29 Indian Journal of Science and Technology Vol.2 No. 12 (Dec. 2009) C. Arunachalam1 and K.

Saritha2 ISSN: 0974- 6846

Protease enzyme: an eco-friendly alternative for leather industry

PG and Research Department of Microbiology, Sri Sankara Arts and Science College, Enathur, Kanchipuram, Tamilnadu, India. 2 PG & Research Department of Microbiology, Sri Venkateshwara College of Arts & Science, Peravurani, Thanjavur Dt., Tamilnadu, India
[email protected]
1

Abstract: We report here a novel keratinase from Bacillus subtilis that has the potential to replace sodium sulfide in

the dehairing process of leather industry. The Protease enzyme produced at laboratory condition has been characterized for its rate of enzyme production, and the environmental influencing factors such as pH and temperature on the activity of the enzyme has also been evaluated. The enzyme produced in pilot scale has been subjected to in vitro (spectrophotometrically) as well as in vivo assay (on wet goat skin for hair removal). The organism grown in the Dyes synthetic medium at 5.3mg/ml of cell dry weight produced 548 U/ml of protease. The in vitro enzyme activity increased with temperature within a range of 25oC to 35oC and found maximum at 45 oC and at pH 11. An index of dehairing comparable to the use of conventional sodium sulfide method was achieved in 7 h of its application on wet goat skin.

Keywords: Protease enzyme, leather industry, Bacillus sp, dehairing of skin.

Introduction Enzymes are vitally important to the existence of life itself. Civilizations have used enzymes for thousands of years without understanding what they were or how they work. However, over the past several generations, science has unlocked the mystery of enzymes and has applied this knowledge to make better use in an evergrowing number of applications. Enzymes play crucial roles in producing the food we eat, the clothes we wear, even in producing fuel for our automobiles. Enzymes are also important in reducing both energy consumption and combating environmental pollution. Leather processing is one of the important industries closely related to everyday life. Despite making significant contributions to the economy, the leather industry causes severe environmental pollution owing to the use of various chemicals and the release of a variety of detrimental materials. In leather processing, the first step in the beam house is to remove hairs from hides and skins. Unhairing is defined as the removal of hair from hides. Application of lime sulfide unhairing system, the effluent problems arising from lime are due to its high alkalinity and suspended solids. Besides this sulfide, on the other hand, liberates toxic hydrogen sulfide, a serious hazard for both tannery workers and sewer men. Tanneries are constantly concerned with the obnoxious Research article
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odor and the pollution caused by the extremely toxic sodium sulfide used in the dehairing process. The conventional dehairing method involves the use of high proportions of lime and sulfide, which contributes to 80 90% of the total pollution load in the leather industry and generates noxious gases as well as solid wastes, e.g. hydrogen sulfide and lime (Thanikaivelan et al., 2004). Deaths because of this toxic chemical process have also been reported (Balasubramanian & Pugalenthi., 2000 Gupta et al., 2002). Worldwide, it is estimated that 315 million bovine leathers are produced per year. Considering the waste treatment cost of $0.30 per m2 of leather produced, more than $1 million is spent per day to treat the waste from tanneries around the world (Ramaswamy, (1996). Enzymatic dehairing in tanneries has been envisaged as an alternative to sulfides (Beynon & Bond, 1989; Altschul et al., 1997). Alkaline proteases can be used which enables the swelling of hair roots, and the subsequent attack of protease on the hair follicle protein allowing easy removal of the hair (Gupta et al. 2002). A large proportion of the known alkaline proteases are derived from microorganisms, especially Bacillus strains (Wang et al.,2006). Enzymatic unhairing accomplished by proteolytic enzymes is of great commercial importance contributing to more than 40% of the worlds commercially produced enzymes. Approximately 50% of the enzymes produced is used for industrial process (Pepper et al., 1963). Further, proteolytic enzymes are more efficient in enzymatic dehairing rather than amylolytic enzymes (Puvankrishanan, 2003). The enzymes cause loosening of the hair, without damaging the fibrous collagen of dermis. The advantages of enzymatic dehairing are as follows: 1) significant reduction or even complete elimination of the use of sodium sulfide, 2) total recovery of hair resulting good quality with good saleable value, and 3) creation of an ecologically conducive atmosphere for the workers. The chemical composition of fresh hides and skins falls approximately within the following limits: water 6065%; protein 25-30; fats 5-10% and small amounts of minerals. The chief entity of protein present in skin is collagen. Enzyme treated leather has shown better strength properties and greater surface area. Simplification of pre-tanning process by cutting down one step viz bating. Americans have been practicing the sweating process for unhairing in which the depilation is Arunachalam & Sarita
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Bacillus Protease in leather industry

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30 Indian Journal of Science and Technology being achieved by the combined actions of the enzymes of autolytic process and those are secreted by the bacterial species (Tailor et al.,1987). A great deal of work has been reported in the past, since 1960 employing enzymes for unhairing. However, the use of enzymes in leather manufacturing process particularly for unhairing has not been accepted by the industry to the desired level. This is mainly because: a) enzymes are not effective enough to eliminate the sulfide completely, b) there is an apprehension that the enzymes assisted process needs stringent process control, and c) the cost of enzymes is not encouraging. Hence, the present work has focused on screening for proteolytic enzyme from a suitable microorganism, which is economically viable and effective enough to eliminate the sulfide completely. Vol.2 No. 12 (Dec. 2009) ISSN: 0974- 6846

2.5g; potassium phosphate - 0.5g; micronutrient solution0.5g; pH is adjusted to pH 7. Preparation of micronutrient: Ferrous sulfate- 10mg; copper sulfate- 5mg; sodium molybdate- 5mg; manganus chloride -5mg; magnesium sulfate- 10mg; boric acid5mg; zinc sulfate- 5mg; Make up to 10 ml with distilled water. The parameters such as pH, temperature and substrate concentration were altered in the assay procedure one at a time. The conditions that favored maximum activity were taken to be optimum conditions. The standard conditions used were pH 11.0 and temperatures 45oC. pH: In the substrate and the culture filtrate mixture, the pH, was altered from 3.5 to 8.0. Which is achieved by preparing the substrate using different buffers with different pH. For pH 3.5 to 5.5 acetate buffer (0.2M), and for pH 6.0 to 8.0 Phosphate buffer (0.2m) was used. Starch (1%) was prepared using the buffer 0.5ml and added to 0.5ml of culture filtrate for the assay. A graph was plotted taking pH values at X axis against enzymes on Y-axis. The optimum pH for enzyme activity was extrapolated from the graph. Temperature: The incubation temperature ranged from 30oC to 80oC and a graph was plotted taking temperature range in X axis and enzymes activity on Y axis. Activity of enzyme: Protease reacts with the casein liberating tyrosine and the liberated tyrosine in alkaline conditions causes the reduction of Phospho molybdate and Phospho- tungstate with Folin ciocalteau reagent to give blue colour. The colour developed is measured at 620 nm. The absorbance was the parameter for the estimation of tyrosine produced. Stock tyrosine was prepared by dissolving 50 mg of tyrosine in 1N HCl and made up to 100ml using distilled water. 0.5ml of concentrated casein 2% was taken in 2 test tubes labeled test and control (t1 and c1). To this 1ml of citrate phosphate buffer was added. The tubes were incubated at 37oC for 5minutes. Then 2ml of enzyme, (whose activity is to be estimated), was added to the tube labeled test. Again the tubes were incubated at 37oC for 30 minutes. After incubation, 2ml of 10% TCA was added

Protease activity at various environmental conditions

One gram of soil sample was collected from 0.15 cm layer near the mutton stall at Peravurani and dissolved in 100ml of distilled water, marked as 10-2 dilution. The soil suspensions were serially diluted up to 10-7. An aliquot of 0.1ml was drawn from 10-5 and 10-6 dilutions and streaked into sterile petriplates. The plates were incubated at 28-37oC for 24 hours. Identification was done using standard methods based on culture morphology, staining, motility and biochemical characteristics. The identified bacterial isolates were plated on the skim milk agar plates and incubated at 37oC for 24 hours until a clear zone of skim hydrolysis give an indication of protease producing organism. Depending on the zone of clearance and the growth of organism, Bacillus species were selected for further studies.

Isolation and organisms

Materials and methods

identification

of

protease

producing

Screening of protease activity

sample was inoculated in a 100ml of yeast extract glucose broth (Glucose- 0.3g; yeast extract - 0.3g; Peptone- 0.3g; sodium chloride-0.5g; water to make 100ml; pH- 7). The culture was then incubated in a rotary shaker (ORBIT Shaker Incubator, 3. 5 Neo lab) for 72 hours at 37oC. 3 Secondary inoculum: A 50ml Fig. 1. Effect of pH on protease activity medium contains: Bacto 2. 5 peptone or tryptone- 0.25g; sodium chloride- 0.25g; 0.25ml of 2 inoculum was added after 1. 5 strization and incubated for 72 hours at 37oC. 1 Production media: Production pH media (500ml) contains: 0. 5 Groundnut cake powder- 7.5g; 0 horsegram flour- 2.5g; Glucose1 2 3 4 Protease activity (g/ml) Research article
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Inoculums preparation Primary inoculum: The bacterial culture obtained from soil

Bacillus Protease in leather industry

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31 Indian Journal of Science and Technology Vol.2 No. 12 (Dec. 2009) ISSN: 0974- 6846

Enzymes suspension X% (X varies from 0.6, 1.2, 2.4 to both the control and the test tubes and then 2ml of enzyme was added to the control tube. The tubes were and 4.8 respectively on L1, L2, L3, and L4). The skins were piled and the dehairing efficacy was assessed. X centrifuged and supernatant was taken for the assay. Standard tyrosine of 0.2ml, 12 0.4ml, 0.6ml, 0.8ml and 1ml was Fig. 2. Effect of temperature variability on protease taken in five test tubes labelled S1 10 to S5. Then 0.5ml of supernatant was taken in 2 tubes, labeled U1 8 and U2. The volumes in the tubes were made up to 2.4 ml with distilled water. Sodium hydroxide 6 with a concentration of 0.5N of volume 2.0 ml was added to all the 4 tubes followed by the addition of 0.6ml of Folin ciocalteau reagent. 2 The tubes were incubated at room temperature for 10 to 20 minutes. Absorbance was measured at 620 0 nm using red filter. Taking the 30 35 40 45 50 55 concentration along the X- axis Temperature 0C and optical density along the Ypresents the case of dehairing of the enzyme-applied axis a standard graph was prepared. skins with respect to time. The pH of the paste was 11.0 Fermented broth was filtered through sterile 0.22 m hydrophilic durapore members filters, filtrate used as enzyme source. Enzyme suspension is taken in Erlenmeyer flask containing 0.5M Cacl2 in 10% (v/v). The setup is allowed in a condition undisturbed (5h) for settling of suspension. Decant the supernatant from the flask and the solids were used. Again the remaining supernatant is removed by squeezing and sodium chloride is added to the solid sediments and allowed it to dry in a shallow pan for 6hrs.

Enzymes recovery

Protease activity (g/ml)

Application procedure

The dehairing efficacy of the enzymes is being assessed by applying the same on the Goatskins. Wet salted Goat skins numbering four were selected and cut along the backbone. The right half was used for dehairing using sodium sulfide whiles the left half using the enzyme in varying concentration (Toyoda & Futami, 1956). Soaking 1: Four right halves and four left halves viz R1 to R4 and L1 to L4) water 350% - the skins were washed. Soaking II : Water 350% - the skins were soaked for 4hours Soaking III : Water 350% - the skins were soaked for overnight.

Liming (control)

The four right halves were pasted with the following composition and piled for six hours: Water 20%; Lime 7%; Sodium sulfide 2.5%. The skins were dehaired and relimed. Liming (experiment): The four left halves (L1 to L4) were applied with the lime enzyme paste as follows: Water 20%; Lime 7%. Research article Bacillus Protease in leather industry
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Results and discussion Protease enzyme produced from Bacillus species at laboratory condition has been characterized and the protease assay is being carried out. Further, the effect of temperature and pH on the optimum activity of the enzymes requires 11pH at 45oC. Coolbear et al. (1992) has stated the enzyme yields have remained relatively low since little work has been done on strain selection, growth optimization and enzyme yields. The Bacillus species produces a large variety of extra cellular enzymes of which protease is of significant industrial importance. In order to obtain protease on commercial scale the Bacillus species is being given priority in our investigation. Bacillus species grown in Dyes synthetic medium (Dyes 1967) for two days is being used for the production of protease enzyme. The test organism grew well in the medium by producing 5.3mg/ml of cell dry weight producing 548 U/ml. The effect of various parameters on protease activity of Bacillus species has been studied. The activity at pH 6.0 was low, but increased to a maximum at pH 11.0 but slowly decreasing to pH 11.0.(Green, 1961, 1961a; Gupta et al., 2002) have demonstrated the pH optimum for Bacillus alkaline protease varies but generally optimum range is from 8-11 (Fig.1). The enzyme activity increased with temperature within a range of 25oC to 35oC in the present observation as reported by Everett and Cordon (1956) and Joo et al. (2002). However, the maximum enzyme activity could be achieved at 45oC as shown in Fig. 2.

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32 Indian Journal of Science and Technology Vol.2 No. 12 (Dec. 2009) ISSN: 0974- 6846

Application studies

The enzyme was then in a pilot scale following the same procedure and the enzyme was applied on skins for assessing the unhairing efficacy. It could be seen from the results that even with the minimum amount of enzyme fairly good degree of hair loosening could be achieved (Table 1). The potential use of protease enzymes in leather processing eliminates the pollution causing chemicals such as sodium, lime and solvents. Future might witness ecolabelled leather products emerging as niche products by the use of protease enzyme technology and the experience gained by the Indian leather industry in this area might greatly help to emerge as a global leader. Our investigation of a novel keratinase from Bacillus subtilis has the potential to replace sodium sulfide in the dehairing process.
Table 1. Enzymatic de-hairing on wet goat-skin (index 1 to 10)
Experiment L1 L2 L3 L4 After 3h 3 5 5 6 After 4h 4 6 6 7 After 5h 5 7 7 8 After 6h 6 7 8 9 After 7h 7 8 9 10

11. 12. 13. 14. 15.

16.

17.

an extracelluar alkaline protease from Bacillus horikoshi. Process Biochem. 38, 155-159. Pepper, K.W. and wyatt, K.G.E. (1963). Enzymatic unhairing of heavy hides. J. S. L. T. C. Vol-47: 460464 Puvankrishanan. R (2003). Microbial enzyme technology in leather industry. Advanced Biotech, Vol 4: 17-18. Ramaswamy, T. (1996). In: textbook on leather. ILIFO, Vol 1: 12-15 Tailor MM, Bailey DG and Feairheller SH (1987) A review of uses of enzymes in the tannery. J. A. L. C. A. 82, 153. Thanikaivelan P, Rao JR, Nair BU and Ramasami T (2004) Progress and recent trends in biotechnological methods for leather processing. Trends Biotechnol . 22, 181188. Toyoda H and Futami A (1956) Influence of fundamental cultural conditions on the production of protease and depilatory enzyme by aerobic bacteria. J.A.L.C.A. 4, 38. Wang HY, Liu DM, Liu Y, Cheng CF, Ma QY, Huang Q and Zhang YZ (2006) Screening and mutagenesis of a novel Bacillus pumilus strain producing alkaline protease for dehairing. Lett. Appl. Microbiol. 44,16.

References 1. Altschul SF, Madden TL, Schaffer A A, Zhang J, Zhang Z, Miller W and Lipman DJ (1997) Gapped Blast and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 33893402. 2. Balasubramanian S and Pugalenthi V (2000) A comparative study of the determination of sulphide in tannery waste water by ion selective electrode (ISE) and iodimetry. Water Res. 34, 42014206. 3. Beynon J and Bond JS (1989) Proteolytic enzymes, a practical approach. IRL press, New York. 4. Coolbear T, Daniel R and Morgan HW (1992) The enzymes from extreme thermophiles, Adv. Biochem. Eng/Biotech. 45, 4857-4897. 5. Dyes (1967) The use of enzymes in the Beam house to solve the tannery effluent problems. Lea. Sci, 24, 199. 6. Everett AL and Cordon TC (1956) Hydrolytic action of facultatively anaerobic microorganisms of raw skins. J.A.L.C.A. 51, 59. 7. Green GH (1961) Unhairing by means of enzymes. J.S.L.T.C. 36, 127-134. 8. Green GH (1961a) A review of enzymes in the tannery. J.S.L.T.C. 45, 167-169 9. Gupta R, Beg QK and Lorenz P (2002) Bacterial alkaline proteases: molecular approaches and industrial applications. Appl. Microbiol. Biotechnol . 59, 1532. 10. Joo HS, Kuamr G, Park GC, Kim KT, Paik R and Chang CS (2002) Optimization of the production of Research article Bacillus Protease in leather industry
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