United States Patent (19) 11) 4,039,382 Thang et al. 45 Aug. 2, 1977 54 MMOBILIZED RIBONUCLEASE AND -Enzyme Systems, Journal of Food Science, vol. 39, ALKALINE PHOSPHATASE 1974, (pp. 647-652). 75 Inventors: Minh-Nguy Thang, Bagneux; Annie Zaborsky, O., Immobilized Enzymes, CRC Press, Guissani born Trachtenberg, Fresnes, Cleveland, Ohio, 1973, (pp. 124-126). both of France Primary Examiner-David M. Naff 73 Assignee: Choay S. A., Paris, France Attorney, Agent, or Firm-Browdy and Neimark 21 Appl. No.: 678,459 22 Filed: Apr. 19, 1976 57 ABSTRACT An insoluble, solid matrix carrying simultaneously sev 30 Foreign Application Priority Data eral different enzymatic functions, is constituted by the Apr. 23, 1975 France ................................ 75.12667 conjoint association by irreversible binding on a previ ously activated matrix support, of a nuclease selected 51) int. Cl? ........................... C07G 7/02; C12B 1/00 from the group of ribonucleases A, T, T, U, and an 52 U.S. Cl. ................................... 195/28 N; 195/63; alkaline phosphatease. Free activated groups of the 195/68; 195/DIG. 11; 195/116 matrix after binding of the enzymes, are neutralized by 58 Field of Search ................... 195/63, 68, DIG. 11, a free amino organic base. The support is selected from 195/116, 28 N among non-denaturing supports effecting the irrevers 56) References Cited ible physical adsorption of the enzymes, such as sup ports of glass or quartz beads, highly cross-linked gels PUBLICATIONS of the agarose or cellulose type. Polymers AUott, A. Lee, J. C., Preparation and Properties of Water Insolu Cott, AGot and/or oligonucleotides U, C, A or G, of ble Derivatives of Ribonuclease Ti. Biochim. Biophys. predetermined lengths, can be obtained by fractionating Acta, vol. 235, 1971, (pp. 435-441). the polyribonucleotides by incubation with the insolu Woetall, H. H., Alkaline Phosphatase Insolubilized by ble solid matrix. Covalent Linkage to Porons Glass., Nature, vol. 223, 1969, (pp. 959-960). Hultin, H. O., Characteristics of Immobilized Multi 16 Claims, No Drawings 4,039,382 1. 2 tice than previously known enzymes bound individually MMOBILIZED RBONUCLEASE AND to an insoluble support. ALKALINE PHOSPHATASE It is another object of the invention to provide such matrices which permit the fractionation of polyribonu BACKGROUND OF THE INVENTION cleotides into copolymers or into oligonucleotides, in a 1. Field of the Invention single operation, under kinetic conditions which are The present invention relates to matrices carrying very satisfactory and with excellent yields, by the simul simultaneously several different enzymatic functions taneous action of specific enzymes, which enable con and more particularly to complexes constituted by en trol of the fractionation in order to obtain specifically zymes bound irreversibly on insoluble supports, as well 10 sought copolymers or oligonucleotides. as to the process for the preparation of these matrices. Other objects and advantages of the present invention The present invention also relates to a process for the will appear from the description which follows. preparation, by means of said matrices, of oligonucleo According to the invention there is provided a matrix tides and polynucleotides with specific end groups. carrying simultaneously several different enzymatic 2. Description of the Prior Art 15 functions, characterized in that it is constituted by the It is known to fractionate polyribonucleotides by conjoint association of a nuclease and of an alkaline action of enzymes, to obtain various polynucleotides phosphatase, with an insoluble solid matrix, adapted to and oligonucleotides, and notably oligo U (or uridine bind the above-said enzymes irreversibly. oligonucleotides) and oligo G (or guanosine-oligonu According to a preferred embodiment of the matrix cleotides). These enzymatic actions comprise essentially 20 carrying several different enzymatic functions accord two stages, of which the first consists of subjecting the ing to the invention, the nuclease is selected from the polyribonucleotides used to enzymatic hydrolysis by group which comprises ribonucleases, and notably ribo using either the ribonuclease of sheep's kidney K. nucleases A, T, T2, U2. KASA and M. GRUNBERG-MANAGO : Eur, J. According to another advantageous feature of the Biochem. I (1967), 152), or beef pancreatic ribonuclease 25 invention, the insoluble support on which the enzymes (G. SCHMIDT - The nucleic Acids I (1955), p.555, are bound is selected from among non-denaturing sup Chargaff and Davidson Ed.) if it is sought to obtain ports effecting the irreversible physical adsorption of uridine-oligonucleotides (that is to say oligonucleotides the enzymes, such as supports of glass or quartz beads, ended by a pyrimidine group), or ribonuclease T sepa highly cross-linked gels notably of the agarose or, in rated from takadiastase of Aspergillus orizae, if it is 30 particular, of the cellulose type. sought to obtain guanosine-oligonucleotides (that is to According to another feature of the matrix according say oligonucleotides ended by a purine group) Egami to the invention, the amounts of active enzymes fixed on TAKAHASHI and UCHIDA - Progress in nucleic the support are advantageously comprised between Acids and Research and Molecular Biology, XII, 1964, 0.001 and 0.15% of alkaline phosphatase, and between 59). This enzymatic hydrolysis cleaves the polymeric 35 0.01 and 0.05% of nuclease by weight with respect to chain at the 5'-phosphate junction, giving rise to a 3'- the weight of the support. phosphate group, and it is followed by a second stage, It is also an object of the present invention to provide which is a dephosphorylation by the alkaline phospha a process for obtaining matrices carrying several differ tase of Escherichia Coli. ent enzymatic functions, characterized in that the solid The enzymes successively applied are in solution, so support previously activated by means of a suitable that it is necessary, at the end of the operations which activating substance, such as cyanogen bromide, for have just been described, to separate the products ob example, is placed in contact, in a suitable buffer solu tained from the enzymes used, which are often very tion, with enzymes to be bound on said support, namely resistant so that these processes are difficult to apply a nuclease and an alkaline phosphatase, for a time and at and the yield of oligonucleotide obtained is very low. 45 a temperature which are interdependent, after which To avoid these difficult separating processes which the amounts of unbound enzymes are removed by wash are complicated and expensive, it has been proposed to ing by means of a buffer solution identical with that substitute for the enzymes in solution used in the course used for the binding, and the free activated groups of of the two stages of the process which has just been the support are neutralized by means of a free amino described, enzymes rendered insoluble by binding to a 50 organic base such as lysine, arginine, ethanolamine, support. The work of PORATH and KRISTIANSEN aniline and the like. ("The Proteins' vol. I, 3rd Ed., Academic Press) has, in According to an advantageous embodiment of the fact, made known enzymes rendered insoluble by bind process according to the present invention, the buffer ing on a matrix of glass or of agarose. However, the solution applied is at a pH between 8.1 and 8.6. processes proposed, which successively apply insoluble 55 According to another preferred embodiment of the enzymes, if they resolve in part the difficulties of separa process of the present invention, the organic base used tion of the soluble enzymes, also however involve a as neutralizing agent for the free activated groups of the considerable number of operations, so that it is still support after binding of the enzymes, is at a pH between necessary, in any case, to resort to processes involving 8 and 10, long and complicated operations, which are of little According to another preferred embodiment of the practicality on the industrial scale. process according to the invention, the duration of contact between the enzymes to be bound and the bind OBJECTS AND GENERAL DESCRIPTION OF ing support is between 1 and 16 hours for temperatures THE INVENTION comprised between 20 C and 4° C. Accordingly it is an object of the present invention to 65 Another advantageous feature of the process accord provide matrices carrying simultaneously several differ ing to the invention provides for the contacting, in a ent enzymatic functions and processes for preparing buffer solution at pH 8.1 - 8.6 of the support with them, which respond better to the requirements of prac amounts of enzymes comprised between 0.001 and 4,039,382 3 4. 0.15% by weight of alkaline phosphatase and between dium bicarbonate in order to remove all traces of ace 0.001 and 0.05% by weight of nuclease, with respect to tone. the weight of the support. - 2. Binding of the enzymes According to the invention, the amount of active Binding of the enzyme is then carried out on the enzymes bound on the support, measured after washing treated matrix, by proceeding as described below. and neutralization, is comprised between 43 and 85% The enzymes to be bound on the matrix are selected with respect to the amount of alkaline phosphatase respectively from among nucleases, on the one hand, initially used and between 47 and 95% with respect to and from among alkaline phosphatases on the other the amount of nuclease initially used. hand. The alkaline phosphatases utilized can be of vege According to the present invention there is also pro 10 table or animal origin, but alkaline phosphatases of bac vided a process for obtaining polymers, AUou.A.Con terial origin are preferred and notably alkaline phospha AGot and/or oligonucleotides U, C, A, or G, of pre tase from Escherichia Coli, on account of its particu determined lengths, characterized in that polyribonu larly high activity.