Method for producing 5-hydroxytryptophan

Abstract

This invention involves to Bacillus licheniformis JSC-69 for producing the 5-HTP, deposited as CGMCC NO: 13533; and a method for the producing 5-HTP using Bacillus licheniformis. Bacillus licheniformis JSC-69 said in this invention produces 5-HTP using tryptophan as the substrate, and the transformation efficiency is 95%˜100%.

Claims

1. A method for producing 5-HTP, by Bacillus licheniformis JSC-69 deposited under accession number CGMCC 13533, said method comprising the following steps: Step a) culturing of Bacillus licheniformis said Bacillus licheniformis JSC-69; Step b) adding tryptophan to said culture of step a) and transforming the tryptophan to 5-HTP by said Bacillus licheniformis JSC-69; Step c) collecting the 5-HTP.

2. The method of claim 1, wherein the culturing step comprising any one or a combination of slant culturing, shake flask culturing, seed tank culturing and fermenter culturing.

3. The method of claim 2, wherein the fermenter culturing is carried out under 30-35° C. and pH 6.5-7.0 for 30-50 h.

4. The method of claim 2, wherein the medium of fermenter culturing comprises sucrose, bean pulp, (NH.sub.4).sub.2SO.sub.4, K.sub.2HPO.sub.4.3H.sub.2O and KH.sub.2PO.sub.4.

5. The method of claim 1, wherein the tryptophan concentration in Step b is 0.2-10 g/L.

6. The method of claim 1, wherein the transforming in Step b is carried out under 25-45° C. and pH 6-8 for 40-60 h.

7. The method of claim 1, wherein the collecting described in Step c includes the following method: centrifuging the transformed product obtained in Step b and collecting the supernatant, which is the product 5-HTP.

8. The method of claim 1, further comprising Step d, that is purifying the product 5-HTP, the “purifying” process includes solid-liquid separation, cation exchange resin adsorption separation, concentration, ethanol dissolution and distillation steps in order of sequence.

Description

EXPERIMENTAL EXAMPLES

Examples 1

Screening of Bacterial Strains

(1) Plate screening medium: 5 g/L beef extract, 10 g/L soy peptone, 5 g/L NaCl, and 15 g/L agar. Liquid screening medium: 20 g/L sucrose, 20 g/L bean pulp, 3 g/L (NH.sub.4).sub.2SO.sub.4, 5 g/L K.sub.2HPO.sub.4.3H.sub.2O, 1 g/L KH.sub.2PO.sub.4, 2 g/L beef extract, 4 g/L soy peptone, 5 g/L NaCl, 8 g/L tryptophan; pH is 7.0.

(2) More than 100 soil or water samples were collected from Nanjing and surrounding areas to prepare appropriate concentrations of dilutions, the prepared samples are directly applied to screening medium plates, cultured at 37° C. for 1 day. Then, single colony of different morphologies are selected and inoculated into test tubes containing liquid medium. After shake culture at 30° C. and 200rpm for 2 days, fermentation supernatant is centrifuged. The 5-HTP content in the supernatant sis determined. 5-HTP production is detected in 4 of more than 500 colonies.

(3) The strains obtained after initial screening are separately inoculated into test tubes containing 5m1 liquid medium and shake-cultured at 30° C. for 2 days. It is then transferred into a flask containing 50m1 liquid medium and fermented at 30° C. 200rpm for 2 days. The content of 5-HTP in fermentation supernatant is accurately determined using HPLC. See Table 1 for results.

(4) TABLE-US-00001 TABLE 1 5-HTP production status of screened strains Strain No. 5-HTP content (g/L) JSC-24 1.2 JSC-69 7.7 JSC-137 6.8 JSC-403 0.4

(5) The results show that 5-HTP content in JSC-69 and JSC-137 fermentation broth is significantly higher than in the fermentation broth of the other two strains.

Examples 2

Identification of Strain JSC-69

(6) The morphological, physiological and biochemical characteristics of JSC-69 are determined according to “Bergey's Manual of Determinative Bacteriology” (2nd Edition 2, 2004). After streak culture on plate medium for 1 day, the colonies are characterized by approximately round shape, milky white color, dark surface, opaque, irregular edge, coarse, sticky and spreading appearance. The colonies tightly adhere to the culture medium and are difficult to pick; in liquid culture, it has membrane without turbidity or precipitation. Individual bacillus should be 0.6×2.0 μm, this Gram-positive and aerobic bacterium is able to move without capsule or chain. The elliptical or cylindrical spore is 0.9×1.5 μm sitting in the middle or at one end with enlarged middle portion. The physiological and biochemical properties JSC-69 are determined, the results should be: the results show that this strain is catalase test positive. Gelatine liquefication, amylolysis and V.P test should also be positive, with citrate utilization. The methyl red test and lecithinase test are negative; it is able to utilize nitrate, but it is also able to utilize D-glucose, L-arabinose, D-xylose and D-mannitol to produce acid.

(7) Based on the analysis of morphological, physiological and biochemical characteristics, JSC-69 is considered to be a new strain of Bacillus licheniformis and named as Bacillus licheniformis JSC-69. This strain was deposited at China General Microbiological Culture Collection Center, the number is CGMCC NO: 13533.

Examples 3

Fermentation Culturing of Strain JSC-69 and Producing the Target Product

(8) 1. Culturing (1) Primary seed culture: Monoclonal strains that have been freshly cultured in plate seed culture medium for ˜18 h are inoculated into 6 ml of liquid seed culture medium and cultured at 37° C. 225 rpm for ˜24 h. Plate seed medium composition (/L): 5 g beef extract, 10 g soy peptone, 5 g NaCl, 1 L water and 15 g agar. Liquid seed medium composition (/L): 5 g beef extract, 10 g soy peptone, 5 g NaCl and 1 L water, pH is7.0. Sterilization is carried out under 0.1 MPa for 20 min. (2) Secondary seed culture: 6m1 of the primary seed culture medium is inoculated into a 1000 ml shake flask containing 200 ml of secondary seed culture medium and cultured at 30° C., 225 rpm for ˜24 h. OD600 is 6.0-10. Secondary seed culture medium composition is: 0.1% peptone, 0.2% corn syrup, 0.5% glucose and 0.5% yeast extract, pH is 8.0. (3) Culture in fermentation tank 1)Fermentation medium composition: See Table 2. Initial pH is 6.5˜7.0.

(9) TABLE-US-00002 TABLE 2 Composition of fermentation medium Name Use amount (g/L) Sucrose 40 Bean pulp 40 (NH.sub.4).sub.2SO.sub.4 5 K.sub.2HPO.sub.4•3H.sub.2O 8 KH.sub.2PO.sub.4 1.8 2) Primary technical parameters and optimization and screening Basic technical parameters: Inoculum size is 1%; fermentation temperature is 31° C., pH is 6.7; and fermentation time is 40 h.

(10) Based on these basic technical parameters, shake flask test is carried out; inoculum size, fermentation temperature, pH, fermentation time and other factors are optimized and screened. Shake flask test method: The strain is inoculated into a test tube containing 5 ml of seed medium to shake-culture at 30° C. for 2 days. It is then transferred into a shake flask containing 50 ml of fermentation medium, in which 8 g/L tryptophan substrate is added. The content of 5-HTP in fermenting supernatant is accurately determined with HPLC.

(11) a) Effect of Inoculum Size

(12) The effects of different inoculum sizes on catalytic synthesis of 5-HTP are examined. Other technical parameters: fermentation temperature is 31° C., pH is 6.7; and fermentation time is 40 h. See Table 3 for results.

(13) TABLE-US-00003 TABLE 3 Effect of different inoculum size on 5-HTP catalytic synthesis Inoculums size (%) 5-HTP content (g/L) 0.5 7.3 1.0 7.8 1.5 7.7 2.0 7.6

(14) It can be seen in Table 3 that too low (0.5%) or too high (2.0%) the inoculum size has adverse effect on catalyzed synthesis of 5-HTP with tryptophan, and 1.0% inoculum size is preferred. It is possible that low inoculum size affects bacterial growth; when the inoculum size is too high, rapid bacterial growth facilitates aging to further affect catalytic efficacy.

(15) b) Effect of Fermentation Temperature

(16) The effects of different fermentation temperatures on catalytic synthesis of 5-HTP are investigated. Other technical parameter conditions: inoculum size is 1.0%; pH is 6.7; and fermentation time is 40h. The results are shown in Table 4.

(17) TABLE-US-00004 TABLE 4 Effect of different fermentation temperature on 5-HTP catalytic synthesis Fermentation temperature (° C.) 5-HTP content (g/L) 30 7.6 31 7.7 32 7.8 33 7.6 34 7.4 35 7.3

(18) It can be seen in Table 4 that the fermentation temperature has a certain effect on catalytic synthesis of 5-HTP with tryptophan. The catalytic effect is poorer below 32° C. than at 32° C.; and the efficacy becomes poorer with higher temperature. The optimal fermentation is observed at 32° C.

(19) c) Effects of pH

(20) The effects of different pH on catalytic synthesis of 5-HTP are examined. Other technical parameter conditions: inoculum size is 1.0%; fermentation temperature is 32° C., and fermentation time is 40 h. See Table 5 for results.

(21) TABLE-US-00005 TABLE 5 Effect of different pH on 5-HTP catalytic synthesis pH 5-HTP content (g/L) 6.5 7.2 6.6 7.3 6.7 7.7 6.8 7.9 6.9 7.7 7.0 7.3

(22) It can be seen in Table 5 that pH has a certain effect on catalytic synthesis of 5-HTP with tryptophan. The catalytic efficacy is poorer below or above pH 6.8 than at pH 6.8. The optimal catalytic efficacy is observed at pH 6.8.

(23) d) Effect of Fermentation Time

(24) The effect of different fermentation times on catalytic synthesis of 5-HTP are examined. Other technical parameter conditions: inoculum size is 1.0%; fermentation temperature is 32° C., pH is 6.8. See Table 6 for results.

(25) TABLE-US-00006 TABLE 6 Effect of different fermentation time on 5-HTP catalytic synthesis Fermentation time (h) 5-HTP content (g/L) 30 6.7 35 7.0 40 7.8 45 7.9 50 7.8 55 7.8

(26) It can be seen in Table 6 that there are certain differences in the catalytic synthesis of 5-HTP with tryptophan under different fermentation times, and the catalytic efficacy gradually increases with longer fermentation time. However, the catalytic efficacy peaks in 45 h without subsequent increase. Therefore, the optimal fermentation time is 45 h.

(27) Therefore, the optimized technical parameters after optimization and screening: inoculum size is 1%; fermentation temperature is 32° C., pH is 6.8; and fermentation time is 45 h.

(28) 3) Process and Procedures 1. Seed preparation requirements: Strain identification conforms to its properties without variations or other bacteria. 2. Medium preparation: The ingredients are accurately weighed according to the formula. Sucrose is fully dissolved with appropriate amount of water; (NH4)2504, K2HPO4.3H2O and KH2PO4 are fully dissolved with appropriate amount of water. The amount of defoaming agent should be appropriate. 3. Sterilization of empty fermentation tank: Temperature is 121˜130° C. with circulating steam, tank pressure is 0.09˜0.15 Mpa, and the maintenance time is 30 min. 4. Feeding materials and adding water: The total volume is adjusted. 5. Sterilization of filled fermentation tank: Temperature is 121° C. tank pressure 0.08˜0.15Mpa, ventilation volume is 0.8˜1.0 vvm, the maintenance time is 30 min with circulating steam; the temperature is finally cooled down to 30° C. 6. Inoculation: inoculum size is 1%. 7. Culture: The temperature is 32° C., the time is 45 h, the tank pressure is 0.05˜0.08 Mpa, pH is 6.8, DO is 25% or higher. Spore formation rate is 98%; when the culture is terminated, the bacterial suspension for JSC-69 transformation is obtained; bacteria density is≥2.0×10.sup.9 cfu/ml. It should cool down and maintain the pressure.

(29) 2. Transformation and Production of Target Product (1) Collecting the bacterial cells: The product produced from culturing Bacillus licheniformis JSC-69 for transforming is centrifuged at 4000 r/min for 10 min to collect bacterial cell sediment. (2) Transforming: 0.2M phosphate buffer (pH 7.4) is added to the bacterial cell sediment to form a bacterial density of 3.0×10.sup.7˜8.0×10.sup.7/ml; then, 8.0 g/L tryptophan is added to carry out transformation for 45 h at 32° C., 1.0 L/min, and 225 r/min. Preparation of 0.2M phosphate buffer (pH 7.4): 71.6 g Na2HPO4.12H2O is dissolved in 1000 ml water to obtain 0.2M Na2HPO4; then 31.2 g NaH2PO4.2H20 is dissolved in 1000m1 water to obtain 0.2M NaH2PO4; finally, 19m1 of 0.2M NaH2PO4 and 81m1 of 0.2M Na2HPO4 are evenly mixed. (3) Solid-liquid separation: The transformation liquid is centrifuged at 4000r/min for 10min to obtain 5-HTP-containing supernatant. Bacillus licheniformis JSC-69 produces 5-HTP using tryptophan as the substrate, and the 24 h transformation rate is 95%˜100%.

Example 4

Purification and Extraction of the Product

(30) 1. Solid-liquid separation of fermentation broth: Bacterial cells are collected using centrifugation or ceramic membrane separation method. 2. The 001×7 cation-exchange resin (732) is used for adsorption and separation, followed by elution with ammonia water. Based on screening, 001×7 cation-exchange resin results in the best adsorption separation efficiency, suitable for large-scale production of 99% specifications of 5-HTP. The adsorption separation conditions of 001×7 resin are as follows: At room temperature, 5-HTP mass concentration of the loading solution is 10.8 mg/ml, pH is adjusted to 3.5, and the sample is loaded at 4.0 ml/min, 7.0% ammonia water with a volume 3 times the resin is used for elution at 3.0 ml/min. 5-HTP mass fraction is >99.0% and the ash content is <1.0% after concentration and crystallization of the eluate. 3. Concentrating Into Solid S5tate

(31) Double-effect or multi-effect evaporation of the eluate is carried out to concentrate into solid, followed by drying to remove the moisture. 4. Hot water dissolution, high-selectivity resin adsorption and separation.

(32) The concentrated solid is dissolved in hot water, followed by further high-selectivity resin adsorption and separation of 5-HTP to remove impurities. After elution with aqueous ammonia, it is concentrated and crystallized to obtain high-purity 5-HTP.

Examples 5

Product Analysis Method

(33) Product analysis method employs HPLC. The standard solutions and samples of tryptophan and 5-HTP are chromatographically determined with a Shimadzu C18 reverse-phase column, using 0.05% trichloroacetic acid (TCA)-methanol (68.75:31.25, v/v) as the mobile phase. Injection volume: 10 μL, mobile phase flow rate: 1.5 ml/min gradient elution, the wavelength of UV spectrophotometric detector is 275 nm. All solutions are filtered through a 0.45 μm filter before use. External standard method is used for calculation.