FIELD OF INVENTION:
This invention relates to a process for synthesizing higher chain chitooligosaccharides.
BACKGROUND OF THE INVENTION:
Production of specific oligosaccharides by chemical methods is challenging due to the need to selectively protect and manipulate chemically quite similar saccharide donors and acceptors. For this reason, development of methods for enzymatic synthesis of oligosaccharides is desirable. The transglycosylation (TG) activity of family 18 chitinases is of special interest because there are numerous potential applications for chitooligosaccharides [i.e., homo- or hetero-oligomers of glucosamine (GlcN) and N-acetylated glucosamine (GlcNAc)], especially in the food, medical, and agriculture fields. The bioactivities of chitooligosaccharides (CHOS) are thought to depend on a combination of oligomer length, degree of acetylation and acetylation pattern (sequence). Transglycosylating chitinases have the potential to play a central role in the development of new well-defined mixtures of CHOS with new or improved biological activity, by coupling smaller CHOS building blocks to each other or to other functional groups.

Chemical processes have several disadvantages compared to bioprocesses because of large pollution and the products obtained through are chemical process are not unique products, difficult to control the reaction to obtain a specific products.

OBJECTS OF THE INVENTION:

An object of this invention is to propose a process for synthesizing higher chain chitooligosaccharides (CHOS);

Another object of this invention is to propose a process for synthesizing higher chain CHOS from their lower chain counterparts using highly efficient chitinase;

Further, object of this invention is to propose a process which is less expensive;

Yet another object of this invention is to propose a process for synthesizing higher chain CHOS which are highly specific in their biological activity.

BRIEF DESCRIPTION OF THE INVENTION:

According to this invention there is provided a process for synthesizing higher chain chitooligosaccharides (CHOS) comprising:

amplifying chitinase D (chiD) from S. proteamaculans (Sp);

subjecting the amplified chitinase D to the step of cloning;

purifying the recombinant chitinase D and analyzing products of Sp ChiD from CHOS.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:

FIG 1: Amplification and cloning of Sp ChiD from S. proteamaculans 568

A) S. proteamaculans 568, Sp chiD (Lane A1) was PCR amplified with gene specific primers using genomic DNA (gDNA) as template and resolved on a 1% agarose gel. The molecular weight marker was DNA ladder mix (100 bp-10kb; lanes A2).

B) The amplicon was ligated to Nco I and Xho I sites pET 22b(+). The presence of insert was confirmed by double digestion with Nco I and Xho I. Lane B2: Sp chiD (1.221 kb). The molecular weight marker was DNA ladder mix (100 bp - 10 kb; lanes B1).

FIG 2: SDS-PAGE analysis of purified Sp ChiD. Recombinant Sp ChiD was (MW 44.75 kDa) purified by Ni-NTA agarose affinity chromatography using elution buffer containing 250 mM imidazole. The sizes of the standards are indicated in kDa. Lane 1: Purified Sp ChiD, Lane 2: Prestained protein molecular weight marker.

FIG 3: Time course of chitooligosaccharides (CHOS) hydrolysis of Sp ChiD as analyzed by TLC.
A reaction mixture, containing 560 nM of enzyme and 2.0 mM substrate in 50 mM sodium acetate buffer pH 6.0, was incubated at 40°C and each indicated time point (0,1,3,5,15,30,45,60,120,180 and 720 min), 10 μ l of reaction portion was withdrawn and added 10 μ l of 0.1 N NaOH to stop the reaction. The reaction solution (20 μ l ) was analyzed on TLC and products were detected with aniline-diphenylamine reagent. (A-E) hydrolysis of CHOS of DP6-DP2. Lane S: a standard mix of chitooligosaccharides (CHOS) (DP1-DP6), Lanes C: Substrate control without enzymatic treatment, Lanes 0-720: Reaction incubation time of 0, 1, 2, 3, 5, 15, 30, 45, 60,120,180, and 720 min. Dotted lines with arrow indicates transglycosylation (TG) products.

FIG 4: Time course of DP3 hydrolysis and TG catalyzed by Sp ChiD. The reaction mixture containing 3.5 mM DP3 in 50 mM sodium acetate buffer pH 6.0 was incubated with 560 nM Sp ChiD for different time periods starting from 0-720 min at 40°C. Each time point, 75 uL of reaction mixtures were withdrawn and equal amount of 75% acetonitrile was added to stop the reaction. The reaction solution (20 uL) was analyzed by isocratic HPLC using Shodex Asahipack NH2P-50 4E column and eluted CHOS were monitored by recording absorption at 210 nm.

A) The top most profile shows a standard mixture of CHOS ranging from DP1-DP6. The other profiles shows how the reaction proceeded (incubation times indicated). Insets show the magnified view of the low peak area products.

B) Overview of concentrations of CHOS products generated during reaction time course. Products were quantified from respective peak areas of HPLC profiles using standard calibration curves of CHOS ranging from DP1-DP6.

C) Magnification for quantifiable TG products (DP4, DP5 and DP6).

FIG 5: Time course of DP4 hydrolysis and TG catalyzed by Sp ChiD. The reaction mixture containing 3.5 mM DP4 in 50 mM sodium acetate buffer, pH 6 was incubated with 560 nM Sp ChiD for different time periods starting from 0-720 min at 40cc. Each time point, 75 uL of reaction mixtures were withdrawn and equal amount of 75% acetonitrile was added to stop the reaction. The reaction solution (20 uL) was analyzed by isocratic HPLC using Shodex Asahipack NH2P-50 4E column and eluted CHOS were monitored by recording absorption at 210 nm.

A) The top most profile shows a standard mixture of chitooligosaccharides (CHOS) ranging from DP1-DP6. The other profiles show how the reaction proceeded (incubation times indicated). Insets show the magnified view of the low peak area products.

B) Overview of concentrations of CHOS products generated during reaction time course. Products were quantified from respective peak areas of HPLC profiles using standard calibration curves of CHOS ranging from DP1-DP6.

C) Magnification for quantifiable TG products (DP5 and DP6).

FIG 6: Time course of DP5 hydrolysis and TG catalyzed by Sp ChiD. The reaction mixture containing 3.5 mM DP5 in 50 mM sodium acetate buffer, pH 6 was incubated with 560 nM Sp ChiD for different time periods starting from 0-720 min at 40°C. Each time point, 75 uL of reaction mixtures were withdrawn and equal amount of 75% acetonitrile was added to stop the reaction. The reaction solution (20 uL) was analyzed by isocratic HPLC using Shodex Asahipack NH2P-50 4E column and eluted CHOS were monitored by recording absorption at 210 nm.

A) The top most profile shows a standard mixture of chitooligosaccharides (CHOS) ranging from DP1-DP6. The other profiles show how the reaction proceeded (incubation times are indicated). Insets show the magnified low peak area products.

B) Overview of concentrations of chitooligosaccharides (CHOS) products generated during reaction time course. Products were quantified from respective peak areas of HPLC profiles using standard calibration curves of CHOS ranging from DP1-DP6.

C) Magnification for quantifiable TG product (DP6).

FIG 7: Time course of DP6 hydrolysis and TG catalyzed by Sp ChiD. The reaction mixture containing 3.5 mM DP6 in 50 mM sodium acetate buffer, pH 6 was incubated with 560 nM Sp ChiD for different time periods starting from 0-720 min at 40°C. Each time point, 75 uL of reaction mixtures were withdrawn and equal amount of 75% acetonitrile was added to stop the reaction. The reaction solution (20 uL) was analyzed by isocratic HPLC using Shodex Asahipack NH2P-50 4E column and eluted CHOS were monitored by recording absorption at 210 nm.

A) The top most profile shows a standard mixture of CHOS ranging from DP1-DP6. The other profiles show how the reaction proceeded (incubation times indicated). Insets show the magnified view of the low peak area products.

B) Overview of concentrations of CHOS products generated during reaction time course. Products were quantified from respective peak areas of HPLC profiles using standard calibration curves of CHOS ranging from DP1-DP6.

FIG. 8: MALDI-TOF-MS analysis of products from DP3 hydrolysis and TG catalyzed by Sp ChiD.
After HPLC analysis of reaction products from DP3 (15th min sample), 40 uL of reaction sample was concentrated under reduced pressure without heating till complete evaporation of solvent and finally dissolved in 4 uL of MilliQ H2O. The reaction mixture (2 uL) was mixed with an equal volume of 2, 5-dihydroxy benzoic acid (2,5-DHB) and the resultant solution was employed for mass measurements using a Ultraflex MALDI-TOF/TOF instrument (Bruker Daltonics GmbH, Germany) with an autoflex 123 smart beam. Peaks in all MALDI-TOF MS spectra were labeled according to their observed atomic mass and the degree of polymerization (DP) of the chitooligosaccharides (CHOS). Each of the CHOS species was observed as Na adducts of the oligosaccharide Na-salt.

A) MALDI-TOF-MS spectrum of the enzymatic products from DP3 substrate.

B) Magnification for low peak area.

FIG 9: MALDI-TOF-MS analysis of products from DP4 hydrolysis and TG catalyzed by Sp ChiD.

After HPLC analysis of reaction products from DP4 (30th min sample), 40 uL of reaction sample was concentrated under reduced pressure without heating till complete evaporation of solvent and finally dissolved in 4 uL of MilliQ H2O. The reaction mixture (2 uL) was mixed with an equal volume of 2,5-dihydroxy benzoic acid (2,5-DHB) and the resultant solution was employed for mass measurements using a Ultraflex MALDI-TOF/TOF instrument (Bruker Daltonics GmbH, Germany) with a autoflex 123 smart beam. Peaks in all MALDI-TOF MS spectra are labeled according to their observed atomic mass and the degree of polymerization (DP) of the oligosaccharide. Each of the oligosaccharide species except DP2 was observed as Na adducts of the oligosaccharide Na-salt.

C) MALDI-TOF-MS spectrum of the enzymatic products from DP4 substrate.

D) Magnification for low peak area.

FIG 10: MALDI-TOF-MS analysis of products from DP5 hydrolysis and TG catalyzed by Sp ChiD.

After HPLC analysis of reaction products from DP5 (15th min sample),40 uL of reaction sample was concentrated under reduced pressure without heating till complete evaporation of solvent and finally dissolved in 4 uL of MilliQ H20. The reaction mixture (2 uL) was mixed with an equal volume of 2,5-dihydroxy benzoic acid (2,5-DHB) and the resultant solution was employed for mass measurements using a Ultraflex MALDI-TOF/TOF instrument (Bruker Daltonics GmbH, Germany) with a autoflex 123 smart beam. Peaks in all MALDI-TOF MS spectra are labeled according to their observed atomic mass and the degree of polymerization (DP) of the oligosaccharide. Each of the oligosaccharide species except DP2 was observed as Na adducts of the oligosaccharide Na-salt.

A) MALDI-TOF-MS spectrum of the enzymatic products from DP5 substrate.

B) Magnification for low peak area.

FIG 11: MALDI-TOF-MS analysis of products from DP6 hydrolysis and TG catalyzed by Sp ChiD.

After HPLC analysis of reaction products from DP6 (30th min sample), 40uL of reaction sample was concentrated under reduced pressure without heating till complete evaporation of solvent and finally dissolved in 4 uL of MilliQ H20. The reaction mixture (2 uL) was mixed with an equal volume of 2,5-dihydroxy benzoic acid (2,5-DHB) and the resultant solution was employed for mass measurements using a Ultraflex MALDI-TOF/TOF instrument (Bruker Daltonics GmbH, Germany) with a autoflex 123 smart beam. Peaks in all MALDI-TOF MS spectra are labeled according to their observed atomic mass and the degree of polymerization (DP) of the oligosaccharide. Each of the oligosaccharide species except DP2 was observed as Na adducts of the oligosaccharide Na-salt.

A) MALDI-TOF-MS spectrum of the enzymatic products from DP6 substrate.

B) Magnification for low peak area.

DETAILED DESCRIPTION OF THE INVENTION:

PCR amplification and cloning of chitinase D from Serratia proteamaculans 568 (S. proteamaculans 568).
Based on ORF sequences available in S. proteamaculans 568 genomic data base (http://aenome.iai-psf.org/finished microbes/serpr/serpr.home.htmL).

primers were designed for the amplification of chitinase encoding gene (Sp ChiD) from the genomic DNA of S. proteamaculans 568. The gene encoding for Sp ChiD was PCR amplified from S. proteamaculans 568 genomic DNA by referring the annotated sequence (GenBank accession no. ABV41826.1). The respective genes was amplified using PCR with gene-specific forward and reverse primers using Pfu DNA polymerase at 58°C annealing temperature with 4 min polymerization time. Amplicon 1.221 kb was gel extracted using Qiagen Gel Clean up kit. Bacterial expression vector pET 22b(+) and the amplicon was double digested with Nco I and Xho I, gel purified both the amplicon and vector was ligated using T4 DNA ligase at 16°C for 16 h. The resultant plasmid was designated as pET 22b(+) -Sp chiD to express Sp ChiD. The resulting plasmid was transformed into E. coli Rosette-gami 2 (DE3) (Novagen) and transformants were selected on ampicillin (100 ug/ml) and chloramphenicol (25 ug/ml) containing LB plates.

Protein expression and purification of Sp ChiD

Overnight culture grown from -80°C stocks of E. coli Rosette-gami 2 (DE3) cells containing pET 22b(+) -Sp chiD was used to inoculate 500 ml_ of Luria -Bertani medium containing ampicillin (100 ug/ml) and chloramphenicol (25 ug/ml). The culture was incubated at 37°C and 200 rpm. When the cell density reached 0.6 (O.D600 ), isopropyl thio-p-D-galactoside was added to a final concentration of 0.5 mM, and the culture was further incubated for overnight at 18°C, followed by harvesting by centrifugation (9,000 g for 10 min at 4°C). The cell pellet was processed for isolation of periplasmic fraction as described in pET manual (Novagen, Darmstadt, Germany), concentrated with buffer exchange using Macrosep Centrifugal Devices (Mr 30000 cut-off, Pall carporation, USA). Crude protein mixture was instantly subjected to Ni-NTA agarose affinity chromatography following the Qiagen's protocol. After SDS-PAGE analysis, the chitinase containing fractions were pooled and then applied to Macrosep Centrifugal Devices to concentrate the protein to remove imidazole and to buffer exchange with 50 mM sodium acetate buffer pH 6.0. Finally protein was stored at 4°C until use. Concentration of the protein was determined by BCA protein estimation kit (Novagen, USA) using a standard calibration curve constructed from BSA.

Analysis of products of Sp ChiD from chitooligosaccharides (CHOS)

Thin layer chromatography (TLC)

In a 150 uL reaction mixture, containing 2 mM of substrate (CHOS; DP2-DP6) and 5 ug of purified Sp ChiD in 50 mM buffer sodium acetate pH 6.0. Reaction mixtures were incubated at 37°C and each time period starting from 0 (just after addition of enzyme), 1,2,3,5,15,30,45,60, 120, 180 and 720 min. 10 uL of the reaction mixture was transferred to an eppendorf tube containing 10 uL of 0.1 N NaOH, to stop the reaction and samples were stored at -20°C until analysis. Aliquots (20 uL)

of the reaction mixtures were chromatographed on a silica gel plate (TLC Silica gel 60, Merck Co., Germany) with a solvent system containing n-butanol, methanol, 25% ammonia solution-water (5:4:2:1[v:v:v:v:]), and the products were detected by spraying the plate with aniline-diphenylamine reagent (400 uL aniline, 400 mg of diphenylamine, 20 ml_ of acetone, and 3 mL of 85% phosphoric acid) and baking it at 180°C using hot air gun (Black & Decker, Germany) for 3 min.

HPLC analysis of DP3-DP6 substrates hydrolysis/transglycosylation (TG) catalyzed by Sp ChiD.

To detect the range of products (especially TG products) generated by Sp ChiD from DP3-DP6 substrates by combining 560 nM Sp ChiD and 3.5 mM of each individual substrate ranging from DP3-DP6 in a reaction solution (800 uL) containing 50 mM sodium acetate buffer pH 6.0. Reaction mixtures were incubated at 40°C and each time period starting from 0 (just after addition of enzyme), 1,2,3,5,15, 30, 45, 60, 120, 180 and 720 min, 75 uL of the reaction mixture was transferred to an eppendorf tube containing 75 uL of 70% acetonotrile, to stop the reaction and samples were stored at -20°C until analysed by isocratic HPLC at 25°c using a Shimadzu 10ATvp UV/VIS HPLC system (Shimadzu corporation, Tokyo, Japan) equipped with a Shodex Asahipack NH2P-50 4E column (4.6 ID x 250 mm) (Showa Denko K.K, USA). Twenty microliter of the reaction mixture was injected in to the HPLC using Hamilton syringe (HAMILTON Bonaduz, Switzerland). The liquid phase consisted of 67% acetonitrile: 33% MilliQ H20 and flow rate was set to 0.70 mL/min, eluted CHOS were monitored by recording absorption at 210 nm. Based on the peak areas obtained from HPLC profiles, chitooligosaccharides (CHOS) concentrations were calculated using authentic oligosaccharide solutions. CHOS HPLC mixture, which contains the equal weights of oligomer ranging from DP1-DP6 were used for standard graph preparation. Standard calibration curves of CHOS moieties were constructed separately for each oligosaccharide. These data points yielded a linear curve for each amino standard sugar with the R2 values of 0.997-1.0, thus allowing molar concentrations of CHOS to be determined with a confidence.

MALDI-TOF-MS

After HPLC analysis, the remaining reaction mixtures (15th min sample for DP3, DP5 and 30th min sample for DP4, DP6) were analyzed by MALDI-TOF-MS to determine the mass dependent product identity. A portion of reaction mixture (40 uL) was concentrated in a concentrator till the complete evaporation of the solvent and dissolved in 4 μ L of HPLC grade MilliQ water. Two microliter of a 9 mg/mL mixture of 2,5-dihydroxybenzoic acid (DHB) in 30% acetonitrile was applied to a MTP 384 target plate ground steel TF (Bruker Daltonics). To this, 2 uL sample was then mixed into the DHB droplet and dried under a stream of air. Then the samples were analyzed with an Ultraflex MALDI-TOF/TOF instrument (Bruker Daltonics GmbH, Germany) with a autoflex 123 smart beam. The instrument was operated in positive acquisition mode and controlled by the FlexControl 3.0 software package. All spectra were obtained in the reflectron mode with an acceleration voltage of 25kV, a reflector voltage of 26, and pulsed ion extraction of 40 ns in the positive ion mode. The acquisition range used was from m/z 50 to 4000. The data were collected from averaging 500 laser shots, with the lowest laser energy necessary to obtain sufficient signal to noise ratios. Peak lists were generated from the MS spectra using Bruker FlexAnalysis software (Version 3.0).

Cloning and characterization of Sp ChiD.

Amplification and cloning of Sp ChiD

Sp ChiD was amplified using gene specific primers with gDNA as template. The gene, ChiD were predicted (http://www.cbs.dtu.dk/services/SiqnalP/) to contain N-terminal leader peptides directing sec-dependent secretion. So the gene was cloned without the signal peptide-encoding part (Sp chiD: 57 bp). The 1.22 kb amplicon of Sp chiD, was cloned in the Nco I and Xho I sites pET 22b (+), respectively (Fig. 1A). The clone was confirmed by double digestion with Nco I and Xho I enzymes (Fig. 1B) and the insert sequence was confirmed by automated DNA sequencing (Europhins, India).

Expression and purification of Sp chitinases D

Sp ChiD gene over expressed in E. coli, was having C-terminal His-tag. The expressed chitinases, extracted from periplasmic fractions (Sp ChiD), was obtained in soluble form. The extracted proteins were purified using Ni-NTA agarose chromatography. SDS-PAGE analysis (Fig. 2) of the purified Sp ChiD revealed a molecular weight of 44 kDa.

Hydrolytic activities of Sp ChiD on chitooligosaccharides (CHOS) and Product analysis by thin-layer chromatography Sp ChiD released TG products from DP3-DP5 substrates and produced DP1 as
major end product (Fig. 3).

DP3-DP6 hydrolysis/TG catalyzed by Sp ChiD as analyzed by HPLC.

DP3 as substrate

The hydrolysis of DP3 substrate yielded DP1-DP6 products among which DP1 DP2, and DP4-DP6 were hydrolysis and TG products, respectively (Fig. 4). Formation of DP4 and DP5 started from 0 min, while DP6 started from 3 min and concentrations of these TG products were steadily increased till 15 min. After 15 min, the TG products concentration decreased and complete hydrolysis of DP4 and DP5 was observed after 45 min, while DP6 was hydrolyzed completely after 30 min. DP2 was released as a major product from DP3 substrate up to 120 min. At 0 min of reaction time, the yields of DP1, DP4 and DP5 compared to the DP2 were 62.4%, 10.6%, and 1.9%, respectively. At 15 min of reaction time, the maximum yields of DP4, DP5 and DP6, synthesized relatively to the major hydrolytic product, DP2, were 14.2%, 4.2% and 1.2%, respectively. By 720 min, DP1 and DP2 products only were present in the reaction. The DP1 formation was 4.11- fold higher than that of DP2.

DP4 as substrate

Sp ChiD released DP1-DP9 products from DP4 substrate among which DP1-DP3 and DP5-DP9 were hydrolysis and TG products, respectively (Fig. 5). Formation of DP5-DP8 products started from 0 min and present till 120 min, while formation of DP9 started from 1 min and present till 90 min along with DP1-DP8 products. Hydrolysis of DP4 mainly resulted in formation of DP2 right through the start of the reaction till 120 min. At 0 min of reaction time, the yields of DP1, DP3, DP5 and DP6 compared to the DP2 were 54.6%, 54.8%, 6.2% and 6.5%, respectively. From DP4 substrate, DP5 and DP6 were the quantifiable TG products and these products reached maximum concentration at 45 min and 30 min, respectively. At 45 min of reaction time, the maximum yield of DP5 synthesized, relatively to the major hydrolytic product DP2, was 16.1% while, DP6 was 10.6% at 30 min. By 720 min, only DP1 and DP2 were present in the reaction among which the DP1 product formation was 6.6-fold higher than that of DP2.

DP5 as substrate

Sp ChiD released DP1-DP10 products from DP5 substrate among which DP1-DP4 and DP6-DP9 were hydrolysis and TG products, respectively (Fig. 6). Formation of DP6-DP9 products started from 0 min and present till 120 min while the formation of DP 10 started from 1 min and present till 90 min along with DP1-DP9 products. In contrast to the other oligomeric substrate, DP5 hydrolysis resulted in formation of DP3 as a major product starting from 0-45 min. The yields of DP1, DP2, DP4 and DP6, compared to the DP3 at 0 min, were 19.25%, 68.44%, 18.26% and 5.07% respectively. The product, DP6 was the only quantifiable TG product from the standard curve obtained with authentic oligosaccharide solutions (DP1-DP6). The maximum yield of DP6 synthesized, relatively to the major hydrolytic product DP2, was 11.25% at 90 min. At the end of the reaction (720 min), only DP1 and DP2 products were present in the reaction among which the DP 1 formation was 2.7- folds higher than that of DP2 product.

DP6 as substrate

Sp ChiD formed DP1-DP10 products from DP6 substrate among which DP1-DP5 and DP7-DP10 products were hydrolysis and TG products, respectively (Fig.7). Formation of TG products (DP7-DP10) started from 0 min onwards and present till 90 min, after wards the TG products got degraded. Hydrolysis of DP6 mainly yielded DP2 product starting from 0-120 min. At 0 min of reaction time, the yields of DP1, DP3, DP4 and DP5, compared to the DP2, were 56.0%, 58.0%, 36.8% and 17.8%, respectively. At the end of the reaction (720 min), DP1-DP5 products were present in the reaction among which the DP1 formation was 9.0, 232.16, 475.6 and 973.8-folds higher than that of DP2, DP3, DP4, and DP5, respectively.

Determination of products identity from DP3-DP6 substrates by MALDI-TOF –MS

Mass-based identities of Sp ChiD products, especially to know the TG products that were higher than the DP6, were analyzed with MALDI-TOF-MS. Reaction products from DP3, DP4, DP5 and DP6 substrates showed products ranging from DP2-DP7 (Fig.8), DP2-DP10 (Fig.9), DP2-DP12 (Fig. 10) and DP2-DP13 (Fig. 11), respectively. Majority of the oligosaccharide species formed as Na adducts of the oligosaccharide Na salt.

WE CLAIM:

1. A process for synthesizing higher chain chitooligosaccharides (CHOS) comprising:

amplifying chitinase D (chiD) from S. proteamaculans (Sp);

subjecting the amplified chitinase D to the step of cloning;

purifying the cloned chitinase D to analyzing products of Sp chiD from chitooligosaccharides (CHOS).

2. The process as claimed in claim 1, wherein the said step of amplification and cloning is preferred by encoding the gene for Sp chiD which was PCR amplified from S. proteamaculans 568 genomic DNA, the said genes were amplified with gene-specific forward and reverse primers using Pfu DNA polymerase at 58°C annealing temperature with about 4 min polymerization time, the Amplicon and vector was ligated into PET 22b(+) using T4 DNA ligase to form the recombinant plasmid which was then transformed into E. coli.

3. The process as claimed in claim 1, wherein the step of purification is preferred by inoculating a medium using cultures of 22b(+) -Sp chiD,

incubating the cultures at 37°C and 200 rpm;

harvesting the incubated culture by centrifugation;

applying the chitinase containing fractions to Macrosep centrifugal Devices to concentrate the protein to remove imidazole and to buffer exchange with sodium acetate buffer.

4. The process as claimed in claim 3, wherein the said medium contains ampicillin and chloramphenicol.

5. The process as claimed in claim 1, wherein the step of analysis of the products of Sp chiD is preferred by thin layer chromatography and HPLC.