World

Journal

of Microbiology

& Biotechnology

12. 284-291

Short Communication: Production of alkaline protease by a new Bacillus subfilis isolate for use as a bating enzyme in leather treatment

A. Hameed, M.A. Natt and C.S. Evans” Bacillus subtilis, isolated from tannery waste, produced an alkaline protease at optimal activity when grown in a casein/gelatine medium in a stirred tank fermenter at 3 7°C with the dissolved oxygen tension at 40% air saturation. Optimum protease activity (223 U ml-‘) was at pH 8.5 and was stable for 1 h up to 45°C but at 60°C lost 80% activity. Use of the crude protease as a bating agent for producing high quality leather is indicated. Tensile strength, bursting strength, tear strength and elongation at break of prepared leather were increased with increasing amounts of protease used for bating. Key words:

Alkaline

protease, BacilLs subtilis,

bating enzyme, leather treatment.

Enzymes have considerable potential in the bioprocessing of skins and hides for leather production, offering effective biotreatments particularly for the dehairing and bating processes (Taylor et al. 1987). Leather preparation includes soaking, dehairing and bating (removal of unwanted interfibrillary material) of skin, in which proteolytic activity is important to effect clean, supple pelts. Chemical processing (lime-sulphide) is responsible for 100% of sulphide and 80% of suspended solids in tannery effluent waste causing major environmental pollution (Malathi and Chakraborti 1991). Although less polluting, biotreatment has been more costly than chemical processing (Taylor et al. 1987). Bating following the dehairing process produces softer, stretchier, silkier leather. Pancreatic protease is often used as trypsin shows good bating action through protein hydrolysis at pH 8.5. Fibrous proteins (98% collagen, 1% keratin and 1% elastin) form 96.5% of skin proteins. A major objective in bating is to effect an internal separation of collagen fibres by degrading keratin, to expose a maximum reactive surface for subsequent tanning agents. It allows the removal of ‘scud’, a mixture of epidermal matter, hair roots, pigments, fat, glands and decomposed cellular debris, so cleaning the pelt. Bating effects on elastin increase suppleness, pliability, elasticity and feel of the leather (Boehne 1993). Depending on its proposed use (for clothing, car seats etc.) different enzyme treatments are required. Several species of bacteria including B. strbtilis have been isolated which produce neutral or alkaline proteases

A. Hameed and MA. Natt are with the Department of Biological Sciences, Quaid-i-Azam University, Islamabad. Pakistan. C.S. Evans is with the School of Biological and Health Sciences, University of Westminster, London, UK; fax: 0171 911 5087. * Corresponding author. @ 1996 Rapid

Science

suitable for bating (Puvanakrishnan & Dhar 1986). Bating enzyme preparations from microbial sources commercially available in Western Europe are not an economic proposition for use in Pakistan. Scale-up and production of microbial proteases in Pakistan would enable the replacement of traditional chemical methods and pancreatic proteases with a commercially competitive process, and improve environmental quality.

Materials

and Methods

Source of Organism and Culftrre Conditions Bacillus subfilis was isolated from tannery waste in Pakistan and identified by the National Collection of Industrial and Marine Bacteria Ltd (NCIMB), Aberdeen, UK. Cultures were routinely maintained on nutrient agar slants with 0.5% peptone, 0.3% beef extract at pH 7.0. Serial dilution was used to estimate c.f.u. ml.-’ Growth of 8. subtilis in shake flasks. To prepare the inoculum, ml of a casein/gelatine medium was prepared in I 1 flasks from 0.48% casein hydrolysate, 6% gelatine at pH 7 to which 3 ml of 20% glycerol, sterilized in a dry oven at 121°C for 30 min, was added. After inoculation agitated cultures were incubated at 37°C for 24 h. Batch cultures of I 1 casein/gelatine medium were inoculated with 50 ml of prepared inoculum with a colony and incubated at 37”C, shaken at count of I.9 X IO6 c.f.u. ml-’ 150 rpm for 72 h. 250

Growth of B. subtilis in Biofermenfers. Casein/gelatine medium (3 I) was sterilized for a 6 1 Eyela fermenter (M-160; aspect ratio 3:l) with a variable pitch impeller (73 mm diameter). Before inoculation 36 ml of sterilized glycerol was added. Initial parameters were set at 37”C, dissolved 0, tension (D.O.T.) at 30% air saturation, pH 7. Samples were collected every 24 h for 3 days. D.O.T. was measured with a polarographic 0, electrode.

Publishers World]oumal

of Microbiology

6 Bmtechnology. Vd 12, 1996

289

A. Hameed, M.A. Naff and C.S. Evans Table crude

1. Quantitative assessment of the bating efficiency of the alkaline protease on pelts processed to finished leather.

Test

Directional of leather

Tensile

strength

(kg cm-*) Tear

strength

(kg cm-‘) Elongation Break

at (%)

cut

Proteoiytic units (PU ml-l) 900

1800

2700

Perpendicular parallel

141 152

170 175

199 173

Perpendicular

36

39

41

parallel

36

41

42

Perpendicular parallel

10 13

15 19

21 28

217

Bursting strength (kg cm-‘) Data

presented

are

means

of triplicate

242

Reprodttcibility All experiments were carried out occasions or as denoted in the text.

Results

254

experiments.

ProfeaseActivity Assay Proteolytic activity with casein as substrate was measured by the method of Kunitz (1947). To 1 ml of 1% casein solution (pH 8.5). I ml of crude enzyme solution was added, and incubated for 30 min at 40°C. Protein was precipitated with 3 ml of 0.3 M trichloroacetic acid (TCA). After 30 min, the supematant was separated by centrifugation at 10,000 X g for 30 min and absorbance at 260 nm determined. Blanks were prepared in which 3 ml TCA was added before incubation. All assays were made in triplicate. One unit of activity is defined as the amount of enzyme which releases I pg of tyrosine under the assay conditions (pH 8.5, 4O’C,

30 min).

Application of Bate to Delimed Goat Pelt Delimed goat pelts, soaked and dehaired by the lime-sulphide process were used for bating trials. Twelve pieces of pelt (40 g each) cut from a completely delimed pelt were agitated with deliming liquor in separate bottles. Crude bating enzyme preparations containing 220 U ml -’ were added for 1 h. Qmlitative For thumb

Testsfor EfFcacyof Eating impression

test, the time

taken

by the bated

pelt

25 mm) with a 20 mm long cut in the centre was determined. The thickness of each piece was measured before attachment to the hooks of the tensile machine which was run until the pieces tore apart. The highest load at break was recorded (kg cm-‘). Bursting strength (kg cm-‘) was measured with a Lastometer (STD-104).

to

regain its original shape after a thumb was pressed on the grain side, and the intensity of the impression were noted. The ease of scud (hair root, degraded protein, etc.) removal by scraping with finger nails was assessed. The degree of slipperiness of the bated pelt pieces was categorized. Quantitative Tests for Efficacyof Batkg After bating with the crude protease, goat pelts were processed to prepared leather. The leather was cut into pieces which were

tested for tensile strength, percentage elongation at break, tear and bursting strengths. Tensile strength (kg cm-‘) was measured with a Priffen and Messon type 85654 tensile machine with a uniform speed of separation of the jaws (100 f 20 mm min-‘), and the load at break noted. A section around the central 50 mm portion of pieces of leather (110 cm long) was tested, 10 mm from the central portion and 25 mm from the outer margins. The mean thickness of the pieces, measured at three different places from the centre was determined. Percentage elongation at break was measured with the jaws set at 50 mm distance. Elongation at break = lOO(A - B) + B where A = length at break; B = initial length of sample (50 mm). Tear strength of pieces of leather (50 x

in triplicate

on at least three

and Discussion

Quantitative assessment of proteolytic activity of Bacillus subfilis K2 was measured in shake flask cultures for 72 h. Protease activity was detected in the culture medium throughout growth, with the optimum activity (112 U ml-‘) correlating with optimum cell number (5.9 X 10’ c.f.u. m-l) 48 h after inoculation. With less inoculum, the optimum cell number and protease activity occurred later at 72 h. On addition of glycerol, the pH of the medium was 5.3 rising to 8.0 after growth for 48 h. In a 3 1 culture in a biofermenter at 37”C, incubated at 150 rev.min-’ agitation, protease activity after 48 h growth was 156 U ml-l and cell number was 2.8 x lo9 c.f.u. ml-‘. Bacterial dry weight could not be accurately determined due to the particulate nature of casein in the medium. After 48 h protease activity was maximal at 37°C (156 U ml-‘) compared with 30°C (90 U ml-I) and 45°C (88 U ml-‘), at a 150 rev.min-’ agitation rate and dissolved D.O.T. of 30% air saturation. At an agitation rate of 180 rev.min - I, activity was 13% and 5% higher than at respectively for cultures at 37°C. 150 and 210 rev.min-I, Raising the D.O.T. to 40% air saturation produced an increase in protease activity to 223 U ml-’ at 37°C and 180 rev.min-‘. The pH optimum of activity was 8.5 within a range from pH 6 to 10. More than 98% of activity was stable for I h from 15 to 45°C but 80% activity was lost at 60°C. A qualitative assessment of the effect of the crude protease as a bating enzyme gave a highly slippery feel to the leather with a very fine texture of the grain. Scud loosening was very easy and the intensity of thumb impression was very deep, taking 25 set to reshape after imprint. These data were confirmed quantitatively. Three different bates containing 900, 1800 and 2700 U ml-‘, respectively were applied to pieces of pelts for 1 h replacing the usual bating treatment after which the leather was fully processed. Table 1 shows that the tensile strength, tear strength, and percentage elongation at break of the leather increased in both perpendicular and parallel directions of the leather with increasing concentrations of crude alkaline protease used in bating. Bursting strength was also increased. The strain of B. stlbfilis K2 was considered suitable for

Profease

development as a new bating enzyme. Proteolytic activity compared favourably with that used in bating with pancreatic protease. This strain also produced lipolytic and amylolytic enzymes (data not given), which although not playing a significant role in bating or dehairing can aid in leather making by degradation of starch and fatty acid esters. It would be more viable to use a crude culture filtrate for bating than undergo further processing to produce pure protease. Production of the crude alkaline protease is straightforward and amenable to scale up, giving increased activity by raising the D.O.T. The commercial potential is promising with production costs lower than using pancreatic protease for bating. If combined with dehairing enzymes for a one-step dehairing and bating process, the use of the limesulphide treatment can be eliminated, so reducing environmental pollution from tanneries.

from

Bacillus subtilis for leather

freafment

References Boehne, H.J. 1993 New enzyme mixtures for bating. Pakistan Leather Trades journal

3, 79-84.

Kunitz, M. 1947 Crystalline soybean trypsin inhibitor. Journal of General Physiology

30, 291-310.

Malathi, S. & Chakraborty, T. 1991 Production of alkaline protease by a new Aspergillusj7aow isolate under solid substrate fermentation conditions for use as a depilation agent. Applied and Environmental

Microbiology

57, 712-716.

Puvanakrishnan, R. & Dhar, SC. 1986 Recent advances in the enzymatic depilation of hides and skins. Leather Science 33, 177-191.

Taylor, M.M., Bailey, D.G. & Feairheller, S.H. 1987 A review of the use of enzymes in the tannery. journal of the Americau Leather Chemistry

Association

82, 153-165.

Acknowledgements We thank The British Council, Pakistan Institute of Leather Technology, Gujranwala, this work.

Directorate & for supporting

(Received

in revised

form

7 February

World

of Mrcrobiology

1996;

accepted

7 February

1996)

loamal

6 Biotechnology.

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