Purification and characterization of a keratinolytic protease produced by probiotic Bacillus subtilis

Ferrareze, Patrícia Aline Gröhs; Corrêa, Ana Paula Folmer; Brandelli, Adriano

doi:10.1016/j.bcab.2016.05.009

Cited by 35 publications

(16 citation statements)

References 51 publications

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“…Yadav et al ( 2015 ) reported that protease activity is increased by Na 2+ (140%) and Fe 2+ (156%) at 2 mM; and, furthermore, that Sn 2+ , Cu 2+ , Pb 2+ and Al 3+ exert stimulating effects at 0.2 mM concentration, however there are few corroborating reports of these findings. In most studies, Cu 2+ has been reported as a strong inhibitor; for example, when adding 1–5 mM Cu 2+ , the protease activity decreases by between 12% to 98% (Divakar et al 2010 ; Yu et al 2011 ; Yadav et al 2015 ; Ferrareze et al 2016 ; Abidi et al 2011 ; Hsiao et al 2014 ; Khaled et al 2011 ). Protease inhibition by Mg 2+ , Ba 2+ and Zn 2+ were also reported by Norifumi et al ( 2012 ), who suggested that these metal ions chemically react with the protein group in the polypeptide chain of the enzyme, resulting in the loss of protease activity (Kamran et al 2015 ).…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of neutral protease from Aspergillus oryzae Y1 isolated from naturally fermented broad beans

et al. 2018

AMB Expr

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The strain Y1, with a notably high production of neutral protease, was isolated from naturally fermented broad beans and subsequently identified as Aspergillus oryzae, through the analysis of its morphology characteristics and 18S rDNA sequence. Naturally fermented broad beans are the main raw material in Sichuan broad-bean sauce. The neutral protease from Aspergillus oryzae Y1 was purified using ammonium sulphate precipitation and DEAE-Sepharose Fast Flow chromatography, which resulted in a 10.0-fold increase in the specific activity (2264.3 U/mg) and a recovery rate of 21%. The estimated molecular mass of the purified protease was approximately 45 kDa. The optimal pH and temperature of the purified protease were 7.0 and 55 °C, respectively. The heat resistance of the purified protease was significantly higher than the commercial protease. The effect of metal ions on the activity of the purified protease approximated that of commercial neutral protease. Furthermore, the maximum hydrolysis rate (Vmax) and apparent Michaelis–Menten constant (Km) values of the purified protease were 256.4103 μg/mL min and 20.0769 mg/mL, respectively. The purified protease had a higher affinity for the substrate than the commercial neutral protease. All the results suggest that this neutral protease exhibits the potential for application in industry due to its good resistance to high temperatures and wide range of acids and bases.

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Section: Discussionmentioning

confidence: 99%

Purification and characterization of neutral protease from Aspergillus oryzae Y1 isolated from naturally fermented broad beans

et al. 2018

AMB Expr

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“…Indeed, it has been previously shown that Stenotrophomonas and Bacillus strains proteases belong to different families with specific catalytic mechanisms (Yamamura et al, 2002;Ferrareze et al, 2016), thus making them potentially complementary if applied together. Therefore, if grown successfully with the right condition and partners, the remarkable mesophilic keratinase activity of St may be potentially interesting for saving energy purposes (Ferrareze, Correa and Brandelli, 2016). For instance, it has been suggested to include glucose during the incubation period to help Stenotrophomonas species growth and optimize keratinase production (Fang et al, 2013).…”

Section: Azokeratin Assay Testing On Bacterial Strainsmentioning

confidence: 99%

Azo dying of α‐keratin material improves microbial keratinase screening and standardization

Gonzalo

Espersen

Al‐Soud

et al. 2020

Microbial Biotechnology

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Microbial conversion through enzymatic reactions has received a lot of attention as a cost-effective and environmentally friendly way to recover amino acids and short peptides from keratin materials. However, accurate assessment of microbial keratinase activity is not straightforward, and current available methods lack sensitivity and standardization. Here, we suggest an optimized Azokeratin assay, with substrate generated directly from azo-dyed raw keratin material. We introduced supernatant filtration in the protocol for optimal stopping of keratinase reactions instead of the widely used trichloroacetic acid (TCA), as it generated biases and impacted the sensitivity. We furthermore suggest a method for standardization of keratinase activity signals using proteinase K, a well-known keratinase, as a reference enabling reproducibility between studies. Lastly, we evaluated our developed method with several bacterial isolates through benchmarking against a commercial assay (Keratin Azure). Under different setups, the Azokeratin method was more sensitive than commonly used Keratin Azure-based assays (3-fold). We argue that this method could be applied with any type of keratin substrate, enabling more robust and sensitive results which can be used for further comparison with other studies, thus representing an important progress within the field of microbial keratin degradation.

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“…B. invictae enzymatic preparation retained 94% of its original activity after 60 min at 50°C [46]. In the case of B. subtilis FTC02PR1 keratinolytic enzymes showed a rapid activity loss after incubation for 15 min at 55°C [47]. In addition, Gegeckas et al [48] reported that the half-life time of BPKer from Bacillus sp.…”

Section: Discussionmentioning

confidence: 99%

The keratinolytic bacteria Bacillus cytotoxicus as a source of novel proteases and feather protein hydrolysates with antioxidant activities

Cavello

Bezus

Cavalitto

2021

Journal of Genetic Engineering and Biotechnology

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Background Argentina’s geothermal areas are niches of a rich microbial diversity. In 2020, species of Bacillus cytotoxicus were isolated for the first time from these types of pristine natural areas. Bacillus cytotoxicus strains demonstrated the capability to grow and degrade chicken feathers with the concomitant production of proteases with keratinolytic activity, enzymes that have multitude of industrial applications. The aim of this research was to study the production of the proteolytic enzymes and its characterization. Also, feather protein hydrolysates produced during fermentation were characterized. Results Among the thermotolerant strains isolated from the Domuyo geothermal area (Neuquén province, Argentina), Bacillus cytotoxicus LT-1 and Oll-15 were selected and put through submerged cultures using feather wastes as sole carbon, nitrogen, and energy source in order to obtain proteolytic enzymes and protein hydrolysates. Complete degradation of feathers was achieved after 48 h. Zymograms demonstrated the presence of several proteolytic enzymes with an estimated molecular weight between 50 and > 120 kDa. Optimum pH and temperatures of Bacillus cytotoxicus LT-1 crude extract were 7.0 and 40 °C, meanwhile for Oll-15 were 7.0 and 50 °C. Crude extracts were inhibited by EDTA and 1,10 phenanthroline indicating the presence of metalloproteases. Feather protein hydrolysates showed an interesting antioxidant potential measured through radical-scavenging and Fe3+-reducing activities. Conclusion This work represents an initial approach on the study of the biotechnological potential of proteases produced by Bacillus cytotoxicus. The results demonstrated the importance of continuous search for new biocatalysts with new characteristics and enzymes to be able to cope with the demands in the market.

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Purification and characterization of a keratinolytic protease produced by probiotic Bacillus subtilis

Cited by 35 publications

References 51 publications

Purification and characterization of neutral protease from Aspergillus oryzae Y1 isolated from naturally fermented broad beans

Purification and characterization of neutral protease from Aspergillus oryzae Y1 isolated from naturally fermented broad beans

Azo dying of α‐keratin material improves microbial keratinase screening and standardization

The keratinolytic bacteria Bacillus cytotoxicus as a source of novel proteases and feather protein hydrolysates with antioxidant activities

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