GB2031905A - Preparing glucose isomerase - Google Patents

Abstract

The enzyme glucose isomerase is prepared by culturing a host microbe having a suitable vehicle containing a gene for glucose isomerase. A genetic element coding for an enzyme such as glucose isomerase comprises joining two DNA sequences, one cut from a suitable cloning vehicle and the other from a chromosome DNA carrying the enzyme gene. New plasmids and E. coli strains are described.

Description

SPECIFICATION Preparing glucose isomerase In order to produce fructose, glucose has been subjected to alkaline isomerisation but this has been found to be commercially unfeasible because of the lack of selectivity, allowing the production of non-metabolisable materials such as psicose and objectionable, coloured materials which are costly to remove.

Subsequently, the microbiological production of glucose isomerase, the enzyme which catalyses the conversion of glucose to fructose, was developed and has achieved commercial importance. As disclosed in U.S. Patent Specification No. 3,645,848, microorganisms which have been used are those from the genera Lactobacillus, Pseudomonas, Pasteurella, Leuconostoc, Streptomyces, Aerobacter and Arthrobacter. Known microbiological processes for preparing glucose isomerase achieve their goal by using microbes having a glucose isomerase gene as a chromosomal gene.

According to a first aspect of the present invention, a process for preparing glucose isomerase comprises culturing a host microbe having a suitable vehicle containing a gene for glucose isomerase.

According to a second aspect of the present invention, a process for constructing a genetic element coding for an enzyme, preferably glucose isomerase, comprises (a) cutting a suitable cloning vehicle to obtain a first linear DNA sequence; (b) cutting a chromosome DNA carrying the desired enzyme gene to obtain a second linear DNA sequence containing the desired enzyme gene; and (c) joining the first and second linear DNA sequences.

It has been found that, using the recombinant DNA methodology described below, a genetic element coding for the glucose isomerase enzyme can be constructed. This genetic element can be inserted into a host microbe which, upon culturing, produces greater amounts of glucose isomerase than was previously possible by the donor strain.

Recombinant plasmids carrying the gene for the enzyme xylose (glucose) isomerase have been constructed by ligating linear DNA fragments prepared by Hind Ill limit digestion of pMB9 DNA (the replicon) and total Escherichia coli K12 DNA (the source of the isomerase gene). The host bacterium was also an E. coli K12 strain (strain JC1553) which was defective in the enzyme xylose (glucose) isomerase and which was transformed by the recombinant plasmids from the phenotype of Xyl-TcS to Xyl+Tcr. The selection for clones carrying the desired recombinant plasmid was based upon the facts that (1) the plasmid pMB9 codes for resistance to the drug tetracycline (Tc), and (2) the recipient E. coli strain JC1553 has a mutation in its xylose isomerase gene and hence cannot grow on this substrate.This strain also cannot isomerise glucose, as the same enzyme carries out both reactions. However, JC1553 grows normally on glucose since, unlike xylose, the major metabolic pathways for the utilisation of glucose do not involve this enzyme.

Xylose-induced cells carrying the recombinant plasmid pUC1002 exhibit an amplification of glucose isomerase activity of more than 500% relative to the level shown by the donor strain.

It should be noted that the glucose isomerase gene which occurs in nature is a chromosomal gene, one copy per chromosome. The glucose isomerase gene of the present invention is inserted into a plasmid which exists in a host in multiple copies. This insertion of the gene into a plasmid, or other suitable vehicle as defined herein, results in an amplication of the gene and the desired gene product, i.e., glucose isomerase.

Examples of other vehicles which can be used in the invention are plasmids such as pBR322 and pBR313 which code for ampicillin and tetracycline resistance, pSC101 which codes for tetracycline resistance, pCR1 1 which codes for kanamycin resistance and yeast 2 plasmid DNA, and bacteriophage vectors, for example, charon X phages.

Examples of other hosts for the vehicle are any E. coli K-12 derivative (see Bacteriological Reviews, Dec.

1972, pages 525-557; these have been approved by the NIH Guidelines) and yeasts, other fungi, and other bacteria. It is recognised that these latter hosts would also have to be approved, at least for use in U.S.A., by the NIH Guidelines. All the enzymes which have been used can be obtained from New England Biolabs, Beverly, Massachusetts, U.S.A.

Examples of other gene sources which can be used in processes of the invention are any glucose isomerase gene, microbial or otherwise.

By foilowing the procedures of this invention, particularly those disclosed in the Examples, genetic elements coding for other enzymes such as, for example, alkaline protease, amylase or cholesterol oxidase, can be constructed.

The plasmid pUC1002 can be isolated from its host bacterium as described for plasmid pMB9 in Example 1.

The work described herein was conducted in conformity with physical and biological containment requirements specified in the NIH Guidelines.

The E. collstrains described herein, including those containing plasmids, have been desposited in the permanent collection of the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Illinois, U.S.A. Their accession numbers in this repository are as follows: JC1553 - NRRLB-11391 K37 - NRRLB-11392 HB129(pMB9) - NRRLB-11390 JC1553(pUC1002) - NRRLB-11393 pUC is the official designation for a plasmid owned by The Upjohn Company.

The accompanying drawing illustrates the steps for making the recombinant plasmid carrying the E. cell gene(s) coding for the production of the enzyme glucose isomerase. Though the abbreviations used are conventional and well known to those skilled in the art, they are redefined here to facilitate a clear understanding of the invention.

Hind Ill, EcoRI, BamHI, Sal I, Hpa I - restriction endonuclease cleavage sites.

Tc - gene coding for resistance to the antibiotic tetracycline.

AGCT - single-stranded DNA fragment containing the bases adenine, guanine, cytosine and thymine.

T4 DNA ligase - a enzyme coded for by bacteriophage T4 which catalyses the covalent joining of polydeoxynucleotides.

Xyl - xylose.

pMB9 - a nontransmissible autonomously replicating E. coli plasmid.

The following Examples are illustrative of the process and products of the subject invention but are not to be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

Example 1- DNA preparation E. coli K37 chromosomal DNA is isolated as described by Marmur [J. Mol. Biol. 3: 208-218(1961)]. The culture is grown in L broth [Lennox, E.S. (1955) Virology 1: 190-206] with vigorous agitation at 370C. until the optical density at 590 nm reaches 0.8. Cells are harvested by centrifugation at 10,000 x g for 10 minutes, washed once with cold 50 mM Tris.HCl, pH 8.0, 20 mM EDTA, and then resuspended in 1/5 volume of the same buffer. Lysis is effected by the addition of sodium dodecyl sulfate so a final concentration of 1.5% followed by heating at 60"C. for 10 minutes. The resulting viscous solution is cooled to room temperature and then sodium perchlorate is added to a final concentration of 1 M.The whole mixture is then shaken with an equal volume of chloroform-isoamyl alcohol (24:1 v/v) for twenty minutes. The resulting emulsion is separated into 3 layers by a 5 minute centrifugation at 10,000 x g. The upper layer containing the DNA is removed to a narrow vessel and overlayed with 2 volumes of cold 95% ethanol. The DNA is then "spooled" onto a glass rod by gentle stirring. The precipitated DNA is dissolved in 15 mM NaCI, 1.5 mM trisodium citrate, and deproteinization repeated as above until little or no protein appears at the interface after centrifugation. Finally, the purified DNA is dissloved in 15 mM NaCI, 1.5 mM trisodium citrate, and 0.5 mM EDTA.

pMB9 plasmid DNA is isolated from E. coli Hub129 grown as above in L broth supplemented with tetracycline at 25 llg/ml. Crude lysates are prepared by resuspending the cells in 1/10 volume of 50 mM Tris-HCI (pH 8.0), 20 mM EDTA plus 500 Ag/ml of predigested (90 minutes at 37"C.) Pronase (Calbiochem, La Jolla, Calif.) and 1% sodium dodecyl sulfate, and incubating them at 37 C. until the suspension clears.

Chromosomal DNA is then sheared by drawing the lysates through a 13 gauge needle, and plasmid DNA is isolated by equilibrium centrifugation (Spinco 50 Ti roter, 15", 40,000 rpm, 48-60 hr.) of the sheared lysates in cesium chloride-ethidium bromide density gradients. The plasmid DNA is removed by collecting fractions through a needle puncture in the bottom of the gradient. Fractions containing the plasmid DNA are pooled and extracted three times with equal volumes of isoamyl alcohol to extract the ethidium bromide. The DNA solution is then dialyzed against 15 mM NaCI, 1.5 mM trisodium citrate, 0.5 mM EDTA, ethanol precipitated, and finally dissolved in a small volume of the above buffer.

Example 2 - Restriction Endonuclease Digestion Hind Ill cleavage of a mixture of E. coli K37 and pMB9 DNA (3:1), prepared as described in Example 1, is done in a reaction mixture containing 7 mM Tris-HCI, pH 7.9, 7 mM MgC12, 60 mM NaCI, 100 llg/ml autoclaved gelatin and 1 unit of Hind Ill endonucleaseqtg of DNA. After incubation at 37"C. for 60 minutes, the reaction is stopped by heating at 65"C. for 5 minutes.

Example 3 - T4 DNA Ligase Reaction The stopped reaction mixture, from Example 2 above, is supplemented to contain a final concentration of 5 mM MgCl2, 10 mM dithiothreitol, 50 I1M ATP and 1 unit of T4 DNA ligase/g of DNA. The reaction is incubated at 0 for approximately 16 hours.

Example 4 - Transformation of E. coli Transformation of E. coil JC1 553 by plasmid DNA is done as described by Cohen et al. [PROC. Natl. Acad.

Sci. USA 69: 2110-2114(1972)1. One hundred ml of L broth are inoculated with JC1553, NRRL B-1 1391, and grown to a optical density of 0.85 at 590 nm. The cells are then chilled, sedimented and washed with 1/2 volume of cold 10 m M NaCI. After centrifugation the cells are resuspended in 1/2 volume of cold 0.03 M CaCI2, held at 0" for 20 minutes sedimented, and then resuspended in 1/10 the original volume of 0.03 M CaCI2. Then 0.2 ml of these cells are mixed with 0.1 ml of the ligated DNA reaction mixture which is previously supplemented with CaCI2 to a concentration of 0.03 M. The resulting suspension is held at 0 for 60 minutes and then subjected to a 42" heat pulse.Finally, the cells are diluted 10 fold into L broth, incubated at 37"C. with vigorous agitation for 2-4 hours to allow for expression of drug resistance, and then plated onto a minimal salts medium containing xylose as the sole source of carbon and energy plus tetracycline at 5 Fg-ml. Growth on this medium distinguishes transformants carrying the desired recombinant genetic element from JC1553 (which cannot grow in the presence of tetracycline or use xylose as its sole source of carbon and energy) and from JC1553 carrying intact pMB9 (which will grow in the presence of tetracycline but again cannot use xylose as its sole source of carbon and energy).

Example 5 - Glucose Isomerase Activity in Crude Extracts of JC1553 EpUC1002) One hundred ml portions of Fraser's medium (Fraser, D. and E. A. Jerrel. 1953 J. Biol. Chem. 205; 291-295) supplemented with xylose at 0.2% (w/v) are inoculated with 5 ml portions of JC1553 (pUC1002), NRRL B-1 1393, grown overnight in Fraser's medium supplemented with tetracycline at 5 Fg/ml. The flasks are incubated at 37"C. with vigorous agitation for six hours after which the cultures are sedimented and washed with cold 30 mM potassium phosphate buffer (pH 7.2). The cells are resuspended in 1/20 volume of the same buffer and crude extracts prepared by sonic disruption.Cellular debris is removed by centrifugation at 10,000 x g for 10 minutes and the supernatants assayed for glucose isomerase activity. The standard assay contains 201moles of potassium phosphate buffer (pH 7.2), 2.5 Cimoles of CoCI2, 5 moles of MgC12, 1 mmole of D-glucose and crude extract in a total volume of 1 ml. The optimum temperature for the bioconversion under these conditions is about 60"C. The extent of conversion of glucose to fructose is determined by taking samples of 0.02 ml at various times and adding these to tubes containing 0.98 ml H20 plus 3.0 ml of concentrated HCI. One ml of a 0.05% solution of resorcinol in ethanol is added to the tubes which are then heated at 77"C for 8 minutes. The tubes are then cooled in an ice-water bath and the optical density at 420 nm measured.

Although the above procedure uses the enzyme in a cell-free form (crude extract) to convert glucose to fructose, the conversion also can be conducted with the enzyme in an immobilised form using procedures known in the art.

Claims (15)

1. A process for preparing glucose isomerase, which comprises culturing a host microbe having a suitable vehicle containing a gene for glucose isomerase.

2. A process according to claim 1 wherein the host microbe is a bacterium.

3. A process according to claim 2 wherein the bacterium is an E. coli K-12 derivative.

4. A process according to claim 3 wherein the E. coli K-12 derivative is strain JC1553, NRRL B-1 1393.

5. A process according to any preceding claim wherein the vehicle is a plasmid.

6. A process according to claim 5 wherein the plasmid is pUC1002.

7. A process according to claim 1 substantially as described in the Examples.

8. A process for constructing a genetic element coding for glucose isomerase, which comprises (a) cutting a suitable cloning vehicle to obtain a first linear DNA sequence; (b) cutting a chromosome DNA carrying a glucose isomerase gene to obtan a second linear DNA sequence containing a glucose isomerase gene; and (c) joinihg the first and second linear DNA sequences.

9. A process according to claim 8 wherein the cloning vehicle is a plasmid.

10. A process according to claim 9 wherein the plasmid is pMB9.

11. A process according to any of claims 8 to 10 wherein the chromosome DNA is E. coli K37.

12. A process for constructing plasmid pUC1002, which comprises (a) cutting plasmid pMB9 to obtain a first linear DNA sequence; (b) cutting E. coli K37 to obtain a second linear DNA sequence containing a glucose isomerase gene; and (c) joining the first and second linear DNA sequences.

13. A process for constructing a genetic element coding for an enzyme, which comprises (a) cutting a suitable cloning vehicle to obtain a first linear DNA sequence (b) cutting a chromosome DNA carrying the desired enzyme gene to obtain a second linear DNA sequence containing the desired enzyme gene; and (c) joining the first and second linear DNA sequences.

14. Plasmid pUC1002.

15. E.collJC1553(pUC1002), NRRLB-11393.