BACKGROUND OF THE INVENTION
Penicillin compounds are widely used as antibacterial drugs. These compounds consist of a ß-lactam ring fused to a thiazolidine ring; hence they are referred as ß-lactam antibiotics. Typical examples of penicillins which are commonly used in clinical practice are benzylpenicillin (penicillin G)  phenoxylmethylpenicillin (penicillin V)  ampicillin and carbenicillin.
Despite of their wide usage as chemotherapeutic usage they suffer a major drawback that some of the microorganisms on which they are acted upon produce ß-lactamase which cleave the ß-lactam ring of penicillin compounds to produce compounds which are devoid of antibacterial activity. There are certain compounds which are well capable to deal with such problem by inhibiting ß-lactamases. These compounds are known as semi synthetic antibiotics or isoxazolyl peniciilins in specific which are in the same class as penicillin. Some of the examples of the semi synthetic antibiotics are cloxacillin  dicloxacillin  flucloxacillin  oxacillin and their respective pharmaceutical salts thereof.
There are certain methods in the prior art which produces isoxazolyl penicillin derivatives such as oxacillin  cloxacillin  dicloxacillin or flucloxacillin. Generally  the method to prepare these isoxazolyl penicillins is a multi-step process which involves preparation of intermediates viz Penicillin G and then 6-aminopenicillanic acid which is subsequently followed by isolation of the intermediates and thereby purification and crystallization of the intermediates. After that  6-aminopenicillanic acid is reacted with a carboxylic acid chloride to form the isoxazolyl penicillin.
Main problem faced during this process is the high usage of power consumption  stability and purity of the intermediates. The industrial scale production of these compounds entails greater investment attributable to a multi-step process. This multi step process involves production of intermediates followed by their addition of an organic solvent to commence the purification and crystallization procedure. Filtration  purification and crystallization procedure are extensive  costly and time consuming processes. This multi step process demands an increased cost of production primarily due to isolation and complex purification methods employed for the intermediates.
Hence  there is a need to provide an in-situ process for the preparation of the isoxazolyl penicillins to avoid these issues of concern. The present invention provides a cost effective  simplified and less laborious in-situ process for the preparation of isoxazolyl penicillins viz cloxacillin  dicloxacillin  flucloxacillin  oxacillin and their respective pharmaceutical salts thereof without isolation of the intermediates Penicillin G and 6-aminopenicillanic acid.

SUMMARY
It is an object of the invention to provide a process for the preparation of Isoxazolyl Penicillins from sugarcane juice.
It is another object of the invention to provide an in-situ process for the preparation of Isoxazolyl Penicillins wherein the process is characterized in the steps comprising:
preparation of a first reaction mixture comprising Penicillin G  by fermentation of sugarcane juice with a salt of phenylacetic acid to form a product comprising Penicillin G and thereby purification of the product to form the first reaction mixture which is used for the preparation of a second reaction mixture;
preparation of second reaction mixture by enzymatic reaction of the first reaction mixture wherein the preparation is characterized in the steps comprising: reacting first reaction mixture with an enzyme to form a solution comprising 6-APA and phenylacetic acid (PAA). Then  adding a first organic solvent in presence of an acidic solution to it at a temperature of 0-40°C to form a first organic layer comprising phenylacetic acid and a first aqueous layer comprising 6-APA. After that  extracting the first organic layer to form a third reaction mixture comprising phenylacetic acid and thereby obtaining the second reaction mixture comprising 6-APA;
in-situ addition of a second organic solvent and a carboxylic acid chloride to the second reaction mixture at a temperature of 0-30°C to obtain a second organic layer and a second aqueous layer; and
in-situ separation of the second organic layer and addition of a sodium salt complex to form the Isoxazolyl Penicillin.
It is yet another object of the invention to provide an in-situ process for the preparation of Isoxazolyl Penicillins wherein the process comprises preparation of a first reaction mixture comprising Penicillin G  then preparation of a second reaction mixture by enzymatic reaction of the first reaction mixture without isolation of Penicillin G and finally  using the second reaction mixture for the preparation of the Isoxazolyl penicillins without isolation of 6-APA from the second reaction mixture.
It is yet another object of the invention to provide a process in which phenylacetic acid (PAA) is recovered and thereby recycled to the fermentation step. This recycling helps in better yield of the Isoxazolyl Penicillins and less consumption of the reactant in fermentation step  hence makes the process cost effective.
It is yet another object of the invention to provide a process for the preparation of Isoxazolyl Penicillins such as cloxacillin  dicloxacillin  flucloxacillin  oxacillin and their respective pharmaceutical acceptable salts thereof.
It is yet another object of the invention to provide a cost effective process for the preparation of isoxazolyl penicillin which requires no isolation step  no crystallization step  and no purification step for intermediates i.e. penicillin G and 6-aminopenicillanic acid formed during the reaction.
DETAILED DESCRIPTION OF THE INVENTION
In the detailed description of the invention  numerous specific details are described to provide a thorough understanding of the various embodiments of the invention. However  one skilled in the relevant art will recognize that an embodiment of the invention can be practiced without one or more of the specific details  or with other apparatus  systems  assemblies  methods  components  materials  parts  and/or the like. In other instances  well-known structures  materials  or operations are not specifically shown or described in detail to avoid obscuring aspects of embodiments of the invention.
The various aspects of the present invention leading to a process for the preparation of sodium salt of various isoxazolyl penicillins are detailed below.
The invention provides to the prior art  an in-situ process for the preparation of beta-lactamase inhibitor semi synthetic antibiotics more specifically it provides a process for the preparation of isoxazolyl penicillins. Isoxazolyl penicillins come in same class as penicillin and are used as penicillinase-resistant penicillin compounds. Penicillinase or beta-lactamase enzyme is an enzyme which is produced by certain strains of bacteria that can deactivate certain types of penicillin antibiotics. Thus  penicillinase-resistant antibiotics such as isoxazolyl penicillins and their pharmaceutical acceptable salts are useful in fighting certain bacterial infections such as Staphylococcus when other penicillins wouldn""t work.
Isoxazolyl penicillins such as cloxacillin  dicloxacillin  flucloxacillin  oxacillin and their respective pharmaceutical acceptable salts thereof are used against bacteria that produce beta lactamase or penicillinase  which affects the antimicrobial activity of the antibiotics.
The invention provides a process for the preparation of sodium salt isoxazolyl penicillins such as cloxacillin sodium  dicloxacillin sodium  flucloxacillin sodium  and oxacillin sodium involving in-situ reaction. The term "in situ" is defined herein as performing two or more reaction sequences without isolating any of the intermediates that is produced during the reaction sequence.
The invention provides an in-situ process for the preparation of isoxazolyl penicillins  which is quite efficient and cost effective and which proves to generate significant feasibility for an industrial scale production of sodium salt of isoxazolyl penicillins without involving separate preparation of the intermediates like Penicillin G and 6-aminopenicillanic acid.
The present invention provides an in-situ process for the preparation of various Isoxazolyl Penicillins from sugarcane juice in which intermediates like penicillin G and 6-aminopenicillanic acid produced during the reaction are not isolated from the reaction sequence. The process is characterized in the steps comprising:
preparation of a first reaction mixture comprising Penicillin G by fermentation of sugarcane juice with a salt of phenylacetic acid to form a product comprising Penicillin G and thereby purification of the product to form the first reaction mixture which is used for the preparation of second reaction mixture wherein the preparation is in-situ;
preparation of a second reaction mixture by enzymatic reaction of the first reaction mixture wherein the preparation is in-situ and is characterized in the steps comprising: reaction of the first reaction mixture with an enzyme to form a solution comprising 6-APA and phenylacetic acid (PAA) without isolation of penicillin G from the first reaction mixture. Then  adding a first organic solvent in presence of an acidic solution to it at a temperature of 0-40°C to form a first organic layer comprising phenylacetic acid and a first aqueous layer comprising 6-APA. After that  extracting the second organic layer to form a third reaction mixture comprising phenylacetic acid (PAA) and thereby obtaining the second reaction mixture comprising 6-APA;
in-situ addition of a second organic solvent and a carboxylic acid chloride to the second reaction mixture (without isolation of 6-APA) at a temperature of 0-30°C to obtain a second organic layer and a second aqueous layer; and
in-situ separating the second organic layer and then adding a sodium salt complex to it to form the Isoxazolyl Penicillin.
In the processes known in the prior art  6-aminopenicillanic acid (6-APA) is prepared separately and is reacted with carboxylic acid chlorides to form Isoxazolyl penicillins but in the present process  6-APA is produced in-situ (within the system) by enzymatic reaction of the first reaction mixture comprising penicillin G generated after the fermentation of sugarcane with a salt of phenylacetic acid. This 6-APA is then reacted with the carboxylic acid chlorides to the Isoxazolyl Penicillins. The in-situ preparation avoids the extra cost of purification  crystallization  equipments  storage of separately produced 6-APA  thus this process comes out to be cost effective. During the in-situ preparation of second reaction mixture  phenylacetic acid is recovered from the system which is recycled to the fermentation step. This recycling makes the process cost effective and allows less consumption of the reactant.
In an embodiment of the invention  the preparation of isoxazolyl penicillins is carried out directly from the sugarcane juice.
In an embodiment of the invention  first reaction mixture comprising Penicillin G is prepared by fermentation of sugarcane juice with a salt of phenylacetic acid. In the fermentation process  2nd generation spore is inoculated into a seed fermentor having sterilized media at a pH of 6 to 6.1 to form a broth. 10% of the broth is transferred to the production fermentor. Into the production fermentor  50% sugarcane juice and 17% metallic salt of phenylacetic acid is added along with 20% ammonia solution to maintain the pH in range of 6.4 to 6.55 to obtain a product which is allowed to go through the purification steps for the production of the first reaction mixture comprising Penicillin G. In the processes known in prior art  penicillin G is produced from sugar solution.
In another embodiment of the invention  the salt of the phenylacetic acid used is the metallic salt and the metal of the metallic salt is selected from the group comprising of sodium  magnesium  potassium and aluminum.
In another embodiment of the invention  the purification of the product produced after fermentation is done by ultra-filtration and nano filtration. The ultra filtration is done to remove the unwanted mycelium and microorganisms from the product. After that  nano-filtration is done to concentrate the purified product to form the first reaction mixture comprising penicillin G.
In an exemplary embodiment of the invention  the ultra filtration is done twice followed by nano filtration to purify and concentrate the product produced after the fermentation step.
In another embodiment of the invention  the enzyme used to react with the first reaction mixture is selected from the group consisting of Penicillin G Acylase or Penicillin Amidase. In present invention  the enzyme used is Penicillin G Acylase and the reaction between the enzyme and the first reaction mixture takes place at a temperature of 28-29°C.
In another embodiment of the invention  the enzymatic reaction of the first reaction mixture takes place at a pH of 6.8 to 8.5 to form a solution comprising 6-APA and PAA. An alkali solution is added to the first reaction mixture along with the enzyme to maintain the pH in range of 6.8 to 8.5. The alkali is selected from the group comprising of sodium bicarbonate  ammonium carbonate  caustic solution and ammonia solution. The solution comprising 6-APA and PAA is allowed to settle and is filtered. The filtrate is then washed with de-mineralized water and is cooled at a temperature of 0-30°C. In present invention the alkali added is 10% ammonia solution.
In another embodiment of the invention  the acidic solution is added with the first organic solvent till the pH of the solution reaches in range of 1 to 3.5 and is selected from the group consisting of HCl  H2SO4  Acetic Acid and any other low pH acid. In the present invention  HCl is added with the first organic solvent to maintain the pH in range of 1 to 3.5.
In another embodiment of the invention  the first organic solvent and the second organic solvent are independently selected from the group consisting of toluene  butyl acetate  methylene chloride  methyl isobutyl ketone  ethyl acetate and any other water immiscible solvent.
In another embodiment of the invention  the carboxylic acid chloride that reacts with the aqueous layer formed after addition of first organic solvent is selected from the group consisting of 3-(2-chlorophenyl)-5-methylisoxazole-4-carbonyl chloride (CMIC chloride)  3-(2 6-dichlorophenyl)-5-methylisoxazole-4-carbonyl chloride (DICMIC chloride)  3-(2-cholor-6-florophenyl)-5methylisoxazole-4 carbonyl chloride (FCMIC chloride)  phenyl-5-methylisoxazole-4-carbonyl chloride (PMIC chloride) and any other side chain isoxazoles depending upon the isoxazolyl penicillin to be prepared.
In an exemplary embodiment of the invention  the carboxylic acid chloride used for the preparation of cloxacillin sodium is CMIC chloride.
In an exemplary embodiment of the invention  the carboxylic acid chloride used for the preparation of dicloxacillin sodium is DICMIC chloride.
In an exemplary embodiment of the invention  the carboxylic acid chloride used for the preparation of flucloxacillin sodium is FCMIC chloride.
In an exemplary embodiment of the invention  the carboxylic acid chloride used for the preparation of oxacillin sodium is PMIC chloride.
In another embodiment of the invention  separation of second organic layer after the addition of second organic solvent is aided with the addition of a salt solution and a solid salt. In the present invention  the salt solution is selected from the group consisting of sodium bicarbonate or any other suitable alkali  whereas the solid salt used is sodium chloride. After the separation of the second organic layer de-moisturization of the second organic layer is done by the addition of sodium chloride.
In another embodiment of the invention  the sodium salt complex added to the de-moisturized second organic layer to form Isoxazolyl Penicillin is selected from any sodium salt  preferably sodium 2-ethylhexanoate. Sodium salt complex is added till the pH of the solution reaches to 7.5-8.5. The solution obtained after the addition of the sodium salt complex is filtered  washed with ethyl acetate (250*3ml) and finally dried at a temperature of 50-55°C to form the Isoxazolyl Penicillin.
In another embodiment of the invention  the process for the preparation of Isoxazolyl Penicillins is an in-situ process in which  neither Penicillin G produced after fermentation of sugarcane with the salt phenylacetic acid in the first reaction mixture is isolated for further reactions  nor 6-aminopenicillanic acid produced after the enzymatic reaction of the first reaction mixture is isolated for further reactions.
In another embodiment of the invention  the process for the preparation of Isoxazolyl Penicillins involves recycling of third reaction mixture comprising phenylacetic acid to the fermentation step. This recycling of phenylacetic acid helps in better production of Penicillin G which in turns helps producing more 6-APA. This more 6-APA then reacts with the carboxylic acid chloride to produce more Isoxazolyl Penicillin. Also  due to recycling  recovery of the reactant i.e. phenylacetic acid can be done which in turns less consumption of the reactant; hence the whole process becomes a cost effective process.
In an exemplary embodiment of the present invention  cloxacillin sodium is prepared from sugarcane juice. The process comprises: in-situ preparation of a first reaction mixture comprising Penicillin G wherein the process comprises fermentation of sugarcane juice with a salt of phenylacetic acid to form a product and thereby purification of the product by ultra-filtration and nano-filtration to form the first reaction mixture;
in-situ preparation of the second reaction mixture by enzymatic reaction of the first reaction mixture to prepare a second reaction mixture comprising 6-APA wherein the preparation comprising: reacting the first reaction mixture with Penicillin acylase enzyme to form a solution comprising 6-APA and phenylacetic acid; adding an alkali to maintain the pH in range of 6.8-8.5 preferably 8; filtering and washing the filtrate; adding a first organic solvent in presence of HCl to the filtrate to obtain a first aqueous and a first organic layer; extracting the first organic layer comprising phenylacetic acid and recycling it to the fermentation step and thereby obtaining the second reaction mixture comprising 6-APA;
adding a second organic solvent and CMIC chloride to the second reaction mixture without isolating 6-APA from it to obtain a second aqueous layer and a second organic layer;
adding sodium bicarbonate and sodium chloride to the solution formed for better separation of the second organic layer; de-moisturizing the second organic layer and reacting it with sodium 2-ethylhexanoate to form the cloxacillin sodium.
In another embodiment of the invention  the preparation of the isoxazolyl penicillins is done without isolation of the intermediates i.e. penicillin G and 6-APA at any stage i.e. reaction is carried out in an in-situ manner  which is very useful on commercial scales. It incurs less capital costs since the cost of equipments / machinery used otherwise for purification of the intermediates is completely eliminated.
The process for preparation of isoxazolyl penicillins as described in various embodiments of the present invention is achieved by in-situ preparations of intermediates i.e. Penicillin G and 6-APA conferring high purity without the loss of the intermediate during its isolation  purification and crystallization procedure. As a consequence  there is a considerable improvement in the process efficacy in contrast to the preparation process already available in the prior art. Also  the industrial applicability of the process is enhanced significantly since it can be easily set up at an industrial scale without the use of complex purification methods or equipments  which thereby increase the efficiency and substantially reduce the cost of industrial production.
It is further mentioned that  if the processes vide the prior art were to be used to isolate  purify  crystallize the Penicillin G and 6-APA formed during the reaction sequence  all or most of the machinery/equipment will need to be used thereby increasing running expenditure on the bulk production of the named products. It is classified here  that the improvements are not limited to just the factors as mentioned herein.
Several variations in the processes and the methods herein disclosed will suggest themselves to those skilled in the art. However  it is to be understood that the present disclosure relates to the preferred embodiment of the invention which is for purposes of illustration only and not to be construed as limiting the scope of the invention.
A pharmaceutical composition containing the product obtained according to the process of the invention has no need to be formulated with additional auxiliaries.
The present invention will now be illustrated in greater detail with reference to Examples  but the present invention should not be interpreted as being restricted thereto.
EXAMPLES:
Example 1
Preparation of Cloxacillin Sodium
6.2ml of 2nd generation spore was inoculated into a seed fermentor and 16L media is prepared for it at a pH of 5.9-6.1. Batch was matured till log 55 hr with pH 5.25 and 10% of it was transferred to the production fermentor. In the production fermentor 100L media was prepared at a pH of 6.9. At temperature 25C matured seeds were transferred to it. From log 3 hr onwards  41 L of 50% sugarcane juice was fed along with 1.4 L of 17% sodium phenylacetate into the production fermentor. From log 4 hr onwards 20 % ammonia solution was added to maintain the pH in the range of 6.4 to 6.55 to obtain a product comprising penicillin G. After ultra-filtration and nano-filtration the concentration of the product was made to 31 mg/ml to form the first reaction mixture. 50gm equivalent of first reaction mixture comprising Penicillin G was dissolved in 450ml of de-mineralized water. At 28-29°C Penicillin acylase enzyme was added. The pH was maintained at around 8 by the addition of 10% ammonia solution. Reaction was continued till completion. The solution was allowed to settle for 5 min and then filtered. The filtrate was then washed with 90ml de-mineralized water. The temperature of the resulting solution was then cooled to 4-5°C and 170ml of toluene was added in presence of HCl at a pH in range of 1-1.4 to obtain a solution comprising 6-aminopenicillanic acid (6-APA) and phenylacetic acid (PAA). The solution obtained was stirred for 5 min to form a first organic layer and a first aqueous layer. The first organic layer comprising PAA was separated to form a third reaction mixture and recycled into the system whereas the first aqueous layer was filtered and wash with 10ml de-mineralized water to form the second reaction mixture. The temperature of the solution was maintained at 0-5°C and 400ml ethyl acetate and 31.5gm of CMIC chloride were added to the filtered/washed second reaction mixture. The pH of the solution was raised to 1.85 with 9% NaHCO3 solution in 2 hrs and then further raised to 2.4 in next 2 hrs. After the completion of 4hrs  NaCl was dissolved to obtain a second aqueous layer and a second organic layer. The second organic layer was then separated and was de-moisturized with saturated NaCl solution (m/c < 2.5%). The temperature was raised to 15-18°C and 50-60 ml of 50% Sodium 2-ethylhexanoate was added to the de-moisturized second organic layer till the pH of the final solution reaches to 8-8.2 to get the solution of cloxacillin sodium. The resultant solution was then filtered  washed with ethyl acetate (250ml*3) and dried at a temperature of 50-55°C. The yield of the Cloxacillin sodium obtained was more than 50gm. The product obtained complies with the pharmacopeia requirement.

RAW MATERIALS AND THEIR INPUTS REQUIRED FOR EXAMPLE 1
Raw material Input
2nd Generation spore 6.2ml
Media 16L
50 % Sugarcane juice 20L
17% Phenylacetic acid 4L
20% Ammonia solution (Till pH is adjusted)
De-mineralized water 550 ml
Toluene 170ml
30% HCl 44 ml
Enzyme 100gm (wet)
Ethyl acetate 1150ml
CMIC chloride 31.5gm
Sodium chloride 70 gm
10% NH3 (Ammonia) 27-30ml
9% NaHCO3 240-250ml
50% Sodium 2-ethylhexanoate 50-60ml
Yield 50gm-minimum

CLAIMS
We Claim:
1. A process for the preparation of isoxazolyl penicillins wherein the process is characterized in the steps comprising:
a) preparation of a first reaction mixture comprising penicillin G from sugarcane juice by fermentation of sugarcane juice with a salt of phenylacetic acid to obtain a product comprising Penicillin G and thereby purification of the product to form the first reaction mixture  wherein the preparation is in-situ;
b) preparation of a second reaction mixture by enzymatic reaction of the first reaction mixture  wherein the preparation is in-situ and is characterized in the steps comprising:
i) reacting the first reaction mixture with an enzyme to form a solution comprising 6-aminopenicillanic acid and phenylacetic acid;
ii) adding a first organic solvent in presence of an acidic solution to form a first organic layer and a first aqueous layer; and
iii) extracting the first organic layer to form a third reaction mixture comprising phenylacetic acid and thereby obtaining the second reaction mixture comprising 6-aminopenicillanic acid;
c) in-situ addition of a second organic solvent and a carboxylic acid chloride to the second reaction mixture to form a second organic layer and a second aqueous layer; and
d) in-situ separation of the second organic layer and addition of a sodium salt complex to it to form the Isoxazolyl Penicillin.
2. The process according to claim 1  wherein the Isoxazolyl Penicillin is selected from the group consisting of cloxacillin sodium  dicloxacillin sodium  flucloxacillin sodium and oxacillin sodium.
3. The process according to claim 1  wherein the salt of phenylacetic acid in step (a) is a metallic salt and wherein the metal of the metallic salt is selected from the group comprising of sodium  magnesium  potassium and aluminum.
4. The process according to claim 1  wherein the purification of the product obtained after fermentation is done by ultra-filtration followed by nano-filtration.
5. The process according to claim 1  wherein the enzymatic reaction in step (i) takes place at a pH of 6.8-8.5.
6. The process according to claim 1  wherein the enzyme used is selected from Penicillin G Acylase and Penicillin Amidase.
7. The process according to claim 1  wherein the acidic solution is selected from the group consisting of HCl  Acetic Acid  Sulphuric acid and any low pH acid.
8. The process according to claim 1  wherein the acidic solution is added till the pH reaches in the range of 0.3-4.5.
9. The process according to claim 1  wherein the first organic solvent and the second organic solvent are independently selected from the group consisting of toluene  Butyl acetate  Methylene chloride  Methyl isobutyl ketone  Ethyl acetate and any other water immiscible solvent.
10. The process according to claim 1  wherein the third reaction mixture is recycled into step (a) for fermentation of sugarcane juice with salt of phenylacetic acid;
11. The process according to claim 1  wherein the carboxylic acid chloride used for the preparation of cloxacillin sodium is CMIC chloride.
12. The process according to claim 1  wherein the carboxylic acid chloride used for the preparation of dicloxacillin sodium is DICMIC chloride.
13. The process according to claim 1  wherein the carboxylic acid chloride used for the preparation of flucloxacillin sodium is FCMIC chloride.
14. The process according to claim 1  wherein the carboxylic acid chloride used for the preparation of oxacillin sodium is PMIC chloride.
15. The process according to claim 1  wherein the sodium salt complex is any sodium salt preferably sodium 2-ethylhexanoate.
16. A process for the preparation of isoxazolyl penicillins wherein the process is characterized in the steps comprising:
a) preparation of a first reaction mixture comprising penicillin G from sugarcane juice by fermentation of sugarcane juice with a salt of phenylacetic acid to obtain a product comprising Penicillin G and thereby purification of the product to form the first reaction mixture wherein purification is done by ultra-filtration followed by nano filtration and wherein the preparation is in-situ;
b) preparation of a second reaction mixture by enzymatic reaction of the first reaction mixture  wherein the preparation is in-situ and is characterized in the steps comprising:
i) reacting the first reaction mixture with an penicillin G acylase to form a solution comprising 6-aminopenicillanic acid and phenylacetic acid;
ii) adding a first organic solvent in presence of HCl to form a first organic layer and a first aqueous layer; and
iii) extracting the first organic layer to form a third reaction mixture comprising phenylacetic acid and thereby obtaining the second reaction mixture comprising 6-aminopenicillanic acid;
c) in-situ addition of a second organic solvent and a carboxylic acid chloride to the second reaction mixture to form a second organic layer and a second aqueous layer; and
d) in-situ separation of the second organic layer and addition sodium 2-ethylhexanoate to it to form the Isoxazolyl Penicillin.
17. The process according to claim 16  wherein the Isoxazolyl Penicillin is selected from the group consisting of cloxacillin sodium  dicloxacillin sodium  flucloxacillin sodium and oxacillin sodium.
18. The process according to claim 16  wherein the salt of phenylacetic acid in step (a) is a metallic salt and wherein the metal of the metallic salt is selected from the group comprising of sodium  magnesium  potassium and aluminum.
19. The process according to claim 16  wherein the enzymatic reaction in step (i) takes place at a pH of 6.8-8.5.
20. The process according to claim 16  wherein HCl is added till the pH reaches in the range of 0.3-4.5.
21. The process according to claim 16  wherein the first organic solvent and the second organic solvent are independently selected from the group consisting of toluene  Butyl acetate  Methylene chloride  Methyl isobutyl ketone  Ethyl acetate and any other water immiscible solvent.
22. The process according to claim 16  wherein the third reaction mixture is recycled into step (a) for fermentation of sugarcane juice with the salt of phenylacetic acid;
23. The process according to claim 16  wherein the carboxylic acid chloride used for the preparation of cloxacillin sodium is CMIC chloride.
24. The process according to claim 16  wherein the carboxylic acid chloride used for the preparation of dicloxacillin sodium is DICMIC chloride.
25. The process according to claim 16  wherein the carboxylic acid chloride used for the preparation of flucloxacillin sodium is FCMIC chloride.
26. The process according to claim 16  wherein the carboxylic acid chloride used for the preparation of oxacillin sodium is PMIC chloride.