CS259581B1 - A method of purifying the extracellular endo-1, 4-6-xylanase of Cryptococcus albidus - Google Patents

Abstract

The solution relates to a single-step purification of extracellular endo-1,4-xylanase of the yeast Cryptococcus albidus induced by the synthetic inducton methyl β-D-xylopyranoside. The essence of the solution is that the enzyme. is obtained from the yeast induction medium as a homogeneous protein in one purification step by ion exchange chromatography on a column of carboxymethyl derivative of fused bacterial dextran (CM-Sephadex C-50) equilibrated in acetate buffer at pH 4.5 to 5.5, so that the induction medium freed from yeast is poured over a prepared column of carrier, on which the enzyme is captured and from which the enzyme is then released by elution with an increasing sodium chloride gradient to a concentration of 1.0 to 1.5 M. The enzyme is used in the sphere of basic and applied research oriented towards the utilization of lignocellulosic wastes and the release of cellulose fibers from plants such as flax and cotton by the action of enzyme preparations. It can also be used for the preparation of rare 1,4-[beta]-xylooligoisosaccharides from aryl /3-D- -xylopyranosides.

Description

239381

The present invention relates to a single-stage purification of the extracellular endo-1,4-β-xylanase of Cryptococcus albidus induced by the synthetic inducer methyl β-D-xylopyranoside (P. Biely, Z. Krátký and M. Vršanská, CS AO). 206 914).

Endo-1, 4/3-xylanases (EC 3.2.1.8) microorganisms are generally cell-secreted enzymes during growth of xylanase, cellulose and other hemi-cellulose-containing plant materials. They are isolated and purified from the cell-free paste media of the producer. In the first purification step, the growth medium is thickened by ultrafiltration, vacuum distillation, lyophilization or drying. From the concentrated preparations, the proteins are precipitated by organic solvents or ammonium sulphate. Concentrated extracellular proteins or proteinaceous fractions obtained are fractionated by gel filtration of crosslinked polymers, or by ion exchange chromatography on various druzochanexes and cation exchangers such as diethyl aminoethyl and carboxymethyl cellulose and dextran derivatives, and hydroxy-apatite and many others are also used carriers. An overview of the methods used for the purification of xylanases can be found in the article by R. F. H. Dekker and G. N. Richards (Adv. Carbohydr. Chem. Biochem. 32,277, 1976). The purification of xylanases from growth media in which xylan or an inorganic material is used as a carbon source has been complicated by the fact that the carbon source is not completely consumed by the microorganism and its residues in the growth medium are interfered with! with the purification of the α 1 enzyme due to enzyme-substrate interactions. An example of the interaction of the remaining xylan can also be found in the case of purification of yeast xylanase (P.Biely, M. Vrsanska, Z. Kratky, Eur. J. Bio-chem. 108, 313, 1980), of which the present invention is devoted. These difficulties are caused by the production of the enzyme in the cells by low molecular weight substances which are not simultaneously its substrate. Some microorganisms such as Strep-tomyces (K. Nakanishi, T. Yasui and T. Kobay-ashi, J. Ferment, Technol., 34, 801, 1976), Cryptococcus albidus yeast (P. Biely, Z. Kratky and M. Vrsanska, CS AO 206 914) and Cryptococcus flavus (T. Yasui, BT Nguyena K. Nakanishi, J. Ferment 62, 533, 1984), the production of xylanase can be induced by methyl (3-D-xylopyranoside. In the case of Streptomyces, the induced xylanases were purified by classical chromatographic methods in combination with pre-selective isoelectric focusing (M Marui, K. Nakanishi and T. Yasui, Agr. Biol. Chem., 49, 3, 399, 1985. The advantage of inducing oxylalanase by a low blood vessel inducer has been utilized for the first time in the case of the Cryptococcus fla-vus enzyme. single stage chromatography with a vacuum-packed medium on a SP-SephadexC-25 column (K. Nakanishi, H. Arai and T. Yasui, J. Ferment. Technol. 62, 361 (1984). The release of the enzyme from the cation exchanger is carried out at a relatively low concentration of sodium chloride (0.05 M), which places relatively high demands on the removal of salts from the inducing media which prevents the enzyme from attaching to the carrier. Extracellular xylase of Cryptococcus albidus yeast, patriaca among the best studied enzyme (P. Biely, Z. Kratky and M. Vrsanska, Eur. J. Biochem. 119, 559, 1981). induction media with methyl (3-D-xylopyranoside in the past has not been purified. Purification of the enzyme thus induced by the present invention.

The invention is based on the fact that the methyl- / S-D-xylopyranoside-induced cryptococcus albidus extracellular eindo-1,4 / 3-xylanase as described in CSAO 206 914 is obtained from homogeneous induction media as homogeneous protein in a single purification step by ion exchange chromatography on a carboxymethyl derivative of the entrapped bacterial dextran (CM-Sephadex C-50, Pharmacia Uppsala, Sweden) equilibrated in acetate buffer, pH 4.5-5.5, so that or, after concentration by vacuum distillation or ultrafiltration, a carrier is passed over the prepared column, and the enzyme is captured from which the enzyme is then released by elution with a sodium chloride gradient of 1.0 to 1.5 M concentration. When using 0.05 M acetate buffer pH 5.0, the saenzyme is displaced from the column as a sharp white-grained fraction at 0.45 M sodium chloride concentration, which is either directly or after concentration by distillation or ultrafiltration. optionally, desalting by dialysis or ultracentrifugation is used as a source of purified xylanase. The advantage of this method of purifying extra-cellular endo-1,4 / 3-xylanase by Cryptococcus albidus is that - it is simple and fast, above all, the purification of growth media enzymes containing plant-oxygenases as a carbon source, - the enzyme is displaced from an ion exchange carrier with a high salt concentration (0.45 Sodium Chloride), allowing the enzyme to bind ion exchange directly by spilling cell-depleted induction media through it; the salt concentration in the induction media is lower than the concentration required to displace the adsorbed enzyme, the enzyme is obtained as an electrophoretic homogeneous preparation that provides an isoelectric foaming time in the pH3 to 7, three to four protein zones showing simultaneous enzymatic activity proportional to representation of individual proteins. 259581 Example 1

Cryptococcus albidus CCY 17-4--1 (CCY, Czechoslovak Yeast Collection, Chemical Institute of CCHV SAV, Bratislava) grown on liquid synthetic) containing 0.67% of Ivasic nitrogen base, 0.2% L-asparagine , 0.5% of potassium dihydrogen phosphate, 1.0% of D-xylose on a rotary shaker at 27 ° C for 24-48 hours are centrifuged and transferred to the same synthetic medium as above except that D-xylose is charged with methyl β-D-xylopyranoside at a concentration of 0.5 mg / ml. After 24-48 hours incubation on a rotary shaker at 27 ° C, the yeast sususpension is centrifuged and the supernatant containing approximately 1.4 units of xylanase in 1 ml is concentrated on a rotary vacuum evaporator at 35 ° C to 1/5 to 1/10 of the pore volume, dialysed against distilled water at 4 ° C (20 hours) and passed through a CM-Sephadex C-50 (1.8 X 20 cm) column equilibrated 0.05 M acetate buffer pH 5.0. After overflow of the sample, the column was eluted with a linear chloride chloride gradient in 0.05 M acetate buffer (240 mL) to 1.5 M sodium chloride concentration. 3.5 ml fractions are collected and analyzed for enzyme activity and protein. Figure 1 shows the elution of xylanase from a CM-Sephadex C-50 column. In the collected fractions, proteins were determined by measuring absorbance at 280 mm (dotted line), xylanase activity (solid line through points) and sodium chloride concentration (broken line). The actively fragrances decompose, dialyzed against distilled water (4 ° C, 20 h) and used as enzyme preparation. The enzyme is obtained in 35% yield with a specific activity of 42.6 units per mg of protein (Table 1).

Table 1

Purification of methyl / S-D-xylopyra-nosid-induced Cryptococcus albidus xylanase

Purification stage Volume Total activity Total protein Specific activity Yield Inductive ml unit 'mg single / mg% medium Concentrated induction 480 816 120 6.8 100 medium 80 760 104 7.3 93 CM-Sephadex 33 290 6.8 42.6 35.5 aDetermination of enzyme activity and definition of enzyme activity unit is described in the literature (P. Biely, M. Vršanská and Z. Krátký, Eur. J. Biochem. 108, 313, 1980).

The methyl β-D-xylopyranozide-induced enzyme is identical to the enzyme produced by Cryptococcus albidus yeast during xylan growth and purified according to the published procedure (P. Biely, M. Vršanská aZ. Krátký, Eur. Biochem. 108, 313, 1980). Both preparations give single and equally motion-free diffusion zones of protein and enzyme activity during electrophoresis in a polyacrylamide gel. Enzymes appear viscous during isoelectric focusing of the thick polyacrylamide gel layer in the pH range from 3 to 7. Several forms of Comassie stained white brilliance Brillant Blue R-250 exhibited enzyme activity which was detected by covalently stained oxylan (P. White, O. Markovic, and D. Mislodic, Anal. Biochem., 144, 147, 1985.

The xynalase-induced supernatant obtained by centrifugation of a yeast suspension incubated with methyl β-D-xylopyranoside as described in Example 1 is not thickened but is directly passed through a CM-Sephadex C-50 column. The captured xylanase from the column displaces and further treats as described in Example 1. The enzyme purification yield and step correspond to those in Table 1. Example 3

The enzyme is induced in yeast as described in Example 1 and applied to CM-Sephadex C-50 as described in Example 2. The xylanase is sealed from the column as follows: the column is first washed with 0.25 M solutions. sodium chloride in 0.05 M acetate buffer pH 5.0 (75 mol) and then with a linear gradient of sodium chloride to 1.0 M concentration in the same acetate buffer (150 ml). Xylanase is isolated by concentration and desalting of the fraction eluted at 0.45 M concentration of chloride. The yield and degree of purification of the enzyme corresponds to the values in Table 1,

Claims (4)

SUBJECT A method for purifying extracellular endo-1,4- / 3-xylanase of yeast Cryptococcus albidus induced by] methyl β-D-xylopyranoside, characterized in that the induction medium containing the enzyme is contacted with a carboxymethyl derivative of a cross-linked bacterial dextran dextran derivative. to which the enzyme is adsorbed and then released by increasing sodium chloride gradient up to 1.5 M concentration. INVENTION 2. The method of claim 1, wherein the chromatography support is equilibrated with an acetate buffer of pH 4.5 to 5.5. 3. Purification method according to items 1 and 2, characterized in that 0.05 M acetate buffer at pH 5.0 is used and the enzyme is released at 0.45 M sodium chloride concentrations. 4. The process of claims 1 to 3 wherein the enzyme solution is further dehydrated and concentrated. 1 sheet of drawings