Keratinases and sulfide from Bacillus subtilis SLC to recycle feather waste

Cedrola, Sabrina Martins Lage; Melo, Ana Cristina Nogueira de; Mazotto, Ana Maria; Lins, Ulysses; Zingali, Russolina B.; Rosado, Alexandre Soares; Peixoto, Raquel S.; Vermelho, Alane Beatriz

doi:10.1007/s11274-011-0930-0

Cited by 71 publications

(48 citation statements)

References 51 publications

(48 reference statements)

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“…Peptidases from extremophiles such as Fervidobacterium and Thermoanaerobacter genera have been described (Brandelli 2008;Cedrola et al 2012;Friedrich and Antranikian 1996;Riessen and Antranikian 2001;Suzuki et al 2006). Most keratinolytic microorganisms degrade chicken feather but dermatophyte fungi are frequently been associated to with human hair degradation (Brandelli et al 2010;Gopinath et al 2015;Grumbt et al 2013;Gupta and Ramnani 2006;Mazotto et al 2011).…”

Section: Discussionmentioning

confidence: 99%

Extracellular peptidases from Deinococcus radiodurans

et al. 2015

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The extremophile Deinococcus radiodurans wild type R1 produces peptidases (metallo- and serine-) in TGY medium and in the media supplemented with human hair (HMY) and chicken feathers (FMY). Enzymatic screening on agar plates revealed peptidase activity. In TGY medium metallopeptidases were detected corresponding to a molecular mass range of 300-85 kDa (gelatinases); 280-130 (caseinases) and a 300 and a 170 kDa (keratinases); and a gelatinolytic serine peptidase (75 kDa). In HMY medium after 144 h, D. radiodurans produced keratinase (290 U/ml), gelatinase (619 U/ml) and sulfite (26 µg/ml). TGY medium produced higher proteolytic activity: 950 U/ml of gelatinolytic (24 h); 470 U/ml of keratinolytic (24 h) and 110 U/ml of caseinolytic (72 h). In the FMY medium, we found gelatinolytic (317 U/ml), keratinolytic (43 U/ml) and caseinolytic (85 U/ml) activities. The sulfite had a maximum release at 48 h (8.1 µg/ml). Enzymography analysis revealed that the keratinases degraded keratin after 24 h of reaction. The addition of sodium sulfite (1.0 %) improved the keratin degradation. Environmental Scanning Electron microscopy revealed alterations such as damage and holes in the hair fiber cuticle after D. radiodurans growth. This work presents for the first time D. radiodurans as a new keratinolytic microorganism.

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Section: Discussionmentioning

confidence: 99%

Extracellular peptidases from Deinococcus radiodurans

et al. 2015

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“…The protease sample was stored in storage buffer, pH 8.0 (25 mM phosphate, 50% glycerol, 1 mM CaCl 2 ). Finally, the purified protease was analyzed by 12.5% SDS-PAGE and subjected to zymogram analysis with casein as the substrate (Cedrola et al, 2012).…”

Section: Purification Of the Serine Proteasementioning

confidence: 99%

Improving production of extracellular proteases by random mutagenesis and biochemical characterization of a serine protease in Bacillus subtilis S1-4

et al. 2016

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ABSTRACT. The feather is a valuable by-product with a huge annual yield produced by the poultry industry. Degradation of feathers by microorganisms is a prerequisite to utilize this insoluble protein resource. To improve the degrading efficiency of feathers, mutagenesis of the bacterium Bacillus subtilis S1-4 was performed. By combining ultraviolet irradiation and N-methyl-N'-nitro-N-nitrosoguanidine treatment for mutagenesis, a high protease-producing mutant (UMU4) of B. subtilis S1-4 was selected, which exhibited 2.5-fold higher extracellular caseinolytic activity than did the wild-type strain. UMU4 degraded chicken feathers more efficiently, particularly for the release of soluble proteins from the feathers, compared to the wild-type strain. Furthermore, an extracellular protease with a molecular weight of 45 kDa, as determined by SDS-PAGE, was purified from UMU4. Biochemical characterization indicated that the caseinolytic activity of the protease was largely inhibited by phenylmethanesulfonyl fluoride, suggesting that the purified enzyme is a serine protease. This protease was highly active over a wide range of pHs (6.0 to 12.0) and temperatures (50° to 75°C) with an optimal pH and temperature of 8.0 and 65°C, respectively. The purified enzyme exhibited good thermostability with a 72.2 min half-life of thermal denaturation at 60°C. In addition, this protease was not sensitive to heavy metal ions, surfactants, or oxidative reagents. In conclusion, strain improvement for protease production can serve as an alternative strategy to promote feather degradation. The UMU4 mutant of B. subtilis and its serine protease could be potentially used in various industries.

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“…Occasionally, combined chemical and enzymatic hydrolysis also proves to be advantageous [10,19]. Bacteria-mediated keratin breakdown is known to be a cumulative effect of proteolytic cleavage as well as auxiliary reducing factors, including released reduced thiols or indigenous disulfide reductase enzymes [20,21]. Nevertheless, enzymatic keratinolysis in vitro, conducted in the absence of red-ox potential of living microbial cells usually requires additional reducing agents to support disulfide bonds cleavage.…”

Section: Introductionmentioning

confidence: 99%

Enzymatic Degradation of Pretreated Pig Bristles with Crude Keratinase of Bacillus cereus PCM 2849

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et al. 2016

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Purpose The aim of the study was to apply and optimize the process of bioconversion of pig bristle waste using keratinolytic enzymes of Bacillus cereus PCM 2849, and to evaluate the amino acid composition of the resultant hydrolysate. Methods Hydrolysis with concentrated culture fluid of B. cereus was applied for bioconversion of pig bristles, after thermo-chemical pretreatment with sulfite. The effect of substrate concentration, sulfite concentration during pretreatment and reaction temperature on the release of amino acids was determined using Box-Behnken design. Amino acid composition of the obtained hydrolysate was determined by HPLC. Structural condition and substructural changes of the residual substrate were evaluated with SEM microscopy and FTIR spectroscopy. Results The applied enzymatic preparation for bristle biodegradation was verified to contain multiple proteases of a wide molecular weight range. A regression model was developed, in which influential parameters were: linear effect of substrate concentration, followed by quadratic effects of reaction temperature, substrate concentration and pretreatment. Optimum reaction conditions were also determined. The resultant hydrolysate was rich in branched-chain amino acids. Residual substrate was detriorated and sulfitolytic cleavage of disulfides and alteration of protein secondary structures was confirmed. Conclusions Application of B. cereus crude keratinase allowed for partial hydrolysis of pig bristles, preceded by sulfitolytic pretreatment. A regression model was built to describe the process of hydrolysis to release free amino acids, at constant enzyme load. Hydrolysis in given conditions allowed to obtain hydrolysate rich in branched chain amino acids. The presented process poses an alternate way of management over pig bristles, a hard-to-degrade keratinous waste.

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Keratinases and sulfide from Bacillus subtilis SLC to recycle feather waste

Cited by 71 publications

References 51 publications

Extracellular peptidases from Deinococcus radiodurans

Extracellular peptidases from Deinococcus radiodurans

Improving production of extracellular proteases by random mutagenesis and biochemical characterization of a serine protease in Bacillus subtilis S1-4

Enzymatic Degradation of Pretreated Pig Bristles with Crude Keratinase of Bacillus cereus PCM 2849

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