JPS6119496A - Method for producing L-phenylalanine - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to a method for producing L-phenylalanine.

(Prior Art) It is known to produce L-phenylalanine by reacting cinnamic acid and ammonia in the presence of ammonia lyase produced by microorganisms.

Such microorganisms include, for example, Sporobolomyces roseus (Sporoboromyces roseus).
eus) Kokoku (FERN P-JIS), Rhodotorula grac
ilis) (F100j!?), Rhodotorul'a marina (Rhodotorul'a marina)
(Engineering FOOza79), Fusarium oxysborum (Fu
sarilmoxyaporum ) (ATOOko0
39, etc. (for example, British Patent No. 4 Dag Suda No. 6g Specification, Yi Temple, Yan Zhaoke-xl, /qri Publication-
-) However, it is said that the ammonia lyase obtained from these microorganisms does not necessarily have sufficient activity and sustainability.

An object of the present invention is to provide a microorganism that produces ammonia lyase with high activity and good persistence.

(Means for Solving the Problems) The present invention provides a method for producing L-phenylalanine by reacting cinnamic acid with ammonia in the presence of ammonia lyase.
orium), which is characterized by carrying out the reaction using ammonia lyase, which has the ability to produce L-phenylalanine from cinnamic acid and ammonia.
- Summary of the method for producing phenylalanine.

The present invention will be explained in detail below.

First, the ammonia-lyase used in the present invention is obtained from a microorganism that belongs to the genus Glatosborium and has the ability to produce L-phenylalanine from ammonia cinnamate.

(ljRM-P, 770 (f,).

The microbiological properties, identification, and reasons for this bacterium are as follows.

(1) Microscopic properties a) Characteristics of MA on malt agar medium Cronyi has moderate growth after one week of culture, with a diameter of approx.
It reaches 30m. The color of the colony is olive-green; the underside is dark green to black. The vegetative hyphae are initially colorless and then turn brown, and their width is up to 728 m;
Aerial mycelium is well developed. Conidiophores arise from vegetative hyphae; usually unbranched; length FiJj~30μm, width 0~6.0μm; smooth, septated, brown, dendriticly branched conidia form the head.

Conidia are budding conidia, oval, lemon-shaped.

Oval, cylindrical, light brown, smooth, unicellular.

Co1/μmXJ~daμm; basal conspores cylindrical, sometimes -13 cells.

No sexual reproductive organs were observed.

mouth) Potato dextrose agar (PDA)
Characteristics on the above ・Similar to the properties on malt agar medium.

(:1) Physiological properties Growth temperature: 10°C to JO°C Optimum temperature = 2. o~26℃ Growth pH: Col 10 Optimum pH: Da~7 (,7) Taxonomic considerations This strain (0-1016) ij, olive-tinged green showing budding conidiation mode ~ Memorize It has been found.

Demat of K11ie (/??/, /976)
According to iaceousHyphomycetea, O
There are 43 species described in the genus Iadosporium. These species are distinguished by differences in colony color, parasitic plant specificity, conidia size, shape, surface structure, cell number, presence or absence of complete generations, etc.

The culture and morphological characteristics of this strain (a-hos6) are:
01adosp listed in 111g (/9?/)
The characteristics matched well with the characteristics of orium dladosporioidea.

Therefore, this strain (o-sor6) is Cladosporium cladosporioides (0: Ladosporium cl.
adoaporioides).

Ammonia-lyase can be obtained from the culture solution of the above-mentioned microorganisms, microbial material obtained by centrifugation, Fi, washed microbial cells obtained by processing the same, dried microbial cells or solidified microbial cells, and enzyme solution or It can be used in any form such as immobilized enzyme.

The medium used for culture is not particularly limited. Examples of non-carbon substances include various carbohydrates, organic acids, etc., and organic ammonium salts, inorganic ammonium salts, urea, etc. can be used as nitrogen sources. In addition, various phosphates, sulfates, etc. may be used as inorganic substances, and various organic nutrients may be added as necessary.

Cultivation is usually carried out under aerobic conditions for about 6 hours to 5 days. The pH and temperature of the culture medium are selected from about 10-00C.

In the method of the present invention, the polyhydric alcohol is present when cinnamic acid and ammonia are reacted using the ammonia lyase.

This polyhydric alcohol is usually added to the reaction solution, and its amount is generally o, i to
t o wt%, preferably 2-. ? It is about % by weight.

The ammonia used in the above reaction includes aqueous ammonia or an ammonia precursor. Examples of this precursor include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium acetate. The reaction can be carried out batchwise, semi-batchwise or continuously. During the reaction, the cinnamic acid concentration is usually 0.
/~! i w/V 14% Ammonia concentration is l
~/j w/v% is adopted. The reaction temperature is usually selected from 20 to 60° C., the pH from about g-ii, and the reaction time varies depending on the reaction conditions, but is usually selected from several hours to several days in the case of a batch method. After the reaction is completed, separation and purification of the reaction product 1) L-phenylalanine can be carried out by conventional methods.

Effects According to the method of the present invention, the ammonia lyase activity is high and the activity persistence is also good, so the improved L-
A method for producing phenylalanine (7)ff can be provided.

Example Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 Cladosborium cladoborioides C-5osb<
ynRM-p 77o←) in the following medium (pH 6, O
7 platinum loops were inoculated into a large Erlenmeyer flask) and cultured with shaking at Colo°C for 3 days.

Medium composition (in lOOInl) Peptone 3.op Yeast extract o, zi NaOA' 0. kjl Cells are collected by centrifugation from ioOml of the culture solution thus obtained. Next, cinnamic acid 0. After dissolving Jll in 3.3H of Yuzl aqueous ammonia and adjusting PR to lθ and θ with hydrochloric acid, 〇g was suspended in the above-mentioned wet bacterial cells. Add water to this bacterial cell suspension to make a total volume/Ill, and react at 30° C. (Koda time).

After the reaction, bacterial cells were removed by filtration and the reaction solution was examined.
It was observed that L-phenylalanine was accumulated.

Note that two analyzes were performed by high performance liquid chromatography. The column used was Zorbax OD S, and the eluent used was a mixture of water at 60θ, methanol (ooml), and phosphoric acid (0.3d1-1-pentanesulfonate sodium somg).

Example 1 Culture was carried out in the same manner as in Example 1 except that the following medium components were different to obtain a culture solution.

Medium composition (100-medium) Peptone 3. OII yeast extract o, zg lJa 073, θmj F glucose
0.39 b-7E; Realanine 0.31 Next, a reaction was carried out under the same conditions as in Example 1. and 441
After a period of time, centrifuge and remove L-? The skewer on which the production of enylalanine was measured (l-phenylalanine was produced o, ttt - 1st time).

Next, after washing the bacterial cells, a new reaction solution was added in the same manner as in Example No., and the bacterial cells were reused to carry out the reaction. After the reaction, the mixture was centrifuged, and when the supernatant was examined, it was found that L-phenylalanine was produced (O, *I...second time).

Comparative Example 1 The bacterial strain was Rhodotorula glutinis FD 03! R
hodotorula glutinis current Rho
a08pori(Liulntoruloi+iθ8)
The reaction was carried out once in the same manner as in Example 1, except that the reaction was carried out once.

The seventh production was 0. /KoI! , the Jth time was 0.0/11.

Sender: Mitsubishi Chemical Industries, Ltd.

Agent Patent Attorney Hase - Procedural Amendment (Voluntary) 1985 as Month/Yuhi, Director General of the Patent Office Corridor 1 Case Display Showa! 9th year patent application No. 1393g7
No. 2 Title of the invention Process for producing It-phenylalanidium 3 Idea for amendment Page 596 Ushibanka Shokoshu Co., Ltd. Taisha 4 Agents 〒100 (and 1 other person) 5 Subject of amendment Specification Contents of the amendment in Column 6 of "Detailed Description of the Invention" (1) Correction of the statement in the specification to the following errata (2) Between line It and line 79 on page 1 O of the specification to “
Example 3 A culture solution was obtained by culturing in the same manner as in Example 1 except for the following components.

Medium composition (in lθθgo) Glucose 1. Ojl Peptone 0-z-1 Yeast Extract o, sy Corn-Steep-1J Car o-5lL-Phenylalanine 0+j1 (PH6゜S) Next, the culture solution lθθMl j NibThe same procedure as Example-1 was carried out except that the entire amount of the obtained bacterial cells was used. The reaction was carried out in the same manner.

As a result, L-phenylalanine was present in the first reaction solution.
99. 1.3 g was produced in the λth reaction solution. ” is inserted.

that's all

Claims (1)

[Claims] (1) In a method for producing L-phenylalanine by reacting cinnamic acid and ammonia in the presence of ammonia lyase, Cladosporium
A method for producing L-phenylalanine, characterized in that the reaction is carried out using ammonia lyase obtained from a microorganism belonging to the genus n) and having the ability to produce L-phenylalanine from cinnamic acid and ammonia.