Number be CCTCC NO:M 2018816, the deposit date is on November 23rd, 2018；Preservation address is that Wuhan City, Hubei China province is military Chinese university.

The Luo Dannuo Bacteriaceae gamboge Bacillus CsE7 is preparing the application in L-thiamine.

The application: CsE7 bacterial strain is in 28 DEG C of shaken cultivations to OD₆₀₀When being 1.0, paddy ammonia is added simultaneously into culture solution Amide and ethamine cultivate 1~15d, take culture solution 100 DEG C of water-baths cracking 30min, and in -20 DEG C after multigelation 2~3 times, 12000rpm is centrifuged 15min, takes supernatant after 0.22 μm of water phase membrane filtration, carries out liquid chromatography mass spectrometric detection；Obtain tea containing L- The supernatant of propylhomoserin.

The final concentration of 20mM of glutamine is added in the application.Add the final concentration of 20mM of ethamine.

The application, preferably culture 7d.

The present invention produces the bacterial strain CsE7 of L-thiamine, for the production theanine endophyte separated from tea tree for the first time, 16S RDNA is accredited as Luo Dannuo Bacteriaceae gamboge Bacillus (Rhodanobacteraceae Luteibacter sp.).The CsE7 bacterium The well-grown on LB culture medium, 28 DEG C of culture 2-3d, single bacterium completion glassy yellow.16S rDNA is after amplification, sequencing, comparison It was found that with Luo Dannuo Bacteriaceae gamboge Bacillus (Rhodanobacteraceae Luteibacter sp.) in ncbi database (GenBank accession no.KM187159) homology is high, up to 99% or more.In conjunction with morphological features, item is grown Part is determined as Luo Dannuo Bacteriaceae gamboge Bacillus, and classification naming is Luo Dannuo Bacteriaceae gamboge Bacillus (Rhodanobacteraceae Luteibacter sp.)CsE7。

The utility model has the advantages that compared with prior art, the present invention produces the bacterial strain of L-thiamine, for the production separated from tea tree for the first time Theanine endophyte Luo Dannuo Bacteriaceae gamboge Bacillus CsE7.Bacterial strain CsE7 is in the LB training for being added to glutamine and ethamine Support the theanine that can synthesize L-thiamine on base, and synthesize can rapid secretion to extracellular, secernment efficiency has up to 90% or more There is good practicability.

Detailed description of the invention

Fig. 1 is CsE7 bacterium colony photo figure；

Fig. 2 is CsE7 bacterium colony gram stain microscopy figure；

Fig. 3 is the Mass Spectrometer Method result figure of CsE7 synthetic product theanine.

Fig. 4 is the theanine synthetic quantity trend chart of CsE7 culture different time.

Fig. 5 is CsE7 when cultivating 5d and 7d, theanine secernment efficiency result figure.

Specific embodiment

The present invention is described further combined with specific embodiments below.

Embodiment 1

1) separation of CsE7 bacterial strain

Picking tea-tree tissue culture seedling secret out of mixing endophyte on solid LB plate carry out monoclonal separation, and to point The monoclonal separated out is numbered to be detected with theanine synthesis capability, and finally selected CsE7 is purpose strain.Later, to purpose strain System carries out the extraction of genomic DNA, and 16S rDNA (the sequence such as SEQ ID of the bacterial strain is carried out using molecule clone technology Shown in NO.1) identification, the bacterial strain, which is compared, through ncbi database belongs to Luo Dannuo Bacteriaceae gamboge Bacillus.

2) Gram's staining and Morphological Identification of CsE7 bacterial strain

The bacterial strain bacterial plaque is rounded, and surface is smooth, wet, raised, opaque yellow, neat in edge (as shown in Figure 1). It takes on a red color after Gram's staining rod-shaped, identifies that the bacterial strain is Gram-negative bacteria (as shown in Figure 2).

3) CsE7 synthesizes the sample preparation and detection process of theanine

The bacterial strain is in 28 DEG C of shaken cultivations to OD₆₀₀When being 1.0, final concentration of 20mM paddy is added simultaneously into LB culture solution Glutamine and 20mM ethamine, while respectively not add the same concentration bacterium solution of any substrate and add the blank training with concentration of substrate Feeding base as a control test, under the conditions of same 28 DEG C of shaken cultivations, continues after cultivating 1d, 3d, 5d, 7d, 10d, 15d, respectively 100 DEG C of water-baths of culture solution that different time points are obtained crack 30min, and in -20 DEG C after multigelation 2~3 times, 12000rpm is centrifuged 15min, takes supernatant after 0.22 μm of water phase membrane filtration, carries out ultra high efficiency liquid chromatography mass spectrometric (UPLC-MS/ MS it) detects.

Second order ms qualification result shows: (mass spectral results are shown in figure to the stable theanine containing various concentration in culture solution 3), and at the 7th day after substrate adds, theanine content averagely about 3.8mg/L (see Fig. 4), and in control group, it does not add and appoints The same concentration bacterium solution of what substrate only detects denier theanine, and tea ammonia is not detected in the blank cultures added with concentration of substrate The presence of acid.Illustrated with this, which has the ability for efficiently synthesizing theanine.

And the theanine of bacterial strain synthesis can rapid secretion to extracellular, secernment efficiency is up to 90% or so (see Fig. 5).

Sequence table

<110>Agricultural University Of Anhui

<120>one plant heights produce tea tree endophyte CsE7 and its application of theanine

<130> 100

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1346

<212> DNA

<213> Rhodanobacteraceae Luteibacter sp.

<400> 1

gggataacgt agggaaactt acgctaatac cgcatacgtc ctacgggaga aagcggggga 60

tcgcaagacc tcgcgcggtt ggatggaccg atgtgcgatt agctagttgg tgaggtaacg 120

gctcaccaag gcgacgatcg ctagctggtc tgagaggatg atcagccaca ctgggactga 180

gacacggccc agactcctac gggaggcagc agtggggaat attggacaat gggcgcaagc 240

ctgatccagc aatgccgcgt gtgtgaagaa ggccctcggg ttgtaaagca cttttatcag 300

gagcgaaatc tgcaaggtta atacctttgc agtctgacgg tacctgagga ataagcaccg 360

gctaactccg tgccagcagc cgcggtaata cggagggtgc aagcgttaat cggaattact 420

gggcgtaaag cgtgcgtagg cggttcgtta agtctgttgt gaaagccccg ggctcaacct 480

gggaatggca atggatactg gcgagctaga gtgtgtcaga ggatggtgga attcccggtg 540

tagcggtgaa atgcgtagag atcgggagga acatcagtgg cgaaggcggc catctgggac 600

aacactgacg ctgaggcacg aaagcgtggg gagcaaacag gattagatac cctggtagtc 660

cacgccctaa acgatgcgaa ctggatgttg gtctcaactc ggagatcagt gtcgaagcta 720

acgcgttaag ttcgccgcct ggggagtacg gtcgcaagac tgaaactcaa aggaattgac 780

gggggcccgc acaagcggtg gagtatgtgg tttaattcga tgcaacgcga agaaccttac 840

ctggccttga catgtccgga atccagcaga gatgcaggag tgccttcggg aatcggaaca 900

caggtgctgc atggctgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 960

agcgcaaccc ttgtccttag ttgccagcga gtaatgtcgg gaactctaag gagactgccg 1020

gtgacaaacc ggaggaaggt ggggatgacg tcaagtcatc atggccctta cggccagggc 1080

tacacacgta ctacaatggt cggtacagag ggttgcgata ccgcgaggtg gagctaatcc 1140

cagaaagccg atcccagtcc ggattggagt ctgcaactcg actccatgaa gtcggaatcg 1200

ctagtaatcg cagatcagct atgctgcggt gaatacgttc ccgggccttg tacacaccgc 1260

ccgtcacacc atgggagtga gctgctccag aagccgttag tctaaccgca agggggacga 1320

cgaccacgga gtggttcatg actggg 1346

Claims

1. a plant height produces the tea tree endophyte of theanine, classification naming is Luo Dannuo Bacteriaceae gamboge Bacillus (Rhodanobacteraceae Luteibacter sp.) CsE7, is preserved in China typical culture collection center, and preservation is compiled Number be CCTCC NO:M 2018816, the deposit date is on November 23rd, 2018；Preservation address is that Wuhan City, Hubei China province is military Chinese university.

2. Luo Dannuo Bacteriaceae gamboge Bacillus CsE7 described in claim 1 is preparing the application in L-thiamine.

3. application according to claim 2, it is characterised in that: CsE7 bacterial strain is in 28 DEG C of shaken cultivations to OD₆₀₀When being 1.0, Glutamine and ethamine are added simultaneously into culture solution, cultivates 1~15d, take 100 DEG C of water-baths cracking 30min of culture solution, and in- 20 DEG C after multigelation 2~3 times, 12000rpm is centrifuged 15min, takes supernatant after 0.22 μm of water phase membrane filtration, carries out liquid Phase Mass Spectrometer Method；Obtain the supernatant containing L-thiamine.

4. application according to claim 3, it is characterised in that: add the final concentration of 20mM of glutamine.

5. application according to claim 3, it is characterised in that: add the final concentration of 20mM of ethamine.

6. application according to claim 3, it is characterised in that: culture 7d.