Approach towards sustainable leather: Characterization and effective industrial application of proteases from Bacillus sps. for ecofriendly dehairing of leather hide.

Tanneries are one of the most polluted industries known for production of massive amount of solid and liquid wastes without proper management and disposal. In this project we demonstrated the ecofriendly single step dehairing of leather hides with minimum pollution load. In this study, Bacillus species (Bacillus paralicheniformis strain BL.HK, Bacillus cereus strain BS.P) capable of producing proteases was successfully isolated by employing the new optimized selective media named M9-PEA as confirmed by 16sRNA genes sequencing. Sequence of 1493 bp long 16S rRNA genes of Bacillus paralicheniformis strain BL.HK and Bacillus cereus strain BS. P was submitted to GenBank under the accession number OP612692.1, OP612721.1 respectively The Bacillus paralicheniformis strain BL.HK, Bacillus cereus strain BS.P produced extracellur proteases of 28 and 37 KDa as resolved by SDS-PAGE respectively. The enzymes showed temperature optima at 50 °C and 55 °C and pH optima at 8.5, 9.5 respectively. The Proteases of Bacillus paralicheniformis strain BL.HK, Bacillus cereus strain BS.P were employed for dehairing of animal hides. The process resulted in significant removal of interfibriller substances without damage to collagen layer after one hour treatment, which was confirmed by histology, scanning electron microscopy. The quantification of various skin constituents (collagen, uronic acid, hexosamines, and GAGs) and pollution load parameters revealed that enzymatic treatment are more reliable. The results of skin application trials at industrial level with complete elimination of chemicals remark the biotechnological potential of these proteases for ecofriendly dehairing of animal hides without affecting the quality of the leathers produced.

Characterization and Pilot-Scale Application of the ZMS-2 Serine Protease with Novel Properties for the Eco-friendly Leather Processing.

Microbial alkaline proteases are dominating the global enzyme market with a share of over 65% due to their multifarious catalytic potentials. Hence, production of proteases with novel properties of commercial significance is highly desirable to meet the global enzyme demand. Here, we report the purification, characterization, and pilot-scale application of a serine protease from the desert soil bacterium Bacillus subtilis ZMS-2 with novel properties as dehairing agent in leather processing. The enzyme was purified 16.5-fold with a specific activity of 1543.5 U mg-1 and recovery percentage of 33.6% using ammonium sulfate precipitation, ion exchange, and gel filtration chromatography. The purified enzyme was characterized as a metal ion-, surfactant-, and denaturant-compatible alkaline serine protease having a molecular weight of 36.1 kDa with an optimum activity at pH 8.5 and 60 °C. The catalytic activity of the enzyme was enhanced by Zn+2 (204%), Ag+ (110%), H2O2 (123%), Triton X-100 (110%), iso-octane (109%), chloroform (110%), ethanol (105%), ethyl acetate (110%), and acetonitrile (128%). During pilot-scale applications, the optimum condition was found to be a combination of enzyme (1.5%, 460 U mL-1), sodium sulfide (2%), and calcium hydroxide (lime) (3%). Under this condition, the time required for complete dehairing was 90 min. Chemoenzymatically processed skins exhibited better physical properties than chemically processed skin, including tensile strength (16.35 ± 6.68 N/mm), ball burst (452.88 ± 6.06 N/mm), percent elongation (38.85 ± 1.06 N), tear strength (50.16 ± 4.42 N/mm), and softness (6.5 mm). Electron microscopy analysis of the treated skin showed complete removal of hairs with roots, confirming the keratin specificity of the enzyme. Moreover, the enzyme-assisted dehairing process reduced chemical oxygen demand (COD), biochemical oxygen demand (BOD), total dissolved solids (TDS), and total suspended solids (TSS) by 68, 77, 34, and 39%, respectively. Thus, the alkaline serine protease from B. subtilis ZMS-2 is a potential dehairing agent for the eco-friendly processing of animal skins on industrial scales.

Time of dehairing alters pork quality development.

Studies investigating the effect of scald time on pork quality are confounded with time of dehairing. To understand better pork quality development and two-toning in hams, twenty-four carcasses were assigned to an 8- or 16-min dwell time prior to the dehairing, with or without scalding (n = 6 per trt). Semimembranosus (SM) muscles were collected following dehairing and at 24 h postmortem. Protracted time to dehair improved ultimate pH (pHu; P < 0.005) and reduced (P < 0.05) color variation. One hundred forty-two carcasses were then subjected to protracted (control, 10-min) dwell times (15-min, or 20-min) in an industrial setting. Lightness was improved with 15-min dwell times compared to control, however 20-min dwell decreased the pHu (P < 0.001), increased lightness (P < 0.05), and percent purge (P < 0.001) in the SM. Also, lightness of the longissimus muscle (LM) increased (P < 0.001) with dwell time. These data show time to dehairing impacts pork quality development and suggest dehairing may be critical to quality development in a muscle-dependent manner.

An efficient dehairing system supported by oxidative-enzymatic auxiliary towards sustainability.

A method of dehairing of goat skins using oxidative chemicals and protease enzymes has been attempted. The dehairing process is one of the important and essential steps in leather making, where hair is removed by lime and sodium sulphide in the conventional process. This conventional dehairing system generates a higher amount of pollution problem as compared to the other unit operations and unit processes. In this work, dehairing of the goat skins through oxidative agents namely magnesium peroxide and protease enzyme has been attempted. For this, protease has been produced from Bacillus sp. at the laboratory level and the activity was found. The dehairing of goat skins takes place for the duration of 14-16 h. The leather produced with the experimental sample showed comparable organoleptic and strength properties with the conventional sample. This method paved the way for the reduction of pollution loads especially BOD, COD, and TDS to the level of 59, 27, and 77%, respectively, in comparison with the control sample. The reaction kinetics for the formation of the ligand-macromolecular complex is found in the isothermal titration calorimetry (ITC) experiment and a mathematical model has been formulated. The dyed crust leather showed comparable colour properties. In addition to that, there is a reduction in processing time for leather making through skipping reliming and deliming processes which are said to be another advantage of this method. The physical strength properties of the experimental leather were also comparable with conventionally produced leather.

Optimized production of keratinolytic proteases from Bacillus tropicus LS27 and its application as a sustainable alternative for dehairing, destaining and metal recovery.

The present study describes the isolation and characterization of Bacillus tropicus LS27 capable of keratinolytic protease production from Russell Market, Shivajinagar, Bangalore, Karnataka, with its diverse application. The ability of this strain to hydrolyze chicken feathers and skim milk was used to assess its keratinolytic and proteolytic properties. The strain identification was done using biochemical and molecular characterization using the 16S rRNA sequencing method. Further a sequential and systematic optimization of the factors affecting the keratinase production was done by initially sorting out the most influential factors (NaCl concentration, pH, inoculum level and incubation period in this study) through one factor at a time approach followed by central composite design based response surface methodology to enhance the keratinase production. Under optimized levels of NaCl (0.55 g/L), pH (7.35), inoculum level (5%) and incubation period (84 h), the keratinase production was enhanced from 41.62 U/mL to 401.67 ± 9.23 U/mL (9.65 fold increase) that corresponds to a feather degradation of 32.67 ± 1.36% was achieved. With regard to the cost effectiveness of application studies, the crude enzyme extracted from the optimized medium was tested for its potential dehairing, destaining and metal recovery properties. Complete dehairing was achieved within 48 h of treatment with crude enzyme without any visible damage to the collagen layer of goat skin. In destaining studies, combination of crude enzyme and detergent solution [1 mL detergent solution (5 mg/mL) and 1 mL crude enzyme] was found to be most effective in removing blood stains from cotton cloth. Silver recovery from used X-ray films was achieved within 6 min of treatment with crude enzyme maintained at 40 °C.

Production of dehairing protease by Bacillus cereus VITSN04: a model cradle-to-cradle approach for sustainable greener production of leathers.

Despite several attempts over decades, process scalability and sustainability remain a challenge to produce an environmental-friendly enzyme to gain industrial attention. In the present study, microbial degradation of chrome shavings (chromium-collagen leather waste) and the resulting collagen hydrolysate for producing the dehairing protease by Bacillus cereus VITSN04 were investigated in a lab-scale fermentor. Scale-up degradation of shavings resulted in higher recovery of collagen hydrolysate (76%) within 72 h compared to shake flasks (68% in 120 h). Earlier achieved medium composition of collagen hydrolysate (12 g L-1) and molasses (15 g L-1) appeared to induce amylase at the high rate, despite the maximal production of protease (203.8 ± 0.18 U mL-1), which was analysed by ANS fluorescence spectroscopy. Optimization of the media containing collagen hydrolysate (12 g L-1) and molasses (5 g L-1) was effective in producing protease (170.6 ± 0.1 U mL-1) and reduced the co-synthesis of amylase (48.2 ± 0.09 U mL-1). The controlled fermentation process by feeding molasses during the exponential growth phase had enhanced the dehairing protease production (∼2.96 fold). The produced protease then partitioned through the biphasic system and showed significant dehairing of goat skins on the pilot scale. Thus, the scalability of the process to produce dehairing enzymes using waste, generated at the site of its use, offers hope for sustainable greener production of leathers.

High-expression and characterization of a novel serine protease from Ornithinibacillus caprae L9T with eco-friendly applications.

In the current work, a novel thermophilic serine protease gene (P3862) from Ornithinibacillus caprae L9T was functionally expressed in Bacillus subtilis SCK6. The monomeric enzyme of about 29 kDa was purified to homogeneity with 43.91% of recovery and 2.81-folds of purification. Characterization of the purified protease revealed the optimum activity at pH 7 and 65 °C. The protease exhibited excellent activity and stability in the presence of Na+, Mg2+, Ca2+, ethanediol, n-hexane, Tween-20, Tween-80 and Triton X-100. P3862 displayed favorable caseinolytic activity, moderate keratinolytic activity but no collagenolytic activity. Besides, the homology model of P3862 possessed a globular configuration and characteristic of α/ß hydrolase fold, and displayed stable interactions with casein, glycoprotein and keratin rather than collagen. Moreover, the crude enzyme could completely dehair goatskin within 6 h, resulting in decrease in BOD5, COD and TSS loads by 72.86, 74.07, and 73.79%, respectively, as compared with Na2S treatment. Biocatalytic applications revealed that it could effectively remove egg-stains from fabrics at 37 °C for 30 min with low supplementation (300 U/mL), and was able to degrade the feathers of duck and chicken. Overall, these outstanding properties make P3862 valuable in the development of environmentally friendly biotechnologies .

Exoproduction and Biochemical Characterization of a Novel Serine Protease from Ornithinibacillus caprae L9T with Hide-Dehairing Activity.

This study is the first report on production and characterization of the enzyme from an Ornithinibacillus species. A 4.2-fold increase in the extracellular protease (called L9T) production from Ornithinibacillus caprae L9T was achieved through the one-factor-at-a-time approach and response surface methodological optimization. L9T protease exhibited a unique protein band with a mass of 25.9 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This novel protease was active over a range of pH (4-13), temperatures (30-80°C) and salt concentrations (0-220 g/l), with the maximal activity observed at pH 7, 70°C and 20 g/l NaCl. Proteolytic activity was upgraded in the presence of Ag+, Ca2+ and Sr2+, but was totally suppressed by 5 mM phenylmethylsulfonyl fluoride, which suggests that this enzyme belongs to the serine protease family. L9T protease was resistant to certain common organic solvents and surfactants; particularly, 5 mM Tween 20 and Tween 80 improved the activity by 63 and 15%, respectively. More importantly, L9T protease was found to be effective in dehairing of goatskins, cowhides and rabbit-skins without damaging the collagen fibers. These properties confirm the feasibility of L9T protease in industrial applications, especially in leather processing.

Optimization of fermentation conditions for production of neutral metalloprotease by Bacillus subtilis SCK6 and its application in goatskin-dehairing.

In this study, a high protease-producing strain was screened by spread plate method and identified by molecular biology and morphological identification. It was identified as Bacillus sp. LCB14. A neutral protease gene was cloned and heterologous expressed by B. subtilis SCK6. Then, the recombinant protease was used to dehair the goat skins. The fermentation conditions of neutral protease production by B. subtilis SCK6 were optimized. The single factor experiments, Plackett-Burma experiment, and response surface method were conducted to determine fermentation medium and culture conditions. The optimized medium contained corn meal 49 g/L, soluble starch 28 g/L, soybean meal 17 g/L, corn steep liquor powder 8 g/L, yeast extract 10 g/L, Na2HPO4 2.3 g/L, KH2PO4 1.9 g/L, MgSO4 0.5 g/L, MnCl2 0.1 g/L and ZnSO4 0.05 g/L. The optimized culture conditions were 35 °C and pH 7.0. Under the optimum conditions, the recombinant strain reached 33467.28 U/mL after 72 hr ferment. Moreover, by fed batch in 30 L fermenters, neutral protease production reached 39,440.78 U/mL and shortened fermentation time from 72 hr to 46 hr. Finally, the crude enzyme was utilized to replace sodium sulfide for dehairing of goatskins. The enzymatic dehaired pelts were white, smooth, and soft; the grain side of enzymatic dehaired pelts were clear; there was no obvious damage to the grain side of enzymatic dehaired pelts by visual observation and tactile test. Furthermore, there were no hair roots, hair follicles and other glands in enzymatic dehaired belts, and the collagen fibers of enzymatic dehaired belt were dispersed well by histological analysis.

A substrate protection approach to applying the calcium ion for improving the proteolysis resistance of the collagen.

Enzymatic dehairing, as a crucial part of cleaner leather processing, has reached processive advancement with potentially replacing the traditional hair removal due to increasing pressure from environmental demand. However, this cleaner technology based on proteases has a problem that the hide grain (collagen-rich structure) is susceptible to be hydrolyzed, decreasing the quality of finished leather. From the perspective of improving the stability of collagen fibers and their resistance to proteolysis, a method for protecting the hide grain during the enzymatic dehairing process was developed. The results showed that calcium ions had a swelling effect on collagen fibers under near-neutral conditions (pH 6.0-10.0), decreasing the thermal stability of collagen and the proteolysis resistance of collagen significantly. The alkaline environment (pH 10.0-12.0) will promote the dissociation of carboxyl groups in hide collagen, promoting the combination of calcium ions and carboxyl groups. This strategy can change the surface charge of collagen fibers and strengthen the connection between collagen fibers, thus improving protease resistance and the thermal stability of collagen. However, collagen fibers could swell violently once the alkalinity of the solution environment was extreme. Despite the above situation, calcium ion was still conducive to maintain the structural stability of collagen fibers. At pH 10.0-12.0, pretreating animal hide with a solution containing calcium ions can improve the protease resistance of hide grain, making the hide grain well-protected. This method provided an effective way to establish a safer enzymatic unhairing technology based on substrate protection. KEY POINTS: • A collagen protection method for hair removal of animal hide was developed. • This method applied calcium ions to collagen at alkaline conditions (pH 10.0-12.0). • Pretreatment results of calcium ions at different pH values on animal hide were compared.

Insights into substrate specificity of proteases for screening efficient dehairing enzymes.

Though numerous proteases have been isolated and screened for the dehairing purpose, their use in the leather industry is limited mainly due to high cost, the need for expertise, and control during unit operation and alterations in the quality of leather due to lack of the right kind of substrate specificity of the enzymes used. This paper deals with the comparative specificity and dehairing efficiency of proteases isolated from Bacillus cereus VITSP01 (PE2) and Brevibacterium luteolum VITSP02 (PE). PE2 and PE were found to be trypsin-like and elastase-like serine proteases respectively. The protease of VITSP02 degraded the proteoglycans efficiently in comparison to that of VITSP01. The results suggest that the possible targets of the studied proteases might be skin proteoglycans, including those cementing the hair root bulb. Hence, an in-depth study on the substrate specificity of the dehairing proteases would help in designing an improved screening method for isolating potent dehairing enzymes.

Identification of the Source for Salmonella Contamination of Carcasses in a Large Pig Slaughterhouse.

To identify the major source of Salmonella contamination in a pig slaughterhouse, samples were collected from the clean and unclean area and Salmonella isolates were further typed. Carcasses entering the clean area showed a Salmonella contamination rate of 96.7% in the oral cavity and 55.0% in the rectum content samples. Evisceration seemed not to be critical as the contamination rate of the carcasses was similar before (16.7%) and after (18.3%) this slaughter step. In the unclean area, a limited number of oral cavity samples were positive after bleeding, while a dramatic increase of positives was observed after dehairing. Salmonella was detected in up to 0.01 mL of the recycled water collected from the dehairing machine. Genotyping of Salmonella isolates showed that similar pulsotypes were present in the oral cavity and recycled water. Based on these observations it can be concluded that the recycled water used in the dehairing machine was the major source for the carcass contamination in this slaughterhouse.

Heterologous expression and purification of keratinase from Actinomadura viridilutea DZ50: feather biodegradation and animal hide dehairing bioprocesses.

The keratin-degrading bacterium Actinomadura viridilutea DZ50 secretes a keratinase (KERDZ) with potential industrial interest. Here, the kerDZ gene was extracellularly expressed in Escherichia coli BL21(DE3)pLysS using pTrc99A vector. The recombinant enzyme (rKERDZ) was purified and biochemically characterized. Results showed that the native and recombinant keratinases have similar biochemical characteristics. The conventional dehairing with lime and sodium sulfide degrades the hair to the extent that it cannot be recovered. Thus, these chemical processes become a major contributor to wastewater problem and create a lot of environmental concern. The complete dehairing was achieved with 2000 U/mL rKERDZ for 10 h at 40 °C. In fact, keratinase assisted dehairing entirely degraded chicken feather (45 mg) and removed wool/hair from rabbit, sheep, goat, or bovine' hides (1.6 kg) while preserving the collagen structure. The enzymatic process is the eco-friendly option that reduces biological (BOD) (50%) and chemical (COD) oxygen demands (60%) in leather processing. Consequently, the enzymatic hair removal process could solve the problem of post-treatments encountering the traditional leather processing. The enzymatic (rKERDZ) dehaired leather was analyzed by scanning electron microscopic (SEM) studies, which revealed similar fiber orientation and compactness compared with control sample. Those properties support that the rKERDZ enzyme-mediated process is greener to some extent than the traditional one.

Biochemical characterization of a partially purified protease from Aspergillus terreus 7461 and its application as an environmentally friendly dehairing agent for leather industry.

Proteases can be used in several biotechnological processes including detergent, food and leather industries. In the leather industry, dehairing is carried out by chemicals, which pollute the environment. Therefore, to make the hair removal process environmentally friendly, a protease produced by Aspergillus terreus has been purified, biochemically characterized and had an efficient ability to remove hair from bovine leather. The protease was produced using 1% wheat bran and was purified 2.3-fold using two chromatographic steps showing a molecular weight of 90 kDa. Optimal temperature and pH were 50 °C and 6.5, respectively. Thermal stability was up to 1 h at 50 °C. Protease was stable to detergents like Tween 80 and to organic solvents. The activity was activated by Ca2+ and inhibited by Hg2+ and Cu2+. The enzyme was classified as serine protease, by the inhibition by PMSF and was stable to reducing agents. It hydrolyzed casein, azocasein, BSA, egg albumin and BTpNA. The Km and Vmax values were 0.65 ± 0.03 mg/mL and 3.66 ± 0.18 µmol/min, respectively. Remarkable properties about temperature, pH, stability to detergents and reducing agents ensure that the protease from A. terreus can be an excellent candidate for industrial applications, particularly in the leather industry.

Production and characterization of a novel thermo- and detergent stable keratinase from Bacillus sp. NKSP-7 with perceptible applications in leather processing and laundry industries.

Keratinase has the ability to degrade the recalcitrant keratinous wastes that cannot be degraded by conventional proteases. The present study describes a novel hyperstable keratinolytic enzyme from Bacillus sp. NKSP-7, which has excellent efficiency of keratin-feather biodegradation, washing and dehairing. The production of extracellular keratinase was improved by 3.02-fold through optimization of various parameters. Purified keratinase (25 kDa) showed optimal activity at 65 °C and pH 7.5, and displayed stability over a range of pH (5.5-9.5) and temperature (20-60 °C) for 8 h. No conspicuous effect was perceived with various chemicals and organic solvents, however, the catalytic efficiency was enhanced in the presence of Ca2+, Cd2+, Na+, Mn2+, sodium sulfite, and ß-mercaptoethanol. The enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF), suggesting that this keratinase belongs to serine protease family. It displayed prodigious stability and compatibility to salinity and commercial detergents. Enzyme exhibited great substrate specificity but high affinity was observed with keratin-rich substrates. Crude and purified keratinase revealed perceptible potential for destaining of blood-stained fabric (10 min), and dehairing of hide (8 h) without any damage. All these auspicious features make this enzyme a promising candidate for various industrial applications, especially in keratin-waste management, detergent formulations and leather processing.

Microbial Bioremediation of Feather Waste for Keratinase Production: An Outstanding Solution for Leather Dehairing in Tanneries.

In leather industries and tanneries, large amount of wastes has been disposed; which polluting water, soil, and atmosphere and causing serious human health problems. In particular, chemical dehairing process of leather industries produces fair amount of toxic wastes. It is, thus, urgently needed to use alternative processes free from pollution. As more than 90% of keratin is contained in feather, it is desirable to develop bioremediation process using keratinolytic microorganisms. In the present investigation, therefore, we first identified Bacillus cereus and Pseudomonas sp. to be able to produce keratinase. Then, the optimization was performed to maximize the keratinase activity with respect to cultivation temperature, pH, and incubation time. Moreover, the effects of metal ions and various substrates on keratinase activity were also investigated. The result indicates that keratinase activity became maximum at 50°C for both strains, whereas the optimal pH was 10.0 for B. cereus and 7.0 for Pseudomonas sp. The highest keratinase activity of 74.66 ± 1.52 U/mL was attained by B. cereus, whereas 57.66 ± 2.52 U/mL was attained by Pseudomonas sp. Enzymatic dehairing efficiency of leathers was also compared with chemical dehairing (Na2S and CaO), where complete dehairing was achieved by treating them with crude keratinase. Partial enzyme purification was performed by acetone precipitation. Batch cultivation of B. cereus using 1 L fermentor indicates a potential candidate for large-scale keratinase production. Thus, keratinase enzyme by degrading poultry wastes (feather) can be an alternative approach to chemical dehairing in leather industries, thus preventing environmental pollution through bioremediation.

Laundry Detergent Compatibility and Dehairing Efficiency of Alkaline Thermostable Protease Produced from Aspergillus terreus under Solid-state Fermentation.

Aspergillus terreus was chosen for production of alkaline protease using solid-state fermentation (SSF). The maximum enzyme yield reached about 34.87 U/mg protein after optimization of fermentation parameters. The produced alkaline protease was purified by precipitation with iso-propanol and then purified through gel filtration and ion exchange column chromatography with a yield of 53.58% and 5.09- fold purification. The enzyme has shown to have a molecular weight of 35 kDa. Optimal pH and temperature for the enzyme activity were 9.5 and 50°C respectively. The highest activity was reported towards casein, with an apparent Km value of 6.66 mg/mL and Vmax was 30 U/mL. The enzyme activity was greatly repressed by phenylmethylsulfonyl fluoride (PMSF). Sodium dodecyl sulfate (SDS) caused activation in enzyme activity. The enzyme retained about 83.8, 70.6, 74.5, 76.4 and 66.4% of its original activity after incubation with Aerial, Leader, Oxi, Persil and Tide, respectively for 8 h at 60°C. Adding of the enzyme in detergents improved the cleansing performance to the blood stains and suggested to be used as a detergent additive. Our outcomes showed that protease could be used as environment green-approach in dehairing process.

High-expression keratinase by Bacillus subtilis SCK6 for enzymatic dehairing of goatskins.

An extracellular keratinase gene from Bacillus sp. LCB12, isolated from saline-alkali soil, was cloned and high-effectively expressed in Bacillus subtilis SCK6. The enzyme was purified by ammonium sulphate precipitation and cation-exchange chromatography. Its molecular mass was estimated to be 30.95 kDa by SDS-PAGE. The optimum conditions for catalytic activity of the enzyme were pH 10.0 and 60 °C. The enzyme activity was completely inhibited by PMSF, while it was slightly inhibited by EDTA. Moreover, the surfactants Tween 20, Tween 80 and Triton X-100, showed little effect on enzyme activity respectively. The crude enzyme was used as an alternative to sodium sulfide for dehairing of goat skins. The goatskin was dehaired by the enzyme at 33-35 °C in 6 h. The enzymatic dehaired pelt showed better general appearance and better whiteness by visual tests, and the grain surface of enzymatic dehaired pelt revealed absence of hair shaft with empty follicles by stereoscopic observation. Meanwhile, the epidermis was completely removed and the collagen fiber structure of enzymatic dehaired pelt was more opened, regular and even in dermis comparing with conventional dehaired pelt by histological analysis.

Thermostable and halotolerant keratinase from Bacillus aerius NSMk2 with remarkable dehairing and laundary applications.

Keratinases hydrolyze structural protein called keratin into constituent peptides. The present study reports excellent washing efficiency and dehairing properties of thermostable and halotolerant keratinase from Bacillus aerius NSMk2. Alkaline keratinase with molecular mass of 9 kDa displayed remarkable thermostability. K+ , Na+ , Ca2+ , Mn2+ , ß-mercaptoethanol, sodium sulfite, dithiothreitol, ethanol, isopropanol, Tween-20, and Tween-80 stimulated keratinase activity, while Hg2+ and Ba2+ were found to be inhibitory. The enzyme efficiently hydrolyzed a variety of complex protein substrates and exhibited high catalytic efficiency toward keratin-rich substrates and least toward collagen. Keratinase showed exceptional stability to salinity and was found to be compatible with most of the commercial detergents. Efficient removal of chocolate, blood, and egg albumin stains from clothes and tolerance to elevated temperature and salinity potentiated the suitability of keratinase from B. aerius NSMk2 as laundary additive. Keratinase could efficiently dehair goat skin after 15 hr of incubation without damaging the grain structure and collagen layers that assures its use as a promising contender for leather industry.

Cloning, expression, and characterization of an alkaline protease, AprV, from Vibrio sp. DA1-1.

A novel alkaline protease (named AprV) gene from Vibrio sp. DA1-1 was cloned and expressed in Escherichia coli BL21 (DE3) pLysS. The sequence analysis showed the highest homology of 68% with the characterized protease from Alkalimonas collagenimarina AC40T. The recombinant AprV was purified with the molecular weight of 28 kDa. The optimum temperature and pH were determined to be 55 °C and 10.0, respectively. The enzyme activity was slightly enhanced by Ca2+, Mg2+, Zn2+, Ba2+, and, however, was highly inhibited by Sn2+ and EDTA. The AprV was stable in the presence of some surfactants and oxidizing agents, such as 1% Tween 20-80, 1% JFC-2, and 5% JFC-2. Casein was found to be the ideal substrate with specific activity of 1139 U/mg. Moreover, we found that AprV (10,000 U), together with commercial detergent, could completely remove the blood on the cotton. Furthermore, AprV also demonstrated dehairing activity on goat and bull skin. These results indicated that the alkaline protease AprV might be a potential candidate for applications in the detergent and leather industries.