WO2020026274A1 - Process for producing mature serratiopeptidase - Google Patents

Abstract

The present disclosure provides a method for producing recombinant mature serratiopeptidase in E. coli. The present disclosure also provides a method for purifying the recombinant serratiopeptidase to obtain a purified protein having high yield, activity, and purity.

Description

PROCESS FOR PRODUCING MATURE SERRATIOPEPTIDASE

FIELD OF INVENTION

[001] The present disclosure broadly relates to the field of recombinant protein production and protein functioning. The disclosure particularly relates to a process for producing recombinant mature serratiopeptidase in Escherichia coli.

BACKGROUND OF INVENTION

[002] Apart from few genera of Enterobacteriaceae such as Erwinia, Serratia, Pseudomonas, etc.; gram-negative bacteria are not renowned for their protein secretion ability. They either do not secrete protein molecules or have very poor extracellular secretion mechanism. The extracellular secretion of these bacteria usually consists of protein molecules which function as a virulence factor and either show toxicity or possess hydrolytic nature.

[003] Serratia marcescens, a known opportunistic pathogen common in nosocomial infections, secretes at least five different types of hydrolases including at least two proteases. The major protease in secretion has molecular weight of around 50kDa and is commonly known as serrapeptase, serralysin protease or serratiopeptidase. Presence of serratiopeptidase in bacterial secretion has its own role for organism survival as a virulence factor. However, the protein molecule has well known therapeutic properties such as anti-inflammatory, analgesic and anti-edemic properties and is used in various drug combinations as an active ingredient.

[004] The bulk production of serratiopeptidase for commercial purposes is done by routine fermentation using native Serratia marcescens strains. Media modification and optimization of physicochemical parameters are used to enhance the yield (Ruchir C Pansuria et. al. Effects of Dissolved Oxygen and Agitation on Production of Serratiopeptidase by Serratia Marcescens NRRL B- 23112 in Stirred Tank Bioreactor and its Kinetic Modeling. J. Microbiol. Biotechnol. 2011, 21(4): 430-437; Dilbaghi et. al. Production, purification and characterization of fibrinolytic enzyme from Serratia sp. KG-2-1 using optimized media; 2017, 3 Biotech.7 (3): 184). The current approach of serratiopeptidase production has its own limitations, such as, the limited scope of yield enhancement and a significant amount of biomass disposal. Moreover, since the source microorganism is a nosocomial pathogen associated with many severe infections such as pneumonia, lower respiratory tract infection, bacteremia, and rare cases of endocarditis, it is not advisable to use such source for production, as the process would result in the production of large bacterial load, potentially hazardous for people associated with industrial operations.

[005] Employing recombinant DNA technology approach is seen as a boon to surpass these hurdles, wherein a non-pathogenic bacterial strain; i.e., E. coli could be used as host carrying serratiopeptidase gene. Overexpression of gene in the presence of suitable inducer and optimization of physicochemical parameters could result in enhanced yield and economical way for the production of serratiopeptidase. Initial cloning and sequencing for production of recombinant serratiopeptidase has been done in the late 80s (Nakahama el al. Cloning and sequencing of Serratia protease gene; Nucleic Acid Res., 1986, 14:5843-5855, and C. Braungel et.al. The metalloprotease gene of Serratia marcescens strain SM6. Molecular & general genetics: 1990, MGG. 222. 446- 51) and even several patents are existing; (i) Recombinant gene in E. coli (patent id: EP0226800A2); (ii) Genetic recombinant Pichia pastoris for serrapeptase gene (patent id: CN 103289978B). In spite of the available technology, industries are preferring the wild strain and traditional approach because of the difficulty associated with the expression and stability of protease genes in the recombinant host cells. Since serratiopeptidase is a broad specificity protease, overexpression of the protein often leads either to the cell toxicity resulting in death of host cells harboring recombinant construct, or the protein becomes inactive forming insoluble aggregates known as inclusion body, along with very low yield (250pg/l in E. coli, 7.0mg/l in S. marcescens). Therefore, novel methods are sought to address the lacunae existing in the prior art.

SUMMARY OF THE INVENTION [006] In an aspect of the present disclosure, there is provided a process for producing recombinant mature serratiopeptidase in E. coli, said process comprising: (a) obtaining recombinant E. coli cells capable of expressing recombinant mature serratiopeptidase having amino acid sequence as set forth in SEQ ID NO: 2; (b) growing the cells for a time period in a range of 14-18 hours to obtain an inoculum; (c) culturing the inoculum in a growth medium for a time period in a range of 2-4 hours to attain an optical density in a range of 0.6-0.8 to obtain a cell culture; (d) inducing the cell culture of step (c) with IPTG at a final concentration in a range of 0.1-2 mM with respect to the medium, to obtain an induced cell culture; and (e) culturing the induced cell culture of step (d) for a time period in a range of 2-16 hours at a temperature in a range of 16- 37 °C for producing recombinant mature serratiopeptidase in E. coli.

[007] In an aspect of the present disclosure, there is provided a process for obtaining purified recombinant mature serratiopeptidase from E. coli, said process comprising: (a) obtaining E. coli cells expressing recombinant mature serratiopeptidase having amino acid sequence as set forth in SEQ ID NO: 2; (b) harvesting the cells to obtain a pellet and contacting the pellet with a buffer TS comprising: Tris: l0-l00mM, NaCl: l00-500mM, Beta-mercaptoethanoh l- lOmM, having pH in a range of 6.8-8.0, to obtain inclusion bodies; (c) contacting the inclusion bodies with a buffer TEST comprising: Tris: l0-l00mM, NaCl: l00-600mM, Beta-mercaptoethanoh l-lOmM, EDTA: l-5mM, glycerol: 0.5-3%, Triton-X-l00: l-3%, urea: 0.5-4M, having pH in a range of 6.8-8.0, to obtain washed inclusion bodies; (d) contacting the washed inclusion bodies with a buffer D comprising: Tris: l0-l00mM, NaCl: l00-600mM, EDTA: l-5mM, glycerohO.1-1.0%, urea:4-8M or guanidinium hydrochloride:3-7M, having pH in a range of 6.0-8.0, to obtain solubilized inclusion bodies; (e) contacting the solubilized inclusion bodies with a buffer R comprising: Tris: l0-l00mM, CaCl₂:5-20mM, ZnCl₂: l-5mM, NaCh lOO-lOOOmM, glycerohO.1-1.0%, having pH in a range of 6.0-8.0, to obtain refolded inclusion body; and (f) performing gel filtration chromatography using the refolded inclusion body with a purification buffer comprising: Tris:50mM, NaCl: l50mM, CaCl₂:5mM, ZnCl₂:2mM, having pH-7.4, to obtain purified recombinant mature serratiopeptidase.

[008] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

[009] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0010] Figure 1 depicts a graphical abstract of mature serratiopeptidase enhanced production and purification through recombinant approach, in accordance with an embodiment of the present disclosure.

[0011] Figure 2 depicts a map of the recombinant construct of the present disclosure, in accordance with an embodiment of the present disclosure.

[0012] Figure 3 depicts isolated genomic DNA of Serratia marcescens MTCC7298 which is used as template for amplification of mature serratiopeptidase gene, in accordance with an embodiment of the present disclosure.

[0013] Figure 4 depicts agarose gel (1%) showing result of temperature gradient PCR for amplification of mature serratiopeptidase gene. T1-T8 shows different annealing temperature points in the range of 55-65 °C. C lane shows the negative control while R lane shows the PCR amplification reaction at desired temperature range, in accordance with an embodiment of the present disclosure.

[0014] Figure 5 depicts colony PCR results of positively transformed colonies after ligation. The result shows Cl, C2, C4, C6, C7, C8 colonies positive for the presence of mature serratiopeptidase gene while colony C3 and C5 are negative, in accordance with an embodiment of the present disclosure.

[0015] Figure 6 depicts agarose gel showing the restriction digestion of mature expression construct of serratiopeptidase. Lane M-is lKb DNA ladder, Lane-P is the intact plasmid construct, Lane-pSD is single digest product of pMsrp7298 in presence of enzyme Ndel giving band at 5Kb which is equivalent to the expression plasmid size (3.6Kb) + gene insert size (l464bp). Similarly, in pDD lane in which plasmid is digested using two different restriction enzymes (Ndel and Xhol) clearly shows a fall equivalent to 1.5kb which is the cloned gene insert for serratiopeptidase, in accordance with an embodiment of the present disclosure.

[0016] Figure 7 depicts a graph showing the transformants obtained in case of different strains I to IV, in accordance with an embodiment of the present disclosure.

[0017] Figure 8 depicts protein overexpression of mature version serratiopeptidase construct checked in three different commercially available E. coli expression hosts; viz-strain-I, strain-II, and strain-III. Gel shows the marker lane (M), uninduced sample (UI) and normalized amount of induced sample (I), supernatent (S) and insoluble pellet fraction (P), in accordance with an embodiment of the present disclosure.

[0018] Figure 9 depicts SDS-PAGE analysis of overexpression of serratiopeptidase in presence of variable concentration of IPTG which acts as an inducer. Cl -20 denotes different concentration of IPTG used for Inducer concentration optimization ranging in between O. lmM to 2.0mM, in accordance with an embodiment of the present disclosure.

[0019] Figure 10 depicts SDS-PAGE is performed for crude inclusion bodies and different collected fractions during washing of inclusion bodies. IB- inclusion body, Wl-wash-l, W2-wash-2, W3-wash-3, W4-wash-4, and cleared IB- cleaned inclusion body after washing, in accordance with an embodiment of the present disclosure. [0020] Figure 11 depicts SDS-PAGE gel image showing purified mature serratiopeptidase fraction after size exclusion chromatography, in accordance with an embodiment of the present disclosure.

[0021] Figure 12 depicts SDS-PAGE gel image showing comparative expression of serratiopeptidase in wild type Serratia marcescens MTCC7298 and in recombinant system, in accordance with an embodiment of the present disclosure.

[0022] Figure 13 depicts a standard curve for determining the activity of serratiopeptidase protein, in accordance with an embodiment of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

[0023] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.

Definitions

[0024] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0025] The articles“a”,“an” and“the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0026] The terms“comprise” and“comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as“consists of only”. [0027] Throughout this specification, unless the context requires otherwise the word“comprise”, and variations such as“comprises” and“comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0028] The term“including” is used to mean“including but not limited to”. “Including” and“including but not limited to” are used interchangeably.

[0029] Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

[0030] For the purposes of the present disclosure, the term ‘mature serratiopeptidase’ refers to the mature form or the active form of serratiopeptidase displaying protease activity. It is not a pro-peptide form which needs processing/cleaving to obtain active form of the protein.

[0031] For the purposes of the present disclosure, the term‘recombinant’ refers to the version of an amino acid sequence or nucleotide sequence which does not occur naturally. It is synthesised for the purpose of cloning and protein expression.

Sequences

[0032] SEQ ID NO: 1 depicts a nucleotide sequence of recombinant mature serratiopeptidase gene.

ATG GCC GCG ACA ACC GGC TAC GAT GCT GTA GAT GAC CTG CTG CAT TAT CAT GAG CGG GGT AAC GGG ATT CAG ATT AAT GGC AAG GAT TCA TTT TCT AAC GAG CAA GCT GGG CTG TTT ATT ACC CGC GAG AAC CAA ACC TGG AAC GGT TAC AAG GTA TTT GGC CAG CCG GTC AAA TTA ACC TTC TCC TTC CCG GAC TAT AAG TTC TCT TCC ACC AAC GTC GCC GGC GAT ACC GGG CTG AGC AAG TTC AGC GCG GAA CAG CAG CAG CAG GCT AAG CTG TCG CTG CAG TCC TGG GCT GAC GTC GCC AAT ATC ACC TTC ACC GAA GTG GCT GCC GGT CAA AAG GCC AAT ATC ACC TTC GGC AAT TAC AGC CAG GAT CGT CCC GGC CAC TAT GAT TAT GGT ACC CAG GCC TAC GCC TTC CTG CCG AAC ACC ATT TGG CAG GGC CAG GAT TTG GGC GGC CAG ACC TGG TAC AAC GTC AAC CAA TCC AAC GTG AAG CAT CCG GCG ACC GAA GAC TAC GGC CGC CAG ACG TTC ACC CAT GAG ATT GGC CAT GCG CTG GGC CTG AGC CAC CCG GGC GAC TAC AAC GCC GGT GAG GGC AAC CCG ACC TAT AAC GAC GTC ACC TAT GCG GAA GAT ACC CGC CAG TTC AGC CTG ATG AGC TAC TGG AGT GAA ACC AAC ACC GGT GGC GAC AAC GGC GGT CAC TAT GCC GCG GCT CCG CTG CTG GAT GAC ATT GCC GCC ATT CAG CAT CTG TAT GGC GCC AAC CTG TCG ACC CGC ACC GGC GAC ACC GTG TAC GGC TTT AAC TCC AAT ACC GGT CGT GAC TTC CTC AGC ACC ACC AGC AAT TCG CAG AAA GTG ATC TTT GCG GCC TGG GAT GCG GGT GGC AAC GAT ACC TTC GAC TTC TCC GGT TAT ACC GCT AAC CAG CGC ATC AAC CTG AAT GAG AAA TCG TTC TCC GAC GTG GGC GGC CTG AAG GGC AAC GTC TCG ATC GCC GCC GGT GTG ACC ATT GAG AAC GCC ATC GGC GGT TCC GGC AAC GAC GTG ATC GTC GGC AAC GCG GCC AAC AAC GTG CTG AAA GGT GGC GCG GGT AAC GAC GTG CTG TTA GGC GGC GGC GGG GCG GAT GAA CTG TGG GGC GGT GCC GGC AAA GAC ATC TTT GTG TTC TCT GCC GCC AGC GAT TCC GCA CCG GGT GCT TCC GAC TGG ATC CGC GAC TTC CAG AAA GGG ATC GAC AAG ATC GAC CTG TCG TTC TTC AAT AAA GAA GCG AAT AGC AGT GAT TTC ATC CAC TTC GTC GAT CAC TTC AGC GGC ACG GCC GGT GAG GCG CTG CTG AGC TAC AAC GCG TCC AGC AAC GTG ACC GAT TTG TCG GTG AAC ATC GGC GGG CAT CAG GCG CCG GAC TTC CTG GTG AAA ATC GTC GGC CAG GTA GAC GTC GCC ACG GAC TTT ATC GTG TAA.

[0033] SEQ ID NO: 2 depicts amino acid sequence of recombinant mature serratiopeptidase protein. MAATTGYDAVDDLLHYHERGNGIQINGKDSFSNEQ AGLFITRENQTWNGYKVFGQPIKLTFSFPDYKFSST NVAGDTGFSKFSAEQQQQAKFSFQSWADVANITFT EVAAGQKANITFGNYSQDRPGHYDYGTQAYAFFPN TIWQGQDFGGQTWYNVNQSNVKHPATEDYGRQTF THEIGHAFGFSHPGDYNAGEGNPTYRDVTYAEDTR QFSFMSYWSETNTGGDNGGHYAAAPFFDDIAAIQH FY GANFSTRTGDTVY GFN SNTGRDFFSTTSNSQKV IFAAWDAGGNDTFDFSGYTANQRINFNEKSFSDVG GFKGNVSIAAGVTIENAIGGSGNDVIVGNAANNVF KGGAGNDVFFGGGGADEFWGGAGKDIFVFSAASD SAPGASDWIRDFQKGIDKIDFSFFNKEAQSSDFIHF VDHFSGAAGEAFFSYNASNNVTDFSVNIGGHQAPD FFVKIVGQVDVATDFIV.

[0034] SEQ ID NO: 3 depicts a forward primer sequence for amplification of recombinant mature serratiopeptidase gene.

TATATTCATATGCCGCGACAACC .

[0035] SEQ ID NO: 4 depicts a reverse primer sequence for amplification of recombinant mature serratiopeptidase gene.

ATGT ACCTC G AGTT AC AC GAT A A AGT CC

[0036] Serratiopeptidase, a broad specificity metalloprotease of around 50kDa molecular mass is the major protease in extracellular secretion of Serratia marcescens. Apart from being a virulence factor, serratiopeptidase shows potent anti-inflammatory, analgesic, anti-edemic effects and constitutes an active ingredient in various drug combinations. In the present disclosure, the terms purified recombinant mature serratiopeptidase, purified serratiopeptidase, and recombinant serratiopeptidase have been used interchangeably.

[0037] The conventional approach of serratiopeptidase production requires large-scale fermentation using the wild type species of Serratia marcescens. Since the bacteria itself is a pathogen and associated with multiple modes of serious infections like pneumonia, empyema, urinary tract infection, corneal keratitis, meningitis, endocarditis, and septic arthritis; it is not advisable to use it as a source for serratiopeptidase production.

[0038] In addition to the drawbacks mentioned in the previous sections, purifying proteins from inclusion bodies is often a tedious job and has its own disadvantages, such as, washing, cleaning, solubilizing, refolding, and purification, additional tag processing, etc., thereby leading to additional costs and making the process less adaptive and economical. The present disclosure addresses the problem by disclosing a process for producing mature functional serratiopeptidase in E. coli. The process also discloses a stable E. coli based recombinant expression system producing functional serratiopeptidase. The present disclosure also discloses optimization method for the protein production to get an enhanced yield and a cost-effective purification procedure from the inclusion bodies. Since, E. coli is a well-established organism for commercial scale production of proteins and is not associated with risk of infections as compared to S. marcescens, the process as disclosed in the present disclosure holds significant advantage.

[0039] The present disclosure discloses a process for producing mature serratiopeptidase protein and specific parameters for getting desired yield of mature serratiopeptidase. The present disclosure also discloses a process for obtaining purified recombinant mature serratiopeptidase. The process discloses specific buffer compositions for purifying the recombinant serratiopeptidase protein.

[0040] In an embodiment of the present disclosure, there is provided a process for producing recombinant mature serratiopeptidase in E. coli, said process comprising: (a) obtaining recombinant E. coli cells capable of expressing recombinant mature serratiopeptidase having amino acid sequence as set forth in SEQ ID NO: 2; (b) growing the cells for a time period in a range of 14-18 hours to obtain an inoculum; (c) culturing the inoculum in a growth medium for a time period in a range of 2-4 hours to attain an optical density in a range of 0.6-0.8 to obtain a cell culture; (d) inducing the cell culture of step (c) with IPTG at a final concentration in a range of 0.1-2 mM with respect to the medium, to obtain an induced cell culture; and (e) culturing the induced cell culture of step (d) for a time period in a range of 2-16 hours at a temperature in a range of 16- 37 °C for producing recombinant mature serratiopeptidase in E. coli.

[0041] In an embodiment of the present disclosure, there is provided a process for producing recombinant mature serratiopeptidase in E. coli as described herein, wherein the recombinant E. coli cells is E. coli C43 (BL21-DE3).

[0042] In an embodiment of the present disclosure, there is provided a process for producing recombinant mature serratiopeptidase in E. coli as described herein, wherein the recombinant E. coli cells comprise a recombinant vector comprising a recombinant nucleotide having a sequence as set forth in SEQ ID NO: 1, operably linked to a promoter.

[0043] In an embodiment of the present disclosure, there is provided a process for producing recombinant mature serratiopeptidase in E. coli as described herein, wherein the recombinant E. coli cells comprise a recombinant vector comprising a recombinant nucleotide having a sequence as set forth in SEQ ID NO: 1, operably linked to a promoter, and wherein the promoter is either a T7 promoter or a pB AD promoter. In another embodiment of the present disclosure, the promoter is a T7 promoter.

[0044] In an embodiment of the present disclosure, there is provided a process for producing recombinant mature serratiopeptidase in E. coli as described herein, wherein the recombinant vector is selected from a group consisting of pET 23b, pET 28a, pET 28b, pET 23a, pET 22a, and pBAD vectors. In another embodiment of the present disclosure, the recombinant vector is pET 23b.

[0045] In an embodiment of the present disclosure, there is provided a process for producing recombinant mature serratiopeptidase in E. coli as described herein, wherein the recombinant E. coli cells comprise a recombinant vector comprising a recombinant nucleotide having a sequence as set forth in SEQ ID NO: 1, operably linked to a promoter, wherein the promoter is T7 promoter, and the recombinant vector is pET 23b.

[0046] In an embodiment of the present disclosure, there is provided a process for producing recombinant mature serratiopeptidase in E. coli as described herein, wherein the recombinant E. coli cells is E. coli C43 (BL21-DE3), and wherein the cells comprise a recombinant vector comprising a recombinant nucleotide having a sequence as set forth in SEQ ID NO: 1, operably linked to a promoter, and wherein the promoter is T7 promoter, and the recombinant vector is pET 23b.

[0047] In an embodiment of the present disclosure, there is provided a process for producing recombinant mature serratiopeptidase in E. coli as described herein, wherein culturing the cells to obtain an inoculum is at a temperature in a range of 25-37°C. In another embodiment of the present disclosure, culturing the cells to obtain an inoculum is at a temperature of 37°C.

[0048] In an embodiment of the present disclosure, there is provided a process for producing recombinant mature serratiopeptidase in E. coli as described herein, wherein the growth medium comprises: (a) tryptone having w/v percentage in a range of 0.1 -2.0% with respect to the medium; (b) yeast extract having w/v percentage in a range of 0.5-2.0% with respect to the medium; and (c) NaCl having weight percentage in a range of 0.2- 1.0% with respect to the medium. In another embodiment of the present disclosure, the growth medium comprises: (a) tryptone having w/v percentage in a range of 0.5- 1.5% with respect to the medium; (b) yeast extract having w/v percentage in a range of 0.75-1.5% with respect to the medium; and (c) NaCl having weight percentage in a range of 0.4-0.8% with respect to the medium.

[0049] In an embodiment of the present disclosure, there is provided a process for producing recombinant mature serratiopeptidase in E. coli as described herein, wherein culturing the induced cell culture is for a time period in a range of 4-6 hours at a temperature of 30°C.

[0050] In an embodiment of the present disclosure, there is provided a process for obtaining purified recombinant mature serratiopeptidase from E. coli, said process comprising: (a) obtaining E. coli cells expressing recombinant mature serratiopeptidase having amino acid sequence as set forth in SEQ ID NO: 2; (b) harvesting the cells to obtain a pellet and contacting the pellet with a buffer TS comprising: Tris: lO-lOOmM, NaCl: l00-500mM, Beta-mercaptoethanol: 1- lOmM, having pH in a range of 6.8-8.0, to obtain inclusion bodies; (c) contacting the inclusion bodies with a buffer TEST comprising: Tris: lO-lOOmM, NaCl: l00-600mM, Beta-mercaptoethanol: l-lOmM, EDTA: l-5mM, Glycerol: 0.5- 3%, Triton-X-lOO: 1-3%, Urea: 0.5-4M, having pH in a range of 6.8-8.0, to obtain washed inclusion bodies; (d) contacting the washed inclusion bodies with a buffer D comprising: Tris: lO-lOOmM, NaCl: l00-600mM, EDTA: l-5mM, Glycerol: 0.1-1.0%, Urea: 4-8M or Guanidinium hydrochloride: 3-7M, having pH in a range of 6.0-8.0, to obtain solubilized inclusion bodies; (e) contacting the solubilized inclusion bodies with a buffer R comprising: Tris: lO-lOOmM, CaCl₂: 5-20mM, ZnCl₂: l-5mM, NaCl: lOO-lOOOmM, Glycerol: 0.1- 1.0%, having pH in a range of 6.0-8.0, to obtain refolded inclusion bodies; and (f) performing gel filtration chromatography using the refolded inclusion bodies with a purification buffer comprising: Tris: 50mM, NaCl: l50mM, CaCl₂: 5mM, ZnCl₂: 2mM, having pH-7.4, to obtain purified recombinant mature serratiopeptidase.

[0051] In an embodiment of the present disclosure, there is provided a process for obtaining purified recombinant mature serratiopeptidase from E. coli, said process comprising: (a) obtaining E. coli cells expressing recombinant mature serratiopeptidase having amino acid sequence as set forth in SEQ ID NO: 2; (b) harvesting the cells to obtain a pellet and contacting the pellet with a buffer TS comprising: Tris: 30-70mM, NaCl: 200-400mM, Beta-mercaptoethanol: 2- 8mM, having pH in a range of 6.8-8.0, to obtain inclusion bodies; (c) contacting the inclusion bodies with a buffer TEST comprising: Tris: 30-70mM, NaCl: 200- 500mM, Beta-mercaptoethanol: 3-7mM, EDTA: 2-4mM, Glycerol: 1-2.5%, Triton-X-lOO: 1.5-2.5%, Urea: 1.5-3M, having pH in a range of 6.8-8.0, to obtain washed inclusion bodies; (d) contacting the washed inclusion bodies with a buffer D comprising: Tris: 30-70mM, NaCl: 200-500mM, EDTA: 2-4mM, Glycerol: 0.2-0.7%, Urea: 5-8M or Guanidinium hydrochloride: 4-6M, having pH in a range of 6.0-8.0, to obtain solubilized inclusion bodies; (e) contacting the solubilized inclusion bodies with a buffer R comprising: Tris: 30-70mM, CaCl₂: 7-l5mM, ZnCl₂: 2-4mM, NaCl: 200-800mM, Glycerol: 0.2-0.7%, having pH in a range of 6.0-8.0, to obtain refolded inclusion bodies; and (f) performing gel filtration chromatography using the refolded inclusion bodies with a purification buffer comprising: Tris: 50mM, NaCl: l50mM, CaCh: 5mM, ZnCl₂: 2mM, having pH-7.4, to obtain purified recombinant mature serratiopeptidase.

[0052] In an embodiment of the present disclosure, there is provided a process for obtaining purified recombinant mature serratiopeptidase from E. coli, said process comprising: (a) obtaining E. coli cells expressing recombinant mature serratiopeptidase having amino acid sequence as set forth in SEQ ID NO: 2; (b) harvesting the cells to obtain a pellet and contacting the pellet with a buffer TS comprising: Tris- 50mM, NaCl- 350mM, Beta-mercaptoethanol- 5mM, pH-8.0, to obtain inclusion bodies; (c) contacting the inclusion bodies with a buffer TEST comprising: Tris- 50mM, EDTA- 5mM, NaCl- 500mM, Glycerol: 2%, Beta-mercaptoethanol- 5mM, Triton-X-lOO- 1.5%, ETrea: 2.5M, pH- 8.0, to obtain washed inclusion bodies; (d) contacting the washed inclusion bodies with a buffer D comprising: Tris- 50mM, EDTA- 5mM, NaCl- 500mM, Glycerol: 0.5%, Urea: 8M/ Guanidinium hydrochloride: 7M, pH-6.8, to obtain solubilized inclusion bodies; (e) contacting the solubilized inclusion bodies with a buffer R comprising: Tris- 50mM, CaCl₂- lOmM, ZnCl₂- 2mM, NaCl- 500mM, Glycerol: 0.5%, pH-6.8, to obtain refolded inclusion bodies; and (f) performing gel filtration chromatography using the refolded inclusion bodies with a purification buffer comprising: Tris- 50mM, NaCl- l50mM, CaCl₂- 5mM, ZnCl₂- 2mM, having pH-7.4, to obtain purified recombinant mature serratiopeptidase.

[0053] In an embodiment of the present disclosure, there is provided a process for obtaining purified recombinant mature serratiopeptidase from E. coli as described herein, wherein the purified serratiopeptidase has a purity in a range of 90%-98%. In another embodiment of the present disclosure, the purified serratiopeptidase has a purity in a range of 92%-96%.

[0054] In an embodiment of the present disclosure, there is provided a process for obtaining purified recombinant mature serratiopeptidase from E. coli as described herein, wherein the purified serratiopeptidase has an activity in a range of 2100 - 2400 EU/mg. In another embodiment of the present disclosure, the purified serratiopeptidase has an activity in a range of 2200 - 2300 EU/mg.

[0055] In an embodiment of the present disclosure, there is provided a process for obtaining purified recombinant mature serratiopeptidase from E. coli as described herein, wherein the purified serratiopeptidase has a viable bacterial count in a range of 0-20. In another embodiment of the present disclosure, the purified serratiopeptidase has a viable bacterial count in a range of 5-15.

[0056] In an embodiment of the present disclosure, there is provided a process for obtaining purified recombinant mature serratiopeptidase from E. coli as described herein, wherein the recombinant mature serratiopeptidase has a yield in a range of 30-40mg/litre. In another embodiment of the present disclosure, the recombinant mature serratiopeptidase has a yield in a range of 32-38mg/litre.

[0057] Although the subject matter has been described in considerable detail with reference to certain examples and implementations thereof, other implementations are possible.

EXAMPLES

[0058] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may apply.

[0059] The present section discloses the importance of steps involved in the process of the present disclosure for obtaining the desired yield of serratiopeptidase and process for purifying the serratiopeptidase protein. Described herein is the entire process starting from development of the construct for protein expression to the purification steps along with the crucial parameters.

Example 1

Development of recombinant construct

[0060] Serratia marcescens MTCC7298 strain initially isolated from the Tasar silkworm gut by Dr. N. Suryanaryan; Central Tasar Research & Training Institute, Piska Nagri, Ranchi, Jharkhand was procured from MTCC, Institute of Microbial Technology Chandigarh. Strain shows casein hydrolysis because of its protease secretion ability. Initial screening by SDS-PAGE, in-gel trypsin digestion and mass spectroscopy of digested fragments confirms serratiopeptidase is the major protease in secretion.

[0061] Genomic DNA of the Serratia marcescens MTCC7298 was used for gene amplification and further for developing recombinant construct.

Gene amplification

[0062] Primers specific to mature serratiopeptidase gene was used for gene amplification by PCR. The protocol follows initial denaturation at 98 °C for 3 minutes and then cycle of denaturation, annealing and extension with a final round of extension for 10 minutes. Annealing temperature for successful amplification falls in between 55-65 °C.

Restriction digestion and ligation

[0063] Amplified gene product was cleaned, and restriction digestion was performed using suitable enzymes and then ligated following the conventional T4 DNA ligase-based ligation at 16 °C for overnight duration. The ligation mixture was used as genetic material for transforming high efficiency E. coli competent cells.

Verification of cloning

[0064] Transformants on the plate were selected and screened for presence of mature serratiopeptidase gene through colony PCR and further, plasmid preparation from these positive clones were analyzed by restriction digestion. [0065] Additional confirmation of successful cloning and to screen the clones for not having any mutation or discrepancies, recombinant construct was sequenced using T7 promoter and terminator as specific primer.

Results

[0066] Figure 1 depicts a flowchart for obtaining a mature recombinant serratiopeptidase protein. As a first step, a stable clone was obtained following a method for optimizing protein expression as described above. After optimizing the expression, a process was optimized to obtain a high yield of active purified protein.

[0067] Figure 2 depicts a map of the recombinant construct comprising the gene encoding mature serratiopeptidase protein in pET23b(+) vector.

[0068] Figure 3 depicts genomic DNA of Serratia marcescens MTCC7298 which was used as a template for amplification of mature serratiopeptidase gene. PCR was performed using the primer sequences as depicted in SEQ ID NO: 3 and SEQ ID NO: 4. Desired amplicon was obtained at a range of different temperatures (55-65 °C) (Figure 4).

[0069] The insert was cloned in the vector pET23b (+), and successfully transformed into E. coli DH5-a cells. The clone was confirmed by performing colony PCR and Figure 5 depicts the positive clones obtained by the process.

[0070] The obtained clone was also confirmed by performing restriction digestion of the intact plasmid obtained with the insert encoding the recombinant mature serratiopeptidase protein. The enzymes used for digestion were Ndel and Xhol. The insert was observed after digesting the plasmid which confirmed the presence of the clone in the vector (Figure 6).

Example 2

Optimization of protein expression

Transformation and protein expression

[0071] The recombinant expression construct designated as pMHSrp7298 containing the gene for mature serratiopeptidase product was transformed into different E. coli expression hosts, and strains were selected on the basis of successful transformation. Transformation was repeated in triplicates independently. The result suggests the expression construct was either unstable or toxic causing the cell death and further optimization of expression and transformation conditions is required.

Optimization of expression conditions

[0072] Host optimization: Different commercially available E. coli expression hosts were used in host optimization. Criteria of successful transformation and expression of correct product was taken into consideration for selecting out the best host.

[0073] Induction point: IPTG based induction was chosen for overexpression of protein and different optical density points; ranges between ODeoo_nm 0.1- 1.0 were taken as time point to induce the growing culture of pMSrp7298 harboring E. coli cells. Overexpression of protein was analyzed by SDS-PAGE for each condition after growing for certain time period (constant for each induced fraction). Densitometric analysis of the obtained protein band was taken as criteria to determine the correct optimized induction point.

[0074] Media composition: Varying concentration of carbon, nitrogen and salts at different variations were tried to optimize the growth media for overexpression of mature serratiopeptidase. Culture was induced in each growth media as pre-determined induction point and grown for certain time period (2- 16 hours). Overexpression of recombinant protein was analyzed through SDS- PAGE, further densitometric analysis of overexpressed protein bands was taken as analysis criteria to select out the best growth medium for mature serratiopeptidase production. The optimal growth medium for maximal expression has following composition as described in Table 1.

Table 1:

[0075] Temperature optimization: The next physicochemical criteria after growth media was temperature for optimizing the overexpression of serratiopeptidase. Mature serratiopeptidase recombinant construct transformed into proper host cells was allowed to grow in media until induction point was reached. Thereafter, IPTG was used for induction and small aliquots were transferred at different temperature points (l6-37°C) under shaking conditions for certain time period.

[0076] Protein overexpression was checked on SDS-PAGE and densitometric analysis of overexpressed protein band was used to select the best temperature condition for maximal overexpression of serratiopeptidase.

[0077] Inducer concentration: Mature serratiopeptidase expression construct harboring host cells were grown in optimized growth media until ODeoo_nm of culture reached to induction point. Culture was transferred in equal amount in different culture tubes and induced with variable concentration of IPTG inducer ranging between O.lmM to 2.0mM. After certain period of time, induced samples were collected, and SDS-PAGE analysis was done to determine the optimal IPTG concentration for maximal intracellular overexpression of mature serratiopeptidase.

[0078] Post induction duration: Post induction duration is an important criterion when expressing a recombinant protein in any heterologous expression system. It is required to arrive at the correct post induction duration for protein over-expression to ensure maximal expression obtained without any degradation/ loss of the desired protein product. Transformed E. coli cells were allowed to grow at optimized conditions and induced using optimal concentration of IPTG. Further culture was allowed to grow for overexpression of recombinant protein. The culture was allowed to induce and grow for 2-20 hours duration. SDS-PAGE of collected samples at different time points after induction was analyzed for maximal protein expression and best post induction duration for serratiopeptidase overexpression was taken.

Results

[0079] The present study tried to express the construct having mature serratiopeptidase gene in different host cells as depicted in Table 2:

Table 2:

[0080] The construct was transformed independently in strains I to IV and checked for transformants and subsequent expression of desired protein. Since serratiopeptidase is a known broad specificity protease expressed extracellularly in native strains, overexpression intracellularly would cause the degradation of inner proteome of transformed bacteria. This will not only hamper the growth but will be lethal for viability and survival of bacteria. Similar results were obtained when the construct was transformed into different host systems, the number of transformants were very less in comparison to only vector, suggesting either the gene or gene product is toxic for cell survival (Figure 7). To circumvent the problem of expression, the transformation was checked in different strains. In the present disclosure, it was observed that maximum number of transformants was obtained in strain IV - E. coli C43 as compared to the other host strains (Figure 7). As a next step, protein expression was checked in the three strains. Figure 8 depicts protein gels showing expression of proteins in strains II, III, and IV, no expression was observed in strain I. It was observed that protein of lower molecular weight than expected was obtained in case of strains II and III. Only strain IV expressed the protein of correct molecular weight. Therefore, E. coli C43 was used as the host cell for expression of recombinant serratiopeptidase for further studies.

[0081] The expression of the protein was optimized for different parameters of induction point, media used, temperature for incubation, concentration of inducer, and post-induction time period.

[0082] The parameters were optimized, and the optimized conditions are summarized below:

• Inoculum: Overnight (16 hour) grown culture of single individual transformed colony (ODeoo_nm >2.0), inoculum size: 2%

• Pre-induction duration: 2-4 hour until ODeoo_nm reaches >0.6 but less than 0.8.

• Point of Induction: OD₆oo_nm 0.1- 1.0 (0.8>POI>0.6).

• Post induction duration: 2-16 hours (4-6 hours).

• Pre-induction temperature: 37 °C

• Post-induction temperature: 16-37 °C (30 °C).

• Inducer Used: Isopropyl b-D-l-thiogalactopyranoside (IPTG)

• Inducer concentration: Range O.lmM to 2.0mM (0.6mM)

[0083] The protein expression was optimized, and it can be observed in Figure 9 that in the presence of different concentrations of IPTG, the protein expression was obtained. In Figure 9, the Lanes Cl to C20 relates to different concentration of IPTG used for inducer concentration optimization ranging in between 0. lmM to 2.0mM.

[0084] The protein expression obtained after optimization was further considered for purification.

Example 3

Purification of mature recombinant serratiopeptidase

[0085] One-liter nutrient broth was inoculated using 1-3% of 12 hours grown primary culture having ODeoo_nm >2.00 as inoculum. The culture was allowed to grow until mid-late log phase at 37 °C at 220rpm, and then IPTG was added for induction post which the culture was transferred for desired time period at optimized temperature point. Bacterial cells were separated though centrifugation at 23000xg for 20 minutes. The supernatant was discarded, and the pellet was stored at -20 °C until further used.

[0086] Cell lysis: Obtained cell pellet was resuspended in TS buffer (pH-6.8- 8.0) using 20ml resuspension buffer for per gram of cell pellet. Resuspended cell pellet was frozen at -20 to -80 °C for one hour and then quickly allowed to thaw. Lysozyme stock was added to the final concentration of 0.2-lmg/ml and the whole resuspension was incubated at 4 °C for 30 minutes to 2 hours on Rotaspin (5-20rpm). Final cell lysis was performed by sonication (15-35% amplitude ten cycles 10 seconds ON 50 second OFF). Supernatant was separated from the insoluble pellet by centrifuging the solution at 23000x g for 20 minutes at 4 °C and location of protein was determined by SDS-PAGE analysis. The densitometric analysis revealed that the protein was in insoluble fraction and purity falls between 65-70%. TS buffer comprises: Tris- 50mM, NaCl- 350mM, Beta-mercaptoethanol- 5mM, pH- 8.0.

[0087] Washing of inclusion bodies: Inclusion bodies were washed with 30ml volume of TEST buffer (pH 6.8-8.0,) and then further washed twice using resuspension buffer. A small fraction of inclusion body was run on SDS-PAGE gel and purity was determined, which comes equivalent to 80-89%. TEST buffer comprises: Tris- 50mM, EDTA- 5mM NaCl- 500mM, Glycerol- 2%, Beta- mercaptoethanol- 5mM, Triton- X-100- 1.5%, Urea- 2.5Molar, pH- 8.0.

[0088] Refolding conditions: Washed inclusion bodies were dissolved in 2ml/gram buffer-D (pH 6.8-8.0), and total protein concentration was determined using Bradford assay. Dissolved inclusion bodies were found to contain about 90-1 lOmg of protein per liter of culture volume. The buffer D comprises: Tris- 50mM, EDTA- 5mM NaCl- 500mM, Glycerol: 0.5%, Urea: 8M/ Guanidinium hydrochloride: 7M, pH- 6.8.

[0089] The solubilized inclusion bodies were diluted further up to lOmg/ml concentration and then allowed to refold by dialysis against buffer-R (pH- 6.8) for overnight duration. The refolded inclusion body preparation was stored at 4 °C. The buffer R comprises: Tris- 50mM, CaCl₂- lOmM, ZnCl₂- 2mM, NaCl- 500mM, Glycerol: 0.5%, pH-6.8. The process of refolding of inclusion body is highly important for proper conformation and functional activity of mature serratiopeptidase. Proper concentration of proteins in solubilized inclusion bodies sample is necessary to allow optimal folding. The composition of refolding buffer (Buffer-R) and buffer used during gel filtration is highly specific for this protein and any difference results in the precipitation/ aggregation of protein leading to the formation of non-functional protein precipitate.

[0090] The size exclusion chromatography using Sephadex G-75 column was performed for further purification from refolded inclusion body. The single peak on FPLC showing serratiopeptidase has been collected, and protein purity was >95% on SDS-PAGE through Coomassie staining. Purified fractions were stored at 4 °C and further lyophilized after dialysis against water.

[0091] Yield: Yield of serratiopeptidase was compared to yield from Serratia marcescens MTCC 7298 strain extracellular production.

[0092] Cost Effectiveness: Since industrial production of serratiopeptidase employs Serratia marcescens wild strains and production is extracellular, it leads to a large accumulation of biomass (cellular debris) and requires additional cost to dispose of. Additional key cost criteria and their comparison is given in Table 3.

[0093] Table 3:

[0094] Bacterial Viable count: According to Indian Pharmacopeia; 2010 the number of viable bacteria per ml of a solution of serratiopeptidase should not contain >100 bacteria. Standard spread plate method was used for analyzing the bacterial load on the purified recombinant serratiopeptidase obtained by the disclosed process.

[0095] Activity Assay: Serratiopeptidase is a proteolytic enzyme used as an anti-inflammatory drug. The concentration of refolded serratiopeptidase solution was measured using an A₂so method using the extinction coefficient value obtained from protparam tool according to the sequence. The concentration was further verified using Bradford assay. The activity assay was determined as mentioned in Indian pharmacopeia (2010) according to Hammersten (Hammersten (1883); Zeitschr. f. physiol. Chem., vii, p. 227), using casein as substrate. The standard curve was plotted using tyrosine solution and the enzyme units were determined using suitable dilutions of the purified enzyme. 1EU is equivalent to the amount of enzyme liberating lpg tyrosine under reaction conditions (Shimogaki et al.; Purification and properties of a novel surface- active agent- and alkaline-resistant protease from Bacillus sp. Y. Agri. Biol Chem. Sep. 55(9):225l-8, 1991).

[0096] Protocol for determining the activity: Protease activity was determined as described by Hagihara et al. (1958), 500ul of suitably diluted enzyme was mixed with 500ul of casein (1% w/v prepared in 50 mM carbonate -bicarbonate buffer of pH- 10.0). The mixture was incubated at 37 °C for 10 min. The reaction was quenched by adding 1.5 ml of 10 % pre-chilled trichloroacetic acid (TCA). The reaction was allowed for 30 min to completely precipitate the proteins. The contents of the reaction tubes were centrifuged and filtered through 0.2um syringe filter. The absorbance of the filtrate was read at 280 nm which was extrapolated against tyrosine standard curve (Figure 13). A unit of protease activity was defined as the amount of enzyme liberating lpg tyrosine/ml/min under the assay conditions. Results

[0097] Figure 10 depicts the presence of protein after washing of the inclusion bodies. As the protein was intact, further steps were followed.

[0098] Figure 11 depicts the purified protein as obtained after applying size exclusion chromatography. It can be appreciated that intact protein was obtained after the chromatography. Bradford assay was used for protein estimation and total protein concentration obtained from one liter of culture ranged between 30- 40mg/liter.

[0099] The yield measured through densitometric analysis as well as Bradford assay suggests, 69-folds increase (Table 4) and 10 times respectively.

Table 4:

* Measured using 66.2 Kda marker band as reference in given range

#Measured by taking Bradford assay of extracellular media in case of wild

Serratia and whole cell lysate for recombinant cells and then multiplying with percentage of serratiopeptidase in whole protein content.

[00100] For the analysis of bacteria viable count, standard spread plate method was used, and the number of the viable counts was less than lOOCFU/ml.

[00101] For the analysis of serratiopeptidase activity assay, the refolded serratiopeptidase was active and activity was found to be ranging in between 2 l00-2400U/mg which is equivalent or higher than the standard value mentioned

(2000U/mg). Table 5 describes the activity of recombinant protein obtained for triplicates. Table 5:

[00102] Figure 12 depicts a comparison of protein expression between that of the wild type S. marcescens to that of the recombinant system of the present disclosure. The analysis was done using gel documentation through densitometry. In brief, protein sample obtained from 6ml supernatant of wild Serratia marcescens was dissolved in 60pl of tris buffer and then 2x loading dye was added. Of which 20m1 was loaded on SDS-PAGE gel. Similarly, cell pellet of 60ul of induced bacteria was resuspended in 20m1 lx loading dye and loaded. Image was taken from biorad gel documentation system. Lanes and band tool were used to mark band and lanes. Background was normalized and band intensity of both was taken using quantitate tool. 66.2Kda marker band was taken as reference. The total protein concentration was measured using Bradford assay and multiplied with the intensity of individual protein band of interest, that is, serratiopeptidase.

[00103] The present disclosure discloses a method for recombinant production of mature form of serratiopeptidase in its functional state. The process could aid in circumventing the use of already established use of wild pathogenic strains of Serratia marcescens for mass production. Additionally, use of mature product gene also by passes the further processing of protein molecules to remove the pro-peptide part from the N-terminal. Overall, it can be appreciated that the process of the present disclosure leads to production of active serratiopeptidase which has desirable purity, yield, and viable bacterial count.

Advantages of the present disclosure:

[00104] The present disclosure discloses a process for producing a recombinant mature serratiopeptidase protein from E. coli expression host cell. Also, the disclosure describes a method for purifying the recombinant serratiopeptidase using optimized buffer compositions to obtain purified protein having desirable and acceptable properties. Significant advantages of the present process are: (i) it provides a process which utilizes non-pathogenic stable E. coli expression system which is easy to handle and not associated with issues of pathogenicity and disposal of increased biomass, as these issues brings down the viability of the process; (ii) the present disclosure describes a process for obtaining active mature recombinant serratiopeptidase thereby, avoiding the steps involving processing to make the protein active; (iii) the present disclosure provides a relatively simple method for purifying serratiopeptidase from the inclusion bodies, as the conventional process of purifying can be tedious and time consuming; and (iv) the present process provides recombinant mature serratiopeptidase that has improved yield, activity, and very less viable bacterial count. Thus, it can be summarized that the present disclosure provides a method for production of mature serratiopeptidase and purification of the protein which is economically favorable and desirable, and also circumvents the problem that occurs in the prior art.

Claims

I/We Claim:

1. A process for producing recombinant mature serratiopeptidase in E. coli, said process comprising:

a) obtaining recombinant E. coli cells capable of expressing recombinant mature serratiopeptidase having amino acid sequence as set forth in SEQ ID NO: 2;

b) growing the cells for a time period in a range of 14-18 hours to obtain an inoculum;

c) culturing the inoculum in a growth medium for a time period in a range of 2-4 hours to attain an optical density in a range of 0.6- 0.8 to obtain a cell culture;

d) inducing the cell culture of step (c) with IPTG at a final concentration in a range of 0.1-2 mM with respect to the medium, to obtain an induced cell culture; and

e) culturing the induced cell culture of step (d) for a time period in a range of 2-16 hours at a temperature in a range of 16-37 °C for producing recombinant mature serratiopeptidase in E. coli.

2. The process as claimed in claim 1, wherein the recombinant E. coli cells is E. coli C43 (BL21-DE3).

3. The process as claimed in claim 1, wherein the recombinant E. coli cells comprise a recombinant vector comprising a recombinant nucleotide having a sequence as set forth in SEQ ID NO: 1, operably linked to a promoter.

4. The process as claimed in claim 3, wherein the promoter is either a T7 promoter or a pBAD promoter.

5. The process as claimed in claim 1, wherein the recombinant vector is selected from a group consisting of pET 23b, pET 28a, pET 28b, pET 23a, pET 22a, and pBAD vectors.

6. The process as claimed in claim 1, wherein growing the cells to obtain an inoculum is at a temperature in a range of 25-37 °C.

7. The process as claimed in claim 1, wherein the growth medium comprises: (a) tryptone having w/v percentage in a range of 0.1-2.0% with respect to the medium; (b) yeast extract having w/v percentage in a range of 0.5-2.0% with respect to the medium; and (c) NaCl having weight percentage in a range of 0.2- 1.0% with respect to the medium.

8. The process as claimed in claim 1, wherein culturing the induced cell culture is for a time period in a range of 4-6 hours at a temperature of 30 °C.

9. A process for obtaining purified recombinant mature serratiopeptidase from E. coli, said process comprising:

a) obtaining E. coli cells expressing recombinant mature serratiopeptidase having amino acid sequence as set forth in SEQ ID NO: 2;

b) harvesting the cells to obtain a pellet and contacting the pellet with a buffer TS comprising: Tris: lO-lOOmM, NaCl: 100- 500mM, Beta-mercaptoethanol: l-lOmM, having pH in a range of 6.8-8.0, to obtain inclusion bodies;

c) contacting the inclusion bodies with a buffer TEST comprising:

Tris: lO-lOOmM, NaCl: l00-600mM, Beta-mercaptoethanol: 1- lOmM, EDTA: l-5mM, Glycerol: 0.5-3%, Triton-X-lOO: 1-3%, Urea: 0.5-4M, having pH in a range of 6.8-8.0, to obtain washed inclusion bodies;

d) contacting the washed inclusion bodies with a buffer D comprising: Tris: lO-lOOmM, NaCl: l00-600mM, EDTA: 1- 5mM, glycerol: 0.1 -1.0%, Urea: 4-8M or Guanidinium hydrochloride: 3-7M, having pH in a range of 6.0-8.0, to obtain solubilized inclusion bodies;

e) contacting the solubilized inclusion bodies with a buffer R comprising: Tris: lO-lOOmM, CaCl₂: 5-20mM, ZnCl₂: l-5mM, NaCl: lOO-lOOOmM, Glycerol: 0.1-1.0%, having pH in a range of 6.0-8.0, to obtain refolded inclusion bodies; and f) performing gel filtration chromatography using the refolded inclusion bodies with a purification buffer comprising: Tris: 50mM, NaCl: l50mM, CaCh: 5mM, ZnCb: 2mM, having pH- 7.4, to obtain purified recombinant mature serratiopeptidase.

10. The process as claimed in claim 9, wherein the purified serratiopeptidase has a purity in a range of 90%-98%.

11. The process as claimed in claim 9, wherein the purified serratiopeptidase has an activity in a range of 2100 - 2400 EU/mg.

12. The process as claimed in claim 9, wherein the purified serratiopeptidase has a viable bacterial count in a range of 0-20.

13. The process as claimed in claim 1, wherein the recombinant mature serratiopeptidase has a yield in a range of 30-40mg/litre.