Process for producing D-amino acids with composite immobilized enzyme preparation

#The present invention relates to a composite immobilized enzyme preparation and a process for producing D-amino acids using the preparation. In particular, the composite immobilized enzyme preparation of the present invention is useful for the production of D-.alpha.-amino acids which are intermediate compounds for the production of antibiotics, such as D-(p-hydroxyphenyl)glycine to be used for the production of the antibiotic, amoxycillin and the like.

#Optically-active D-amino acids are important compounds as intermediate compounds for drugs and it has been known that they can be produced efficiently by combining an asymmetric hydrolysis reaction of 5-substituted hydantoins into the corresponding D-N-carbamyl-.alpha.-amino acids with the enzymes, hydantoinases (hereinafter sometimes abbreviated as "Hase") (JP-B 62-30785), and an conversion reaction of the resultant D-N-carbamyl-a-amino acids into the corresponding D-.alpha.-amino acids with the enzymes, D-N-carbamyl-.alpha.-amino acid .alpha.-midohydrolases (hereinafter sometimes abbreviated as "decarbamylase" or "DCase") (PCT/JP91/01696: WO 92/10579).

#In addition, JP-A 63-185382, WO 92/00739 and the like disclose that the respective reactions are carried out more efficiently by using these enzymes in the form of so- called immobilized enzymes wherein they are immobilized on supports such as ion exchange resins and the like.

#FIG. 1 is a graph illustrating the time courses of the reactions for the production of the corresponding D-p-hydroxyphenylglycine from 5-(p-hydroxyphenyl)hydantoin in Example 3 by using the composite immobilized enzyme preparation of the present invention and the mixture prepared by immobilizing hydantoinase and decarbamylase separately to the different resins.

#FIG. 2 is a graph illustrating the stability of the enzymes in repeated reactions by using the composite immobilized enzyme preparation in Example 4.

#FIG. 3 is a graph illustrating the comparison of the activity of the composite immobilized enzyme preparation with the activity of the mixture of the separately prepared immobilized enzyme preparations (the hydantoinase activity is twice as much as the composite immobilized enzyme preparation) in Example 6.

#As for the hydantoinase used in the present invention, the enzymes from animals, plants and microorganisms can be used. The hydantoinases from microorganisms are suitable for industrial production. As such a microorganism, those disclosed in JP-B 62-30758 are exemplified. The bacteria include Achromobacter, Aerobacter, Aeromonas, Aqrobacterium, Alcaliqenes, Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Enterobacter, Erwinia, Escherichia, Klebsiella, Microbacterium, Micrococcus, Protaminobacter, Proteus, Pseudomonas, Sarcina, Serratia, Xanthomonas and the like. The Actinomycetes include Actinomyces, Mycobacterium, Nocardia, Streptomyces, Actinoplanes and the like. The filamentous fungi include Asperqillus, Paecilomyces, and Penicillium and the like. The yeasts include Candida, Pichia, Rhodotorula, Torulopsis and the like.

#Among the above microorganisms, examples of strains which have relatively high hydantoinase activities and are excellent in practical use include Aerobacter cloacae IAM 1221, Agrobacterium rhizogenes IFO 13259, Brevibacterium incertum IFO 12145, Corynebacterium sepedonicum IFO 3306, Microbacterium flavum ATCC 10340, Micrococcus roseus IFO 3764, Pseudomonas striata IFO 12996, Mycobacterium smecmatis ATCC 607, Nocardia corallina IFO 3338, Streptomyces flaveolus IFO 3241, Bacillus sp. KNK108 (FERM P-6056), Bacillus sp. KNK245 (FERM BP-4863) and the like.

#In addition, the enzymes produced by artificial microorganisms in which hydantoinase productivity is imparted or increased by gene recombinant technique, for example, E. coli HB101pTH104 (FERM BP-4864), or the dihydropyrimidinase having similar activity as disclosed in JP-A 53-136583 can be used.