Recombinant Keratinase Research Articles

Biochemical and molecular characterization of novel keratinolytic protease from Bacillus licheniformis (KRLr1).

The keratin-degrading bacterium Bacillus licheniformis secretes a keratinase with potential industrial interest. Here, the Keratinase gene was intracellularly expressed in Escherichia coli BL21(DE3) using pET-21b (+) vector. Phylogenetic tree analysis showed that KRLr1 is closely related to Bacillus licheniformis keratinase that belongs to the serine peptidase/subtilisin-like S8 family. Recombinant keratinase appeared on the SDS-PAGE gel with a band of about 38 kDa and was confirmed by western blotting. Expressed KRLr1 was purified by Ni-NTA affinity chromatography with a yield of 85.96% and then refolded. It was found that this enzyme has optimum activity at pH 6 and 37°C. PMSF inhibited the KRLr1 activity and Ca2+ and Mg2+ increased the KRLr1 activity. Using keratin 1% as the substrate, the thermodynamic values were determined as Km 14.54 mM, kcat 912.7 × 10-3 (S-1), and kcat/Km 62.77 (M-1 S-1). Feather digestion by recombinant enzyme using HPLC method, showed that the amino acids cysteine, phenylalanine, tyrosine and lysine had the highest amount compared to other amino acids obtained from digestion. Molecular dynamics (MD) simulation of HADDOCK docking results exhibited that KRLr1 enzyme was able to interact strongly with chicken feather keratine 4 (FK4) compared to chicken feather keratine 12 (FK12). These properties make keratinase KRLr1 a potential candidate for various biotechnological applications.

Design of 5′-UTR to Enhance Keratinase Activity in Bacillus subtilis

Keratinase is an important industrial enzyme, but its application performance is limited by its low activity. A rational design of 5′-UTRs that increases translation efficiency is an important approach to enhance protein expression. Herein, we optimized the 5′-UTR of the recombinant keratinase KerZ1 expression element to enhance its secretory activity in Bacillus subtilis WB600 through Spacer design, RBS screening, and sequence simplification. First, the A/U content in Spacer was increased by the site-directed saturation mutation of G/C bases, and the activity of keratinase secreted by mutant strain B. subtilis WB600-SP was 7.94 times higher than that of KerZ1. Subsequently, the keratinase activity secreted by the mutant strain B. subtilis WB600-SP-R was further increased to 13.45 times that of KerZ1 based on the prediction of RBS translation efficiency and the multi-site saturation mutation screening. Finally, the keratinase activity secreted by the mutant strain B. subtilis WB600-SP-R-D reached 204.44 KU mL−1 by reducing the length of the 5′ end of the 5′-UTR, which was 19.70 times that of KerZ1. In a 5 L fermenter, the keratinase activity secreted by B. subtilis WB600-SP-R-D after 25 h fermentation was 797.05 KU mL−1, which indicated its high production intensity. Overall, the strategy of this study and the obtained keratinase mutants will provide a good reference for the expression regulation of keratinase and other industrial enzymes.

Molecular strategies to increase keratinase production in heterologous expression systems for industrial applications.

Keratinase is an important enzyme that can degrade recalcitrant keratinous wastes to form beneficial recyclable keratin hydrolysates. Keratinase is not only important as an alternative to reduce environmental pollution caused by chemical treatments of keratinous wastes, but it also has industrial significance. Currently, the bioproduction of keratinase from native keratinolytic host is considered low, and this hampers large-scale usage of the enzyme. Straightforward approaches of cloning and expression of recombinant keratinases from native keratinolytic host are employed to elevate the amount of keratinase produced. However, this is still insufficient to compensate for the lack of its large-scale production to meet the industrial demands. Hence, this review aimed to highlight the various sources of keratinase and the strategies to increase its production in native keratinolytic hosts. Molecular strategies to increase the production of recombinant keratinase such as plasmid selection, promoter engineering, chromosomal integration, signal peptide and propeptide engineering, codon optimization, and glycoengineering were also described. These mentioned strategies have been utilized in heterologous expression hosts, namely, Escherichia coli, Bacillus sp., and Pichia pastoris, as they are most widely used for the heterologous propagations of keratinases to further intensify the production of recombinant keratinases adapted to better suit the large-scale demand for them. KEY POINTS: • Molecular strategies to enhance keratinase production in heterologous hosts. • Construction of a prominent keratinolytic host from a native strain. • Patent analysis of keratinase production shows rapid high interest in molecular field.

Recombinant expression and molecular engineering of the keratinase from Brevibacillus parabrevis for dehairing performance

Keratinase is capable of distinctive degradation of keratin, which provides an eco-friendly approach for keratin waste management towards sustainable development. In this study, the recombinant keratinase (KERBP) from Brevibacillus parabrevis was successfully expressed in Escherichia coli. The purified KERBP had the specific activity of 6005.3 U/mg. It showed remarkable tolerance to various surfactants and also no collagenolytic activity. However, the moderate thermal stability limited its further application. Thus, protein engineering was further adopted to improve its stability. The variants of T218S, S236C and N181D were constructed by site-directed mutagenesis and combinatorial mutagenesis. Compared with the wild type, the t1/2 at 60 °C for the variants T218S, S236C and N181D were 3.05-, 1.18- and 1-fold increase, respectively. Moreover, the double variants N181D-T218S and N181D-S236C significantly improved thermostability with 5.1 and 2.9 °C increase of T50, and prolonging t1/2 at 60 °C with 4.09 and 1.54-fold, respectively. And the catalytic efficiency of the T218S and N181D-T218S variants was also significantly improved. Furthermore, the keratinase displayed favorable ability to dehair wool from skin within 7 h, which showed potential in leather dehairing. Our work contributes to a further insight into the thermostability of keratinase and offers a promising alternative for industrial leather application.

Biotransformation of keratin waste to amino acids and active peptides based on cell-free catalysis

BackgroundKeratin is the primary constituent of the vertebrate epidermis and epidermal appendages, as well as the main waste product generated during poultry processing from feathers, hair, scales, nails, etc. Keratin is generally hard, stubborn and difficult to hydrolyze; however, it is also inexpensive and contains more than 85% protein. Currently, tens of millions of tons of keratin waste are produced each year worldwide; however, no effective methods for the recovery of keratin waste have been reported thus far, making such research urgent. Keratinase has been reported to be useful for keratin waste recovery; however, nearly all keratinases are unable to hydrolyze keratin after they are detached from living cell systems. This may be due to low keratinase activity and lack of synergistic factors.ResultsHerein, the keratinase gene from Bacillus licheniformis BBE11-1 was successfully expressed in Bacillus subtilis WB600, allowing for improved activity of the recombinant keratinase KerZ1 to 45.14 KU/mL via promoter substitution and screening of the ribosome-binding sites. Further, real-time control of temperature, pH, dissolved oxygen, and feed strategy allowed the activity of KerZ1 to reach 426.60 KU/mL in a 15-L fermenter, accounting for a 3552-fold increase compared to the wild-type keratinase (120.1 U/mL). Most importantly, we proposed a method based on the synergistic action of keratinase KerZ1 and sodium sulfite, to hydrolyze feathers into amino acids. In specific, 100 g/L of feather waste can be successfully converted into 56.6% amino acids within 12 h, while supporting the production of dozens of bioactive peptides.ConclusionsThe activity of recombinant keratinase can be greatly enhanced via transcription and translational regulation in Bacillus subtilis. The synergistic action of keratinase and sulfite can rapidly degrade feather waste and produce amino acids and polypeptides.

Efficient keratinase expression via promoter engineering strategies for degradation of feather wastes

Keratinases are promising alternatives over ordinary proteases in several industrial applications due to their unique properties compared with their counterparts in the protease categories. However, their large-scale industrial application is limited by the low expression and poor fermentation efficiency of keratinase. Here, we demonstrate that the expression level of keratinase can be improved by constructing a more efficient enzyme expression system hereby enables the highest production titer as regarding recombinant keratinase production to date. Specially, ten promoters were evaluated and the aprE promoter exhibits a significant promotion of keratinase (kerBv) titer from 165 U/mL to 2605 U/mL in Bacillus subtilis. The batch fermentation mode resulted in a maximum keratinase activity of 7176 U/mL at 36 h in a 5-L fermenter. Furthermore, the extracellular keratinase activity attained up to 16,860 U/mL via fed-batch fermentation within 30 h. The combination of keratinase with l-cysteine brings about 66.4 % degree of degradation of feather. Our work provides a new insight into the development of efficient keratinase fermentation processes with B. subtilis cell factory.

Cloning and expression of a thermostable keratinase gene from Thermoactinomyces sp. YT06 in Escherichia coli and characterization of purified recombinant enzymes.

The feather-degrading strain Thermoactinomyces sp. YT06 secretes an extracellular keratinolytic protease (KERTYT); however, the gene encoding this protease remains unknown. The kerT1 gene (1170bp) encoding keratinase was cloned and expressed in Escherichia coli BL21(DE3). Purified recombinant keratinase (rKERTYT) was achieved at a yield of 39.16% and 65.27-fold purification with a specific activity of 1325 U/mg. It was shown that rKERTYT has many similarities to the native enzyme (KERTYT) by characterization of rKERTYT. The molecular weight of rKERTYT secreted by recombinant E. coli was approximately 28kDa. The optimal temperature and the pH values of rKERTYT were 65°C and 8.5, respectively, and the protein remained stable from 50 to 60°C and pH 6-11. The keratinase was strongly inhibited by phenyl methane sulfonyl fluoride (PMSF), suggesting that it belongs to the serine protease family. It was significantly activated by Mn2+ and β-mercaptoethanol (β-Me). rKERTYT showed stability and retained over 80% activity with the existence of organicsolvents such as acetone, methylbenzene and dimethyl sulfoxide. These findings indicated that rKERTYT will be a promising candidate for the enzymatic processing of keratinous wastes.

Enhanced extracellular recombinant keratinase activity in Bacillus subtilis SCK6 through signal peptide optimization and site-directed mutagenesis.

Keratinase has a great commercial value owing to its applications in the enzymatic dehairing of goatskins. In this study, we adopted a combined strategy to enhance the extracellular recombinant keratinase activity in Bacillus subtilis SCK6. First, nine signal peptides were screened to enhance the expression of extracellular keratinase. The recombinant strain with SPLipA exhibited the highest extracellular keratinase activity of 739.03 U per mL, which was two-fold higher activity of the wild type. Second, based on the multiple sequence alignment with the bacterial alkaline proteases, the mutant (M123L/V149I/A242N) was introduced into the keratinase. Comparing with the wild type of keratinase, the mutant M123L/V149I/A242N showed an increase in the extracellular keratinase activity, which was about 1.2-fold higher activity of the wild type. Finally, the keratinase expression vector with SPLipA and mutant M123L/V149I/A242N was constructed, and the extracellular keratinase activity reported at 830.91 U per mL was a 2.2-fold activity of the wild type. Then, the mutant keratinase was purified and characterized. The mutant exhibited properties similar to those of the wild type at an optimal temperature of 60 °C and pH 10.0. Conclusively, the extracellular expression of keratinase was enhanced via a combined strategy, and the mutant keratinase demonstrated properties similar to that of the wild type of keratinase.

Mining and Expression of a Metagenome-Derived Keratinase Responsible for Biosynthesis of Silver Nanoparticles.

A keratinase gene kerBv was mined from soil metagenomes. The open reading frame consisted of 1149 bp and potentially encoded a protein of 382 amino acid residues. It shared the same active site with several reported typical keratinases via analysis of the amino acid sequence. The keratinase was successfully expressed in B. subtilis WB600 with pMA5 expression vector. The maximum activity of 164.8 U/mL in the fermentation supernatant was observed after incubating for 30 h in Terrifc Broth (TB) medium. The keratinase exhibited outstanding resistance to metal ions and was surfactant-stable. Additionally, the enzyme displayed broad substrate specificity especially toward insoluble substrate feather meal because of its disulfide bond-reducing activity. Furthermore, the reducing power of the recombinant keratinase was investigated. It showed that the protein exhibited a relatively high reducing power, which was subsequently used in the biosynthesis of silver nanoparticles (AgNPs). The biosynthesized AgNPs were characterized by ultraviolet-visible (UV-vis) spectroscopy, dynamic light scattering (DLS), transmission electron microscope (TEM), as well as Fourier transform infrared spectroscopy (FTIR) and displayed obvious antibacterial activities toward Escherichia coli.

A novel alkaline surfactant-stable keratinase with superior feather-degrading potential based on library screening strategy

A novel keratinase was mined and expressed in Escherichia coli BL21 (DE3) via function-driven screening with fosmid library. The catalytic properties of purified keratinase were investigated in detail following enzyme purification. The recombinant keratinase was purified to homogeneity with an estimated molecular weight of 26kDa using nickel affinity chromatography, of which the optimal reaction pH and temperature were 10.0 and 55°C, respectively. It could remain stable at pH 5.0–12.0 and 40–60°C. Metal ions such as Ca2+, Mn2+, Ag+, Na+, Mg2+, Li+, Sn2+ (1mM) displayed positive influence on keratinase, and particularly, Ca2+ exhibited remarkable improvement effect by 2.6 folds. It was strongly inhibited by PMSF as a protease inhibitor. On the contrary, it could be obviously activated by various surfactants, such as Tween 40 and Triton X-114. The recombinant keratinase showed high specificity towards casein, soluble keratin, BSA, and wool. The keratinase could efficiently degrade the feathers, which demonstrated its applicable potential for biodegradation of keratin wastes and regeneration of soluble protein.

Cloning and expression of keratinase genes in Escherichia coli BL21(DE3) from Bacillus species

B. licheniformis and B. subtilis are capable to produce enzyme, which hydrolyzed feather highly. Keratinase is a group of commercial enzyme, that has many important applications in industries of waste management, leather, food. Hydrolyzed keratin is used to create some rare amino acids, such as, serine, cysteine and proline or used as animal feed, fertilizer, glue that contribute to reducing environmental pollution. We carried out cloning and expression of recombinant keratinase from Bacillus licheniformis and Bacillus subtilis in Escherichia coli BL21(DE3). Ker 1 from the strain B. licheniformis HT10 was integrated into pET22b vector and Ker 2 from B. subtilis Đ.nd1.2 was integrated into the pET32a vector. In the same expression conditions at 37°C, the 1mM IPTG, two obtained proteins showed a differ in size and activity of the enzyme respectively is 28 kDa, 23.5 U/ml and 36 kDa, 18.5 U/ml.

Expression of Bacillus pumilus keratinase rK27 in Bacillus subtilis: enzyme application for developing renewable flocculants from bone meal

In this study thermostable keratinase rK27 of Bacillus pumilus KS12 was expressed and secreted in Bacillus subtilis WB980 expression system under the control of xylose promoter (PxylA). The concentration of the recombinant keratinase rK27 produced by B. subtilis reached 4,432 U/mL after 24 h of culture at 37 °C and 200 rpm with 0.5 % xylose at an initial concentration of 0.3 OD600nm. Using the one-factor-at-a-time approach, we achieved an improvement in enzyme yield of up to 3.4-fold (15,390 U/mL) in the presence of 3 % yeast extract and 0.5 % tryptone. The enzyme was purified to homogenity using nickel affinity chromatography with a 3.63-fold purity and 80 % recovery. The purified enzyme rK27 hydrolyzed 1 g bone meal after 12 h at 40 °C, pH 9, with a maximum protein release of 37.3 mg/g bone meal; in comparison subtilisin Carlsberg hydrolyzed 19.3 mg/g bone meal and proteinase K hydrolyzed 6.2 mg/g bone meal. The hydrolysate obtained after hydrolysis of bone by rK27 was found to be effective as a flocculant at 0.1 mg in a 10 % (w/v) kaolin solution when compared with hydrolysates obtained from substilisin Carlsberg and proteinase K, which were effective at 0.5 mg and >2 mg, respectively.

Codon Optimization Significantly Improves the Expression Level of a Keratinase Gene in Pichia pastoris

The main keratinase (kerA) gene from the Bacillus licheniformis S90 was optimized by two codon optimization strategies and expressed in Pichia pastoris in order to improve the enzyme production compared to the preparations with the native kerA gene. The results showed that the corresponding mutations (synonymous codons) according to the codon bias in Pichia pastoris were successfully introduced into keratinase gene. The highest keratinase activity produced by P. pastoris pPICZαA-kerAwt, pPICZαA-kerAopti1 and pPICZαA-kerAopti2 was 195 U/ml, 324 U/ml and 293 U/ml respectively. In addition, there was no significant difference in biomass concentration, target gene copy numbers and relative mRNA expression levels of every positive strain. The molecular weight of keratinase secreted by recombinant P. pastori was approx. 39 kDa. It was optimally active at pH 7.5 and 50°C. The recombinant keratinase could efficiently degrade both α-keratin (keratin azure) and β-keratin (chicken feather meal). These properties make the P. pastoris pPICZαA-kerAopti1 a suitable candidate for industrial production of keratinases.

Expression, Characterization and Fermentation Optimization of Keratinase from Bacillus licheniformis in Escherichia coli

In order to enhance the production of keratinase in Escherichia coli and study the enzymatic properties, the ker gene(1 140 bp) was cloned from a feather-degrading bacterium of Bacillus licheniformis BBE11-1. After the 1 053 bp fragment of signal peptide-truncated ker gene was inserted into the expression vector pET-22b(+), the recombinant plasmid was transformed into E. coli BL21(DE3). The recombinant keratinase purif ied by hydrophobic interaction chromatography using HiTrap Phenyl-sepharose Fast Flow was a serine protease most active at 50 ℃ and pH 10. The activity of keratinase was enhanced by Mg2+ and K+, and inhibited by Mn2+. The highest keratinase activity was 460.2 U/mL in growth medium containing inducer(0.05 mmol/L IPTG, 150 mmol/L glycine and 60 g/L yeast extract) at 20 ℃. This report focused on the expression and characterization of recombinant keratinase in E. coli. We found that optimizing culture conditions could preferably enhance production of keratinase in E. coli. The results would be helpful in producing keratinase by gene engineering strain. Fig 4, Tab 1, Ref 22

Expression and characterization of extreme alkaline, oxidation-resistant keratinase from Bacillus licheniformis in recombinant Bacillus subtilis WB600 expression system and its application in wool fiber processing

A keratin-degrading bacterium of Bacillus licheniformis BBE11-1 was isolated and its ker gene encoding keratinase with native signal peptide was cloned and expressed in Bacillus subtilis WB600 under the strong P HpaII promoter of the pMA0911 vector. In the 3-L fermenter, the recombinant keratinase was secreted with 323 units/mL when non-induced after 24 h at 37 °C. And then, keratinase was concentrated and purified by hydrophobic interaction chromatography using HiTrap Phenyl-Sepharose Fast Flow. The recombinant keratinase had an optimal temperature and the pH at 40 °C and 10.5, respectively, and was stable at 10-50 °C and pH 7-11.5. We found this enzyme can retained 80 % activity after treated 5 h with 1 M H2O2, it was activated by Mg(2+), Co(2+) and could degraded broad substrates such as degraded feather, bovine serum albumin, casein, gelatin, the keratinase was considered to be a serine protease. Coordinate with Savinase, the keratinase could efficient prevent shrinkage and eliminate fibres of wool, which showed its potential in textile industries and detergent industries.

Expression of a keratinase (kerA) gene from Bacillus licheniformis in Escherichia coli and characterization of the recombinant enzymes

The gene kerA (1,047bp) encoding the main keratinase from Bacillus licheniformis was cloned into two conventional vectors, pET30α and pET32α, and expressed in Escherichia coli. From SDS-PAGE analysis, the recombinant keratinases were 45 and 55kDa. They had different optimal pH values (7.5 and 8.5) but the same optimum temperature of 50°C. The recombinant keratinase produced in E. coli pET30α-kerA was more stable than that produced in E. coli pET32α-kerA, and retained approx. 70% of its total enzyme activity after 30min at 70°C.

Extracellular Expression of Keratinase from Bacillus licheniformis ER-15 in Escherichia coli

Keratinase (ker BL) from Bacillus licheniformis ER-15 was cloned into vector pEZZ18 for extracellular expression in Escherichia coli HB101. Recombinant keratinase was secreted with high specific activity (75 units/mg) under non-inducible conditions after 36 h at 37 degrees C and 300 rpm in a shake flask. Protein was concentrated and, subsequently, purified by ion-exchange chromatography using Q-sepharose with 95.8% yield. The recombinant keratinase was a serine protease and most active in the pH range of 8-12 and at 60 degrees C. The enzyme was stable over a wide pH range of 4-12 for 3 h. ker BL degraded bovine serum albumin, casein, azocasein, gelatin, and feather. E. coli HB101 harboring pEZZ18 ker BL2 degraded chicken feather completely within 24 h at 37 degrees C.

Expression and Purification ofPseudomonas aeruginosaKeratinase inBacillus subtilisDB104 Expression System

The DNA encoding Pseudomonas aeruginosa keratinase was ligated into pRPA expression vector and transformed into Bacillus subtilis DB104. Recombinant keratinase (rK), secreted by B. subtilis after 72 h of incubation, was purified to electrophoretical homogeneity by nickel affinity chromatography and found to have a molecular mass of 33 kDa. The rK had an optimal pH and temperature at 8.0 and 60 degrees C, respectively, and was stable at pH 6.0-9.0 and 10-50 degrees C. It was strongly inhibited by Cu(2+), Fe(2+), Hg(2+), Fe(3+), ethylene glycol tetraacetic acid, and ethylene diamine tetraacetic acid but activated by Ca(2+), Mg(2+), Zn(2+), dithiothreitol, glutathione, and beta-mercaptoethanol. According to substrate specificity, the rK was considered to be a metalloprotease.

Functional Expression and Characterization of Keratinase from Pseudomonas aeruginosa in Pichia pastoris

Recombinant keratinase (rK) from Pseudomonas aeruginosa was secreted by Pichia pastoris SMD1168H with a final yield of 580 mg/L (1.03 kU/mL) after 72 h of induction. The rK was purified after nickel affinity chromatography and was stable at pH 6.0-9.0 and 10-60 degrees C. It was nonglycosylated protein with a molecular mass of 33 kDa and had an optimal pH and temperature at 8.0 and 60 degrees C, respectively. Ba(2+), Ca(2+), Mg(2+), Mn(2+), Zn(2+), dithiothreitol, glutathione, and beta-mercaptoethanol activated, while Cu(2+), Fe(2+), Hg(2+), Fe(3+), ethylene glycol tetraacetic acid, ethylene diamine tetraacetic acid, and p-chloromercuribenzoate inhibited its activity. rK could hydrolyze broad substrates and cleave hydrophobic and aromatic amino acids at P(1) position, behaving as those from the wild type strain and E. coli transformant.

Cloning, Expression, and Purification of Pseudomonas aeruginosa Keratinase in Escherichia coli AD494(DE3)pLysS Expression System

The DNA encoding keratinase from Pseudomonas aeruginosa was ligated into pET-43b(+) expression vector and transformed into Escherichia coli AD494(DE3)pLysS. After isopropyl beta-d-thiogalactopyranoside induction, the soluble recombinant keratinase was expressed in E. coli. The keratinase with a molecular mass of 33 kDa was purified to electrophoretical homogeneity after nickel affinity chromatography. It had an optimal pH and temperature of 8.0 and 50 degrees C, respectively, and was stable at pH 6.0-9.0 and 10-60 degrees C. It was highly inhibited by Cd(2+), Cu(2+), Hg(2+), Ni(2+), Fe(3+), ethylene glycol tetraacetic acid, ethylenediaminetetraacetic acid, and p-chloromercuribenzoate, but activated by Ba(2+), Ca(2+), Mg(2+), Mn(2+), Zn(2+), dithiothreitol, glutathione, and beta-mercaptoethanol. According to substrate specificity results, the purified keratinase was considered to be a metalloprotease.