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CHAPTER 6

APPLICATION OF PROTEASE IN LEATHER

PROCESSING AND TISSUE CULTURE

6.1 DEHAIRING OF GOAT SKINS AND COW HIDES

6.1.1 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

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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

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the de-haired pelt has been assessed for quality by scanning electron

microscopy (SEM) and histological studies.

6.1.2 Materials and Methods

6.1.2.1 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 7514. Other chemicals used in this study were of

commercial and analytical grade.

6.1.2.2 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.

6.1.2.3 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

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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.

6.1.2.4 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.

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6.1.2.5 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 cm2

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.

6.1.2.6 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.

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6.1.2.7 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.

6.1.3 Results and Discussion

6.1.3.1 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.

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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.

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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

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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.

6.1.3.2 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.

The formulations used were: Crude (40 U/mL); UF (165 U/mL);

SD (274 U/g)

Table 6.1 Depilation of cow hide with different enzyme formulations

Trial

no.

Experimental

conditions

Remarks

1 5.0 kg of hide + 750

mL water + 750 mL

Crude enzyme (15%

v/w)

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.

2 5.5 kg of hide + 1650

mL Crude enzyme

(30% v/w)

Observation was same as in trial no. 1

3 5.5 kg of hide + 2750

mL Crude enzyme

(50% v/w)

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

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Table 6.1 (Continued)

4 5.2 kg of hide + 1200

mL water + 210 mL

UF enzyme (4% v/w)

Depilation started after 3 h of incubation,

removing 50% of hair in 3 h and

completed in 5 h.

5 4.0 kg of hide + 900

mL water + 120 mL

UF enzyme (3% v/w)

Depilation started after 3 h of incubation,

removing 50% of hair in 3 h and

completed in 6 h.

6 5.8 kg of hide + 1500

mL water + 4% (w/w)

SD enzyme (274 U/g)

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.

6.1.3.3 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

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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 2011.

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

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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

6.1.3.4 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.

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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

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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.

6.1.3.5 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.

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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.

No.Parameter Unit Conventional Enzymatic

%

Reduction

1 TSS mg/L 10964 330 97

2 TDS mg/L 12964 3232 75

4 Sulfide mg/L 1360 32 97.7

6 BOD mg/L 3500 470 86.6

7 COD mg/L 10880 1792 83.5

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.

6.1.4 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

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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

6.2.1 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 (www.carlroth.com/website/fr-

fr/pdf/Zellpassage_Trypsin_E.pdf). 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).

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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.

6.2.2 Materials and Methods

6.2.2.1 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).

6.2.2.2 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 CO2 incubator.

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6.2.2.3 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.

6.2.2.4 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.

6.2.2.5 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

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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.

6.2.3 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

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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

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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 lines

Cell Number (X105)

Control (with trypsin)Protease enzyme

5X 10X 15X 20X

L6 4.2 3.3 3.63 4.221 4.062

C6 10.7 8.49 9.31 10.09 9.64

NIH 3T3 7.4 5.64 5.73 5.81 6.81

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

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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.

6.2.4 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.