DD249712B1 - 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 is to breed the strain Acinetobacter calcoaceticus on water-soluble fatty acid esters as the only C-Queile, in whose degradation fatty acids are formed.

Description

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 bacterial and yeast strains or fungi on complex media (including bouillon, whey, corn steep liquor) are cultured, the bacteria separated by centrifugation and the culture supernatants for lipase production prepared (Literature z, B. Lipases, ed. B. Borgström and H. Brockmann, Elsevier, Amsterdam, 1984; 0 .Herbburg, HP Kleber, Acta Blotechnol. 2,1982,4, p.337-342; this, supra, 3,1983,2, pp. 185-186). Strains used for lipase production are e.g. Those of the genus Rhizopus (Rh. Spec.), Candida (C. cylindrical, Pseudomonas, Mucor, Aspergillus, Acinetobacter and others. The presented methods have the disadvantage that by culturing on complex media, the lipase is heavily contaminated and several to their isolation elaborate purification steps are necessary.

Object of the invention

The invention aims to provide a process for enzyme production which allows to obtain an enzyme of substantially higher purity and specific activity and thus to facilitate 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 commercially 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 to 40 ° C, preferably at 3O 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. B. Polyoxyethylene sorbitan laurate, -plamitat or oleate, inter alia, as the N-source NH ₄ Cl, (NH ₄ I ₂ SO ₄ or the like.) After a growth time of 5 to 40 hours - depending on the physiological state and The amount of seed material is separated by centrifugation and the cell-free culture supernatant obtained The enzyme obtained in the cell-free culture supernatant in cultivation according to the invention is in contrast to cultivations on complex media contaminated only slightly with foreign substances and has a high specific activity (Table 1).

The isolation of the enzyme from the culture supernatant is carried out on a hydrophobic support material, e.g. Octyl-Sepharose, as follows: The obtained cell-free Kuttur supernatant is added at 4 ° C with crystalline NaCl to a concentration of 1 mmol / l. The solution is placed on a glass column filled with octyl-Sepharose. The lipase adsorbs to the hydrophobic carrier. Foreign proteins are bound only slightly. 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 doubly enriched. The lipase preparation thus obtained is diskelectrophoretically pure. The invention will be explained in more detail below using an exemplary embodiment.

embodiment

Acinetobacter calcoaceticus (69 V) is grown at 30 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 0.7.5mg FeSO ₄ 7H ₂ 0.075mg MnSO ₄ .4H ₂ 0, 0.75 mg of ZnSO ₄ .7H ₂ .0.15 mg of CuSO ₄ .5H ₂ .0.15 mg of CoCl ₂ .6H ₂ O and 0.15 mg of boric acid.

Ats vaccine suspension is a flush of bouillon agar. The C source used is polyoxyethylene sorbitan monooleate in a concentration of ЮTІ / І as sole carbon source and NH ₄ Cl (3 g / l) as 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 recovered. NaCl is added to this culture supernatant to a concentration of 1 mol / l and the solution is applied to an octyl-Sepharose column at 4 ° C. The lipase activity remains on the carrier material. l phosphate buffer, pH 7.5) and phosphate buffer, pH 7.5 until no protein is detectable in the eluate The enzyme is then treated with Triton X-100 (5 g / l in 50 mmol / l phosphate buffer, pH 7.5) from The enzyme obtained is tablet-electrophoretically pure The isolation of the lipase is shown in Table 2.

Table 1

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

specific activity

(цтоі / тіп / тд protein) _

26 24 78

Culture substrate volume activity protein (Pmol / min / ml) (Mg / ml) bouillon 0.3 9.5 Polyoxyethylene sorbitan monolaurate 6.5 0.25 Polyoxyethylene sorbitan monopalmitate β, ο 0.25 Polyoxyethylene sorbitan monooleate 26.6 0.34

Table 2

Purification of the lipase (growing medium: polyoxyethylene sorbitan monooleate) of Acinetobacter calcoaceticus

Reinig.-stage Volume (ml) Protein (mg / ml) Total act, (цтоі / тіп) spec. Activity (цтоі / тіп / тд protein) Clean, (-fold) Yield (%) Culture supernatant Triton X-100 fraction 190 30 0.34 0.13 5054 3040 78.2 779.2 9.96 60

Claims (4)

1. A process for the preparation of lipase by culturing the strain Acinetobacter calcoaceticus, characterized in that water-soluble fatty acid esters are used as the sole C source. 2. The method according to claim 1, characterized in that is cultivated at temperatures of 20 to 40 ⁰ C in submerged culture in a liquid minimal medium, after a growth time of 5 to 40 hours, the bacteria are separated and the enzyme is separated from the culture supernatant. 3. The method according to claim 1 and 2, characterized in that the water-soluble fatty acid esters Polyqxyethylensorbitanmonooleat, laurate-pal mi at, -öleat od. Like. Be used. 4. The method according to claim 1 to 3, characterized in that from the cell-free culture supernatant by chromatography on a hydrophobic support such. B. Octylsepharose an isolation of the enzyme takes place up to the disk electrophoretic purity.