SU975797A1 - Method for isolating leucineaminopeptitdase from aspergillus oryzae - Google Patents

Description

The invention relates to methods for producing the enzyme leucine aminopept Dases (K.F.3.4.11.1.), used in molecular biology to study the amino acid sequence in protein molecules, to obtain amino acids and in medicine.

Known methods for producing leucine aminopeptides from animal and plant materials, for example, from the kidney. pigs £ 1J and from pea seeds £ 2 J

However, these methods are based on the use of scarce or food raw materials and do not have industrial significance.

The closest in technical essence is a method for producing peycinaminopeptidase using microorganisms, in particular from mold Aspergillus oryzal [3] ·

The method consists in preliminarily scouring the crude enzyme solution by group treatment with amberlite, fractionating with ammonium sulfate, and precipitating with rivanol. The resulting precipitate is extracted twice.

E, 5 N acetate buffer with a pH of 5.0. Proteins are precipitated in the ⁵ chilled extract by adding chilled acetone to a 67% ** concentration. The precipitate is dissolved in water. Subsequent ion-exchange chromatography on DEAE-cellulose is carried out with a linear 0-0.4 M NaCt gradient in 0.01 N phosphate buffer with a pH of 7> 0.

After concentrating on ultrafilter · leucine aminopeptidase fraction, it is applied to a Sephadex 0-100 column and re-gel filtered on a Sephadex G ”-200 column in 0.1 M acetate buffer with a pH of 6.5. Then, chromatography was performed on diethylaminoethyl-seph ₂₀ dex A-50 with phosphate buffer (pH

7.0) with a linear gradient of 0-0.4 M NaC |. The purification is completed by repeated gel filtration on Sephadex G-200 in 0 _r 1 M acetate buffer with a pH of 6.5.

The resulting preparation is frozen to -15 ° C.

The total activity of leucine aminopeptidase (in the extract - 2090 units, in the resulting preparation - 262 units) - 13%. 5

The specific activity of leucine aminopeptidase in the extract is 0.181 units / mg protein, in the resulting preparation 4.54 units / mg protein (Lei-Gly-Gly tripeptide substrate). The calculated degree of purification is 25.

Along with providing high specific activity, the known method has drawbacks - a relatively low yield in total activity caused by a 15 ₍ long and multi-stage separation process in which losses occur and an insufficiently high degree of purification,

The purpose of the invention is to increase the yield and degree of purification, simplifying the isolation process.

The goal is achieved by a method of producing leucine aminopeptidase ₂₅ Aspergillus oryzae, comprising extracting the enzyme buffer, its precipitation with an organic solvent followed by chromatography on DEAE cellulose eluting with buffer, _Μ containing NaS1, wherein the organic solvent used in the precipitation at a concentration of ethanol 60-67%; chromatography on DEAE-cellulose is carried out twice, while the sorption of the enzyme is carried out in static ³⁵ conditions with a volume ratio of Enzyme: sorbent 1 :(. 1.3-1.5), elution of the enzyme they are carried out under dynamic conditions with a stepwise NaCl gradient from 0.2 to 0.5 I in the buffer, and between the chromatographic stages the enzyme-containing solution is subjected to heat treatment at 60-62 ^σ 0 for ΙΟΙ 2 min.

Typically, 0.005 M vero is used. ⁴⁵ buffer with pH 7.9 “B, O ·

This improves the quality of the sorption of the enzyme on ion-exchange cellulose, which is carried out by mixing the enzyme solution with sorbent ⁵⁰ under static conditions. _K pro- conducting desorption enzyme under dynamic conditions with a stepped gradient separation of proteins achieved clarity. The differences in the conditions of ⁵⁵ tion sorption and desorption steps reduce the time of ion exchange chromatography and rehromatografii. An increase in the degree of purification at these stages of sweat eliminates gel filtration at Sephadex and simplifies the process.

The proposed method is as follows.

Amilorizin P 10 X is extracted for 3 hours with a 0.00-5 M veronal buffer with a pH of 7–9 “§j1 at room temperature. Centrifuge 25 “35 min at 5.500-6000 rpm. The centrifugate is cooled and ethyl alcohol cooled to -20 ^ C is added to a final concentration of 60-6?%. After 20 minutes, the precipitate was separated by centrifugation, which was carried out for 20-25 minutes at 5500-6000 rpm and 0-4 ° C, and dissolved in a double volume of 0.005 M veronal buffer with a pH of 7.9 "8.1. The undissolved precipitate is separated by centrifugation under the same conditions. The resulting centrifugate is mixed with a balanced buffer DEAE-cellulose in a volume ratio of 1: (1.3-1.57 and fill the column. Non-absorbed proteins and leucinamine-peptidine-inactive proteins are washed with a 0.2 N solution of NaC1 in a 0.005 M Verona buffer with pH 7, 9-8,1. the active fraction was desorbed with 0.5 M solution PaS1 in the same buffer, dialysed and heated to 6D-62 ^C. C., kept [at this temperature for 10-12 min, rapidly cooled to 0-4 = 0, mixed with buffer balanced DEAE-cellulose in a volume ratio of 1: (1.3 “1” 5) and filled into the column. and the desorption of the enzyme is carried out as the first chromatography. The resulting preparation is concentrated with dry Sephadex G-75.

The yield in activity is 25% (activity in the extract is 8.68 units, in the resulting preparation - 2.17 units).

The degree of purification is 38 (the specific activity of the extract is 0.00815, the obtained preparation is 0.31 u / mg protein on a Lei-I-nitroanilide substrate).

Example 1. 50 g of amylorizin P 10 X was extracted in 250 ml of 0.005 M veronal buffer with a pH of 7.9–8.1.1 for 3 hours at room temperature. Centrifuged 25 “35 min at 5” 56.0 thousand rpm.

The extract (215 ml) was cooled to 0-4 ° C and 502 ml of 96% ethanol cooled to -20 ° C were added to a final concentration of 67%. _s After 20 min, the precipitate is separated by centrifugation at 5.5 “6.0 thousand rpm at (-2) 2) ° C and dissolved in 100 ml

0.005 M veronal buffer with a pH of 7.9 8.1. Insoluble precipitate is separated by centrifugation at the same conditions.

The resulting 135 ml of centrifugate was mixed for one hour with 175 ml of buffer-balanced DEAE-cellulose (in a volume ratio of 1: 1.3) and filled into a chromatographic column. Unsorbed and ballast proteins are washed with two liters of a 0.2 N solution of NaC1 in a 0.005 N veronal buffer with a pH of 7.9-8.1. The active fraction 15 is desorbed by passing through a column of 1 L of a 0.5 N NaCl solution in the same buffer. The first 20 ml of the eluate is discarded (do not have leucine aminopeptidase activity), the next 320 ml, containing the maximum active enzyme, are collected and dialyzed against 5 L of 0.0005 I veronal buffer with a pH of 7.9 “8.1 for 24 hours. During dialysis, the buffer solution is changed 4 times! fifteen

The dialyzed solution is heated to 6O-62 ° C and maintained at this temperature for 10 minutes, then quickly cooled to 2-6 ° C.

Inactivated proteinases 30 are separated by rechromatography on ion exchange cellulose. The dialyzed enzyme solution was mixed for 1 hour with DEAE-cellulose-balanced buffer in a volume ratio of 1: 1.3 and filled into the chromatographic column. Unsorbed proteins are washed with two liters of a 0.2 I solution of NaCl in a 0.005 N veronal buffer with a pH of 7.9 “8.1. The active fraction _{0 is} stripped with one liter of 0.5 AND NaCl solution in the same buffer. The first 20 ml of the eluate is discarded (do not have leucine aminopeptidase activity), the next 320 ml containing the maximum 'active enzyme is collected and dialyzed against 5 L of 0.0005 I veronal [buffer with pH 7.9-θ, Ι for 24 hours. IВо dialysis time, the buffer solution is changed 4 times. _I

In order to concentrate in a 350 ml dialyzed enzyme solution, a cellophane bag with 40 g of dry Sephadex G> ~ 75 is placed. After 24 hours, 2.36 g of dry ammonium sulfate (up to 39 от from 0.7 saturation) and 0.0012 magnesium chloride are added to 5 ml of concentrated enzyme ^Si solution.

The total yield according to the activity of the obtained drug is 25? (the activity of the extract is 8.84 units, the obtained preparation is 2.21 units). The degree of purification is 32 (specific activity of the extract is 0.0079, the resulting preparation 0.25 units / mg protein on the Lei-nitroanilide substrate).

Example 2. 50 g of amylorizin P 10 X is extracted in 250 ml of 0.005 M veronal buffer with a pH of 7.9-8.1 for 3 hours at room temperature. Centrifuged, precipitated proteins with alcohol, centrifuged, dissolved the precipitate and centrifuged again, as in example 1.

The resulting 130 ml of a centrifugate was mixed for one hour with 195 ml of DEAE-celluloea equilibrated buffer (in a volume ratio of 1: 1.5) and the chromatographic column was filled. Unsorbed and ballast proteins are washed with two liters of a 0.2 M NaCI solution in 0.005 And veronal buffer with a pH of 7.9 “8.1. The active enzyme fraction of desorbiruft, passing through a column of 1 l of 0.4 K NaCl solution in the same buffer. The first 30 ml of the eluate is discarded. Sleep has leucine aminopeptidase activity, the next 340 ml, containing the most active enzyme, is collected. Dialysis and heat treatment are carried out similarly to the first example.

During rechromatography, the dialyzed Enzyme solution is mixed for one hour with DEAE-cellulose balanced buffer (in volume ratios of 1: 1.5) and the chromatographic column is filled. Unsorbed and ballast proteins are washed with two liters of a 0.2 I solution of NaCI in a 0.005 M veronal buffer with a pH of 7.9–8.1. The active fraction is stripped with one liter of a 0.4 M NaCl solution in the same buffer. The first 30 ml of the eluate is discarded (do not have leucine aminopeptidase activity), the next 330 ml containing the most active enzyme is collected. Dialysis and concentration of the final product are carried out similarly to the first example.

The total yield of the activity of the obtained product is 25? (the activity of the extract is 8.91 units, the obtained preparation is 2.23 units). The degree of purification - 34 (specific activity of the extract of 0.0093, the resulting drug 975797

0.30 units / mg protein on a Lei-P-nitroanilide substrate).

Example 3 · Extraction and precipitation of proteins with ethanol is carried out analogously to the first example. In ion-exchange chromatography, 130 ml of the enzyme solution are mixed for one hour with 180 ml of DEAE-cellulose balanced with buffer (in volume ratios ι 1: 1.4) and the chromatographic column is filled in · ♦ · column. Unsorbed and ballast proteins are washed with two liters of a 0.3 M NaC1 solution in a 0.005 M veronal buffer with a pH of 7.9 “θ, 1. The enzyme fraction is desorbed by passing ¹⁵ through a column of 1 L of a 0.4 And NaCl solution. in the same buffer. The first 30 ml of the eluate is discarded (do not have leucine aminopeptidase activity), the next 330 ml of the eluate, containing the maximum active enzyme, is collected. Dialysis and thermal inactivation of ballast proteins is carried out similarly to the first example. During rechromatography, the dialyzed enzyme solution is mixed for 1 hour with DEAE-cellulose balanced buffer (in volume ratios of 1: 1.4), the column is filled, washed with 0.3 I with a NaC1 solution in 0.005 I of a verine buffer with a pH of 7.9- 8) 1 and de-30 sorb 0.4 And a solution of NaCl in the same buffer. 330 ml (from the 31st to the ZBO-th ml) of the eluate containing the most active enzyme are collected. The final product is obtained after dialysis and concentration '35.

The total yield according to the activity of the final product is 25% (the activity of the extract is 8.77 units, the obtained preparation is 2.79 units). The degree of cleaning is 40 (the specific activity of the extract is 0.00815, the resulting preparation of 0.31 units / mg protein on the Leir-nitroanilide substrate).

Determination of enzyme activity.

One unit of activity leytsinamino · ¹ peptidase take such amount of enzyme which hydrolyses 1.0 micromole leucine-I-nitroanilide per minute at _and at 37 ° C and pH 8,5 «

The specific activity was calculated by the formula leytsinaminopeptidazsch ^{Λ Τ ^ '^, ΤΓ ·} χ ^Knitted - ^{/ u protein'} "where oSGen 3.5 g of the reaction mixture in the cuvette cm ^;

3.9 extinction coefficient. , 1 μmol 'of I-nitroaniline under experimental conditions;

t is the time of incubation of the enzyme with the substrate;

0.1 · * · the volume of the drug solution taken for analysis, cm · **;

x is the protein concentration in the initial solution, mg / cm ^ (determined by Lowry,).

The activity of leucine aminopeptides was determined on a domestic production Lei-I-nitroanilide substrate.

The technical and economic effect of the invention is to ensure the production of the enzyme leucine aminopeptidase, used in molecular biology and medicine from cheap microbial raw materials ;, increasing the overall yield by activity, which according to the known method is 13%, and according to the proposed one, 25%. In addition, the degree of purification increases from 25 (by a known method) to 38 (by the proposed). During the isolation, gel-filtration steps on Sephadex R-100 and Sephadex G-200 are also excluded.

Claims (1)

1. Spacknan, PI, Snith E.L., 3. Hakadnl, T., Hasuno S ,, Iguchf, C. Prown D.U. Leuclne anfnopeptldase IV. j Purification of leuctne anonopepttdeOsolatlon and properties of the engy-se II frori Asp, oryzoe. Atyr. Blol. jme from swine RMnftv. J. Rlol. Chen., Chen, 1973, Vol. 37, Mr, ii, p. 76719 $ 5, voK 212, Nr. 1, p. 255 .; 77. Nr. 1, p. 113-1t8,