DD249712A1 - PROCESS FOR PREPARING LIPASE - Google Patents

Abstract

The object of the invention is to provide a method which provides an enzyme of high specific activity and enrichment. The object is seen to produce the desired enzyme by culturing a bacterial strain on a previously unused culture substrate. The essence of the invention consists in cultivating the Acinetobacter calcoaceticus strain on fatty acid esters of water-soluble fatty acids as the sole source of C, fatty acids being produced during their degradation.

Description

embodiment

Acinetobacter calcoaceticus (69V) is grown at 3O ⁰ C in submerged culture in a liquid minimal medium. The medium contains per liter: 6.97K ₂ HPO ₄ , 1.5g NaH ₂ PO ₄ , 0.2g MgSO ₄ - 7H ₂ O, 7.5mg FeSO ₄ .7H ₂ O, 0.75mg MnSO ₄ .4H ₂ O , 0.75 mg of ZnSO ₄ - 7H ₂ O, 0.15 mg of CuSO ₄ - 5H ₂ O, 0.15 mg of CoCl ₂ · 6H ₂ O and 0.15 mg of boric acid. As a vaccine suspension is a flush of bouillon agar. Used as the C source such as polyoxyethylene sorbitan monooleate at a concentration of 10 ml / l as the sole carbon source and NH ₄ Cl ( ₃ g / l) as the N source.

After a growth time of 12 h, the bacteria are separated by centrifugation (2000 × g, 20 min) and the cell-free culture supernatant is obtained. NaCI is added to this culture supernatant to a concentration of 1 mol / l and the solution is applied at 4 ° C. to an octyl-Sepharose column. The lipase activity remains on the carrier material. Successively, the carrier is washed with 1 mol / l sodium chloride solution (in 50 mmol / l phosphate buffer, pH 7.5) ^xl until no protein is detectable in the eluate. The enzyme is then dissolved with Triton X-100 (5 g / l in 50 mmol / l phosphate buffer, pH 7.5) from the carrier. The recovered enzyme is discrete electrophoretically pure. The isolation of the lipase is shown in Table 2, x) and phosphate buffer, pH 7.5

Table 1

Volume activity, protein concentration and specific activity of the lipids in the culture supernatant of Acinetobacter calcoaceticus after growth on different culture substrates

Culture substrate

Volume activity (μιηοΙ / Γηίη / ΓηΙ)

Protein (mg / l)

specific activity (^ mol / min / mg protein)

bouillon

polyoxyethylene

sorbitan mono-

laurat

polyoxyethylene

sorbitan mono-

palmitate

polyoxyethylene

sorbitan mono-

0.3

6.5

6.0

26.6

9.5

0.25

0.25

0.34

0.03

Table 2

Purification of the lipase (culture substrate: polyoxyethylene sorbitan monooleate) of Acinetobacter calcoaceticus

cleaning stage • Volume protein Total act. spec. Cleaning. yield activity (Ml) (Mg / ml) (Jumol / min) (ΜΐτιοΙ / min / (-subject) (%) mg protein) Culture supernatant 190 0.34 5 054 78.2 Triton X-100 fraction 30 0.13 3 040 779.2 9.96 60

Claims (4)

claims: 1. A process for the preparation of lipase, characterized in that the strain Acinetobacter calcoaceticus is grown and are used as the only C source water-soluble fatty acid esters. 2. The method according to item 1, characterized in that the strain Acinetobacter calcoaceticus is grown at temperatures of 20-40 ⁰ C in Submerskultur in a liquid minimal medium, after a growth time of 5-4Oh the bacteria are separated and the enzyme separated from the culture supernatant becomes. 3. The method according to item 1 and 2, characterized in that the only C source, in the degradation of fatty acids, water-soluble fatty acid esters such. As polyoxyethylene sorbitan monooleate, laurate, palmitate or oleate can be used. 4. The method according to item 1 to 3, characterized in that from the cell-free culture supernatant by chromatography on a hydrophobic support such. B. Octyl Sepharose an isolation of the enzyme to the disk electrophoretic purity. Field of application of the invention The invention relates to a process for the preparation of lipase ₍ which can be used in practice for the hydrolysis of fatty acid esters. Characteristic of the known technical solutions It is already known to produce lipase. For this purpose, various bacteria and yeast strains or fungi on complex media (including bouillon, whey, corn steep liquor) are cultured, the bacteria separated by centrifugation and prepared the culture supernatants for lipase production (literature, for example, Lipases, ed. B. Borgström and H.Brockman , Elsevier, Amsterdam, 1984). The illustrated methods have the disadvantage that the lipase is heavily contaminated by the cultivation on complex media and their isolation several complex purification stages are necessary. Object of the invention The invention aims to provide a process for enzyme production with a hitherto unused bacterial strain, which makes it possible to obtain an enzyme with substantially higher purity and specific activity and thus to simplify the isolation from the culture supernatant. Explanation of the essence of the invention The object of the invention is to modify essential known features of the previous procedure and to provide the desired enzyme with high specific activity by using a hitherto unused strain and special cultivation substrates. The object is achieved in that the enzyme lipase from the strain Acinetobacter calcoaceticus is produced. The stock is preferably on 3% bouillon agar at 4 ° C in the dark. The cultivation of Acinetobacter calcoaceticus takes place after seeding at a temperature of 20-40 ⁰ C, preferably at 30 ⁰ C, in submerged culture in a liquid minimal medium. The cultivation is carried out according to the invention on water-soluble fatty acid esters as the sole C source, in the degradation fatty acids are formed. Such C sources are z. Polyoxyethylene sorbitan laurate, palmitate or oleate, etc. As N-source, NH ₄ Cl, (NH ₄ I ₂ or the like is used, after a growth time of 5-40 hours, depending on the physiological state and amount of Inoculum - the bacteria are separated by centrifugation, for example, and the cell-free culture supernatant is obtained The enzyme obtained in the cell-free culture supernatant in the cultivation according to the invention is only slightly contaminated with foreign substances and has a high specific activity, in contrast to cultivations on complex media (Table 1). The isolation of the enzyme from the culture supernatant is carried out on a hydrophobic carrier material, such as, for example, octyl-Sepharose, as follows: The obtained cell-free culture supernatant is treated with crystalline NaCl at 4 ° C. to a concentration of 1 mmol / l is added to a glass column filled with octyl-Sepharose, whereby the lipase adsorbs to the hydrophobic carrier, and foreign proteins are only slightly bound. Successively, the column is washed with 1 mol / l NaCl in phosphate buffer (50 mmol / l, pH 7.5) and 50 mmol / l phosphate buffer (pH 7.5). The lipase is replaced by a 0.5% solution of Triton X-100 in 50 mmol / l phosphate buffer, pH 7.5, of the octyl Sepharose. 60% of the original lipase are found 10 times enriched. The lipase preparation thus obtained is diskelectrophoretically pure. The invention will be explained in more detail below using an exemplary embodiment.