FORM 2
The Patent Act, 1970 Complete Specification
Section 10


A PROCESS FOR THE PREPARATION OF PURIFIED PENICILLINASE FROM CRUDE EXTRACT USING DYE-LIGAND AFFINITY CHROMATOGRAPHY
SALIM K. MUJAWAR , DEBASHISH DUTTA AND RABINDRA KUMAR NANDA
HINDUSTAN ANTIBIOTICS LIMITED, PIMPRI, PUNE-411018, MAHARASHTRA, INDIA, an Indian Company incorporated under the Companies Act 1956.
The following specifications particularly describe and ascertain the nature of this invention and the manner in which it is to be performed.


This invention relates to the process for the preparation of purified penicillinase, an industrially important enzyme. More particularly it relates to the process for the purification of penicillinase from enzyme extract from Bacillus cereus using derivatized macroporous beaded crosslinked polymers. The purification achieved by a process of this invention is in high yields and is effected in a single operation.
Penicillinase finds great use in medical world. Penicillinase finds application in the Sterility Test of injectable sterile penicillin preparations and also as an enzyme marker in preparation of diagnostic kits based on Enzyme Linked Immunosorbent Assay (ELISA).The enzyme is also administered in penicillin sensitised patients. These applications are developed due to absolute specificity of penicillinase in hydrolyzing cyclic amide bond present in the penicillins to form corresponding penicilloic acid. The action of penicillinase turns the active antibiotic into an inactive molecule. This particular property forms the basis for the use of penicillinase in Sterility Tests wherein the penicillin is inactivated and hence it is possible to detect any contaminants incorporated during the formulations of penicillins. The hydrolysis of penicillins to penicillioc acid alter the iodine binding property of penicillin molecule, which enables the detection of action of penicillinase on penicillin molecules by simple decolourisation of starch iodine solution. The method relies on the reduction of 8 equivalents of iodine by one molecule of penicilloic acid generated by action of penicillinase on penicillins.The simple end point determination has led to use of this enzyme as a marker in ELISA based diagnostic kits.
Different protocols are developed for purification of penicillinase by affinity chromatography such as: Agarose derivatised with 6-amino penicillanic acid or 7-amino desacetoxy cephalosporanic acid as described in Antimicrobial Agent and Chemotherapy, Vol.15, p. 229, 1979; Sepharose 4 B derivatised with cefotaxime as described in Current Chemotherapy and Immunotherapy, Vol.1 p.751, 1982; Purification of Beta-lactamases by affinity chromatography as described in Biochemical Journal, Vol.221, p. 505, 1984; Sepharose 4B derivatised with N-acetyl-D-penicillamine as described in Journal of Chromatography, Vol. 448, p. 109, 1988; Amino benzyl penicillin coupled to macroporous carrier of organic, inorganic or bioorganic materials as described in German Patent DD 260 292, Crosslinked macroporous glycidyl copolymers derivatised with ampicilloic acid as described in Indian Patent No. 178 540.

These protocols for purification of penicillinase suffer from one or more of the following disadvantages: lower yield, use of column chromatographic procedures, bed compression, attrition of the affinity chromatography support when used in batch mode.
In our copending application no. 0271/DEL/2001 we have described and claimed a process for purification of Pen G acylase using derivatised macroporous beaded crosslinked allyl glycidyl ether copolymers by immobilizing dye as a ligand useful as an affinity support.
Derivatised macroporous beaded crosslinked glycidyl copolymers as described in application no. 0271/DEL/2001 have been used for purification of penicillin G acylase.
The main object of the present invention is to provide a process for the preparation of purified penicillinase from the crude enzyme extract of Bacillus cereus using novel derivatised macroporous beaded crosslinked allyl glycidyl ether copolymers.
Another object of the invention is to develop a process, which is simple, rapid and involves one step.
Yet another object is to provide a process by which purified penicillin preparation is useful for the Sterility Test and as an enzyme marker in ELISA.
Accordingly, the present invention provides a process for preparation of purified penicillinase, which comprises suspending the novel macroporous beaded crosslinked copolymers in a crude penicillinase enzyme extract prepared in a buffer solution having a molarity of 0.05 and pH of 7.5, agitating the suspension for a period upto 30 minutes at a temperature of 25°C at an rpm 100. filtering the suspension, washing the beads with buffer solution having molarity of 0.05 and pH.7;5, eluting the adsorbed penicillinase with buffer solution containing metal salt solution followed by desalting the penicillinase by conventional methods.
The present invention also provides the process for preparation of purified penicillinase, comprises suspending the novel crosslinked beaded copolymers in a crude penicillinase enzyme

extract prepared in a solution having molarity in the range of 0.05 to 0.2 and containing 30 % ammonium sulfate and at a pH in the range of 7 to 8, agitating the suspension for a period of 30 minutes at a temperature in the range of 25 to 28 °C at an rpm in the range of 100 to 200, filtering the suspension, washing the beads with buffer solution having a molarity in the range of 0.05 to 0.2 containing 30 % ammonium sulfate at a pH in the range of 7 to 8, eluting the adsorbed enzyme with buffer solution in the range of 7 to 8 devoid of ammonium sulfate.
In another embodiment of the invention, the inorganic buffer solution used may be prepared with phosphate or tris chloride having a preferred molarity of 0.05 and pH at 7.5.
In yet another embodiment of the invention, the alkali metal salt used for this washing may be such as sodium chloride.
Penicillinase and total protein content in the enzyme extract used was 200 IU/ml and 0.2 mg/ml , respectively. Thus, the specific activity of the penicillinase in the enzyme extract was 1000 IU/mg. The enzyme activity was determined using penicillin G as a substrate as described in Hindustan Antibiotics Bulletin Vol. 3, p. 85, 1961. Protein was determined as described in Journal of Biological Chemistry Vol. 193, p. 265, 1951.
The process of the present invention is described with reference to the following examples, which are given by the way of illustrations only and should not construe to limit the scope of the invention in any manner.
Examples for purification of penicillinase by using derivatised macroporous beaded crosslinked copolymers
Example 1
33 mis of crude enzyme extract of penicillinase from Bacillus cereus prepared in phosphate buffer having molarity of 0.05, containing 30% ammonium sulfate at pH 7.5 containing 6660 IU of penicillinase was taken in 100 ml capacity container. 1.5 grams of macroporous beaded

crosslinked copolymer (allyl glycidyl ether - ethylene glycol dimethacrylate) derivatised with Cibacron Blue dye having CLD of 50 was added to the enzyme solution and the suspension was agitated at 100 rpm at 25°C for a period of 30 minutes. The beads were separated by filtration and washed with 20 mis of phosphate buffer having molarity of 0.05 containing 30% ammonium sulfate at pH 7.5. The penicillinase adsorbed onto the beads was eluted in 5 lots of 5 mis of phosphate buffer having a molarity of 0.05, pH 7.5. Fractions containing penicillinase were pooled. The adsorption and recovery were 100 % and 68%, respectively.
Example 2
33 mis of crude enzyme extract of penicillinase from Bacillus cereus prepared in phosphate buffer having molarity of 0.05, containing 30% ammonium sulfate at pH 7.5 containing 6660 IU of penicillinase was taken in 100 ml capacity container. 1.5 grams of macroporous beaded crossl inked copolymer (allyl glycidyl ether - ethylene glycol dimethacrylate) derivatised with Cibacron Blue dye having CLD of 200 was added to the enzyme solution and the suspension was agitated at 200 rpm at 28°C for a period of 30 minutes. The beads were separated by filtration and washed with 20 mis of phosphate buffer having a molarity of 0.05 containing 30% ammonium sulfate at pH 7.5. The penicillinase adsorbed onto the beads was eluted in 5 lots of 5 mis of phosphate buffer having molarity of 0.05, pH 7.5. Fractions containing penicillinase were pooled. The adsorption and recovery were 100 % and 65%, respectively.
Example3
33 mis of crude enzyme extract of penicillinase from Bacillus cereus prepared in phosphate buffer having molarity of 0.2, containing 30% ammonium sulfate at pH 7 containing 6660 IU of penicillinase was taken in 100 ml capacity container. 1.5 grams of macroporous beaded crosslinked copolymer (allyl glycidyl ether - ethylene glycol dimethacrylate) derivatised with Basilen Blue dye having a CLD of 50 was added to the enzyme solution and the suspension was agitated at 100 rpm at 25°C for a period of 30 minutes. The beads were separated by filtration and washed with 20 mis of tris chloride buffer having molarity of 0.2 containing 30% ammonium sulfate at pH 7. The penicillinase adsorbed onto the beads was eluted in 5 lots of 5

mis of phosphate buffer having molarity of 0.05, pH 8. Fractions containing penicillinase were pooled. The adsorption and recovery were 100 % and 59.3%, respectively
Example 4
33 mis of crude enzyme extract of penicillinase from Bacillus cereus prepared in phosphate buffer having molarity of 0.2, containing 30% ammonium sulfate at pH 8 containing 6660 IU of penicillinase was taken in 100 ml capacity container. 1.5 grams of macroporous beaded crosslinked copolymer (allyl glycidyl ether - ethylene glycol dimethacrylatc) derivatised with Reactive Red dye having CLD of 50 was added to the enzyme solution and the suspension was agitated at 100 rpm at 28°C for a period of 30 minutes. The beads were separated by filtration and washed with 20 mis of phosphate buffer having molarity of 0.2 containing 30% ammonium sulfate at pH 8. The penicillinase adsorbed onto the beads was eluted in 5 lots of 5 mis of phosphate buffer having a molarity of 0.05 and at pH 7.8. Fractions containing penicillinase were pooled. The adsorption and recovery were 100 % and 54%, respectively.
Example 5
33 mis of crude enzyme extract of penicillinase from Bacillus cereus prepared in phosphate buffer having molarity of 0.05, at pH 7.5 containing 6660 IU of penicillinase was taken in 100 ml capacity container. 1.5 grams of macroporous beaded crosslinked copolymer (allyl glycidyl ether - ethylene glycol dimethacrylate) derivatised with Reactive Green dye having CLD of 50 was added to the enzyme solution and the suspension was agitated at 100 rpm at 23C for a period of 30 minutes. The beads were separated by filtration and washed with 20 mis of phosphate buffer having molarity of 0.05 at pH 7.5. The penicillinase adsorbed onto the beads was eluted in 5 lots of 5 mis of phosphate buffer having a molarity of 0.2, pH 8 containing 10%(w/v) of sodium chloride. Fractions containing penicillinase were pooled. The adsorption and recovery were 100 % and 78%, respectively. The specific activity of purified penicillinase was 4360 IU/mg.

Advantages of the invention
The invention provides a process for the purification of penicillinase from an enzyme extract of Bacillus cereus cells and offers following advantages:
1. High recovery of more than 78% of penicillinase are obtained.
2. Both purification and concentration are achieved simultaneously.
3. The operations involve single step of affinity interaction chromatography.
4. The process developed is a batch operation and use of column chromatography equipment is not required.
5. The purified penicillinase enzyme obtained is useful in sterility testing of penicillinase
formulations and as an enzyme marker in ELISA.
6. The process is economically feasible due to low cost, ready avaibility, high capacity and
recyclibility of the dye-ligand affinity matrices.
We claim
1. The present invention provides a process for preparation of purified penicillinase, which comprises suspending the novel macroporous beaded crosslinked copolymers in a crude penicillinase enzyme extract prepared in a buffer solution having a molarity of 0.05 and pH of 7.5, agitating the suspension for a period upto 30 minutes at a temperature of 25C at an rpm of 100, filtering the suspension, washing the beads with buffer solution having a molarity of 0.05 and pH of 7.5, eluting the adsorbed penicillinase with buffer solution having a molarity of 0.2 and a pH of 8 containing 10% sodium chloride followed by desalting the penicillinase by conventional methods.
2. The present invention also provides the process for preparation of purified penicillinase, comprises suspending the novel crosslinked beaded copolymers in a crude penicillinase enzyme extract prepared in a solution having a molarity in the range of 0.05 to 0.2 and containing 30% ammonium sulfate and at a pH in the range of 7 to 8, agitating the suspension for a period of 30 minutes at a temperature in the range of 25 to 28 °C at an rpm in the range of 100 to 200, filtering the suspension, washing the beads with buffer solution having a molarity in the range of 0.05 to

0.2 containing 30% ammonium sulfate at a pH in the range of 7 to 8, eluting the adsorbed enzyme with buffer solution at a pH in the range of 7 to 8 devoid of ammonium sulfate.
3. A process claimed in 1 and 2, wherein, the buffer solution used is phosphate and has a preferred molarity of 0.05 and at pH 7.5.
4. A process claimed in 1, wherein, the alkali metal salt used for the eluting is sodium chloride. 5.A process for the purification of penicillinase as herein described with reference to the examples 1 tc S.

( Authorized signatory )
Hindustan Antibiotics Limited.
Dated this 1 day of July 2002