WO2019006700A1 - 一种生产5-羟色氨酸的方法 - Google Patents

一种生产5-羟色氨酸的方法 Download PDF

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WO2019006700A1
WO2019006700A1 PCT/CN2017/091858 CN2017091858W WO2019006700A1 WO 2019006700 A1 WO2019006700 A1 WO 2019006700A1 CN 2017091858 W CN2017091858 W CN 2017091858W WO 2019006700 A1 WO2019006700 A1 WO 2019006700A1
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hydroxytryptophan
culture
jsc
fermentation
bacillus subtilis
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PCT/CN2017/091858
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French (fr)
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孙敬方
赵云现
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保定保利瑞合生物科技有限公司
南京寿柏生物科技有限公司
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Priority to PCT/CN2017/091858 priority Critical patent/WO2019006700A1/zh
Publication of WO2019006700A1 publication Critical patent/WO2019006700A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus

Definitions

  • the invention relates to a Bacillus and a method for producing 5-hydroxytryptophan by fermentation using Bacillus, belonging to the field of biotechnology.
  • 5-hydroxytryptophan is a precursor of the hormone serotonin in the human body. It has a sedative effect and can help the body to produce this hormone and balance itself.
  • 5-HTP is a reaction medium for tryptophan and serotonin (complex amine), that is, 5-hydroxytryptophan is an important precursor for the inhibition of the neurotransmitter complex amine (5-HT).
  • 5-HTP neurotransmitter complex amine
  • 5-hydroxytryptophan can suppress appetite, reduce fat intake, reduce anxiety, control mood, and promote sleep.
  • 5-HTP as a food ingredient is naturally extracted from the seeds of Griffonia simplicifolia, a West African drug plant.
  • 5-hydroxytryptophan is a natural amino acid that can be found in proteins found in many diets.
  • 5-Hydroxytryptophan is a natural alternative to the prescription drug Prozac. Like Prozac, 5-hydroxytryptophan enhances the serotonin activity.
  • Serotonin is a hormone secreted by the brain that affects our mood, sleep and appetite. It requires 5-hydroxytryptophan to maintain normal body serotonin imbalance. Therefore, if the content is too low, it is easy to be depressed, anxious and sleep disorders. Prozac such drugs can prevent brain cells from depleting serotonin too quickly, leading to serotonin deficiency.
  • the efficacy of 5-hydroxytryptophan is slightly different: it increases the amount of serotonin produced by brain cells, which in turn increases the serotonin content.
  • 5-hydroxytryptophan produces serotonin in the human body and is useful for normal neurologically important substances and brain function. Serotonin can help sleep and effectively control pain. Therefore, 5-hydroxytryptophan has also been successfully used to treat sleep disorders. Since serotonin is the precursor of melatonin, a natural hormone that regulates sleep and wake-up cycles, melatonin levels increase as serotonin levels increase. plus. Since the effects of 5-hydroxytryptophan and SSRI are similar, it is better not to take the latter. One of the reasons is that anti-depressant prescription drugs are expensive; second, prescription drugs often cause some undesirable side effects, including dry mouth, anxiety and loss of sexual desire.
  • 5-HTP can help suppress appetite, which can make the body consume less carbohydrates and feel fuller.
  • the daily intake of 750 mg of 5-HTP can reduce the absorption of carbohydrates without increasing weight. In turn, the effect of weight loss is achieved.
  • 5-hydroxytryptophan 1, anti-depression, tranquilizer: 5-HTP can increase the production of serotonin in brain cells, thereby increasing the content of serotonin.
  • 5-hydroxytryptophan produces serotonin in the human body and is useful for normal neurologically important substances and brain function. Serotonin can help sleep, but also can effectively control pain, can treat depression; 2, reduce stress and improve sleep: when the serotonin content increases, the content of melatonin increases, making sleep more sweet; 3, weight loss: can effectively control appetite, improve the sensitivity of the satiety center, in the diet control of the weight loss process, reduce hunger, make the weight loss process easier and easier to achieve, is the current popular diet food supplement.
  • 5- Hydroxytryptophan has been widely used by major health care companies in Europe and America. It is used to increase the production of serotonin in human blood by naturally increasing the level of serotonin. At present, the preparation of 5-hydroxytryptophan is mainly from plant sources, extracted from plants of Ghana in Africa. However, production is limited by resources and the extraction cost is high. Obviously, microbial transformation and fermentation production are a good way.
  • a 5-hydroxytryptophan-producing Bacillus subtilis JSC-137 which has been deposited at the General Collection of Microorganisms and Cultures of China, numbered CGMCC NO: 13534, and the date of deposit is January 5, 2017 .
  • Morphological characteristics of the cells a single bacteria 0.7 ⁇ 2.5 ⁇ m, uniform coloration. No capsule, Zhousheng flagella, can exercise. Gram-positive bacteria, aerobic bacteria. Spore 0.9 ⁇ 1.5 ⁇ m, elliptical to columnar, located in the bacterium The center of the body is slightly or slightly, and the cells do not expand after the spores are formed. The surface of the colony is rough and opaque, stained white or yellowish. The surface of the colony has wrinkles, the colony is large, and the center is raised. It is uniformly turbid in the liquid matrix and does not form a bacterial membrane and a fungus ring.
  • Bacillus subtilis JSC-137 Bacillus subtilis JSC-137) was tested for physiological and biochemical properties. Results: The strain was obligately aerobic. It can use protein, various sugars and starches to decompose glucose to produce acid and not produce gas. It can decompose arabinose, mannitol, casein, gelatin and starch. Catalase test, nitrate reduction test, V-P test positive; phenylalanine deaminase test, egg yolk lecithin enzyme test negative.
  • the present invention further provides a method for producing 5-hydroxytryptophan using the Bacillus subtilis JSC-137, which method comprises the following steps:
  • Step a expanding culture of Bacillus subtilis JSC-137;
  • Step b using step a to expand the cultured Bacillus subtilis JSC-137 to convert tryptophan;
  • step c 5-hydroxytryptophan is collected.
  • the expanded culture described in the step a includes any one or a combination of the slant culture, the shake flask culture, the seed tank culture, and the fermenter culture.
  • the medium used in the expanded culture process is an aqueous medium containing absorbable carbon, nitrogen and phosphorus sources.
  • the medium may also include suitable salts, minerals, metals, and other nutrients;
  • the carbon source is selected from one of glucose, fructose, sucrose, galactose, dextrin, glycerin, starch, syrup, and molasses or a plurality of;
  • the nitrogen source is selected from one or more of ammonium chloride, ammonium sulfate, ammonium nitrate, and potassium nitrate.
  • the culture medium used in the shake flask culture and seed tank culture stages is an aqueous medium containing absorbable carbon, nitrogen and phosphorus sources; the medium may also include suitable salts, minerals, metals and other nutrients
  • the carbon source is selected from one or more of the group consisting of glucose, fructose, sucrose, galactose, dextrin, glycerin, starch, syrup and molasses
  • the nitrogen source is selected from the group consisting of ammonium chloride, ammonium sulfate, ammonium nitrate, potassium nitrate One or more of them.
  • the medium component comprises tryptone, beef extract, sodium chloride; further preferably the seed medium composition is: 15.0 g/L tryptone, 4.0 g/L beef extract, 15.0 g/L chlorination Sodium; initial pH 7.0.
  • the fermentation medium used in the fermentation tank culture stage is an aqueous medium containing absorbable carbon, nitrogen and phosphorus sources.
  • the medium may also include suitable salts, minerals, metals, and other nutrients;
  • the carbon source is selected from one of glucose, fructose, sucrose, galactose, dextrin, glycerin, starch, syrup, and molasses or a plurality of;
  • the nitrogen source is selected from one or more of ammonium chloride, ammonium sulfate, ammonium nitrate, and potassium nitrate.
  • the fermentation medium component comprises glucose, yeast extract, MgSO 4 ⁇ 7H 2 O, FeSO 4 ⁇ 7H 2 O, KH 2 PO 4 , K 2 HPO 4 , ZnSO 4 ; further preferably the fermentation medium composition is : glucose 10g / L, yeast extract 3.0g / L, MgSO 4 ⁇ 7H 2 O 0.6g / L, FeSO 4 ⁇ 7H 2 O 0.01g / L, KH 2 PO 4 0.4g / L, K 2 HPO 4 0.6g /L, ZnSO 4 0.001 g / L, initial pH 6.5 ⁇ 7.0.
  • the expanded culture conditions in the step a are: temperature 25-45 ° C, pH 6.5-7.0, time 20-50 hours.
  • the culture conditions of the shake flask and the seed tank culture stage are: temperature 25-45 ° C, pH 6.5-7.0, time 20-30 hours.
  • the culture conditions of the fermentation tank culture stage are: fermentation temperature 30-35 ° C, pH 6.5-7.0, fermentation time 30-50 hours.
  • the fermentation temperature is 31 ° C; pH 6.8; fermentation time 40 h.
  • the tryptophan concentration in step b is 0.2-10 g/L; the transformation conditions are: conversion at 25-45 ° C for 40-60 hours, pH 6-8.
  • the "collecting 5-hydroxytryptophan" described in the step c includes the following method: the conversion product obtained in the step b is subjected to centrifugation, and the supernatant fraction is taken as the product 5-hydroxytryptophan.
  • the method of the present invention further comprises: step d, purifying the product 5-hydroxytryptophan.
  • the "purification” process includes, in order, a step of solid-liquid separation, cation exchange resin adsorption separation, concentration, ethanol dissolution, and distillation.
  • the strain in the dormant state is activated, and then the culture is gradually expanded to obtain a certain amount and quality of the strain.
  • the general process of expanding the culture is: slant culture, shake flask culture, seed tank stepwise culture, fermenter culture, in actual operation, the seed tank culture grade can be according to fermentation scale, bacterial growth characteristics, inoculum size and the like. And ok.
  • substrate refers to a reactant to be converted
  • transformation refers to the use of a microorganism to convert a reactant to be converted into a desired product by a specific metabolic pathway under suitable conditions. the process of.
  • the present invention uses "tryptophan” as a substrate and is transformed with a specific strain of Bacillus licheniformis JSC-137 to obtain the final product "5-hydroxytryptophan".
  • the Bacillus subtilis JSC-137 of the present invention has a wider fermentation temperature (30 ° C to 37 ° C) than the general Bacillus, and can utilize a complicated nitrogen source in a short time. Growth to a higher biomass, biotransformation of tryptophan to produce 5-hydroxytryptophan. By optimizing the fermentation conditions, Bacillus subtilis JSC-137 produces 5-hydroxytryptophan with tryptophan as a substrate, and the transformation efficiency is 85%-90%, which can further purify the transformant and obtain high purity. 5-hydroxytryptophan.
  • Plate screening medium beef extract 5g / L, soy peptone 10g / L, NaCl 5g / L, 15g / L agar.
  • Liquid medium used for screening sucrose 20g / L, soybean meal 20g / L, (NH 4 ) 2 SO 4 3g / L, K 2 HPO 4 ⁇ 3H 2 O 5g / L, KH 2 PO 4 1g / L, beef cream 2 g / L, soy peptone 4 g / L, NaCl 5 g / L, tryptophan 8 g / L; pH 7.0.
  • More than 100 soil samples or water samples were collected from Nanjing and surrounding areas, and diluted to the appropriate concentration.
  • the plates were directly applied to the screening medium and cultured at 37 ° C for 1 day. Individual colonies of different forms were picked.
  • the test medium containing the liquid medium was shaken at 30 ° C and 200 rpm for 2 days, and the fermentation supernatant was centrifuged. The content of 5-hydroxytryptophan in the supernatant was determined. Four strains were detected from more than 500 colonies to produce 5-hydroxytryptophan.
  • the strains obtained by the initial screening were inoculated into test tubes containing 5 ml of seed medium, and cultured at 30 ° C for 2 days with shaking. It was then transferred to a flask containing 50 ml of fermentation medium and fermented at 30 ° C, 200 rpm for 2 days. The content of 5-hydroxytryptophan in the fermentation supernatant was accurately determined by HPLC. The results are shown in Table 1.
  • Table 1 shows the production of 5-hydroxytryptophan in some strains
  • the cells were streaked on the plate medium for 1 day, and the surface of the colony was rough and opaque, stained white or yellowish.
  • the surface of the colony has wrinkles, the colony is large, and the center is raised. It is uniformly turbid in the liquid matrix and does not form a bacterial membrane and a fungus ring.
  • a single bacterium is 0.7 x 2.5 microns and is evenly colored. No capsule, Zhousheng flagella, can exercise.
  • Gram-positive bacteria aerobic bacteria.
  • the spores are 0.9 ⁇ 1.5 ⁇ m, elliptical to columnar, located in the center of the cells or slightly biased, and the cells do not expand after the spores are formed. JSC-137 was tested for physiological and biochemical properties.
  • the strain was obligately aerobic. It can use protein, various sugars and starches to decompose glucose to produce acid and not produce gas. It can decompose arabinose, mannitol, casein, gelatin and starch. Catalase test, nitrate reduction test, V-P test positive; phenylalanine deaminase test, egg yolk lecithin enzyme test negative.
  • JSC-137 is considered to belong to the new strain Bacillus subtilis, named Bacillus subtilis JSC-137. This strain has been deposited with the China General Microorganisms Collection and Management Center on January 5, 2017, numbered CGMCC NO: 13534.
  • (1) Primary seed culture The Bacillus subtilis JSC-137 slant culture which grew well in a loop was inoculated into a seed culture medium, and cultured at 30 ° C and 225 rpm for 25 hours with shaking.
  • the seed medium composition was: 15.0 g/L tryptone, 4.0 g/L beef extract, and 15.0 g/L sodium chloride. Initial pH 7.0.
  • Secondary seed culture 6 ml of the primary seed culture solution was taken, inoculated into a 1000 ml shake flask containing 200 ml of the secondary seed culture solution, and cultured at 30 ° C, 225 rpm for about 24 hours. The OD 600 value is 8.0-9.0.
  • Secondary seed culture liquid base component The seed culture medium composition was: 15.0 g/L tryptone, 4.0 g/L beef extract, and 15.0 g/L sodium chloride. Initial pH 7.0.
  • Fermentation medium components glucose 10g / L, yeast extract 3.0g / L, MgSO 4 ⁇ 7H 2 O 0.6g / L, FeSO 4 ⁇ 7H 2 O 0.01g / L, KH 2 PO 4 0.4g / L, K 2 HPO 4 0.6 g/L, ZnSO 4 0.001 g/L.
  • the initial pH is 6.5 to 7.0.
  • Shake flask test method Take the strain inoculated with 5ml seeds The medium was cultured in a test tube at 30 ° C for 2 days. It was then transferred to a shake flask containing 50 ml of fermentation medium and fermented. 8 g/L tryptophan substrate was added to the fermentation medium. The content of 5-hydroxytryptophan in the fermentation supernatant was accurately determined by HPLC.
  • the inoculum amount is too low (0.5%), or too high (2.5%) is unfavorable for the catalytic synthesis of 5-hydroxytryptophan by tryptophan, preferably 1.5% inoculum.
  • the amount of inoculation is too low, which is not conducive to the growth of the cells; the inoculum is too high, the growth of the cells is too fast, and the aging is catalyzed.
  • the fermentation temperature has a certain influence on the catalytic synthesis of 5-hydroxytryptophan by tryptophan.
  • the catalytic effect below 31 °C is worse than that at 31 °C, but the catalytic effect above 31 °C is also worse than that at 31 °C.
  • the fermentation time is preferably 40h.
  • inoculum size 1.5%
  • fermentation temperature 31 ° C pH 6.8
  • Fermentation time 40h the optimized technical parameters: inoculum size 1.5%; fermentation temperature 31 ° C; pH 6.8; Fermentation time 40h.
  • Seed preparation requirements The strain identification conforms to the characteristics, no variation, no bacteria.
  • Fermentation empty tank sterilization temperature 121 ⁇ 130 ° C, keep circulating steam, tank pressure 0.09 ⁇ 0.15Mpa, hold time 30 minutes.
  • Feed water adjust the total volume.
  • Fermentation tank sterilization temperature 121 ° C, tank pressure 0.08 ⁇ 0.15Mpa, ventilation 0.8 ⁇ 1.0vvm, maintenance time 30 minutes, to maintain circulating steam; finally cooled to 30 ° C.
  • Inoculation temperature 30 ° C, inoculum 1%.
  • Transformation 0.2 M phosphate buffer (pH 7.4) was added to the cell pellet to form a bacterial suspension having a bacterial concentration of 3.0 ⁇ 10 7 to 8.0 ⁇ 10 7 /m1; Then, tryptophan at a concentration of 8.0 g/L was added, and the aeration amount was 1.0 L/min at a temperature of 31 ° C, and the transformation was carried out at a rotation speed of 225 r/min. After 40 hours, a conversion liquid was obtained.
  • Bacillus subtilis JSC-137 produces 5-hydroxytryptophan by tryptophan as a substrate, and the conversion rate is 85% to 90% in 24 hours.
  • Solid-liquid separation of fermentation broth The cells are collected by centrifugation or ceramic membrane separation.
  • 001 ⁇ 7 cation exchange resin (732) is adsorbed and separated, and ammonia water is eluted.
  • 001 ⁇ 7 cation exchange resin After screening, 001 ⁇ 7 cation exchange resin has the best adsorption and separation effect, and is suitable for large-scale production of 99% 5-hydroxytryptophan.
  • the conditions for adsorption separation of 001 ⁇ 7 resin are as follows: at room temperature, the concentration of 5-hydroxytryptophan in the sample solution is 10.8 mg/ml, the pH is adjusted to 3.5, and the sample is loaded at a loading rate of 4.0 ml/min to 3 The resin volume of 7.0% ammonia water was eluted at an elution rate of 3.0 ml/min. The product obtained after concentration and crystallization of the eluate had a mass fraction of 5-hydroxytryptophan greater than 99.0% and an ash content less than 1.0%.
  • the eluate is concentrated to a solid by double or multiple effect evaporation. Dry to remove moisture.
  • the concentrated solid was dissolved in hot water, and further adsorbed and separated by a resin highly selective for 5-hydroxytryptophan to remove impurities. After elution with aqueous ammonia, it was concentrated and crystallized. High purity 5-hydroxytryptophan.
  • the product analysis method is an HPLC detection method.
  • a standard solution and sample of tryptophan and 5-hydroxytryptophan were chromatographed using a Shimadzu C18 reverse phase column, 0.05% trichloroacetic acid (TCA)-methanol (68.75:31.25, v/v) as the mobile phase.
  • Injection volume 10 ⁇ L
  • mobile phase flow rate 1.5 ml/min gradient elution
  • UV spectrophotometer wavelength 275 nm All solutions were filtered through a 0.45 ⁇ m filter before use. Calculated by the external standard method.

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Abstract

提供了一种生产5-羟色氨酸的枯草芽孢杆菌（Bacillus subtilis）JSC-137以及应用其以色氨酸为底物生产5-羟色氨酸的方法，该枯草芽孢杆菌JSC-137的保藏编号为CGMCC NO：13534。

Description

一种生产5-羟色氨酸的方法 技术领域
本发明涉及一种芽孢杆菌以及利用芽孢杆菌发酵生产5-羟色氨酸的方法，属于生物技术领域。
背景技术
5-羟色氨酸(5-HTP)是人体内激素serotonin的前驱物质，有镇静作用，可以帮助人体内这种激素的生产，使自己体内达到平衡。5-HTP是色氨酸、5-羟色胺(复合胺)的反应媒介，也就是说，5-羟色氨酸是重要的抑制神经传递素复合胺(5-HT)的前体。在脑部、血小板、肠胃、中枢神经系统中均有复合胺。在情绪变化时，会相应分泌复合胺，它是脑部控制情绪、行为、胃口、睡眠、冲动的重要化学物质。5-羟色氨酸能有效帮助产生复合胺，因此可促进情绪、神经系统的健康。研究表明，5-羟色氨酸可抑制食欲，减少脂肪摄取，减少焦虑，控制情绪，促进睡眠。目前国内可稳定生产的有优速达生物，产品质量已经得到欧美等国家的认可。
5-HTP作为食品成分是天然提取来自于Griffonia simplicifolia，一个西非药物植物的种子中得到的。5-羟色氨酸是一个自然氨基的酸性物质，在许多饮食所含的蛋白质中可以找到。5-羟色氨酸是处方药-百忧解(Prozac)的天然替代品。正如百忧解一样，5-羟色氨酸可增强5-羟色胺的活动力。
5-羟色胺是脑部分泌的一种激素，会影响我们的情绪、睡眠和食欲，造成脑中的血清素不平衡时需5-羟色氨酸来维持身体处于正常状态。因此含量太低则容易忧郁、焦虑及睡眠失调。百忧解这类的药物，可防脑细胞太快耗尽血清素，而导致5-羟色胺不足。而5-羟色氨酸的功效稍有不同：它可增加脑细胞的血清素制造量，进而提升5-羟色胺的含量。
5-羟色氨酸在人体中会制造出血清素，对于正常的神经的重要物质和脑的功能有帮助。血清素可以帮助睡眠，也可有效的控制疼痛。因此5-羟色氨酸也成功地被拿来治疗睡眠失调。由于血清素是褪黑激素(一种调节睡眠和清醒周期的天然荷尔蒙)的前身，因此当血清素的含量提高时，褪黑激素的含量也就增 加。既然5-羟色氨酸和SSRI的作用类似，何不服用后者就好。原因之一是抗忧郁处方药贵；二是处方药常会引起一些不良的副作用，包括口干、焦虑及丧失性欲。
此外，使用5-HTP能帮助抑制食欲，它能使人体所摄取的碳水化合物更少且更快感到饱足，每天摄取750毫克的5-HTP可以减少碳水化合物的吸收，并且不使体重增加，进而达到减轻体重的效果。
5-羟色氨酸的临床应用：1、抗忧郁、镇定剂：5-HTP可增加脑细胞的血清素制造量，进而提升5-羟色胺的含量。5-羟色氨酸在人体中会制造出血清素，对于正常的神经的重要物质和脑的功能有帮助。血清素可以帮助睡眠，也可有效的控制疼痛，可以治疗忧郁症；2、减缓压力和改善睡眠方面：当血清素的含量提高时，褪黑激素的含量也就增加，使得睡眠更为香甜；3、减肥：可以有效控制食欲，提高饱食中枢的敏感性，在减肥过程的饮食控制中，降低饥饿感，使减肥过程更轻松容易达成，是当前流行的减肥食品辅助剂。对想减肥的人是一大福音；4、其他：除具有传统的医药功能和抗忧郁症外，还具有减肥、戒瘾、改善睡眠、缓和经前综合症，治疗偏头疼等功能；5-羟色氨酸现已被欧美各大保健品公司广泛应用。用于通过自然地提高血清素的水平，增加人体血液中复合胺的生成。目前，5-羟色氨酸的制备主要由植物来源，提取于非洲的植物加纳种子。但产量受资源限制，并且提取成本高。显然，微生物转化和发酵法生产是一条良好的途径。
发明内容
为解决现有技术存在的问题，本发明的目的在于提供一种芽孢杆菌以及利用该芽孢杆菌发酵生产5-羟色氨酸的方法。
一种生产5-羟色氨酸的枯草芽孢杆菌(Bacillus subtilis)JSC-137，其已在中国普通微生物菌种保藏管理中心保藏，编号为CGMCC NO：13534，保藏日期为2017年01月05日。
所述枯草芽孢杆菌JSC-137的鉴定：
(1)菌体的形态特征：单个细菌0.7×2.5微米，着色均匀。无荚膜，周生鞭毛，能运动。革兰氏阳性菌，需氧菌。芽孢0.9×1.5微米，椭圆到柱状，位于菌 体中央或稍偏，芽孢形成后菌体不膨大。菌落表面粗糙不透明，污白色或微黄色。菌落表面有褶皱，菌落较大，中央突起。在液体基质中呈均匀混浊，不形成菌膜和菌环。
(2)菌体的生理生化鉴定：将枯草芽孢杆菌JSC-137(Bacillus subtilis JSC-137)进行生理生化性状检测，结果：该菌株专性好氧。可利用蛋白质、多种糖及淀粉，分解葡萄糖产酸不产气，能分解阿拉伯糖、甘露醇、酪素、明胶、淀粉。过氧化氢酶实验、硝酸盐还原实验、V-P实验阳性；苯丙氨酸脱氨酶实验、卵黄卵磷脂酶实验阴性。
本发明进一步提供应用所述的枯草芽孢杆菌JSC-137生产5-羟色氨酸的方法，该方法具体包括如下步骤：
步骤a，对枯草芽孢杆菌JSC-137进行扩大培养；
步骤b，用步骤a扩大培养获得到的枯草芽孢杆菌JSC-137转化色氨酸；和
步骤c，收集5-羟色氨酸。
进一步，步骤a所述扩大培养包括斜面培养、摇瓶培养、种子罐培养和发酵罐培养中的任意一种或几种的组合。
所述扩大培养过程所用培养基是含有可吸收的碳、氮和磷源的含水培养基。所述培养基还可包括适宜的盐、矿物质、金属和其他营养成分；所述碳源选自葡萄糖、果糖、蔗糖、半乳糖、糊精、甘油、淀粉、糖浆和糖蜜中的一种或多种；氮源选自氯化铵、硫酸铵、硝酸铵、硝酸钾中的一种或多种。
其中，所述摇瓶培养和种子罐培养阶段所用培养基是含有可吸收的碳、氮和磷源的含水培养基；所述培养基还可包括适宜的盐、矿物质、金属和其他营养成分；所述碳源选自葡萄糖、果糖、蔗糖、半乳糖、糊精、甘油、淀粉、糖浆和糖蜜中的一种或多种；氮源选自氯化铵、硫酸铵、硝酸铵、硝酸钾中的一种或多种。优选所述培养基组分包括胰蛋白胨、牛肉浸膏、氯化钠；进一步优选所述种子培养基组成为：15.0g/L胰蛋白胨、4.0g/L牛肉浸膏、15.0g/L氯化钠；初始pH7.0。
所述发酵罐培养阶段所用发酵培养基是含有可吸收的碳、氮和磷源的含水培养基。所述培养基还可包括适宜的盐、矿物质、金属和其他营养成分；所述碳源选自葡萄糖、果糖、蔗糖、半乳糖、糊精、甘油、淀粉、糖浆和糖蜜中的 一种或多种；氮源选自氯化铵、硫酸铵、硝酸铵、硝酸钾中的一种或多种。优选所述发酵培养基组分包括葡萄糖、酵母膏、MgSO4·7H2O，FeSO4·7H2O，KH2PO4，K2HPO4，ZnSO4；进一步优选所述发酵培养基组成为：葡萄糖10g/L，酵母膏3.0g/L，MgSO4·7H2O 0.6g/L，FeSO4·7H2O 0.01g/L，KH2PO4 0.4g/L，K2HPO4 0.6g/L，ZnSO4 0.001g/L，初始pH6.5～7.0。
进一步，步骤a所述扩大培养条件为：温度25-45℃，pH6.5-7.0，时间20-50小时。
其中，所述摇瓶和种子罐培养阶段培养条件为：温度25-45℃，pH6.5-7.0，时间20-30小时。
所述发酵罐培养阶段培养条件为：发酵温度30-35℃，pH6.5-7.0，发酵时间30-50小时。优选，发酵温度31℃；pH6.8；发酵时间40h。
进一步，步骤b所述色氨酸浓度为0.2-10g/L；转化条件为：25-45℃下转化40-60小时，pH为6-8。
进一步，步骤c所述“收集5-羟色氨酸”包括如下方法：对步骤b得到的转化产物进行离心处理，取上清液部分即为产物5-羟色氨酸。
进一步，本发明方法还包括：步骤d，对产物5-羟色氨酸进行纯化。所述“纯化”过程依次包括固液分离、阳离子交换树脂吸附分离、浓缩、乙醇溶解、蒸馏的步骤。
本发明所述“扩大培养”以菌种扩增为目的，将处于休眠状态的菌种活化后，再经过逐级扩大培养，最终获得一定数量和质量的菌种的过程。所述扩大培养的一般过程为：斜面培养、摇瓶培养、种子罐逐级培养、发酵罐培养，在实际操作中，种子罐培养级数可按照发酵规模、菌体生长特性、接种量等因素而确定。
本发明所述“底物”是指待转化的反应物，所述“转化”是指利用微生物，在适宜的条件下，将待转化的反应物经过特定的代谢途径转化为人类所需要的产物的过程。本发明以“色氨酸”为底物，经过特定菌种地衣芽孢杆菌JSC-137的转化，得到终产物“5-羟色氨酸”。
本发明所述枯草芽孢杆菌JSC-137(Bacillus subtilis JSC-137)，与一般的芽孢杆菌相比，具有更广泛的发酵温度(30℃～37℃)，可以利用复杂氮源，短时间内 生长达到较高的生物量，可生物转化色氨酸生产5-羟色氨酸。通过对发酵条件的优化，枯草芽孢杆菌JSC-137以色氨酸为底物生产5-羟色氨酸，转化效率达85％～90％，可将转化液经进一步纯化及能得到高纯度的5-羟色氨酸。
具体实施方式
实施例1菌株筛选
平板筛选培养基：牛肉膏5g/L，大豆蛋白胨10g/L，NaCl 5g/L，15g/L琼脂。
筛选时所用液体培养基：蔗糖20g/L，豆粕20g/L，(NH4)2SO4 3g/L，K2HPO4·3H2O 5g/L，KH2PO4 1g/L，牛肉膏2g/L，大豆蛋白胨4g/L，NaCl 5g/L，色氨酸8g/L；pH值7.0。
从南京及周边地区共釆集100多份土样或水样，并制成适当浓度的稀释液，直接涂布于筛选培养基平板，37℃培养1天，分别挑取不同形态的单菌落接种于装有液体培养基的试管，30℃、200rpm震荡培养2天，离心取发酵上清液。测定上清中5-羟色氨酸含量。从500多个菌落中检测到4株可以产生5-羟色氨酸。
取初筛得到的菌株分别接种于装有5ml种子培养基的试管中，30℃震荡培养2天。然后转接到装有50ml发酵培养基的三角瓶，30℃、200rpm发酵2天。用HPLC法精确测定发酵上清中5-羟色氨酸的含量。结果见表1。
表1部分筛菌株的产5-羟色氨酸情况
菌株编号 5-羟色氨酸的含量(g/L)
JSC-24 1.2
JSC-69 7.7
JSC-137 6.8
JSC-403 0.4
结果表明，菌株JSC-69和JSC-137发酵液中5-羟色氨酸含量明显高于另外两种菌株。
实施例2菌株JSC-137的鉴定
对JSC-137按《伯杰氏系统细菌学手册》(第二版,2004)进行形态特征及生理 生化特征的鉴定。
在平板培养基上划线培养1天，菌落表面粗糙不透明，污白色或微黄色。菌落表面有褶皱，菌落较大，中央突起。在液体基质中呈均匀混浊，不形成菌膜和菌环。单个细菌0.7×2.5微米，着色均匀。无荚膜，周生鞭毛，能运动。革兰氏阳性菌，需氧菌。芽孢0.9×1.5微米，椭圆到柱状，位于菌体中央或稍偏，芽孢形成后菌体不膨大。将JSC-137进行生理生化性状检测，结果：该菌株专性好氧。可利用蛋白质、多种糖及淀粉，分解葡萄糖产酸不产气，能分解阿拉伯糖、甘露醇、酪素、明胶、淀粉。过氧化氢酶实验、硝酸盐还原实验、V-P实验阳性；苯丙氨酸脱氨酶实验、卵黄卵磷脂酶实验阴性。
基于形态特征、生理生化特性分析，认为JSC-137属于Bacillus subtilis新菌株，以命名为Bacillus subtilis JSC-137。此菌株已于2017年01月05日保藏于中国普通微生物菌种保藏管理中心，编号为CGMCC NO：13534。
实施例3菌株JSC-137的发酵培养与目标产物转化
1、扩大培养
(1)一级种子培养：将一环生长良好的所述枯草芽孢杆菌JSC-137斜面培养物接种于种子培养基，在30℃、225rpm条件下震荡培养25小时。种子培养基组成为:15.0g/L胰蛋白胨、4.0g/L牛肉浸膏、15.0g/L氯化钠。初始pH7.0。
(2)二级种子培养：取6ml一级种子培养液，接种于含200ml的二级种子培养液的1000ml摇瓶中，30℃、225rpm培养约24小时。至OD600值为8.0-9.0。二级种子培养液基成分：种子培养基组成为:15.0g/L胰蛋白胨、4.0g/L牛肉浸膏、15.0g/L氯化钠。初始pH7.0。
(3)发酵罐发酵培养
1)发酵培养基成分：葡萄糖10g/L，酵母膏3.0g/L，MgSO4·7H2O 0.6g/L，FeSO4·7H2O 0.01g/L，KH2PO4 0.4g/L，K2HPO4 0.6g/L，ZnSO4 0.001g/L。初始pH6.5～7.0。
2)主要技术参数及优化筛选
基本技术参数：接种量1％；发酵温度31℃；pH6.7；发酵时间40h。
在此基本技术参数基础上，进行摇瓶试验，对接种量、发酵温度、pH、发酵时间等因素分别进行了优化筛选。摇瓶试验方法：取菌株接种于装有5ml种子 培养基的试管中，30℃震荡培养2天。然后转接到装有50ml发酵培养基的摇瓶中，发酵。发酵培养基中加入8g/L色氨酸底物。用HPLC法精确测定发酵上清中5-羟色氨酸的含量。
a)接种量的影响
考察了不同接种量对5-羟色氨酸催化合成的影响。其它技术参数条件：发酵温度31℃；pH6.7；发酵时间40h。结果见表2。
表2不同接种量对5-羟色氨酸催化合成的影响
接种量(％) 5-羟色氨酸的含量(g/L)
0.5 7.3
1.0 7.5
1.5 7.7
2.0 7.6
2.5 7.6
从表2可以看出，接种量过低(0.5％)，或过高(2.5％)对色氨酸催化合成5-羟色氨酸均不利，以1.5％接种量为佳。可能接种量过低，不利于菌体生长；接种量过高，菌体生长过快容易衰老进而催化效果。
b)发酵温度的影响
考察了不同发酵温度对5-羟色氨酸催化合成的影响。其它技术参数条件：接种量1.5％；pH6.7；发酵时间40h。结果见表3。
表3不同发酵温度对5-羟色氨酸催化合成的影响
发酵温度(℃) 5-羟色氨酸的含量(g/L)
30 7.5
31 7.7
32 7.6
33 7.6
34 7.5
35 7.3
从表3可以看出，发酵温度对色氨酸催化合成5-羟色氨酸有一定的影响，31℃以下较31℃催化效果差，但31℃以上也较31℃催化效果差，而且随温度升高， 效果越来越差。以31℃发酵最佳。
c)pH的影响
考察了不同pH对5-羟色氨酸催化合成的影响。其它技术参数条件：接种量1.0％；发酵温度32℃；发酵时间40h。结果见表4。
表4不同pH对5-羟色氨酸催化合成的影响
pH 5-羟色氨酸的含量(g/L)
6.5 7.1
6.6 7.2
6.7 7.4
6.8 7.8
6.9 7.7
7.0 7.3
从表4可以看出，pH对色氨酸催化合成5-羟色氨酸有一定的影响，pH6.8以下或pH6.8以上均较pH6.8催化效果差，以pH6.8发酵时催化效果最佳。
d)发酵时间的影响
考察了不同发酵时间对5-羟色氨酸催化合成的影响。其它技术参数条件：接种量1.0％；发酵温度32℃；pH6.8。结果见表5。
表5不同pH对5-羟色氨酸催化合成的影响
发酵时间(h) 5-羟色氨酸的含量(g/L)
30 6.5
35 7.2
40 7.8
45 7.8
50 7.8
55 7.8
从表5可以看出，不同发酵时间下色氨酸催化合成5-羟色氨酸有一定的差别，随发酵时间延长，催化效果逐渐增加。但40h以后，催化效果已达到顶峰，之后不再增加。因此发酵时间以40h为佳。
因此，经优化筛选，优化后技术参数：接种量1.5％；发酵温度31℃；pH6.8； 发酵时间40h。
3)工艺操作
种子制备要求：菌种鉴定符合特性，无变异，无杂菌。
培养基配置：按配方准确称量。蔗糖加适量水充分溶解；(NH4)2SO4，K2HPO4·3H2O，KH2PO4，加适量水充分溶解。消泡剂适量。
发酵空罐灭菌：温度121～130℃，保持流通蒸汽，罐压0.09～0.15Mpa，维持时间30分钟。
进料加水：调整总体积。
发酵实罐灭菌：温度121℃，罐压0.08～0.15Mpa，通气量0.8～1.0vvm，维持时间30分钟，保持流通蒸汽；最后降温至30℃。
接种：温度30℃，接种量1％。
培养：温度31℃,时间40小时,罐压0.05～0.08Mpa,pH 6.8，DO25％以上。芽孢形成率98％，培养终止，得到转化用菌液，液体菌数≥2.0×109cfu/ml。降温保压。
3、目的产物转化与生产
(1)收集菌体细胞：取所述枯草芽孢杆菌JSC-137转化用菌液在离心机上以4000r/min的转速高心10min，收集菌体细胞沉淀。
(2)转化：将0.2M的磷酸盐缓冲液(pH7.4)加到所述菌体细胞沉淀中,形成菌体浓度为3.0×107～8.0×107个/m1的菌悬液；然后加入浓度为8.0g/L的色氨酸，在31℃温度下、通气量为1.0L/min，以225r/min的转速进行转化，40h后，得到转化液。
所述0.2M的磷酸盐缓冲液(pH7.4)的配制：将71.6g Na2HPO4·12H2O溶于1000m1水，得到0.2M Na2HPO4；然后将31.2g NaH2PO4·2H2O溶于1000m1水，得到0.2M NaH2PO4；最后，将19m1 0.2M的NaH2PO4和81m1 0.2M的Na2HPO4混合均匀后即得。
(3)固液分离：将所述转化液在离心机上以4000r/min的转速高心10min，得到含5-羟色氨酸上清液。
枯草芽孢杆菌JSC-137以色氨酸为底物的微生物转化法生产5-羟色氨酸，24h转化率达85％～90％。
实施例4产物的纯化与提取
1、发酵液固液分离：采用离心或陶瓷膜分离法收集菌体。
2、001×7阳离子交换树脂(732)吸附分离，氨水洗脱。
经筛选，001×7阳离子交换树脂吸附分离效果最佳，适合于99％规格5-羟色氨酸的规模化生产。001×7树脂吸附分离的条件为：在常温下，上样液5-羟色氨酸质量浓度为10.8mg/ml，调pH为3.5，以4.0ml/min的上样速度上样，以3倍树脂体积的7.0％氨水以3.0ml/min的洗脱速度进行洗脱，洗脱液浓缩结晶后获得的产品中5-羟色氨酸质量分数大于99.0％，灰分小于1.0％。
3、浓缩成固体
将洗脱液进行双效或多效蒸发浓缩成固体。烘干去除水分。
4、热水溶解，高选择性的树脂吸附分离
将浓缩固体用热水溶解，进一步用对5-羟色氨酸高度选择性的树脂吸附分离，去除杂质。氨水洗脱后，浓缩，结晶。得高纯度5-羟色氨酸。
5、液相蒸馏，得固相为5-羟色氨酸。
实施例5产物分析方法
产物分析方法为HPLC检测方法。采用ShimadzuC18反相柱，0.05％的三氯乙酸(TCA)-甲醇(68.75:31.25，v/v)为流动相，对色氨酸和5-羟色氨酸的标准溶液和样品进行色谱测定。进样量：10μL，流动相流速：1.5ml/min等梯度洗脱，紫外分光光度检测器的波长为275nm。所有溶液在使用前均用0.45μm滤膜过滤。用外标法进行计算。

Claims (10)

  1. 一种生产5-羟色氨酸的枯草芽孢杆菌(Bacillus subtilis)JSC-137，其保藏编号为CGMCC NO：13534。
  2. 应用权利要求1所述的枯草芽孢杆菌JSC-137生产5-羟色氨酸的方法。
  3. 如权利要求2所述的方法，其特征在于，该方法包括如下步骤：
    步骤a，对枯草芽孢杆菌JSC-137进行扩大培养；
    步骤b，用步骤a扩大培养获得到的枯草芽孢杆菌JSC-137转化色氨酸；和
    步骤c，收集5-羟色氨酸。
  4. 如权利要求3所述的方法，其特征在于，步骤a所述扩大培养包括斜面培养、摇瓶培养、种子罐培养、发酵罐培养中的任意一种或几种的组合。
  5. 如权利要求4所述的方法，其特征在于，所述发酵罐培养阶段培养条件为：发酵温度30-35℃，pH6.5-7.0，发酵时间30-50小时。
  6. 如权利要求4所述的方法，其特征在于，所述发酵罐培养阶段的培养基组分包括：葡萄糖、酵母膏、MgSO4·7H2O、FeSO4·7H2O、KH2PO4、K2HPO4、ZnSO4。
  7. 如权利要求3所述的方法，其特征在于，步骤b所述色氨酸浓度为0.2-10g/L。
  8. 如权利要求3所述的方法，其特征在于，步骤b所述转化条件为：25-45℃下转化40-60小时，pH为6-8。
  9. 如权利要求3所述的方法，其特征在于步骤c所述“收集5-羟色氨酸”包括如下方法：对步骤b得到的转化产物进行离心处理，取上清液，即为产物5-羟色氨酸。
  10. 如权利要求3所述的方法，其特征在于，所述方法包括：步骤d，对产物5-羟色氨酸进行纯化；所述纯化过程依次包括固液分离、阳离子交换树脂吸附分离、浓缩、乙醇溶解、蒸馏的步骤。
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