CHAPTER 6 APPLICATION OF PROTEASE IN LEATHER PROCESSING AND TISSUE CULTURE

Transcription

1 152 CHAPTER 6 APPLICATION OF PROTEASE IN LEATHER PROCESSING AND TISSUE CULTURE 6.1 DEHAIRING OF GOAT SKINS AND COW HIDES Introduction Leather manufacturing process is one of the highly polluting industrial activities. Dehairing of skins/hides by conventional chemical methods is the major cause of pollution in the leather processing industries. The extensive use of sulfide not only leads to unfavorable consequences on the environment but also undermines the efficacy of the effluent treatment plants (Bailey et al 1982). Hence, rationalization of dehairing process by systemic use of proteases in place of lime and sulfide becomes an issue of primary importance in leather processing (Puvanakrishnan and Dhar 1988). This will lead to a substantial reduction of effluent load and toxicity in addition to improvement in leather quality (Puvanakrishnan and Dhar 1986). Many biological systems and tools had been used in leather manufacturing. The earliest document informs that excretion of animals and birds were used. Americans had been practicing sweating method for depilation of animal skins. Depilation was achieved by the confined action of autolytic enzymes and the enzymes secreted by wild bacterial species grown on the skins (Taylor et al 1987). Practice of this method dates back to paleolithic times and finds a place in commercial application for depilation of sheep skins (Dhar 1974). Use of pancreatic enzymes for depilation had been

2 153 studied first by Rohm (1913). Pancreatic enzyme was used for depilation and the quality of the leather produced was tested. Pig skins were depilated using serine pancreatic enzymes and the depilatory action was attributed to proteolytic, lipolytic and amylolytic properties of the enzyme. Depilation time was considerably shortened by pretreatment of skins with 4% calcium hydroxide followed by 1.5% of ammonium sulfate. Microbial enzymes are by far the most important of all commercial enzymes. It was observed that enzyme from A. oryzae, A. parasiticus, A. fumigatus, A. effusus, A. ochraceus, A. wentii and P. griseofulvum exhibited satisfactory depilatory effect on sheep skins (Gillespie 1953). Bacterial enzymes gain much commercial interest on account of a) easy production by submerged fermentation, b) relatively higher yield, c) relatively shorter production time and d) easy recovery of product. Proteases can be used in major steps of leather processing, such as neutral proteases in soaking (Laxman et al 2004) and alkaline proteases in dehairing (Dayanandan et al 2003). Despite the great deal of developments in the area of depilatory enzymes, enzymatic depilation has not been widely accepted for commercial practice. There are two major factors attributable to this. Primarily the cost of the enzymes is much exorbitant compared to the chemicals presently used for depilation and secondly the enzymatic depilation process demands much stringent process control compared to the chemical depilation systems. Cost of production media is one of the cost centres in enzyme production. Reduction in the cost of media therefore can bring down the cost of the enzyme substantially. In this work, the protease prepared using a cost-effective medium was studied for the application of depilation of cow hides and goat skins and

3 154 the de-haired pelt has been assessed for quality by scanning electron microscopy (SEM) and histological studies Materials and Methods Materials Freshly flayed wet-salted goatskins and cowhides were used for depilation experiments. Protease enzyme used in this study was obtained from Bacillus pumilus MTCC Other chemicals used in this study were of commercial and analytical grade Protease enzyme for dehairing of skins and hides The crude, ultrafiltered and partially purified enzyme (precipitation and flocculation) having activity in the range of 25 U/mL to 1200 U/g was used for depilation of skins/hides. One unit of protease activity was defined as the liberation of one mg of tyrosine equivalent of casein substrate per ml of enzyme solution under standard assay conditions Depilation with precipitated/flocculated enzyme Two sets of experiments, enzymatic and conventional (lime and sulfide) depilation of cow hide and goat skin were carried out simultaneously. Two wet-salted fresh goat skin and cow hides were taken and soaked overnight employing 300% of water. The soaking liquid was maintained at ph 9.0 with sodium carbonate. The skins and hides were washed thoroughly. Cow hides were cut into two parts (right and left) from the middle. The soaked weights of right and left parts were noted. The right part of the cow hide was used for enzymatic depilation whereas the left was used for the conventional depilation. One goat skin was taken for enzymatic and another one for conventional depilation. Enzymatic depilation of goat skin was done

4 155 by paste method while for cow hide drum method was used. For conventional depilation of goat skin and cow hides, dip method was used. For enzymatic depilation of cow hide, protease enzyme at the concentration of 3% (w/w) was taken, dissolved in little amount of water and added to the drum along with 20% of water and cow hides. Drum was rotated at 4 rpm and set to 10 min ON and 50 min OFF per hour for 6 h. Hair loosening was checked at each hour. For enzymatic depilation of goat skin, 3% (w/w) protease was taken and a paste was prepared with sufficient water and applied to the flesh side of goat skin, incubated at ambient temperature (28-32 C) for 6 h and hair loosening was observed at each hour and finally loosened hair was removed using conventional beam and knife method. For the conventional depilation process of cow hides and goat skins 10% lime and 3% sodium sulfide were added along with 200% water and the paddle was run for 10 min for every hour for 18 hours. The pelts were then scudded Depilation with different enzyme formulations Depilation of cow hides was carried out with different formulations of enzyme (Crude, ultra-filtered and spray dried enzyme) as shown in Table 6.1. The crude enzyme having enzyme activity of 40 U/mL was formulated by the addition of 10% (w/v) TATA salt, 0.1 % Bronopol and 0.2% sodium benzoate and stored in a cold room. All the experiments were carried out by drum method with intermittent rotation at 4 rpm, with 10 min ON and 50 min OFF per hour timer control. Hair loosening activity and depilation were monitored. The experimental conditions and results are shown in Table 6.1.

5 Histological analysis Samples from enzymatic as well as conventional de-haired pelt of cow hide and goat skin were cut to a size of 1 cm 2 area, washed thoroughly with distilled water and were fixed in formal saline (0.9% sodium chloride solution in 10% formaldehyde). Samples were then dehydrated with ethanol and used for the histological study. Dehydrated skin/hide samples were fixed in paraffin block and section of 10, 20 and 100 µm were obtained using microtome. The samples were then stained using hematoxylin and eosin to examine histological features. Sections were also stained by the following method to study the skin constituents after dehairing process (Bancroft and Gamble, 2004): Masson s trichrome staining for collagen and Verhoeff s staining for elastin. These sections were examined under microscope and pictures of stained skins were taken Scanning electron microscopic (SEM) analysis of dehaired pelt Samples from enzymatic as well as conventional de-haired pelts of cow hide and goat skin were cut, washed properly with distilled water, fixed in buffered formalin, dehydrated with a series of methanol and then finally with acetone. After that samples were flushed with nitrogen gas to remove the acetone completely and freeze dried. The freeze dried samples were cut into 3-4 mm thickness, mounted vertically or horizontally on copper stubs, coated with platinum. Cross and surface view of samples was examined in a FEI Quanta 200 series Environmental SEM unit operated at an accelerating voltage of 12 kv.

6 Analysis of pollution parameters of effluent generated by depilation process The effluent generated from the dehairing of cow hides by enzymatic as well as conventional method were analysed for pollution parameters viz BOD (Biological oxygen demand), COD (Chemical oxygen demand), TDS (Total dissolved solids), TSS (Total suspended solids), and sulphide. The analysis of effluent was done at Tamilnadu Pollution Control Board, Guindy, Chennai Results and Discussion Depilation of goat skins and cow hides with precipitated enzyme The enzyme (1200 U/g) obtained by precipitation and flocculation was used for dehairing of cow hide as well as goat skin. Enzymatic dehairing of cow hide was done by drum method at room temperature. Dehairing was observed at each hour. Figure 6.1a indicates the raw hide which was processed for depilation by enzymatic and conventional methods. Figure 6.1 b & c shows enzymatic dehairing cow hide at 2 h and 4 h respectively. Approximately 50% of depilation was observed at 2 h of incubation and at the end of three and half hours complete depilation was observed. Figure 6.1d shows depilated pelt of cow hide by lime and sulphide method. Complete depilation was observed at 18 h of incubation. The depilated pelt by enzymatic method was better in quality when compared to lime and sulphide method, which indicated complete hair removal from hair follicles in case of enzymatic method whereas hairs received burns in case of lime and sulphide method which can be observed by the reddish brown color of the depilated pelt.

7 158 Figure 6.1 Dehairing of cow hide: (a) raw hide (b) enzymatic dehairing at 2 h (c) complete enzymatic dehairing at 4 h (d) complete dehairing at 12 h by conventional method Visual observation of the enzymatically dehaired pelts of goatskins and cowhides revealed complete absence of fine hairs and epidermis and more whiteness than the controls due to elimination of sulfide in the process. It was reported earlier that the removal of residual fine hairs remained the greatest obstacle to the development of hair saving enzymatic process (Paul et al 2001). In case of goat skin depilation, hair loosening was observed after incubation for 1 h by enzymatic method and complete depilation was observed at 2-3 h of incubation at room temperature. Conventional depilation (lime and sulphide method) of goat skin was similar to the one observed with cow hides. Complete depilation was observed after 14 h of incubation.

8 159 Application of enzyme by paste method and dehaired pelts of goat skin is shown in Figure 6.2. Figure 6.2 Dehairing of goat skin: (a) application of enzyme by paste method (b) enzymatic dehairing at 1 h (c) complete enzymatic dehairing at 3 h (d) complete dehairing at 12 h by conventional method Enzymatic depilation of goat skin/cow hide has been widely studied by many investigators either by employing crude enzyme (Dettmer et al 2011; Nadeem et al 2010) or enzyme concentrated via precipitation (Sivasubramanian et al 2008; Rajkumar et al 2011; Sundararajan et al 2011). Sivasubramanian et al (2008) have studied the depilation of cow hides using conventional, enzyme assisted and enzyme-only approach and reported that the enzymatic process required shorter duration of 6 h for complete depilation of skins and hides than the control groups (conventional and enzyme assisted). Similarly, Mukhtar and Haq (2008) have also reported

9 160 the enzymatic, enzyme assisted and conventional depilation of skins and reported that the best result with skin processing were obtained when skin was treated with crude enzyme in combination with 7% lime sulphide. In another study Rao et al (2009) has shown the dehairing of goat skin by protease obtained from Bacillus circulans when incubated for 12 h Depilation with different enzyme formulations Further, the depilation of cow hide with different enzyme formulations was studied. It was observed that 50% crude enzyme as such can be used for the depilation process of cow hide. Even 15 and 30% crude enzyme showed depilation but was not able to remove the short hairs. Further the studies showed complete depilation of cow hide in 5-6 h with ultrafiltered (UF) enzyme whereas depilation with spray dried enzyme showed the presence of sort hairs. SD (274 U/g) The formulations used were: Crude (40 U/mL); UF (165 U/mL); Table 6.1 Depilation of cow hide with different enzyme formulations Trial no. Experimental conditions kg of hide ml water ml Crude enzyme (15% v/w) kg of hide ml Crude enzyme (30% v/w) kg of hide ml Crude enzyme (50% v/w) Remarks Depilation started after 3 h of incubation and 50-60% dehairing was observed at the end of 5 h. Depilation process completed in 10 h but sort hairs were still remaining. Observation was same as in trial no. 1 Depilation started after 2 h of incubation and 50-60% dehairing was observed at the end of 4 h. complete depilation was observed in 8 h with absence of sort hairs

10 161 Table 6.1 (Continued) kg of hide ml water ml UF enzyme (4% v/w) kg of hide ml water ml UF enzyme (3% v/w) kg of hide ml water + 4% (w/w) SD enzyme (274 U/g) Depilation started after 3 h of incubation, removing 50% of hair in 3 h and completed in 5 h. Depilation started after 3 h of incubation, removing 50% of hair in 3 h and completed in 6 h. Depilation started after 2 h of incubation, removing 50-60% of hairs in 4 h and copmpleted in 8 h but sort hairs were observed Though crude enzyme at the concentration of 50% can be used for depilation of cow hides, visual observation of the depilated pelt indicated that UF enzyme at the concentration of 3-4% was better in terms of quality as well as incubation time for depilation. Apart from the higher activity in the UF, it is also possible that the absence of some compounds removed during the ultrafiltration could also be responsible for the better leather quality compared to crude and spray dried form of enzymes. The use of UF enzyme for the depilation process can reduce the cost of the tanning process since it does not require any chemicals for the downstreaming process of enzymes. There is no other literature available on the use of ultrafiltered enzyme for depilation process to the best of our knowledge Histological analysis Histological analysis was carried out in order to investigate the efficiency of enzyme on depilation of cow hides as well as goat skin in comparison with conventional depilation. The pelt after depilation was studied for histological characteristics (Figure 6.3). H&E and Masons Trichome staining clearly indicated the difference in pattern of hair removal from the hair follicle of cow hides. Hairs were completely removed from the

11 162 hair follicle in case of enzymatic depilation, whereas in conventional dehairing, hair root was burnt inside the hair follicle as indicated by the black spot inside the hair follicles. Similar result for the enzymatic depilated pelts was reported by Sivasubramanian et al 2008; Jaswal et al 2008 and Sundararajan et al Figure 6.3 Staining of cow hide pelt (a) H&E (b) masson s Trichome and (c) verhoff s staining of conventional dehaired hide; (d) H&E (e) masson s Trichome and (f) verhoff s staining of enzymatic dehaired skin Verhoff s staining indicated that there was mild degradation of elastin in enzymatic dehairing pelt which might be due to presence of elastase activity in the enzyme (Figure 6.3 c & f). Black spot in the Figure 6.3 (c & f) indicates the presence of elastic fibers in the skin. Similarly, Sivasubramanian et al (2008) have also compared the presence of elastic fibers in conventional, enzyme-assisted and enzyme-only depilated pelts and shown that elastic fibers was completely absent in the enzymatic dehaired pelts. Most of the studies do not elaborate the study on the effect of enzymes on the elastic fibers which is one of the important constituent of the skin contributing to the

12 163 leather quality. Similar results were observed for goat skin with H&E, Masson s Trichome and Verhoff s staining of depilated pelt (Figure 6.4). Figure 6.4 Staining of goat skin pelt (a) H&E (b) Masson s trichome and (c) verhoff s staining of conventional dehaired skin; (d) H&E (e) masson s trichome and (f) verhoff s staining of enzymatic dehaired skin Scanning electron microscopic (SEM) analysis of dehaired pelt The pelts of control and experiment were also observed under scanning electron microscope. The scanning electron micrographs of grain at magnification of 80X and cross section at magnification of 500X are given as Figure 6.5 & 6.6. SEM of grain surface of pelt showed that the opening of hair follicles in enzymatically treated cow hides was better than the conventional treatment. Due to swelling effect in lime and sulfide system, the hair follicle could have been closed. SEM of cut surface of pelts indicated that the opening of fiber bundle in enzymatic treated pelts was lesser than that of control pelts. More opening in the case of control might be due to the swelling effect caused by lime and sodium sulfide.

13 164 Figure 6.5 SEM of grain surface of dehaired pelt of cow hide (a) conventional (b) enzymatic and goat skin (c) conventional (d) enzymatic Figure 6.6 SEM of cut surface of dehaired pelt of cow hide (a) conventional (b) enzymatic and goat skin (c) conventional (d) enzymatic

14 165 Saravanabhavan et al (2005) and Sivasubramanian et al (2008) has also studied scanning electron micrograph of grain surface and cross section of depilated pelt of cow hide obtained by chemical as well as enzymatic method Analysis of pollution parameters of effluent generated from depilation process and its environmental impact Pre-tanning processes generally account for 70-80% of the total COD of effluent from all leather making processes (Marsal et al 1999). About 75% of the organic waste from a tannery was from pretanning processes and 70% of this waste was from hair rich in nitrogen (Kamini et al 1999).The most commonly employed methods for depilation rely upon the use of sulphide during liming to destroy keratin, the principal component of hair. This produces an effluent with a chemical oxygen demand of about 60,000 mg/l and constitutes the polluting aspect of leather manufacturing. The use of sulphide in depilation process can be eliminated by the use of proteolytic enzymes (Choudhary et al 2004). The environmental impact of the generated effluent from the depilation process of cow hides by conventional and enzymatic methods was assessed in terms of BOD, COD, TSS, TDS, sulphide and calcium. Samples from the generated effluent from conventional and enzymatic depilation were taken for analysis and the results obtained are shown in Table 6.2. The results show that total suspended solid (TSS) and total dissolved solids (TDS) could be greatly reduced to 95 and 75% respectively by enzymatic process. Biological oxygen demand (BOD) and chemical oxygen demand (COD) was also reduced by 86.6 and 83.5% respectively when compared with conventional process.

15 166 A little amount of sulphide was observed in effluent generated by enzymatic process which comes with enzyme since enzyme was concentrated via precipitation. Table 6.2 Analysis of pollution parameters S. % Parameter Unit Conventional Enzymatic No. Reduction 1 TSS mg/l TDS mg/l Sulfide mg/l BOD mg/l COD mg/l Sivasubramanian et al (2008) have also reported 85 and 90% reduction in BOD and COD by enzymatic depilation of hides whereas TDS and TSS were reduced by 85%. Further, Dayanandan et al (2003) have evaluated the pollution load generated by liming process of goat skin and reported 50, 40, 60 and 20% reduction in BOD, COD, TDS and TSS respectively by enzymatic process CONCLUSIONS In this study an alkaline protease developed from Bacillus pumilus MTCC 7514 was employed for depilation process as an alternative of conventional depilation process. The potential of protease for depilation process was good as it was completed in 2-3 h for goat skin and 4 h for cow hide by employing precipitated protease preparation whereas depilation of cow hide took 4-5 h for UF enzyme and 8 h for crude enzyme. The enzymatic process resulted in complete removal of hair and epidermis layer as observed

16 167 by histological and SEM analysis of the pelt. The enzymatic depilation process was eco-friendly as it greatly reduced the pollution parameters viz. BOD, COD, TDS and TSS to an extent of 86.6, 83.5, 75 and 97%, respectively. Since the protease enzyme was produced by cost effective means and having good efficiency in depilation of cow hide as well as goat skin, it could be commercially exploited. 6.2 USE OF PROTEASE IN DETACHMENT AND PASSAGING OF THE ADHERENT CELL CULTURE Introduction Cell culture is used for a variety of purposes: Recombinant protein production, experimental investigations of cellular behaviour and mechanisms, in vitro toxicity, screening and testing of new drugs etc. the basic tools used for culturing cells are the same regardless of the purpose (Leif and Skriver 1999). In cell culture, passaging is the process of sub-culturing cells in order to produce a large number of cells from pre-existing ones. Passaging (also known as subculture or splitting cells) involves splitting the cells and transferring a small number into each new vessel. Cells can be cultured for a longer time if they are split regularly, as it avoids the senescence associated with prolonged high cell density ( The techniques of passaging cells is very well known and use of porcine derived trypsin in passaging of adherent cell cultures is routinely used (Leif and Skriver 1999). Various enzyme and chelating agents have been studied for use in selectively cultivating a specific group of cells present in a tissue (Irie 1976).

17 168 In the present study, a protease derived from the Bacillus pumilus MTCC 7514 was investigated as a substitute of trypsin (frequently used for detachment of adherent cells) for its application in detachment of animal cells during passaging of cell culture. The compatibility of protease under study for the cell detachment was observed by viable cell count after protease treatment of cells Materials and Methods Cell lines and medium L6 (rat skeletal muscle myoblast), C6 (rat brain glioma cell) and NIH 3T3 (mouse embryonic fibroblast) cell lines were collected from NCCS, Pune. The growth medium for the cell lines used was high glucose (4.5 g/l) Dulbecco's Modified Eagle's Medium (DMEM) (Sigma Aldrich, USA) with 10% fetal bovine serum (FBS) (Pan Biotech, Germany), supplemented with penicillin (120 U/ ml), streptomycin (75 mg/ml), gentamycin (160 mg/ml) and amphotericin B (3 mg/ml) Seeding of cell lines Cell lines grown in T 25 flask were washed with PBS twice, trypsinized with 3 ml of trypsin solution (0.25% trypsin and 0.02% of EDTA). After detachment, trypsin action on the cells is inhibited using 3 ml of serum containing growth medium. The detached cells were collected and centrifuged at 1500 rpm for 3.0 minute. The cell pellet were dissolved in 2.0 ml of media, from which 100 µl was used to seed 6 well plates containing 2.5 ml of media. Cells were distributed uniformly by gentle shaking. The plates were incubated at 37 C in CO 2 incubator.

18 Protease enzyme preparation Crude protease enzyme (300 ml) obtained from Bacillus pumilus MTCC 7514 by submerged fermentation was lyophilized to get the enzyme in powdered form. Protease enzyme (0.2 g) was dissolved in 36.0 ml of phosphate buffer (stock solution) followed by filter sterization. Different dilutions of enzyme were prepared from the stock solution using sterilized PBS. The dilutions were 1X, 5X, 10X, 15X, 20X and 25X Detachment of cell lines and microscopic studies Detachment of cells was observed with time under microscope at different concentration of protease enzyme and trypsin as a control. Magnitude of microscope was set to 100X. Further 1 ml of four concentrations (5X, 10X, 15X and 20X) of protease enzyme was used for detachment of adherent cell lines. The fully grown cells in 6 wells culture plate were subjected for protease treatment and observed for the cellular detachment. Enzyme action on the cells was inhibited using 1 ml serum containing growth medium. The detached cells were collected and centrifuged at 1500 rpm for 3.0 minute. The cell pellets were dissolved in 2.5 ml of growth medium and the same were seeded and subjected for viability assay Viability assay Viability of the cell lines passaged using test enzyme was determined by the 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium (MTT) assay to determine the amount of the active mitochondrial enzymes present in the viable cells which convert MTT in to Formazon, a coloured product. After passaging, cell lines were incubated overnight to adhere. Once the cells adhered to the surface, medium was decanted carefully, washed twice with PBS carefully then 1.0 ml of MTT was added to each wells. After

19 170 incubation, the supernatant of each well was replaced with MTT diluted in serum-free medium and the plates were incubated at 37 C for 4 h. Once the reaction is completed,the MTT solution was aspirated and 3 ml Dimethyl sulphoxide (DMSO) was added to each well and pipetted up and down to dissolve all of the dark blue crystals and then left at room temperature for a few minutes to ensure all crystals are dissolved. Finally, absorbance of the solution was taken at 540 nm in ELISA reader. The obtained readings were taken as the degree of cellular viability as the optical density of the Formazone is directly related to the number of viable cells. The number of viable cells was calculated using standard curve Results and Discussion In cell culture passaging, use of mammalian derived proteases (e.g. porcine derived trypsin) is a potential risk of contaminating cell culture with adventitious agents such as viruses (e.g. porcine parvoviruses). Furthermore, the formation of aggregates in cell suspensions may be a problem with mammalian derived proteases. It would therefore be an advantage if the porcine derived trypsin could be replaced by any other material having the same or substantially the same ability as porcine derived trypsin to detach cell cultures, thereby eliminating the potential risk associated with using animal or human derived material in cell culture (Leif and Skriver 1999). Three cell lines (NIH 3T3, L6 and C6) were treated with trypsin as well as with different concentration of protease under study for the detachment and viability test. The pictures of untreated, trypsin and protease treated cell lines are shown in Figure 6.7 (C6), 6.8 (L6) and 6.9 (NIH 3T3). Visual observation of morphology of the detached cells with protease enzyme was comparable to the trypsinized cells. Enzymes at the concentration of 5X and 10X detached the cells almost immediately after addition while rest of the

20 171 concentration of enzyme took 1-5 min for the detachment. The NIH cell line was detached immediately even at 15X concentration of enzyme. Figure 6.7 Micrograph of the C6 Cell lines (a) adherent cells (b) cells after trypsinization (c) cells after enzyme treatment Figure 6.8 Micrograph of the L6 Cell lines (a) adherent cells (b) cells after trypsinization (c) cells after enzyme treatment Figure 6.9 Micrograph of the NIH 3T3 Cell lines (a) adherent cells (b) cells after trypsinization (c) cells after enzyme treatment

21 172 The viability of cells was carried out by MTT assay and results are shown in Table 6.3. It was observed that viability of cells after protease treatment was equivalent to the viability of trypsin treated cells. 15X concentrated enzyme was optimum concentration for the detachment of L6 and C6 cell line since the viability was more whereas for NIH cell line 20X concentrated enzyme was found suitable. In case of L6 cell line, viability of cells at enzyme concentration of 15X was better than trypsin. The less viability of cells count in case of L6 and C6 at 20X may be the incomplete detachment of the cells which leads to the reduced number of seeding cells. Table 6.3 Viable cell number of different cell line after detachment Cell Number (X10 5 ) Cell lines Protease enzyme Control (with trypsin) 5X 10X 15X 20X L C NIH 3T However, cellular detachment is the desired utility of the enzymes to be used for adherent cell passaging but keeping cells healthy after detachment is also a key issue which is to be noticed. In case of protease detachment, hydrolysis of cellular surface biomolecules (proteins) results into the dissociation of the cells from each other and the substratum. Towards this direction, the optimum concentration of the enzyme which should detach the cells keeping these healthy is the required materials for animal cell and tissue culture. The current study indicates that the protease derived from Bacillus pumilus MTCC 7514 could be used as alternative for the detachment of cells during passaging in animal tissue and cell culture. Leif and Skriver (1999) have also studied the detachment of different cell lines using Fusarium

22 173 protease SP387 at two different concentration (0.25 and 0.5 mg/ml) and reported that no difference in cell number was observed after reseeding in all cell lines regardless of the type of protease (porcine trypsine or fusarium protease) used for passage. In another study, Irie (1976) has also reported a neutral protease obtained from a strain of Bacillus polymyxa useful for animal tissue cell culture for the detachment of adherent cell lines. Furthermore, Shiv Shankar et al (2011) have also shown the separation of cells using an alkaline protease obtained from a new strain of Beauveria sp. but they have not reported about the viability of cells after treatment with alkaline protease which makes it unpredictable to claim about its usefulness in animal tissue cell culture Conclusion This study investigated the suitability of protease obtained from Bacillus pumilus MTCC 7514 in detachment of cells lines during passaging. The results show that detachment efficiency and viability of cells at 15X and 20X enzyme concentrations was comparable to that with trypsin and hence it claims to be a suitable protease which may be as an alternative of trypsin for detachment of cell lines during passaging in animal tissue and cell culture study.