Tanabe Seiyaku Company, Limited

Tnanbe Seiyaku Co., Ltd.

Tetsuya Tosa

Ichiro Chibata

Takao Mori

Tadashi Sato

Taizo Watanabe

Motoki Fujimura

Satoshi Minobe

Isao Takata

Yuhsi Matuo

Takeji Shibatani

Kozo Yamamoto

Makoto Masuda

Ryujiro Sano

Satoru Takamatsu

There is disclosed a method for determining an endotoxin by reacting the endotoxin adsorbed on an adsorbent having specific endotoxin adsorbability with an endotoxin detecting reagent, including the steps of:

Method for determining endotoxin and apparatus therefor

A method for determination of a pyrogen content which comprises bringing a specimen into contact with an adsorbent composed of a nitrogen-containing heterocyclic compound bonded to a water-insoluble carrier directly or through a spacer, and after or without eluting the pyrogen adsorbed on the adsorbent, determining the content of the adsorbed pyrogen by the Limulus method.

Method for determining pyrogen content

An immobilized enzyme having reversible solubility is prepared by attaching an enzyme to a copolymer of two of more monomers selected from the group consisting of acrylic acid or an alkyl ester thereof, methacrylic acid or alkyl ester or dialkylamino-alkyl ester thereof, and a vinylpyridine derivative of the general formula [U]: ##STR1## wherein R is an alkyl. Preferred capolymers are methacrylic acid-alkyl methacrylate-alkyl acrylate, methacrylic acid-alkyl methacrylate, alkyl acrylate-methacrylic acid, alkyl acrylate-methacrylic acid-alkyl acrylate, alkyl acrylate-methacrylic acid-2-methyl-5-vinylpyridine and dialkylaminoalkyl methacrylate-alkyl methacrylate. The immobilized enzyme has a high activity and can dissolve even in an organic solvent or in a solution containing an organic solvent.

Immobilized enzyme having reversible solubility

A process for preparing an immobilized microorganism which comprises cultivating a microorganism in a culture broth, treating the broth with glutaraldehyde when cultivation being completed, collecting microbial cells from the broth, admixing the microbial cells with an aqueous solution of a polysaccharide having 10 w/w % or more of sulfate moiety in the molecule thereof and then gelling the polysaccharide in the resulting mixture to entrap the microbial cells within the gel matrix of the polysaccharide. The process is suitable for the industrial preparation of immobilized microorganisms.

Process for preparing immobilized microorganism

At least one acryl or allyl monomer is polymerized in an aqueous suspension containing a fumarase-producing microorganism. The resultant immobilized fumarase-producing microorganism is contacted with bile acids or salts thereof. Then, the immobilized fumarase-producing microorganism is subjected to enzymatic reaction with fumaric acid or a salt thereof. L-Malic acid is prepared without the production of the by-product succinic acid.

Process for preparing L-malic acid

An immobilized enzymatic active substance having an improved stability of enzymatic activity is prepared which comprises an enzymatic active substance such as microbial cells entrapped within a gel matrix of an unsubstituted amino, monoalkylamino or dialkylamino group-introduced polysaccharide having not less than 10 w/w % of sulfate moiety in the molecule thereof such as carrageenan. The immobilized enzymatic active substance is prepared by admixing the enzymatic active substance and an aqueous solution of the polysaccharide and then allowing to form a gel of the polysaccharide.

Enzymatic active substance immobilized in a polysaccharide gel matrix

An immobilized aminoacylase is prepared by bonding aminoacylase to a water-insoluble porous anion exchanger such as a porous phenolic resin, porous styrene resin, porous silica or porous glass having anion exchange groups and treating the bonded amino-acylase with a crosslinking agent such as an aliphatic dialdehyde. The porous anion exchanger preferably has a pore size of about 150.degree. to 3,000 A.degree., pore volume of about 0.3 to 1.0 m.sup.1 /g, specific surface area of about 10 to 150 m.sup.2 /g and particle size of above 0.1 to 1.2 mm. Preferred anion exchangers are trimethylammonium-introduced styrene resin and trimethylammonium-introduced silica.

Immobilized aminoacylase

A method for removing a pyrogen from a solution of a pyrogen-containing substance such as an amino acid, a nucleic acid base, an antibiotic, a hormone, a vitamin, an enzyme, an antibody or the like comprising contacting the solution with an adsorbent to adsorb the pyrogen, which is characterized in that the adsorbent comprises a water-insoluble carrier and a nitrogen-containing heterocyclic compound of the formula:

Method for reducing the pyrogen content of or removing pyrogens from

An immobilized aminoacylase preparation is obtained by adsorbing tannin on a water-insoluble, hydrophilic carrier to give a water-insoluble tannin preparation, adsorbing aminoacylase on the tannin preparation to give a water-insoluble tannin-aminoacylase preparation, and then binding ferric or ferrous ion thereto. The immobilized preparation obtained above sustains a high rate of enzymatic activity for a long period of time when used in an enzymatic reaction thereof with N-acyl-DL-amino acids.

An enzyme or microorganism is entrapped within the gel matrix of a sulfated polysaccharide (said polysaccharide containing more than 10 w/w% of a sulfate moiety in its molecule) in the presence of ammonium ion, a metal ion, a water-soluble amine or a water-miscible organic solvent. The immobilized enzyme or microorganism thus obtained shows a high level of catalytic activity for a long period of time and can be used for continuous enzymatic reactions with substrates.

Immobilized catalytically active substance and method of preparing the

A water-insoluble tannin preparation is obtained by covalent binding or physical adsorption of tannin onto a water-insoluble, hydrophilic carrier. The preparation has a specific affinity for proteins and can be used as an adsorbent for purification, isolation and/or separation of proteins (e.g., Enzymes, albumin, globulin, hormonal proteins) from a mixture of compounds. Further, the water-insoluble tannin preparation having a catalytically active enzyme absorbed thereon can be used as a heterogeneous catalyst to induce enzymatic reactions.

Water-insoluble tannin preparation for immobilization of proteins

An immobilized glucose isomerase-producing microorganism is reacted with D-glucose to produce D-fructose. Immobilization is carried out by polymerizing at least one acryloyl monomer in an aqueous suspension containing a glucose isomerase-producing microorganism. The acryloyl monomers employed include acryloylamide, N,N'-lower alkylene-bis-acryloylamide, bis(acryloylamidomethyl)ether and N,N'-di-acryloyl-ethyleneurea.

Preparation of d-fructose

A blood purification means whereby blood of a patient to be treated is led around an external circuit connecting to the patient's blood stream and is brought into direct or indirect contact with a fumarate solution, and is further brought into direct or indirect contact with an enzymic preparation, which is suitably an aspartase preparation, which catalyzes a reaction of L-aspartic acid formation from fumaric acid and ammonia. The purification means may further include a preliminary stage or stages whereat an unrequired substance in a patient's blood is decomposed to a non toxic substance and ammonia subsequently convertable to aspartic acid, and may also include a low molecular sieve means preventing re-entry of aspartic acid produced into the patient's blood stream.

Blood purification means

At least one acryloyl monomer is polymerized in an aqueous suspension containing a penicillin amidase-producing microorganism. The acryloyl monomers employed in the present invention include acryloylamide, N,N'-lower alkylene-bis-acryloylamide, bis(acryloylamidomethyl)ether and N,N'-di-acryloylethyleneurea. The resultant immobilized penicillin amidase-producing microorganism is subjected to enzymatic reaction with penicillin. 6-Amino-penicillanic acid is produced.

Process for preparing 6-aminopenicillanic acid

Coenzyme A is purified by a process involving contacting a cyanogen halide-activated water-insoluble polysaccharide with a cell-free extract of a coenzyme A-producing microorganism to immobilize proteins such as enzymes having affinity for coenzyme A on the polysaccharide. The immobilized proteins are then contacted with a coenzyme A-containing solution to absorb coenzyme A followed by eluting coenzyme A from the immobilized proteins. Alternatively, the proteins having affinity for coenzyme A may be isolated from the cell-free extract prior to immobilization by absorbing the proteins on coenzyme A immobilized on a cyanogen halide-activated water-insoluble polysaccharide followed by eluting the proteins from the immobilized coenzyme A.

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Kyoto, Japan

Tetsuya Tosa