微生物转化生产L-5-羟基色氨酸

Process For The Preparation Of
5-H ydrox y-L-Tryptophan
It has now bee n fou nd that gram-positive un icellular bacteria, in particular,现在已经发现，革兰氏阳性单细胞细菌不含使人致病的病原microorga ni sms which are n ot huma n-pathoge nic, of the families 体，而且其中假单胞菌属、棒状杆菌属、肠杆菌属和芽孢杆菌Micrococcus, Pseudomonas, Corynebacterium, Enterobacter and Bacillus 属细菌都有能力将L -色氨酸或wN -乙酰-L -色氨酸转化为5 - capable of converting L-tryptophan an-N-acetyl-L-tryptophan into 羟基-L型色氨酸，而且收率很咼。

5-hydroxy-L-tryptopha n in good yields.
Of these families, a new strain of bacillus has proven to be especially 在这些细菌属中，一个芽孢杆菌新菌株已被证明特别适合进行advantageous for the microbiological hydroxylation. This novel bacillus st「a羟化反应。

这个新颖的芽孢杆菌已被提交给美国典型菌种保藏has been filed with the American Type Culture Collection and has been 中心，并已被编号为枯草芽孢杆菌ATCC 21733。

designated Bacillus subtilis ATCC 21733.
The properties of this bacillus strain are as follows: 这株芽孢杆菌菌株的特性如下：
1. Rods. 1 棒。

2. Mortality: No ne
3. Gram sta in: labile to n egative.
4. Spore shape: large, an gular, occurre nee of shadow forms. 2死亡率：无
3革兰氏染色：不稳定阴性。

4抱子形状：大，棱角分明，有阴影。

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5. Growth on bouillon-agar plates: small colonies after 1-2 days of incubatior肉汤琼脂板培养：在30 "c接种，在37 C培养1-2 天出现较at 30.degree.C, exhibiting a somewhat larger area at an incubating temper大菌苔。

凸黄白色菌落，表面粗糙，底部圆而平滑。

菌落边缘of 37.degree.C. Co nvex yellowish-white col on ies of a rough upper surface,细裂，菌落中心有褶皱，也有的平滑。

with a roun ded and smooth un derside. Edges of colony fin ely lobed, with
colony cen ters partially smooth, partially folded.
6. Growth on special agar plates for Bacillus subtilis （glucose, pept one, mea以枯草杆菌在特殊琼脂上培养（含葡萄糖，蛋白胨，肉提取extract）:colonies of normal size after 1-2 days of incubation at 30.degree.C物）：在30 c或37 c培养1-2天后，菌落正常大小，扁平呈
37.degree.C, flat, and of a yellowish to brownish color, surface creased like黄色至棕褐色，表面有折痕如皮肤，菌落中心发黑，边缘粗裂。

skin, with a dark cen ter, and coarsely lobed edges. Colo ny un dersides are 菌落底部圆润折叠。

roun ded and folded.
7. Growth on corn steep liquor/glucose/yeast extract plates: small colonies T a fl e米浆/葡萄糖/酵母浸膏培养：在30 c接种，在37 c培养1-2 days of incubation at 30.degree.C or 37.degree.C, flat, and of a light to1-2 天出现较大菌苔。

扁平，轻微白色，表面粗糙，中心平整white color, surface rough and lobed with a smooth or folded cen ter, edges或折叠，边缘细裂。

菌落底部圆润有折痕。

fin ely lobed. Un derside of colonies roun ded and creased.
8. Growth on mann itol -- tilted agar tubes: weak.
9. Growth on lactose -- tilted agar tubes: no rmal.
10. Growth on D-glucose -- tilted agar tubes: no rmal.
11. Growth on KNO.sub.2 -- tilted agar tubes: weak to normal.
12. Growth on Voges-Proskauer -- tilted agar tubes: no rmal.
13. Growth on iron （II） -- tilted agar tubes: no rmal. 8甘露醇-琼脂倾斜管：弱。

9乳糖-琼脂倾斜管：正常。

10D -葡萄糖-琼脂倾斜管：正常。

11KNO2 -琼脂倾斜管：弱，正常。

12 Voges-Proskauer -琼脂倾斜管：正常。

13 Fe++ -琼脂倾斜管：正常。

14. No gelati n liquefacti on was observed （ran dom culture, high gelati n layet% 没有观测到明胶液化（随机培养，厚明胶层）。

15. Col on ies grow n on Bacillus subtilis special agar assume a brow nish-pini5 枯草芽抱杆菌在特殊琼脂培养基培养3-5 天后，呈棕粉色color after sta nding for 3 to 5 days and spread an in creas in gly pungent odo并传播越来越刺鼻的气味。

16. Biochemical properties: nitrate is reduced （nitrite indication）. Indole is 16 生化特性：还原亚硝酸盐（亚硝酸盐指示）。

能合成吲哚。

syn thesized.
From the above evaluatio n, the strain is 从上述分析来看，这株菌株应该分类为枯草芽孢杆菌的物种, assigned to the species Bacillus subtilis, with isolated deviations from the 按《Bergey's 细菌鉴定手册》分类。

5 -羟基-L -色氨酸的制备
taxonomic standard (Bergey's Manual of Determinative Bacteriology). Referring to the other hydroxylating microorganism families particularly suitable among the micrococcaceae are Sarcina lutea and Micrococcus lysodeikticus.
Among the pseudomonadaceae, Protaminobacter alboflavus and Pseudomonas facilis are preferred.
Among the coryn ebacteriace ，Cory nebacterium hoagii is especiall preferred. 棒杆菌科，霍氏棒状杆菌(Coiynebacterium hoa g ii 是首选。

Among the enterobacteriaceae, Escherichia coli strains are particularly 在肠杆菌，优先考虑大肠杆菌菌株(
Escherichia coli。

preferred.
When the substrate -N -acyl-L-tryptophan, the acyl group is preferably
当底物是 3- N -酰基-L -色氨酸时，酰基最好是
1-5
个碳原子
aliphatic hydrocarbon carboxyl of one to five especially one to three carbon 或 1-3 个碳原子的脂肪族酰基，如乙酰基，或芳香族酰基，如 atoms, for example, acetyl, or aromatic hydrocarb on carboxyl such as ben 苯甲酰基和甲苯甲酰基。

and toluoyl.
The microbiological hydroxylation is conducted in accordance with 微生物催化的底物羟基化反应按传统的方法进行。

例如，通常
conventional methods. For example, there are usually first conducted
先进行初步实验以确定最佳发酵条件， 包括确定培养基、 底物、
preliminary
experiments to ascertain the optimum fermentation conditions,底物浓度、操作条件----如温度、通风、pH 值、搅拌----和活化、 in cludi ng such parameters as the selection of the n utrie nt medium, the sub 添加底物和底物与微生物活性酶接触的的最佳时间， 底物与微
solvent, the substrate concentration, the operating conditions -- such as
生物活性酶接触的的最佳时间可以通过薄层色谱方法确定。

temperature,
aeration, pH, agitation -- and the optimum times for germination, addition of substrate, and substrate contact with the microorganism enzyme by
means of analytical methods -- particularly by means of thin-layer chromatography.
In this conn ectio n, it was fou nd that the yield is in creased by an addition of 在这方面，发现加入 5-50 毫克 /升的 Fe +呵以提咼产量，而加 Fe.sup.+.sup.+ ions to the fermentation solution preferably in a concentrati 入n 40-80 毫克/升的 VC 可以加速微生物转化。

还发现在微生物 of 5 to 50 mg/liter, and that ascorbic acid preferably in a concentration of 4的生长期结束时，也就是接种后 8-15 小时候加入底物有利于 80 mg/liter acceleratesthe microbiological conversion.It was furthermore
羟化反应。

observed that it is advantageous to add the substrate toward the end of the main growth period of the microorganism, for example, 8 to 15 hours after inoculation.
It is also to be no ted that con ce ntratio ns of about 80-250 mg of substrate p 必须注意的是当底物浓度为 80-250
毫克 /升时，产率最高。

liter of nutrient medium result in the highest yields.
As further preferred conditions, the culture is incubated at°25-4te pH is
进一步优化的条件是在 25-40 °c 接种，pH 值调整到 6-8，通气
adjusted to 6-8, and 1-5 liters of air per mi nute are added. The con version 速度为1-5升/分钟。

底物转换反应可由薄层色谱进行适当控 substrate is suitably controlled by a thin-layer chromatographic analysis of
制，发酵结束后，可以通过过滤或离心除去微生物细胞。

sample extracts. After the fermentation has been terminated, the culture broth is freed of the bacterial cells by filtration or centrifugation.
The isolation of the product of the process is conducted conventionally. In
产品的分离也很容易进行。

首选的方法是先以
H+
形式被新鲜
accorda nee
with a preferred method, the process product is adsorbed on a 再生的阳离子交换树脂吸附，然后用氨或氨盐洗脱。

进一步纯 freshly rege nerated cation excha nger in the H.sup.+ form and then eluted |化可以将洗脱液浓缩，然后葡聚糖凝胶层析。

means of ammonia or an ammonium salt condition. For further purification,
the concentrated eluate can then be chromatographed over dextran gels, for example. EXAMPLE 1
实施例
1
微球菌科中特别适合催化底物羟化反应的是藤黄八叠球菌 (Sarcina lute )a 和溶壁微球菌( Micrococcus lysodeiktic )us 。

在假单胞菌科中，优先选择白黄精朊杆菌( Protaminobacter alboflavuS 和敏捷假单抱菌(Pseudomonas fac )s 。

An Erlenmeyer flask containing 500 ml. of a sterilized aqueous medium, 配制500ml 培养基溶液，组成为0.1%蛋白胨、0.2%玉米浆、
consisting of 0.1 percent peptone, 0.2 percent corn steep liquor, 0.5 percen 0t .5%D ( + )-葡萄糖、 0.5% 酵母提取物(营养液)，调整 pH
D(+)-glucose, and 0.5 percent yeast extract (nutrient solution), adjusted to 值至 8-0，将配制好的培养基溶液倒入锥形瓶中，灭菌后接入
8.0, is inoculated with 3 drops of supernatant broth of a tilted agar culture o 3f 滴枯草芽孢杆菌( Bacillus subtilis ，ATCC 21733 )斜面肉 Bacillus subtilis (ATCC 21733) and shaken for 24 hours at 30.degree. C.汤培养基的上清液，3° °c 振荡培养 24 小时。

The thus-germinated inoculating cultures are centrifuged for 15 minutes at
将得到的培养物在
6000-8000
转/
分的条件下离心
15
分钟，悬
6,000-8,000
g.'s, and respectively two cultures are suspended in 100 ml. o 浮于 100ml 无菌培养基溶液中，得培养物悬浮液。

另取 3°°ml
sterilized nutrient solution. The suspension is transferred into an inoculatio 培养基溶液(含少量 FeSO 4.7H 2O 和 L-色氨酸)，置于接种 flask containing 300 ml. of nutrient solution, as well as minor amounts of FeSO.sub.4 .sup.. 7 H.sub.2 O and L-tryptophan.
By means of this bacterial suspension, 10 l. of sterilized nutrient solution, containing 325 mg. of tryptophan and 18.07 mg. of FeSO.sub.4 .sup.. 7
H.sub.2 O and adjusted to pH 7.5, is inoculated and incubated for 24 hourd 接入枯草芽孢杆菌原液，在 30。

振荡培养 24 小时，通气速度 30.degree. C. at an aeration of 2 l./min. and an agitating speed of 220 r.p.r 2.升/分钟，搅拌速度 220 转/分。

From this preliminary fermentation stage, 0.9 1. is transferred into a main
在发酵初始阶段，补加
0.9
升培养基溶液，在发酵
10
小时后
fermentor
charged with 15 1. of sterilized nutrient solution. During the main 将 PH 值调节到 6.8，并加入 495 毫克 FeSO 4.7H 2O 和 3.3 克 fermentation, the same technical conditions are employed as in the preliminary 氨酸 (先溶于 635 毫升无菌水中，灭菌，以后叫底物溶液)。

fermentation. The pH is adjusted to 6.8 Ten hours after inoculation, 3.3 g. of L-tryptophan and 495 mg. of FeSO.sub.4 .sup.. 7 H.sub.2 O, dissolved in 635 ml. of sterile water, are added thereto.
The progressi on of the microbiological con vers ion is determ ined by an alys 反 应过程中间隔取样检测，以控制微生物转化进度。

samples taken at intervals.
The substrate has bee n con verted, except for trace amou nts, 68.5 hours a 在添加色氨酸 68・5 小时后，色氨酸几乎完全转化为 L-5-
羟基
addition of the substrate.
色氨酸。

The culture broth is freed of the bacillus cells by means of continuous 发酵完毕离心去除菌体， 将上清液和 750
克新鲜再生的阳离子
centrifuging. The cell-free culture broth is mixed with 750 g. of freshly 交换(H +)混合，搅拌 45 分钟。

此后过滤得交换树脂。

regenerated cation exchanger in the H.sup.+ form and agitated for 45 minutes.
Thereafter, the exchange resin is filtered off.
Respectively 20 g. of the excha nge res in, stored in the cold state, is agitate 取 20 克交换树脂，放冷后加入到 25 毫升 1 摩尔醋酸铵溶液中， several times with 25 ml. aliquots of 1-molar ammonium acetate solution fc 搅拌 10 分钟，然后过滤。

重复操作三次， 将 3 次洗脱液合并， 10 mi nu tes and then filtered. The combi ned eluates are lyophilized and the 冻干，或者溶于水后再进行葡聚糖层析，用 0.1摩尔碳酸氢氨 chromatographed in the form of an aqueous solution over a "Sephadex" 溶液洗脱
column with 0.1-molar ammonium bicarbonate solution at 10.degree. C. After combining and lyophilizing the main fractions, 2.56 g. of 将所得产品合并，可以得到
2.56 克 5-羟基-L-色氨酸，m.p.为
5-hydroxy-L-tryptophan is obtained, m.p. 268.degree.-270.degree. C. (unde 2r 68-270 c 。

decomposition). EXAMPLE 2
实施例
2
This example differs from Example 1 only in the manner of adding the 实施例 2
和实施例 1
的主要区别是加入底物方式不同，
另外在
substrate and in the addition of ascorbic acid to the substrate.
培养基溶液中还加入了
VC。

The substrate is added in the form of a sterile stock solution consisting of: 4
底
6
物溶液组成：
mg. iron(ll) sulfate 1 g. ascorbic acid 3.3 g. L-tryptophan in 635 ml. of steril 46 毫克 FeSO 4.7H 2O+3.3 克 L-色氨酸 +1gVC+635ml 水 water.
瓶中，灭菌后将培养物悬浮液移入接种瓶中，得到枯草芽孢杆 菌原液。

制备10升培养基溶液（含 18.07毫克FeSO 4.7H 2O 和325毫
克L-色氨酸），调节PH 为7.5 ,置入15升发酵罐中，灭菌后
灭菌备用
The total amou nt is added as follows: on e-half thereof upon ino culation of将底物溶液分为两份，一份在发酵罐接种后立即加入，一份在main fermentor, and one-half after 10 hours of fermentation time. 发酵10小时候加入。

Concentration At Concentration After Beginning of 10 Hours of Fermentati在发酵开始时色氨酸、FeSO 4和VC 的浓度分别为o/g/L，
-6
Fermentation Tryptophan 0.1 g./l. 0.2 g./l. FeSO.sub.4 5 .sup.. 1O.sup.-.sUjP6l° mol 和0.033g/L ,在发酵10 小时后再加入另一份底物molar 10.sup.-.sup.5 molar Ascorbate 0.033 g./l. 0.067 g./l. 溶液后，浓度分别为ONg/L , 10 5 mol 和0.067g/L。

After a total ferme ntatio n period of 48 hours, i.e. 38 hours after the seco nd 在发酵48小时后(第二份底物溶液加入后38小时)，发酵液addition of substrate, the culture medium con tai ns 125 mg./l. of 中5-羟基-L-色氨酸浓度为125mg/L(理论值的58.1%)，在发酵
5-hydroxy-L-tryptopha n (58.1 perce nt of theory). The final yield after 66 ho§6s 小时后，发酵液中5-羟基-L-色氨酸浓度达到理论值的
of total fermentation time is 62.8 percent of theory. EXAMPLE 362.8% 。

实施例 3
This example differs from Example 1 merely in the concentration of the 实施例 3 与实施例 1 不同之处仅在于底物浓度。

substrate. 在接种并发酵10 小时后，加入色氨酸-硫酸亚铁溶液。

色氨酸
Ten hours after inoculation of the main fermentor, a solution is added -硫酸亚铁溶液组成：
consisting of 1.65 g. of L-tryptophan and 46 mg. of iron(II) sulfate in 635 ml1.65gL-色氨酸+46mg 硫酸亚铁+635ml 无菌水
of sterile water. 总发酵周期为50，58和66小时时，分别检测发酵液5 -羟基-Measurements of the 5-hydroxy-L-tryptophan content after a total fermenta t ic色氨酸含量，结果收率分别是理论收率的100%、96% 和period of 50, 58, and 66 hours, respectively, indicate, in the same sequenc9e9,.4%。

final yields of 100, 96, and 99.4 percent of theory.
EXAMPLE 4 实施例4
In a 500 ml. Erlenmeyer flask, 100 ml. of a nutrient medium according to 将4ml无菌氯化钠溶液加入到试管斜面中，振荡后取1ml枯草Example 1 is inoculated with 1 ml. of a cell suspension of Bacillus subtilis s芽孢杆菌(Bacillus subtilis sp.悬浮液，接入到BOml 实施例
obtained as a supernatant broth of a tilted agar culture with 4 ml. of sodiumi 制备的培养基溶液中，放入500ml 锥形瓶中。

chloride solution.
Thereafter, the culture is shaken for 24 hours at 30.degree. C. and then 然后在30振荡培养24小时后，以6000-8000转/分离心15 cen trifuged at 6,000-8,000 g.'s for 15 minu tes un der sterile con ditio ns. The分钟I，离心时保持无菌状态。

将离心得到的菌体悬浮于100ml sediment is again suspended in 100 ml. of isotonic salt solution, buffered to等渗溶液中，用磷酸溶液缓冲PH至6后，再离心一次。

重复
6 with phosphate, and once more centrifuged. This washing step is repeat©清洗步骤一次。

再次将菌体悬浮于20ml盐溶液中，缓冲至
After once again suspending the washed cells in 20 ml. of salt solution, PH=6，将菌体溶液移入100ml 锥形瓶中，加入0.26ml w-N- buffered to pH 6, the culture is tran sferred into a 100 ml. Erle nm eyer flask,乙酰d-L-色氨酸溶液。

4 mg. of . -N-acetyl-L-tryptophan w-N-乙酰-L-色氨酸溶液制备：取10ml容量瓶，加入
(.beta.-[indolyl-3]-.alpha.-acetylaminopropionic acid) is added thereto in the<50mg w-N-乙酰-L-色氨酸，用2N 氢氧化钠溶液调节PH=4.5，form of 0.26 ml. of a sterile-filtered stock solution, obtained by adjusting an补充水定容至10ml，灭菌待用。

aqueous suspension of 150 mg. of the compound to pH 4.5 (with 2N NaOH)
and replenishing the solution to obtain 10 ml.
After 48 hours of incubation at 30.degree. C. under shaking, the cells are 在30振荡培养48小时后，离心去除菌体。

取100M 上清液removed by centrifuging, and respectively 100 .mu.l. of the overflow is appl用快速硅胶板(Woelm薄层板，5cm宽)进行薄层色谱分析。

directly to instant silica gel plates (Woelm) as a starting line of a width of 5 cm., for analysis by means of thin-layer chromatography.
TLC Evaluation: TLC 分析：
In addition to the fermentation sample, respectively 5 .mu.l. of 0.2 percent 除了发酵样品外，另外各取5M0.2% 浓度的L-色氨酸、--N-
strength methanolic solutions of the following three referenee substances w乙fe-L-色氨酸和5-羟基-L-色氨酸的甲醇溶液作为参比溶液。

applied:
L-tryptophan
. w-N c-Acetyl-L-tryptophan, and
5-Hydroxy-L-tryptophan.
After an in itial dryi ng step, the chromatography was con ducted for 2 hours b展开剂：丙酮：氯仿：冰醋酸：水=40:40:20:5，体积比
the asce nding mode in an elue nt system of acet on e:chloroform:glacial acetl：开时间：2小时。

用不同的可喷雾的染色剂进行染色，得到
acid:water (volume ratio 40:40:20:5). Two chromatograms obtained in this
两种图谱，分别进行分析。

way were evaluated by dyeing with various sprayable reagents, as follows:
Plate 1: Dyei ng with the Ude nfrie nd reage nt (first spraying with a 0.1 percen 先用 O.1%1-亚硝基-2-萘酚的乙醇溶液染色，再用一种混合溶 etha nolic solution of 1-ni troso-2-naphthol, seco nd sprayi ng with a solution 液染色。

(混合液配比：0.亦125% 亚硝酸钠溶液和 5ml2N 盐 mixture of 0.2 ml. 25 percent sodium nitrite solution and 5 ml. of 2N 酸溶液)，这种染色方法对 5
羟吲哚衍生物有很好特异性。

hydrochloric acid),
which is extensively specific for 5-hydroxyindole derivatives.
Plate 2: Spraying and subsequent heating with a 1 percent solution of 2 用 1%对二甲氨基苯甲醛溶液(将对二甲氨基苯甲醛溶于乙 p-dimethylami nobe nzaldehyde in etha nol/co nee ntrated HCl (1:1) = van Urk 醇浓盐酸溶液中，乙醇：浓盐酸 =1：1)染色、加热烘干。

reagent. Results:
结果：
Plate 1 (specific coloring): The fermentation sample shows a single marked 1 发酵样品呈阳性， 染色位置和 R f 值(
0.15
)显示发酵液存在
"Udenfriend-positive" zone having the color and R.sub.f value (0.15) of 5 -
羟基
- L -
色氨酸。

5-hydroxy-L-tryptophan.
2发酵样品薄层分析显示， 除了有5 -HTP ，还有L -色氨酸(R f
Plate 2 (unspecific coloring): The fermentation sample shows, in addition to = 0.24)以及未反应的 N -乙酰- L -色氨酸( R f = 0.75)。

the zone analogous to 5-HTP, pronounced proportions of L-tryptophan (R.sub.f = 0.24), as well as unreacted N-acetyl-L-tryptophan (R.sub.f = 0.75).
EXAMPLE 5
An alogously to Example 4, .-N-be nzoyl-L-tryptopha n is con verted into 5-hydroxy-L-tryptophan.
Characterization of 5-hydroxy-L-tryptophan, accessible by fermentation: colorless crystals, m.p. 268.degree.-270.degree. C. (decomposition); g.' rotation [.alpha.].sub.546.sup.20 = -37.4.degree. (1 percent aqueous soluti Uv data of the aqueous solution; maximum at 276.5 m.mu. EXAMPLE 6
实施例
6
A supernatant broth of a tilted agar culture (0.5 ml.) of Sarcina lutea (ATCC 取 2ml 氯化钠溶液加入到藤黄八叠球菌( Sarcina lutea ， 9341) with 2 ml. of NaCl solution is added to 5 ml. of a nutrient medium in a ATCC9341 )斜面培养试管中， 振荡后取 0.5ml 菌液放入含 5ml centrifugal tube, sealed under sterile conditions.
培养基溶液的离心管中，在无菌状态下密封。

After 42 hours of shaking at 30.degree. C., the cell material is centrifuged off a 0《振荡培养 42 小时后，5000 转离心 10 分钟得菌体，用 5000 g's (10 minutes) and washed twice, each time by re-suspension in 5 mtl l o 磷酸缓冲液(PH=6 )清洗两次、离心，以便清除任何可 phosphate-buffered salt soluti on (pH 6) and subseque nt cen trifugi ng, in ord i 的干扰代谢物产物。

to remove any possibly interfering metabolites.
The thus-washed sediment is suspended in 2 ml. of buffered salt solution (将清洗完毕的菌体悬浮于 2mlPH =6
的缓冲溶液(含 4mg2g /L
6), containing 4 mg. (2 g./l.) of L-tryptophan.
的 L-色氨酸)中。

After 20, 40, and 43 hours of incubation under shaking at 30.degree. C., th 在 30 C 振荡培养 20、40 和 43 小时后，离心得上清液，取 reaction mixture is centrifuged, and respectively 0.5 ml. of the supernatant 0.5ml43 小时发酵上清液冻干。

liquor is withdrawn under sterile conditions and lyophilized.
The lyophilized product of the sample withdrawn after 43 hours is mixed wit 将冻干物溶于 0・2ml 甲醇盐酸溶液(甲醇：°.°1N
盐酸=0.9：
1 ) 0.2
ml. of a 90 percent strength methanolic solution of 0.01N hydrochloric 中，加热到 40 C ，振荡器振荡 2-3 分钟。

实施例 5
与实施例4类似，3-N-苯甲酰-L-色氨酸转化为5-羟基-L-色氨 酸。

通过发酵得到的5-羟基-L-色氨酸特征如下： 无色晶体，m.p. 268- 270 'C （分解）； 旋光光度［%】546 20 =-37.4 o （ 1%水溶液） 水溶液的紫外数据： 最大吸收值波长 中间吸收值波长
shoulder at 295 m.mu.
276.5 m g 295 m g
minimum at 248 m.mu.
最小吸收值波长 248 mg
acid. The mixture is heated to 40.degree. C., and the test sample is shaken for 2-3 minutes with the aid of a vibrating mixer.
The overflow is applied in the form of a dot on the starting line of a CelluloS将发酵液用纤维素-F薄层板进行TLC分析，用5-羟基-L-色氨thin-layer plate (Merck) and chromatographed in the ascending mode agai酸标准品作为对照，层析150分钟。

authentic 5-hydroxy-L-tryptophan as comparison, for 21/2 hours.
Elue nt:aceto ne:chloroform:glacial acetic acid:water, 40:40:20:5 (volume ra展开剂：丙酮：氯仿：冰醋酸：水=40:40:20:5，体积比Identification according to Udenfriend: The dry plate is thoroughly sprayed 按实施例 4 中的 1 操作，用0.1%1- 亚硝基-2-萘酚(溶于95% with a 0.1 perce nt solution of 1-ni troso-2-naphthol in 95 perce nt etha nol an乙醇中)染色，室温迅速干燥。

然后用°・2ml25% 亚硝酸钠溶dried briefly at room temperature. Thereafter, the plate is sprayed with a 丹進和y5ml2N盐酸混合溶液喷雾，得到一个蓝紫色点，R f 值为prepared mixture of 0.2 ml. of 25 percent sodium nitrite solution and 5 ml. o>f06-0.07,5-羟基-L-色氨酸的R f 值为°.°6 2N hydrochloric acid. Already during the second spraying step, the bacterial test solution develops a blue-purple color spot at an R.sub.f value of 0.06 -
0.07. R.sub.f value of 5-hydroxy-L-tryptophan: 0.06.
EXAMPLE 7 实施例7
Under the conditions set forth in Example 6, L-tryptophan is incubated with在实施例6设定的条件下，L-色氨酸用溶壁微球菌(Micrococcus Micrococcus lysodeikticus (IFO 3333) in place of Sarcina lutea. lysodeikticu，IFO3333 进行生物转化，不用实施例 6 所使用的R.sub.f value of the bacterial synthesis product: 0.06-0.07. 藤黄八叠球菌(Sarcina lutep。

细菌合成的产品的R f值：
R.sub.f value of 5-hydroxy-L-tryptophan: 0.05. 0.06-0.07。

5 -羟基-L -色氨酸的R f值：0.05。

EXAMPLE 8 实施例8
Under the conditions set forth in Example 6, L-tryptophan is incubated with在实施例6设定的条件下，L-色氨酸用霍氏棒杆菌Corynebacterium hoagii (ATCC 7005) instead of with Sarcina lutea. (Corynebacterium hoag，ii ATCC 7005)进行生物转化，不用实施R.sub.f value of the bacterial synthesis product: 0.06-0.08. 例6所使用的藤黄八叠球菌(Sarcina lutep。

细菌合成的产品
R.sub.f value of 5-hydroxy-L-tryptophan: 0.05. 的R f 值：0.06-0.08。

5 -羟基-L -色氨酸的R f值：0.05。

EXAMPLE 9 实施例9
Under the conditions set forth in Example 6, L-tryptophan is incubated with在实施例6设定的条件下，L-色氨酸用白黄精朊杆菌Protaminobacter alboflavus (ATCC 8458) in place of Sarcina lutea. (Protaminobacter alboflavus ，ATCC 8458 )进行生物转化，R.sub.f value of the bacterial synthesis product: 0.06-0.08. 不用实施例6所使用的藤黄八叠球菌(Sarcina luteN。

细菌合
R.sub.f value of 5-hydroxy-L-tryptophan: 0.05. 成的产品的R f 值：0.06-0.08。

5 -羟基-L -色氨酸的R f值：0.05。

EXAMPLE 10 实施例10
Under the conditions set forth in Example 6, L-tryptophan is incubated with在实施例6设定的条件下，L-色氨酸用敏捷假单胞菌Pseudomonas facilis (ATCC 11 228) instead of with Sarcina lutea. (Pseudomonas facilis ，ATCC 11 228) 进行生物转化，不用
R.sub.f value of the bacterial synthesis product: 0.07-0.08. 实施例6所使用的藤黄八叠球菌( Sarcina lute)a 。

细菌合成的
R.sub.f value of 5-hydroxy-L-tryptophan: 0.07. 产品的R f 值：0.07-0.08。

5 -羟基-L -色氨酸的R f值：0.07。

EXAMPLE 11 实施例11
Under the conditions set forth in Example 6, L-tryptophan is incubated with在实施例6设定的条件下，L-色氨酸用发明人分离得到的芽孢strain of bacillus isolated by the inventors in place of Sarcina lutea. 杆菌进行生物转化，不用实施例6所使用的藤黄八叠球菌
R.sub.f value of the bacterial synthesis product: 0.05-0.06. (Sarcina luteN。

细菌合成的产品的R f值：0.05-0.06。

R.sub.f value of 5-hydroxy-L-tryptophan: 0.06. 5 -羟基-L -色氨酸的R f值：0.06。