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 Mg²⁺, Co²⁺ 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.     

Improvement of uridine production in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> by metabolic engineering

Objectives

To construct a Bacillus subtilis strain for improved uridine production.

Results

The AAG_2846–2848 fragment of the pyrAB gene, encoding carbamoylphosphate synthetase, was deleted in B. subtilis TD246 leading to a 245% increase of uridine production and the conversion from glucose to uridine increased by 10.5%. Overexpression of the pyr operon increased the production of uridine by a further 31% and the conversion rate of glucose to uridine was increased by 18%. In addition, the blocking of arginine synthesis or disabling of glutamate dehydrogenase significantly enhanced the uridine production. The highest-producing strain, B. subtilis TD297, accumulated 11 g uridine/l with a yield of 240 mg uridine/g glucose in shake-flask cultivation.

Conclusion

This is the first report of engineered B. subtilis strains which can produce more than 11 g uridine/l, with a yield reaching 240 mg uridine/g glucose in shake-flask cultivation.

     

Purification,characterization and partial amino acid sequences of a novel cephalosporin-C deacetylase from Bacillus subtilis

Cephalosporin-C deacetylase [EC 3.1.1.41] was purified electrophoretically to homogeneity from the newly isolated Bacillus subtilis SHS 0133 (FERM BP-2755). The enzyme was purified about 27-fold with a yield of 9 % and a specific activity of 187.4 U/mg protein. The native enzyme (molecular weight, 280,000) was composed of eight identical subunits with apparent molecular weights of 35,000. The cephalosporin-C deacetylase was stable up to 60°C for 30 min at pH 7.0. The enzyme exhibited Michaelis-Menten kinetics with the substrates cephalosporin C, 7-aminocephalosporanic acid (7-ACA) and p-nitrophenyl acetate; the K_m values were 24.0, 7.9 and 1.0 mM, respectively. One of the reaction products from 7-ACA, deacetyl-7-ACA, was a weak non-competitive inhibitor and other product, acetate, was a weak competitive inhibitor; the K_i values were 171 and 290 mM, respectively. However, these weak product inhibitors did not prevent the completion of the deacylation of 7-ACA. The pI value of the enzyme was determined to be 5.3 using isoelectric focusing. The observed data indicate that the enzyme is different from known cephalosporin-C deacetylases. In addition, amino acid sequencing of the N-terminus and Achromobacter proteinase I-digested peptides yielded no sequences with similarities to other known proteins by a computer search.     

Optimization of Microbial Flocculant-Producing Medium for <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

This study aimed to improve microbial flocculant production by optimizing the components of a Bacillus subtilis CZ1003 culture medium. Using the flocculation rate as the dependent variable, single-factor experiments were performed and beef extract at a concentration of 9 g/L was found to be the optimal nitrogen source, while glucose at a concentration of 20 g/L was the optimal carbon source. KCl, MgCl₂, NaCl, and CaCl₂ at concentrations of 0.75, 2.5, 0.5, and 5.0 g/L, respectively, were the optimum inorganic salts, in order of flocculant production activity. Orthogonal experimental demonstrated that KCl played a dominant role for Bacillus subtilis production of bioflocculants, followed by NaCl and CaCl₂. Optimization experiments demonstrated that the optimal combination of the two salts was 0.75 g/L KCl and 0.5 g/L NaCl, resulting in a flocculation rate of 36.2% when included together at these concentrations. The final optimized medium consisted of 20 g/L glucose, 9 g/L beef extract, 0.75 g/L KCl, and 0.5 g/L NaCl. Compared with the initial medium, the optimized medium enhanced the flocculation activity from 12.1 to 36.2%, which equates to an increase of 199.17%. Meanwhile, the flocculant yield was increased from 0.058 g/L to 0.134/L, an increase of 131.03%. The optimized medium could be used to improve microbial flocculant production and provides a basis for further exploration.     

Fermentation production of keratinase from Bacillus licheniformisPWD-1 and a recombinant B. subtilis FDB-29

Bacillus licheniformis PWD-1, the parent strain, and B. subtilis FDB-29, a recombinant strain. In both strains, keratinase was induced by proteinaceous media, and repressed by carbohydrates. A seed culture of B. licheniformis PWD-1 at early age, 6–10 h, is crucial to keratinase production during fermentation, but B. subtilis FDB-29 is insensitive to the seed culture age. During the batch fermentation by both strains, the pH changed from 7.0 to 8.5 while the keratinase activity and productivity stayed at high levels. Control of pH, therefore, is not necessary. The temperature for maximum keratinase production is 37°C for both strains, though B. licheniformis is thermophilic and grows best at 50°C. Optimal levels of dissolved oxygen are 10% and 20% for B. licheniformis and B. subtilis respectively. A scale-up procedure using constant temperature at 37°C was adopted for B. subtilis. On the other hand, a temperature-shift procedure by which an 8-h fermentation at 50°C for growth followed by a shift to 37°C for enzyme production was used for B. licheniformis to shorten the fermentation time and increase enzyme productivity. Production of keratinase by B. licheniformis increased by ten-fold following this new procedure. After respective optimization of fermentation conditions, keratinase production by B. licheniformis PWD-1 is approximately 40% higher than that by B. subtilis FDB-29. Received 16 July 1998/ Accepted in revised form 07 March 1999     

High-level intra- and extra-cellular production of <Emphasis Type="SmallCaps">d</Emphasis>-psicose 3-epimerase via a modified xylose-inducible expression system in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

d-Psicose 3-epimerase (DPEase) converts d-fructose into d-psicose which exists in nature in limited quantities and has key physiological functions. In this study, RDPE (DPEase from Ruminococcus sp. 5_1_39BFAA) was successfully constitutively expressed in Bacillus subtilis, which is the first report of its kind. Three sugar-inducible promoters were compared, and the xylose-inducible promoter P _xylA was proved to be the most efficient for RDPE production. Based on the analysis of the inducer concentration and RDPE expression, we surmised that there was an extremely close correlation between the intracellular RDPE expression and xylose accumulation level. Subsequently, after the metabolic pathway of xylose was blocked by deletion of xylAB, the intra- and extra-cellular RDPE expression was significantly enhanced. Meanwhile, the optimal xylose induction concentration was reduced from 4.0 to 0.5 %. Eventually, the secretion level of RDPE reached 95 U/mL and 2.6 g/L in a 7.5-L fermentor with the fed-batch fermentation, which is the highest production of DPEase by a microbe to date.     

The purification and characterization of a novel alkali-stable pectate lyase produced by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> PB1

Pectinase is an important kind of enzyme with many industrial applications, among which pectinases produced by bacteria were scarce compared with fungal sources. In this study, a novel bacterium which produced extracellular pectinase was firstly isolated from flue-cured tobacco leaves and identified as Bacillus subtilis PB1 according to its 16S rRNA gene. The pectinolytic enzyme was purified by ammonium sulfate precipitation, ion-exchange and gel filtration chromatography, after which molecular weight was determined as 43.1?±?0.5 kDa by SDS–PAGE. Peptide mass fingerprinting of the pectinase by MALDI-TOF MS showed that the purified enzyme shared homology with pectate lyase and was designated as BsPel-PB1. The optimal temperature for BsPel-PB1 was 50 °C. The optimal pH was pH 9.5 for BsPel-PB1 while it had a broad pH stability from 5 to 11. The values of K _m and V _max were 0.312 mg/mL and 1248 U/mL, respectively. Accordingly, the BsPel-PB1 was a novel alkaline pectate lyase which could find potential application as a commercial candidate in the pectinolytic related industries.     

Biodegradation of feather waste by keratinase produced from newly isolated Bacillus licheniformis ALW1

Keratinase are proteolytic enzymes which have gained much attention to convert keratinous wastes that cause huge environmental pollution problems. Ten microbial isolates were screened for their keratinase production. The most potent isolate produce 25.2?U/ml under static condition and was primarily identified by partial 16s rRNA gene sequence as Bacillus licheniformis ALW1. Optimization studies for the fermentation conditions increased the keratinase biosynthesis to 72.2?U/ml (2.9-fold). The crude extracellular keratinase was optimally active at pH 8.0 and temperature 65?°C with 0.7% soluble keratin as substrate. The produced B. licheniformis ALW1 keratinase exhibited a good stability over pH range from 7 to 9 and over a temperature range 50–60?°C for almost 90?min. The crude enzyme solution was able to degrade native feather up to 63% in redox free system.     

Characterization of an alkaline elastase from alkalophilic Bacillus Ya-B

The alkaline elastase produced by alkalophilic Bacillus Ya-B was a new type of proteinase which had a very high optimum pH and high elastolytic activity. It also had a high hydrolyzing activity against keratin and collagen. The molecular weight was determined to be 23 700 and 25 000 by ultracentrifugation analysis and SDS-polycrylamide gel electrophoresis, respectively. The isoelectric point was 10.6. The optimum reaction temperature was 60°C. Like many alkaline proteinases, this enzyme required Ca²⁺ for stability. The optimum reaction pH was 11.75 toward casein and elastin-orcein. The K_cat/K_m values of the enzyme to synthetic substrates were constant from pH 8.5 up to 12.75. The enzyme was stable in the pH range 5.0–10.0. The enzyme was inhibited by alkaline proteinase inhibitors Streptomyces subtilisin inhibitor and microbial alkaline proteinase inhibitor, but not by elastatinal or the metalloproteinase inhibitor metalloproteinase inhibitor. Sodium chloride inhibited the elastolytic activity but not the caseinolytic activity at a concentration below 0.2 M. The inhibitory effect of sodium chloride to elastolytic activity was much more prominent at pH 9.0 than at pH 11.5. More than 50% of the enzyme bound onto elastin in the pH range below the isoelectric point of this enzyme. The amino-terminal sequence of the enzyme was determined, and compared with those of subtilisin BPN′ and subtilisin Carlsberg. Extensive sequence homology was noted among these three enzymes.     

Biodegradation of feather waste by extracellular keratinases and gelatinases from Bacillus spp.

In this study, three feather degrading bacterial strains were isolated from agroindustrial residues from a Brazilian poultry farm. Three Gram-positive, spore-forming, rod-shaped bacteria and were identified as B. subtilis 1271, B. licheniformis 1269 and B. cereus 1268 using biochemical, physiologic and molecular methods. These Bacillus spp. strains grew and produced keratinases and peptidases using chicken feather as the sole source of nitrogen and carbon. B. subtilis 1271 degraded feathers completely after 7 days at room temperature and produced the highest levels of keratinase (446 U ml^?1). Feather hydrolysis resulted in the production of serine, glycine, glutamic acid, valine and leucine as the major amino acids. Enzymography and zymography analyses demonstrated that enzymatic extracts from the Bacillus spp. effectively degraded keratin and gelatin substrates as well as, casein, hemoglobin and bovine serum albumin. Zymography showed that B. subtilis 1271 and B. licheniformis 1269 produced peptidases and keratinases in the 15?C140 kDa range, and B. cereus produced a keratinase of ~200 kDa using feathers as the carbon and nitrogen source in culture medium. All peptidases and keratinases observed were inhibited by the serine specific peptidase inhibitor phenylmethylsulfonyl fluoride (PMSF). The optimum assay conditions of temperature and pH for keratinase activity were 40?C50°C and pH 10.0 for all strains. For gelatinases the best temperature and pH ranges were 50?C70°C and pH 7.0?C11. These isolates have potential for the biodegradation of feather wastes and production of proteolytic enzymes using feather as a cheap and eco-friendly substrate.     

A thermo-halo-tolerant and proteinase-resistant endoxylanase from Bacillus sp. HJ14

A glycosyl hydrolase family 10 endoxylanase from Bacillus sp. HJ14 was grouped in a separated cluster with another six Bacillus endoxylanases which have not been characterized. These Bacillus endoxylanases showed less than 52 % amino acid sequence identity with other endoxylanases and far distance with endoxylanases from most microorganisms. Signal peptide was not detected in the endoxylanase. The endoxylanase was expressed in Escherichia coli BL21 (DE3), and the purified recombinant enzyme (rXynAHJ14) was characterized. rXynAHJ14 was apparent optimal at 62.5 °C and pH 6.5 and retained more than 55 % of the maximum activity when assayed at 40–75 °C, 23 % at 20 °C, 16 % at 85 °C, and even 8 % at 0 °C. Half-lives of the enzyme were more than 60 min, approximately 25 and 4 min at 70, 75, and 80 °C, respectively. The enzyme exhibited more than 62 % xylanase activity and stability at the concentration of 3–30 % (w/v) NaCl. No xylanase activity was lost after incubation of the purified rXynAHJ14 with trypsin and proteinase K at 37 °C for 60 min. Different components of oligosaccharides were detected in the time-course hydrolysis of beechwood xylan by the enzyme. During the simulated intestinal digestion phase in vitro, 11.5–19.0, 15.3–19.0, 21.9–27.7, and 28.2–31.2 μmol/mL reducing sugar were released by the purified rXynAHJ14 from soybean meal, wheat bran, beechwood xylan, and rapeseed meal, respectively. The endoxylanase might be an alternative for potential applications in the processing of sea food and saline food and in aquaculture as agastric fish feed additive.     

Enhanced thermostability of keratinase by computational design and empirical mutation

Keratinases are proteolytic enzymes capable of degrading insoluble keratins. The importance of these enzymes is being increasingly recognized in fields as diverse as animal feed production, textile processing, detergent formulation, leather manufacture, and medicine. To enhance the thermostability of Bacillus licheniformis BBE11-1 keratinase, the PoPMuSiC algorithm was applied to predict the folding free energy change (ΔΔG) of amino acid substitutions. Use of the algorithm in combination with molecular modification of homologous subtilisin allowed the introduction of four amino acid substitutions (N122Y, N217S, A193P, N160C) into the enzyme by site-directed mutagenesis, and the mutant genes were expressed in Bacillus subtilis WB600. The quadruple mutant displayed synergistic or additive effects with an 8.6-fold increase in the t _1/2 value at 60 °C. The N122Y substitution also led to an approximately 5.6-fold increase in catalytic efficiency compared to that of the wild-type keratinase. These results provide further insight into the thermostability of keratinase and suggest further potential industrial applications.     

Functional analysis of the C-terminal propeptide of keratinase from Bacillus licheniformis BBE11-1 and its effect on the production of keratinase in Bacillus subtilis

The keratinase from Bacillus licheniformis BBE11-1 is a serine protease and expressed as a pre-pro-precursor. To produce a mature and active keratinase, the propeptide must be cleaved on the C-terminal via cis or trans. In this study, to enhance the production of keratinase in Bacillus subtilis, single amino acid substitutions, single residue deletions and linkers were introduced at the C-terminus of the propeptide. The results showed that optimizing the residue of cleavage site of propeptide will affect the cleavage efficiency of propeptide, and the mature enzyme yield of Leu(P1)Ala mutant increases 50% compared with the wild-type. In addition, inserting linkers and deleting individual residues at the C-terminal of the propeptide decreases the mature keratinase production. Our results indicated that the primary structure of the C-terminus of propeptide is crucial for the mature keratinase production. Propeptide engineering at C-terminus may be an effective approach to increase the yield of keratinase.     

Novel extremely acidic lipases produced from Bacillus species using oil substrates

The extremely acidophilic microorganisms Bacillus pumilus and Bacillus subtilis were isolated from soil collected from the commercial edible oil and fish oil extraction industry. Optimization of conditions for acidic lipase production from B. pumilus and B. subtilis using palm oil and fish oil, respectively, was carried out using response surface methodology. The extremely acidic lipases, thermo-tolerant acidic lipase (TAL) and acidic lipase (AL), were produced by B. pumilus and B. subtilis, respectively. The optimum conditions for B. pumilus obtaining the maximum activity (1,100 U/mL) of TAL were fermentation time, 96 h; pH, 1; temperature, 50 °C; concentration of palm oil, 50 g/L. After purification, a 7.1-fold purity of lipase with specific activity of 5,173 U/mg protein was obtained. The molecular weight of the TAL was 55 kDa. The AL from B. subtilis activity was 214 U/mL at a fermentation time of 72 h; pH, 1; temperature, 35 °C; concentration of fish oil, 30 g/L; maltose concentration, 10 g/L. After purification, an 11.4-fold purity of lipase with specific activity of 2,189 U/mg protein was obtained. The molecular weight of the extremely acidic lipase was 22 kDa. The functional groups of lipases were determined by Fourier transform-infrared (FT-IR) spectroscopy.     

Molecular Modeling of Cloned <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Keratinase and Its Insinuation in Psoriasis Treatment Using Docking Studies

Present study demonstrated the expression of cloned Bacillus subtilis RSE163 keratinase gene and in silico binding affinities of deduced protein with psoriasis topical drugs for systemic absorption and permeation through skin. The ker gene expressed in E. coli showed significantly higher keratinase activity 450 ± 10.43 U representing 1342 bp nucleotides encoding 447 amino acids with molecular weight of 46 kDa. The modeled structure was validated using ramachandran’s plot showing 305 residues (84.3%) in most favoured region. Docking studies using extra precision (XP) method of Glide showed optimum binding affinities with the drugs Acitretin (? 39.62 kcal/mol), Clobetasol propionate (? 37.90 kcal/mol), Fluticasone (? 38.53 kcal/mol), Desonide (? 32.23 kcal/mol), Anthralin (? 38.04 kcal/mol), Calcipotreine (? 21.55 kcal/mol) and Mometasone (? 28.40 kcal/mol) in comparison to other psoriasis drugs. The results can further be correlated with in vitro enzymatic experiments using keratinase as an effective drug mediator through skin to serve the unmet need of industries.     

Isolation of keratinase from bacterial isolates of poultry soil for waste degradation

Isolation of two keratinolytic bacterial strains from poultry soil as well as purification and properties of keratinase were investigated. Isolates were designated as KI8101 and KI8102 (KI, keratin isolates) and were identified as Bacillus subtilis and B. licheniformis respectively. The purified enzyme from KI8102 exhibited a high specific activity of 500 U/mg with 71‐fold purification and 41% yield. SDS‐PAGE analysis indicated that the purified keratinase had a molecular mass of 32 kDa. The optimum temperature and pH were 50°C and 7.5, respectively. Its K_m was 83.3 μM and V_max was 71.4 μmol/mL min. The bacterium could potentially degrade keratin waste such as human hair, nails, bovine hair and wool. Therefore, the enzyme could improve the nutritional value of meat and poultry‐processing waste containing keratin and could be a potential candidate for biotechnological processing involving keratin hydrolysis.     

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

The gene kerA (1,047 bp) 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 55 kDa. 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 30 min at 70 °C.     

Supplementations of ornithine and KNO3 enhanced bacitracin production by Bacillus licheniformis LC-11

During the fermentative process of Bacillus licheniformis LC-11, the dissolved oxygen (DO) and ornithine (Orn) content in the medium fell to zero, indicating that they were the potential limiting factors for cell growth and/or bacitracin biosynthesis. In addition, given that the nitrate-reducing system existing in B. licheniformis could favor cell anaerobiosis, the impacts of KNO₃ and Orn supplementations were therefore evaluated for improving bacitracin production in both a shaking flask and a 10-L fermentor. Orn supplementation (0.04 g/L) at 16 h or KNO₃ addition (0.5 g/L) in the production medium enhanced bacitracin production by 5.8 and 3.7 %, respectively. The combined effects of KNO₃ and Orn supplementations were then conducted via a two-level central composite design, resulting in a predicted maximum bacitracin yield of 821.81 U/mL if KNO₃ and Orn were supplemented at 514.39 mg/L and 45.35 mg/L, respectively. This predicated yield was then verified experimentally in a 10-L fermentor, achieving a 10.8 % increased bacitracin production (825 U/mL) over that of the control.