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Xylose Isomerase, a Method for Production of Such Xylose Isomerase, Immobilized Xylose Isomerase and a Method for Isomerization of Glucose to Fructose

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Xylose Isomerase, a Method for Production of Such Xylose Isomerase, Immobilized Xylose Isomerase and a Method for Isomerization of Glucose to Fructose Patentamt JEuropaischesEuropean Patent Office © Publication number: 0 194 760 Office europeen des brevets A2 («) EUROPEAN PATENT APPLICATION © Application number. 86301050.0 © Int. CI.4: C 12 N 9/92 C 12. P 19/24 _ © Date of filing: 14.02.86 //C1 2N 1 1/02 © Priority: 12.03.85 DK 1111/85 @ Applicant: NOVO INDUSTRI A/S Novo Alle DK-2880 Bagsvaerd(DK) © Date of publication of application: 17.09.86 Bulletin 86/38 © Inventor: Skot,Georg Nygardsterrasserne 205 G @ Designated Contracting States: DK-3520 Farum(DK) AT BE CH DE FR GB IT LI NL SE © Inventor: Giirtler, Hanne Kulsviervej 81 B, st.t.v. DK-2800 Lyngby(DK) © Representative: Brown, John David et al, FORRESTER & BOEHMERT Widenmayerstrasse 4/I D-8000 Munchen 22(DE) (54) Xylose isomerase, a method for production of such xylose isomerase, immobilized xylose isomerase and a method for isomerization of glucose to fructose. Xylose Isomerase, a Method for Production of such Xylose Isomerase, Immobilized Xylose Isomerase and a Method for Isomerization of Glucose to Fructose. The xylose isomerase can be produced by means of a strain belonging to the Streptomyces murinus cluster. This production method provides a good compromise in regard to fermentation yield, purification yield and possibility for production of an immobilized zylose isomerase product with satisfactory physical and enzyme chemical properties. The invention relates to a xylose isomerase, a method for production of such xylose isomerase, an immobilized xylose isomerase and a method for isomerization of glucose to fructose. Enzymes capable of isomerizing glucose to fructose i.e. glucose isomerases, may be obtained from a large number of different microorganisms, and the enzyme yield and the properties of the enzymes vary from species to species even frequently from strain to strain. In all xylose isomerase producing strains found in nature xylose is required in the medium for maximum enzyme production. All described glucose isomerases fundamentally are xylose isomerases. Also, in regard to enzyme nomenclature they are classified as EC 5.3.1.5, and glucose isomerase is not to be found in the official enzyme nomenclature. Thus, for the sake of clarity the enzyme in the following ordinarily will be identified as xylose isomerase, even if the most important application thereof is the isomerization of glucose to fructose. The economy related to the production of xylose isomerases depends largely on the cost of the substrate and the enzyme yield, and the most important enzyme properties, which are particularly important for the economic use in the isomerization process, are good purification yield and good ability to be converted to an immobilized product with satisfactory physical and enzyme chemical properties, e.g. stability at high tempera- tures, resistance towards physical pressure and a good half life. No one of the known xylose isomerases including the commercial glucose isomerase products do possess the best possible values of all desirable properties. Often it is necessa- ry to compromise. On this basis a great amount of research has been carried out in the field comprising production of xylose iso- merases capable of isomerization of glucose to fructose from different microbiological sources. This research is reflected in the patent litterature, which comprise huge numbers of patents covering different microbial sources of xylose isomerase. Thus, both bacteria, e.g. belonging to the genera Streptomyces, Actino- planes, Bacillus, and Flavobacterium, and fungi e.g. belonging to the class Basidiomycetes, have been described in the patent litterature as xylose isomerase producing microorganisms. Also, just to choose some examples, among many others the following species belonging to the genus Streptomyces have been described in the patent litterature as xylose isomerase producing microorganisms. S. flavovirens S. achromogenus S. echinatus S. wedmorensis S. albus (US 3,616,221), S. olivochromogenes S. venezuelae (US 3,622,463) S. griseoflavus (US 4,351,903) S. glaucescens (US 4,137,126) S. olivaceus (US 3,625,828) S. galbus S. gracilis S. matensis S. niveus S. platensis (Hungarian patent 12,415) S. violaceoniger (German patent 2,417,642) S. acidodurans (US 4,399,222) S. phaeochromogenes S. fradiae S. roseochromogenes S. olivaceus S. californicus S. vinaceus S. virginiae (Japanese patent 69,28,473) Syrups containing a mixture of glucose and fructose are widely used in industry because of their sweet taste and their low tendency to crystallize. Such syrups are preferably produced from glucose syrups using a xylose isomerase with glucose isomer- ase activity to catalyze the isomerization of glucose to fructose. It is important for the economy of this process that the enzyme costs are low and that there be a negligible formation of by-products, which will have to be removed before the syrup can be used. Thus the purpose of the invention is to provide a xylose isomerase, which can be produced with a good compromise in regard to fermentation yield, purification yield and ability of the produced xylose isomerase to be converted to an immobilized product with satisfactory physical and enzyme chemical proper- ties, a corresponding immobilized xylose isomerase, and a corresponding method for isomerization of glucose to fructose. Now, surprisingly according to the invention it has been found that bacteria belonging to the Streptomyces murinus cluster are xylose isomerase producing with high fermentation yield and furthermore that the xylose isomerase can be produced with a good purification yield and an ability to be converted to an immobilized product with satisfactory physical and enzyme chemical properties for application in an industrial glucose isomerization process, and that constitutive and glucose repression resistant strains of the Streptomyces murinus cluster provide special advantages in regard to fermentation economy. In this application with claims the designation Streptomyces murinus cluster is to be understood as defined in Rolf-Dieter HEITZER's dissertation "Numerische Taxonomie der Actinomyceten-Gattungen STREPTOMYCES und STREPTOVERTICILLIUM" (filed January 27, 1981), obtainable from Biological department of the Technical University of Darmstadt, Germany. This means that S. murinus DSM 40091, DSM 40240 (previously named S. roseoluteus), NRRL 8171, FERM-P 3710, DSM 3252, and DSM 3253 all belong to this Streptomyces murinus cluster. It has been found that the xylose isomerase produced by these above indicated six members of the cluster are immunologic- ally identical as determined by tandem crossed immunoelectro- phoresis with antibodies produced against the xylose isomerase produced by means of DSM 3252, this fact emphasizing the close taxonomic relationship between these individual members of the cluster. Thus, the xylose isomerase according to the invention is characterized by the fact that it is identical to the xylose isomerase produced by cultivation of a strain of the Streptomyces murinus cluster. In a preferred embodiment of the xylose isomerase according to the invention the xylose isomerase is produced by cultivation of a strain of the Streptomyces murinus cluster. Despite the fact that a huge number of species are known as xylose isomerase producers, no strain of the Strepto- myces murinus cluster is known as a xylose isomerase producer, and surprisingly it has been found that any strain of the Streptomyces murinus cluster is very well suited for production of xylose isomerase and provides the advantages indicated above. Also the invention comprises a method for production of a xylose isomerase, wherein an aerobic submerged fermentation is carried out with a strain of the Streptomyces murinus cluster on a conventional medium containing carbon and nitrogen, whereafter the thus formed xylose isomerase is recovered. In a preferred embodiment of the method according to the invention the strain of the Streptomyces murinus cluster is DSM 40091, DSM 40240, NRRL 8171, FERM P-3710, or DSM 3252. DSM is Deutsche Sammlung von Mikroorganismen, Gottingen, Germany. NRRL is Northern Regional Research Laboratory, USA. FERM is Fermenta- tion Research Institute, Japan. If xylose is present in the medium, the fermentation yield is satisfactory, and also, a satisfactory immobilized xylose isomerase can be produced. In a preferred embodiment of the method according to the invention the strain of the Streptomyces murinus cluster is constitutive in regard to xylose isomerase production. In this case it is not necessary to incorporate the expensive substrate component xylose in the medium, but any cheap carbon source, e.g. glucose will do. Furthermore, surprisingly it has been found that it is possible to find real constitutive and glucose repression resistant mutants of the S. murinus cluster; thus S. murinus DSM 3253 being a real constitutive and glucose repression resistant mutant of S. murinus DSM 3252 is unique in producing more xylose isomerase in media with glucose than in media with xylose, vide example 1. Prior art mutant strains of xylose isomerase producing Streptomyces species referred to as "constitutive" (U.S. 4.399.222, example 3, and US Re 29.152, table 3) all produced higher enzyme yields in xylose containing media than in media without the xylose inducer. Reference is made to example 4, which represents a comparison between the xylose isomerase producing ability of a constitutive mutant belonging to the S. murinus cluster, which can be used in the method according to the invention, and of prior art "constitutive"
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