JPH0154994B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an affinity adsorbent for affinity chromatography. Affinity chromatography is an excellent method for specifically purifying specific proteins. The present inventors have conducted intensive studies to develop an affinity adsorbent for affinity chromatography that has the potential to be widely applied to the discovery and purification of various enzymes that exhibit the ability to bind xylose, such as glucose isomerase. As a result, the present invention was achieved. The gist of the first invention is that xylose is obtained by reaction between an aldehyde group of xylose and an amino group of a support having an amino group. The gist of the second invention resides in an affinity adsorbent for affinity chromatography, characterized in that xylose is bonded to A substance capable of binding to xylose from a mixture by affinity chromatography using xylose as a ligand and an affinity adsorbent bonded to the N atom of the amino group of the support at the position of the aldehyde group of xylose. The problem lies in the method of separating the To explain the present invention in more detail, the first feature of the affinity adsorbent for affinity chromatography of the present invention is that it uses xylose as a ligand. ) is a widely used adsorbent for the purification of The second feature is that xylose is bonded to a support having an amino group at the aldehyde group of xylose as shown below (in the formula below, the support is polyacrylamide, and the support and xylose Spacer between (CH ₂ ) _o N
= (n=4 to 8). Polyacrylamide -CONH( _CH2 ) _o N=CH-CH(OH) -CH(OH)-CH(OH) _-CH2 (OH) This utilizes the aldehyde intermediate in sugar tautomerism and The NH ₂ group of the sugar is reacted with the aldehyde group of the sugar, and as a result, the sugar can be bonded to the support relatively easily, without aminating the hydroxyl group of the sugar or using reagents such as dicyclohexylcarbodiimide. It can be introduced by The resulting adsorbent has affinity for enzymes that recognize and bind to xylose, and has very similar protein properties to enzymes that do not bind to xylose, even when present in extremely small amounts. It has the advantage that it can be clearly separated as an unadsorbed substance even when it is mixed, and is useful as an adsorbent in affinity chromatography. As the support, polyacrylamide, agarose derivatives, etc. are usually used. The type of support is selected depending on the reaction used to immobilize xylose on the support. In addition to the support having a functional group that reacts with the ligand, those having a spacer between the functional group and the substrate may also be used. As such supports, those commercially available from Pharmacia, Biochemicals, and Bioted can be used. In order to prepare the affinity adsorbent for affinity chromatography of the present invention, a support and a ligand may be coupled together by a conventional method. The coupling reaction may be appropriately selected depending on the type of support. When polyacrylamide is used as the support, a spacer may be introduced as shown in the following formula and then coupled with xylose.

【table】

[Table] The affinity adsorbent for affinity chromatography of the present invention obtained as described above can be used to separate a substance capable of binding to xylose from a mixture. For example, Thermus thermophilus HB8
(ATCC 27634) can be used to separate and purify thermostable glucose isomerase from bacterial cells. As shown in the examples below, high-purity glucose isomerase can be obtained by specifically adsorbing glucose isomerase in a mixture onto the affinity adsorbent of the present invention and desorbing it with a buffer containing sodium chloride or xylose. can be obtained in high yield. Specifically, glucose isomerase can be separated and purified by a three-step process of 50% ammonium sulfate fractionation of the disrupted bacterial cell solution, diaminoethyl cellulose column treatment, and affinity chromatography using the affinity adsorbed solid of the present invention. To explain in more detail, the bacterial cells are ground by alumina grinding in a buffer, the supernatant is obtained by centrifugation, and this supernatant is
Add ammonium sulfate to 30% saturation, let stand, and centrifuge to obtain supernatant. Add ammonium sulfate to this supernatant again until 50% saturation.
Collect the precipitate by centrifugation and dissolve in buffer. After filtering, it was packed into a diethylaminoethyl cellulose column equilibrated in a buffer.
Elute with a linear gradient of 0M to 0.5M sodium chloride. Sodium chloride concentration is 0.15~
Glucose isomerase elutes around 0.25M. The active fractions are collected and dialyzed, then subjected to the affinity chromatography of the present invention equilibrated with the same buffer, and eluted with a buffer containing sodium chloride or xylose to obtain glucose isomerase. Column chromatography of diethylaminoethyl cellulose is not necessarily required, but it is preferable to avoid contamination of the affinity chromatography support. Furthermore, since non-specific adsorption may occur depending on the sample, it is preferable to perform gel filtration or ion exchange chromatography once after affinity chromatography. Hereinafter, the present invention will be explained in more detail with reference to Examples. In addition, the enzyme activity in Examples was measured as follows. First, a reaction solution and a coloring solution having the following compositions are prepared. Reaction solution composition A solution Demineralized water 1 volume 1.0MD-Glucose 2〃 0.1MMgSO ₄・7H ₂ O 1 volume 0.02MCoCl ₂・6H ₂ O １〃 B solution 0.2M Phosphate butter (PH7.0) 5 volumes A solution and B solution Mix immediately before use. Coloring liquid composition Demineralized water 175 volumes Sulfuric acid 271〃 1.5% cysteine hydrochloride aqueous solution 11〃 1.5% carbazole ethanol solution〃 Mix under ice cooling. Next, add 1 volume of enzyme solution to 1 volume of reaction solution and heat at 70℃30
The isomerization reaction was carried out for 1 minute, and the reaction was stopped with 2 volumes _of 0.5MHC1O4. Mix 5 ml of coloring solution with 20μ of this isomerized solution and heat at 60°C.
Perform a color reaction for 10 minutes and measure the absorbance at 560 nm. One unit of enzyme activity is the amount of enzyme that produces 1 mg of fructose from glucose per hour under the above conditions. Example 1 (Preparation of affinity adsorbent) Bio-Gel P-300 (100-200mesh) Bio Rad
Weighed 2 g of 1,4-diaminobutane H ₂ N (CH ₂ ) ₄ NH ₂ in a 100 ml Erlenmeyer flask at 90°C in a closed system equipped with a 1NHCl trap.
Perform the reaction for 8 hours. The progress of the reaction occurs
The remaining amount of 1NHCl that traps NH ₃ was determined by titration. Collect Bio-Gel on a glass filter and add H ₂ O
Remove unreacted H ₂ N(CH ₂ ) ₄ NH ₂ by washing with .
Next, Bio-Gel on a glass filter was added to 50 ml of 0.1M xylose, and cutting was performed by reaction at 90°C for 1 hour. After the reaction, the mixture was collected on a glass filter and washed with H ₂ O to remove xylose, followed by washing with 0.5M NaCl and H ₂ O in this order. Next, suspend this in 35 ml of 0.7M saturated sodium acetate,
The mixture was kept at 0.degree. C. and 1 ml of acetic anhydride was added dropwise over 1 hour. The mixture was further maintained at 4° C. for 1 hour to acetylate the remaining amino groups. After separating the reaction solution, add H ₂ O,
It was washed with 1M NaCl and H ₂ O in this order. The solid affinity adsorbent thus obtained is hereinafter referred to as PA-xylose. Example 2 (Isolation and purification of glucose isomerase) (1) Culture of bacteria Thermus Thermophilus HB8 was mixed with 0.3% yeast extract, 0.5% peptone, 0.1% glucose, and 0.2%.
NaCl, 0.05% inorganic salt vitamin solution (in 1 part water)
_25gMgCl2・_6H2O , _5gCaCl2 , 2g
MnSO ₄・6H ₂ O, 0.5g ZnSO ₄・7H ₂ O, 0.5g
H ₃ BO ₃ , 5g FeCl ₃・6H ₂ O, 15mg CuSO ₄ ,
25mg Na ₂ MoO ₄・2H ₂ O, 50mg CoNO ₃・
_6H2O , 20mg _NiNO3・_6H2O , 80mg pyridoxane, 10mg folic acid, _0.5mlH2SO4 , 10mg thiamine pyrophosphate, _40mg riboflavin, 80mg nicotinamide, 80mg para-aminobenzoic acid, 10mg biotin,
0.4mg cyanocobalamin, 80mg pantothenic acid, 20
mg lipoic acid, 200 mg inositol, 50 mg choline, 50
Using a medium containing (mg orotic acid, 100 mg spermine), aerated culture was carried out at 75° C. for 5 to 6 hours, and the bacteria were collected at around 300 creets. Approximately 10g per medium
HB8 bacterial cells were obtained. (2) Separation and purification of enzyme (A) Extraction The wet weight of the HB8 cells obtained as above is approximately 1 kg.
Add 2 extraction buffer (50mM Tris-HCl (PH
7.5), 14mM Mg( _CH3COO ) ₂ , 1mM EDTA,
0.14mM KCl, 50mM β-mercaptoethanol) was added, and the mixture was ground by a mill to extract the enzyme. The extract was centrifuged at 10,000 g for 20 minutes to separate the precipitate and supernatant, and the precipitate was extracted again with extraction buffer 1 and centrifuged to obtain the supernatant. The supernatants from the two centrifugations were combined to obtain a crude enzyme solution (2.7). (B) Diethylaminoethyl cellulose (DEAE
Add ammonium sulfate to the crude enzyme solution obtained by Cellulose DE-32 (manufactured by Fumarmacia) column chromatography (A) until 30% saturation, leave it overnight, remove the precipitate by centrifugation at 10000 g for 40 minutes, and add Ammonium sulfate was added until % saturation, left to stand, and centrifuged in the same manner to collect the precipitate. Transfer this precipitate to as little buffer as possible (50mM Tris-HCl (PH7.5), 1mM
EDTA) and dialyzed against the same buffer overnight. 600 ml of this dialyzed enzyme solution was added to a DEAE cellulose DE-32 column (inner diameter 5 cm, length 44 cm) equilibrated with the same buffer solution and adsorbed.
Increase the concentration of sodium chloride in the buffer from 0M to 0.5M
Elution was performed with a linear concentration gradient of . The enzyme is eluted at salinity between 0.15M and 0.25M. (C) PA-xylose affinity chromatography The glucose isomerase active fraction obtained in (B) was collected and thoroughly dialyzed against 10 mM Tris-ECl (PH7.5) buffer. This dialyzed enzyme solution was equilibrated with the same buffer solution on a PA-xylose column (φ1
x 10cm) for adsorption, and elution was performed with a buffer containing 0.1M NaCl. The obtained active fraction had a protein content of 4 mg, a total activity of 2250 units, and a specific activity of 560 units/mg protein.

Claims (1)

[Scope of Claims] 1 Obtained by the reaction of the aldehyde group of xylose and the amino group of a support having an amino group,
An affinity adsorbent for affinity chromatography, characterized in that xylose is used as a ligand and is bonded to the N atom of an amino group of a support at the position of an aldehyde group of xylose. 2 Obtained by the reaction of the aldehyde group of xylose and the amino group of a support having an amino group,
A substance capable of binding to xylose is separated from the mixture by affinity chromatography using xylose as a ligand and an affinity adsorbent bonded to the N atom of the amino group of the support at the position of the aldehyde group of xylose. Method. 3. A patent claim in which the mixture is a mixture containing an enzyme capable of binding to xylose obtained by adsorption and separation using an anion exchanger from a crushed bacterial cell extract of Thermus thermophilus, and the substance to be separated is glucose isomerase. The method described in Scope No. 2.