Production, one-step purification, and characterization of a keratinolytic protease from Serratia marcescens P3

Bach, Evelise; Sant’Anna, Voltaire; Daroit, Daniel Joner; Corrêa, Ana Paula Folmer; Segalin, Jéferson; Brandelli, Adriano

doi:10.1016/j.procbio.2012.10.007

Cited by 66 publications

(31 citation statements)

References 33 publications

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“…UFPEDA 485. On the other hand, values of K > 1 in runs 3, 4, 7 and 8 point out that, at the highest PEG concentration (C PEG = 30.0% w/w), the enzyme preferentially partitioned to the top PEG-rich phase likely due its high hydrophobicity [12,31,32] or to a salting out effect [6,31,32]. As shown in Fig.…”

Section: Preliminary Selection Of Atps For Fibrinolytic Protease Extrmentioning

confidence: 85%

“…Partitioning in aqueous two-phase systems (ATPS) is an effective method for separating and purifying mixtures of biomolecules [12][13][14][15], which is suitable for large-scale production in an environmentally friendly way. Traditional techniques such as chromatography and ammonium sulfate precipitation are slowly being replaced, especially in the primary steps of proteases purification, by extraction/purification methods like ATPS, because of their lower costs of production and operation [6,12,16,17].…”

Section: Introductionmentioning

confidence: 99%

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Purification of a fibrinolytic protease from Mucor subtilissimus UCP 1262 by aqueous two-phase systems (PEG/sulfate)

Nascimento

Sales

Porto

et al. 2016

Journal of Chromatography B

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A fibrinolytic protease from M. subtilissimus UCP 1262 was recovered and partially purified by polyethylene glycol (PEG)/sodium sulfate aqueous two-phase systems (ATPS). The simultaneous influence of PEG molar mass, PEG concentration and sulfate concentration on the enzyme recovery was first investigated using a 2(3) full factorial design, and the Response Surface Methodology used to identify the optimum conditions for enzyme extraction by ATPS. Once the best PEG molar mass for the process had been selected (6000g/mol), a two-factor central composite rotary design was applied to better evaluate the effects of the other two independent variables. The fibrinolytic enzyme was shown to preferentially partition to the bottom phase with a partition coefficient (K) ranging from 0.2 to 0.7. The best results in terms of enzyme purification were obtained with the system formed by 30.0% (w/w) PEG 6000g/mol and 13.2% (w/w) sodium sulfate, which ensured a purification factor of 10.0, K of 0.2 and activity yield of 102.0%. SDS-PAGE and fibrin zymography showed that the purified protease has a molecular mass of 97kDa and an apparent isoelectric point of 5.4. When submitted to assays with different substrates and inhibitors, it showed selectivity for succinyl-l-ala-ala-pro-l-phenylalanine-p-nitroanilide and was almost completely inhibited by phenylmethylsulfonyl fluoride, behaving as a chymotrypsin-like protease. At the optimum temperature of 37°C, the enzyme residual activity was 94 and 68% of the initial one after 120 and 150min of incubation, respectively. This study demonstrated that M. subtilissimus protease has potent fibrinolytic activity compared with similar enzymes produced by solid-state fermentation, therefore it may be used as an agent for the prevention and therapy of thrombosis. Furthermore, it appears to have the advantages of low cost production and simple purification.

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Section: Preliminary Selection Of Atps For Fibrinolytic Protease Extrmentioning

confidence: 85%

Section: Introductionmentioning

confidence: 99%

Purification of a fibrinolytic protease from Mucor subtilissimus UCP 1262 by aqueous two-phase systems (PEG/sulfate)

Nascimento

Sales

Porto

et al. 2016

Journal of Chromatography B

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“…The optimum activity of keratinases range between pH 6.0 and 9.0. The enzymes are either neutral or alkaline proteases requiring Ca ++ , as the activity is inhibited in presence of Ca ++ chelating EDTA or EGTA [76]. Keratinases are able to hydrolyze both soluble proteins as well as insoluble, fibrous proteins [77].…”

Section: Microbial Keratinasementioning

confidence: 99%

Keratinaceous Wastes and Their Valorization through Keratinolytic Microorganisms

Ningthoujam¹,

Tamreihao²,

SaikatMukherjee³

et al. 2018

Keratin

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Keratin is a fibrous protein mainly found in higher vertebrates such as mammals, birds, and reptiles. It is also a major constituent of human epithelial tissues. Major keratinaceous wastes include skin, hair, wool, feather, horns, hooves, and nails. Large amounts of such wastes are generated from meat industry, poultry houses, and wool industry etc. Though keratinous wastes contain about 90% protein, keratin is usually recalcitrant to normal proteases. Such wastes have been traditionally digested using physico-chemical methods. But such techniques are energy-intensive and technologically demanding. Also, such approaches lead to degradation of certain amino acids such as lysine. In nature, keratinaceous wastes don't accumulate indicating that keratinolytic microorganisms exist in nature. Keratinase producing strains are distributed among bacteria, fungi, and actinobacteria etc. Hence, potent keratinolytic microbes and their enzymes may be used for valorization of keratinous wastes. Efficient degradation of such wastes may generate value-added products such as feed additives, agricultural biofertilizers, and cosmetics. This chapter will give a comprehensive overview of types of keratinaceous wastes, kinds of keratinolytic microbes and keratinases, and valorization of such wastes using keratinase producing strains and/or keratinases.

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“…Alternatively, the optimal components concentration can be deduced using a central composite design (CCD), followed by analysis using the RSM (Harde et al 2011;Daroit et al 2011;Bach et al 2012). …”

Section: Optimisation Of Keratinase Productionmentioning

confidence: 99%

Microbial keratinases: characteristics, biotechnological applications and potential.

Purchase

2016

The Handbook of Microbial Bioresources

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Keratinases are a group of proteolytic enzymes that can catalyse the cleavage and hydrolysis of the highly stable and fibrous proteins: keratins. A diverse range of microorganisms, including fungi, actinomycetes and bacteria, have been reported to produce keratinases that have biotechnological applications and potential. These keratinases have been usefully applied in agricultural, pharmaceutical, leather and textile processes as well as within environmentally friendly waste management solutions. Potential uses of keratinases include the fields of biomedicine, cosmetics, biological control and the generation of green energy. Herein we aim to provide an overview of the properties of this group of versatile enzymes, including the mechanisms of keratin degradation. The diversity of microbial sources of keratinases is discussed and the optimisation of keratianse production examined.We conclude with an assessment of the established biotechnological applications of keratinases in different industries and current research that highlights other promising potential uses.

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Production, one-step purification, and characterization of a keratinolytic protease from Serratia marcescens P3

Cited by 66 publications

References 33 publications

Purification of a fibrinolytic protease from Mucor subtilissimus UCP 1262 by aqueous two-phase systems (PEG/sulfate)

Purification of a fibrinolytic protease from Mucor subtilissimus UCP 1262 by aqueous two-phase systems (PEG/sulfate)

Keratinaceous Wastes and Their Valorization through Keratinolytic Microorganisms

Microbial keratinases: characteristics, biotechnological applications and potential.

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