Feathers are a major by-product of the poultry industry. They are mainly composed of keratins which have wide applications in different fields. Due to the increasing production of feathers from poultry industries, the untreated feathers could become pollutants because of their resistance to protease degradation. Feathers are rich in amino acids, which makes them a valuable source for fertilizer and animal feeds. Numerous bacteria and fungi exhibited capabilities to degrade chicken feathers by secreting enzymes such as keratinases, and accumulated evidence shows that feather-containing wastes can be converted into value-added products. This review summarizes recent progress in microbial degradation of feathers, structures of keratinases, feather application, and microorganisms that are able to secrete keratinase. In addition, the enzymes critical for keratin degradation and their mechanism of action are discussed. We also proposed the strategy that can be utilized for feather degradation. Based on the accumulated studies, microbial degradation of feathers has great potential to convert them into various products such as biofertilizer and animal feeds.

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Progress in Microbial Degradation of Feather Waste

Proteins are one of the most important and resourceful biomolecules that find applications in health, industry, medicine, research, and biotechnology. Given its tremendous relevance, protein engineering has emerged as significant biotechnological intervention in this area. Strategic utilization of protein engineering methods and approaches has enabled better enzymatic properties, better stability, increased catalytic activity and most importantly, interesting and wide range applicability of proteins. In fact, the commercialization of engineered proteins have manifested in economically beneficial and viable solutions for industry and healthcare sector. Protein engineering has also evolved to become a powerful tool contributing significantly to the developments in both synthetic biology and metabolic engineering. The present review revisits the current trends in protein engineering approaches such as rational design, directed evolution, de novo design, computational approaches etc. and encompasses the recent progresses made in this field over the last few years. The review also throws light on advanced or futuristic protein engineering aspects, which are being explored for design and development of novel proteins with improved properties or advanced applications.

Current Trends in Protein Engineering: Updates and Progress

Proteases are important industrial biocatalysts that constitute the largest group of enzymes acting as proteinases, peptidases, and amidases with a broad range of industrial applications. In this review, particular attention has been given to comprehensively scrutinize the proteases. After the succinct introduction, classification of proteases as exopeptidases (amino and carboxy proteases) and endopeptidases (serine, aspartic, cysteine, and metalloproteases), sources of alkaline, acidic and neutral protease like animal, plant and microbial sources along with the multi-industrial applications have been dissertated. Now a day’s, mostly proteases, which are present in the market, are produced from microbial sources because of the fast production rate and the limited requirement of cultivation. In addition to this, a critique on the applications of proteases in food, detergent, leather, pharmaceutical, cosmetics, silk degumming, silver recovery, chemical industry, and wastewater treatment industries is also concisely addressed. Finally, protein engineering and immobilization strategies to improve the catalytic properties of protease are thoroughly vetted. The quest for novel sources of protease enzyme has been encouraged to fulfill their ever-increasing demands for industrial exploitation.

Protease—A Versatile and Ecofriendly Biocatalyst with Multi-Industrial Applications: An Updated Review

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Enzyme Kinetics A Modern Approach

Bacillus spp. are an affordable source of enzymes due to their wide distribution, safety in work, ease of cultivation, and susceptibility to genetic transformations. Researchers are particularly interested in proteolytic enzymes, which constitute one of the most diverse groups of microbial proteins in terms of properties. Despite the long history of their research, this group of enzymes continue to show great potential for practical application in the biomedical industry, as well as in the agricultural industry. Thus, the unique properties of bacillary proteinases, such as stability in a wide range of temperatures and pH, high specificity, biodegradability of a wide range of substrates, and the high potential of sequenced Bacillus genomes are a powerful foundation for the development of new biotechnologies. The current review aims to discuss recent studies on various enzymes in particular, proteinases produced by bacteria of the genus Bacillus, along with their prospective practical applications. This article also presents an interpretive summary of the recent developments on the usage of probiotic Bacillus strains as potential feed additives.

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The Practical Potential of Bacilli and Their Enzymes for Industrial Production

Mesophilic alkaline serine proteases from various bacteria have been commercially applied in a range of industries owing to their high catalytic efficiency and wide substrate specificity. However, these proteases have an optimal catalytic temperature of approximately 50 °C, and their activity decreases significantly at low temperature. Therefore, to enhance their cold activity, it is necessary to improve the catalytic performance of these proteases at low temperature. The alkaline serine protease (DHAP) from Bacillus pumilus BA06 is a typical mesophilic enzyme, which has demonstrated great potential in various industrial applications. Here we attempted to improve the cold activity of DHAP via directed evolution. Seven variants (P9S, A1G/K27Q, A38V, A116T, T162I, S182R, and T243S) of DHAP from B. pumilus were obtained via directed evolution. The results showed that all of the variants had increased proteolytic activity at 15 °C towards both the casein and synthetic peptide substrates. With the exception of variant T243S, the thermostability of these variants did not decrease in comparison with the wild-type enzyme. Kinetic analysis indicated that the increase in catalytic efficiency was largely attributed to the increase in turnover number (kcat). Furthermore, the combined variants generated by site-directed mutagenesis showed a further increase in specific caseinolytic activity and the kcat value for hydrolysis of the synthetic peptide. The combined variants of P9S/K27Q and P9S/T162I exhibited an approximate 5-fold increase in caseinolytic activity at 15 °C and almost no loss of thermostability. Finally, the possible mechanism responsible for the change in catalytic properties for these variants was interpreted based on structural modeling. Directed evolution and site-directed mutagenesis were combined to engineer variants of the DHAP from B. pumilus. All of the variants exhibited an increase in hydrolytic efficiency at low temperature towards both of the substrates, casein and synthetic peptide, without any loss of thermostability compared with the wild-type. These data suggest that engineering low-temperature activity for a bacterial protease is not always associated with the loss of thermostability. Furthermore, our findings demonstrate that enhanced cold activity and thermostability could be integrated into a single variant.

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Engineering Bacillus pumilus alkaline serine protease to increase its low-temperature proteolytic activity by directed evolution

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

A native plasmid (pSU01) was detected by genome sequencing of Bacillus subtilis strain S1-4 Two pSU01-based shuttle expression vectors pSU02-AP and pSU03-AP were constructed enabling stable replication in B subtilis WB600 These vectors contained the reporter gene aprE, encoding an alkaline protease from Bacillus pumilus BA06 The expression vector pSU03-AP only possessed the minimal replication elements (rep, SSO, DSO) and exhibited more stability on structure, suggesting that the rest of the genes in pSU01 (ORF1, ORF2, mob, hsp) were unessential for the structural stability of plasmid in B subtilis In addition, recombinant production of the alkaline protease was achieved more efficiently with pSU03-AP whose copy number was estimated to be more than 100 per chromosome Furthermore, pSU03-AP could also be used to transform and replicate in B pumilus BA06 under selective pressure In conclusion, pSU03-AP is expected to be a useful tool for gene expression in Bacillus subtilis and B pumilus

Construction of novel shuttle expression vectors for gene expression in Bacillus subtilis and Bacillus pumilus.

To engineer dehairing alkaline protease (DHAP) variants to improve cold activity and increase thermostability so these variants are suitable for the leather processing industry. Based on previous studies with bacterial alkaline proteases, double-site mutations (W106K/V149I and W106K/M124L) were introduced into the DHAP from Bacillus pumilus. Compared with the wild-type DHAP hydrolytic activity, the double-site variant W106K/V149I showed an increase in specific hydrolytic activity at 15 °C by 2.3-fold toward casein in terms of hydrolytic rate and 2.7-fold toward the synthetic peptide AAPF-pN by means of kcat/Km value. The thermostability of the variant (W106K/V149I) was improved with the half-life at 60 and 70 °C increased by 2.7- and 5.0-fold, respectively, when compared with the thermostability of the wild-type DHAP. Conclusively, an increase in the cold activity and thermostability of a bacterial alkaline protease was achieved by protein engineering.

Hong-Yan Zhao

Papers

Engineering Bacillus pumilus alkaline serine protease to increase its low-temperature proteolytic activity by directed evolution

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

Construction of novel shuttle expression vectors for gene expression in Bacillus subtilis and Bacillus pumilus.

Single-site substitutions improve cold activity and increase thermostability of the dehairing alkaline protease (DHAP)