Characterization of a keratinolytic protease produced by the feather-degrading Amazonian bacterium Bacillus sp. P45

Abstract

An extracellular keratinolytic protease produced by Bacillus sp. P45 was purified and characterized. The keratinase had a molecular weight of approximately 26 kDa and was active over wide pH and temperature ranges, with optimal activity at 55°C and pH 8.0. However, this enzyme displayed low thermostability, being completely inactivated after 10 min at 50°C. Keratinase activity increased with Ca²⁺, Mg²⁺, Triton X-100, ethanol and DMSO, was stable in the presence of the reducing agent 2-mercaptoethanol, and was inactivated by SDS. PMSF (phenylmethylsulfonyl fluoride) completely inactivated and EDTA strongly inhibited the enzyme, indicating that the keratinase is a serine protease depending on metal ions for optimal activity and/or stability. Accordingly, analysis of tryptic peptides revealed sequence homologies which characterize the keratinase as a subtilisin-like serine protease. The purified enzyme was able to hydrolyze azokeratin and keratin azure. Casein was hydrolyzed at higher rates than keratinous substrates, and 2-mercaptoethanol tended to enhance keratin hydrolysis. With synthetic substrates, the keratinase showed a preference for aromatic and hydrophobic residues at the P1 position of tetrapeptides; the enzyme was not active, or the activity was drastically diminished, towards shorter peptides. Keratinase from Bacillus sp. P45 might potentially be employed in the production of protein hydrolysates at moderate temperatures, being suitable for the bioconversion of protein-rich wastes through an environmentally friendly process requiring low energy inputs.     

利用定点突变分析海藻糖合酶的功能

我们通过对来自红色亚栖热菌(Meiothermus ruber) CBS-01中的海藻糖合酶(Trehalose synthase)序列比对及三维模型构建, 我们构建了D200G/H165R, R227C, R392A三个定点突变体, 检测其对麦芽糖及海藻糖的转化能力。结果发现: 在50°C时, D200G/H165R、R392A基本失去其原有活性, 而R227C产生海藻糖的能力降低。37°C时, D200G/H165R失去转化能力, 而R392A及R227C保有部分能力。因此我们推测, R392位点可能是维持酶的结构及热稳定性的关键位点, 而D200位点在反应过程中也起重要作用。     

Hydrolysis of native proteins by keratinolytic protease of Doratomyces microsporus

A keratinolytic protease from the fungus Doratomyces microsporus was investigated for its ability to hydrolyse different native proteins. The purified enzyme was incubated for up to 24 h with keratinous substrates as well as with non-keratinous proteins. The results showed that the enzyme was broad specific since it hydrolysed various globular and fibrillar proteins. The hydrolysis of keratinous substrates decreased in the following order: skin keratins > nail keratins > hair keratins. With non-keratinous substrates, the order was: casein > BSA > elastin. Feather keratin and collagen could not be hydrolysed. Comparison of the enzyme with some known proteolytic enzymes showed that on keratin from stratum corneum the activity of the keratinase was comparable to that of proteinase K, other enzymes were less active. Hydrolysis of porcine skin with the keratinase revealed the degradation of the epidermis while dermis was not damaged.     

Secretion of a trypsin-like thiol protease by a new keratinolytic strain of Bacillus licheniformis

When cultured in feather-containing broth with a growth optimum of pH 7.0 and 47 degrees C, a Bacillus licheniformis strain exhibited a high chicken feather-degrading activity. A trypsin-like protease was isolated from its ferment broth and was partially characterized. The enzyme was constitutively secreted and was highly active towards N-benzoyl-Phe-Val-Arg-p-nitroanilide as chromogenic substrate. Its pH optimum was 8.5 and it exhibited the highest activity at 52 degrees C. Fractionation on Sephadex G-100 column revealed that its molecular mass was about 42 kDa. The enzyme, which is new for the genus Bacillus, is a thiol protease, as tosyl-L-phenylalanine chloromethyl ketone, tosyl-L-lysine chloromethyl ketone, phenylmethylsulfonyl fluoride and ethylenediamine tetraacetate did not inhibit it, while HgCl2 and para-chloromercuribenzoate lowered its activity.     

发酵性丝孢酵母蛋白酶稳定性的研究

研究了发酵性丝孢酵母所产蛋白酶的最适作用条件及稳定性。结果表明,发酵性丝孢酵母蛋白酶的最适反应温度为55℃,最适反应p H为7.0;有良好的热稳定性,50℃保温30 min,仍保留78%以上的酶活力;贮存稳定性也较好,10℃贮存60 h,仍保留80%左右的酶活力;5 mmol/L的金属离子Mn2+和Ca2+对蛋白酶有激活作用,其中Ca2+激活作用最显著,使酶活提高了0.87倍;1%(w/w)的表面活性剂吐温-100可以促进酶活力,使酶活提高了24.6%,1%(w/w)的SDS对酶活力有抑制作用,将残余酶活降低为原酶液的67.1%;2.5%(w/w)的葡萄糖、半乳糖和海藻糖等能抑制该蛋白酶活力,其中海藻糖抑制作用最显著,使残余蛋白酶活力降低到原酶液的23.6%;甘油可作为酶保护剂保护蛋白酶稳定性,且浓度为7.5%(w/w)的甘油能明显激活酶活力。     

Degradation of PrP(Sc) by keratinolytic protease from Nocardiopsis sp. TOA-1

A keratinolytic alkaline proteae (NAPase) from Nocardiopsis sp. TOA-1 degraded a scrapie prion without any chemical or physical treatment. Optimal temperature and pH were 60 degrees C and above pH 10.0. The scrapie prion was completely degraded within 3 min under optimal conditions.     

Purification and properties of a collagenolytic protease produced by marine bacteriumVibrio vulnificus CYK279H

A collagenolytic enzyme, produced byVibrio vulnificus CYK279H, was purified by ultrafiltration, dialysis, Q-Sepharose ion exchange and Superdex-200 gel chromatography. The enzyme from the supernatant was purified 13.2 fold, with a yield of 11.4%. The molecular weight of the purified enzyme was estimated by SDS-PAGE to be approximately 35.0 kDa. The N-terminal sequence of the enzyme was determined as Gly-Asp-Pro-Cys-Met-Pro-Ile-Ile-Asn. The optimum temperature and pH for the enzyme activity were 35°C and 7.5, respectively. The enzyme activity was stable within the pH and temperature ranges 6.8∼8.0 and 20∼35°C, respectively. The purified enzyme was strongly activated by Zn²⁺, Li²⁺, and Ca²⁺, but inhibited by Cu²⁺. In addition, the enzyme was strongly inhibited by 1, 10-phenanthroline and EDTA. The purified enzyme was suggested to be a neutral metalloprotease.     

AFM observation of band-like cellulose assemblies produced by Acetobacter xylinum

We succeeded in estimating the thickness of band-like cellulose assemblies by combined use of atomic force microscopy (AFM) and transmission electron microscopy. The thickness of "dense" band-like cellulose assemblies was estimated at 20-30 nm from their AFM height profiles, which was several times greater than that of "coarse" band-like and ribbonlike cellulose assemblies. On the basis of these results, the folded- chain model previously proposed was discussed, and a different organization of TC subunits was suggested for the "dense" band-like cellulose assembly.     

Mature DIABLO/Smac is produced by the IMP protease complex on the mitochondrial inner membrane

DIABLO/Smac is a mitochondrial protein that can promote apoptosis by promoting the release and activation of caspases. To do so, DIABLO/Smac must first be processed by a mitochondrial protease and then released into the cytosol, and we show this in an intact cellular system. We propose that the precursor form of DIABLO/Smac enters the mitochondria through a stop-transfer pathway and is processed to its active form by the inner membrane peptidase (IMP) complex. Catalytic subunits of the mammalian IMP complex were identified based on sequence conservation and functional complementation, and the novel sequence motif RX(5)P in Imp1 and NX(5)S in Imp2 distinguish the two catalytic subunits. DIABLO/Smac is one of only a few specific proteins identified as substrates for the IMP complex in the mitochondrial intermembrane space.     

Sustainable and practical degradation of intact chicken feathers by cultivating a newly isolated thermophilic <Emphasis Type="Italic">Meiothermus ruber</Emphasis> H328

The sustainable and practical degradation of intact chicken feathers by a newly isolated thermophilic bacterium Meiothermus ruber H328 was presented with extensive data. Aerobic cultivation with moderately thermophilic strain H328 at 55°C for 6 days led to the apparently complete decay of the truly intact feathers and provided 1.89 mmol free amino acids and 7.32 mmol acid-hydrolyzed amino acids from 50 ml of culture containing 3% (w/v) intact chicken feathers. The amino acid components in the soluble fraction of the culture conspicuously agreed with those calculated from the intact feathers. This demonstrated that more than 55% of total keratin proteins were solubilized from the intact chicken feathers into the culture in the forms of free amino acid and/or soluble oligopeptide, and most of them are directly derived from the intact feathers by proteolytic digestion. Feather degradation by strain H328 surpasses that by any other microorganisms with regard to degradation efficiency, absence of requirement for pretreatment of the feathers, and product fidelity in the amino acid component. Furthermore, the culture containing the degradative products from the intact feathers was subjected to matrix-assisted laser desorption ionization mass spectrometry-time-of-flight analysis, and it was revealed that the molecular masses of the solubilized products, oligopeptides, were less than 1,000. This result allows us to investigate the bioactivities of oligopeptides derived from the degradation of chicken feathers by cultivation with strain H328 as well as the production of amino acids for feedstuffs.     

Effect of rice--glycerol complex medium on the production of Lovastatin by Monascus ruber

Response surface methodology (RSM) was employed to study the effect of the composition of the rice-glycerol complex medium on the production of lovastatin (Lvs) by the ascomyceteMonascus ruber in mixed solid-liquid (or submerged) cultures at 25°C. Four components (rice powder, peptone, glycerol, glucose) were studied to evaluate, the approximate polynomial for all dependent variables, explaining their effects on the production of Lvs. The best composition derived from RSM regression was (in g/L) rice powder 34.4, peptone 10.8, , glucose 129, KNO₃ 8.0, MgSO₄·7H₂O 4.0 and glycerol 36.4 mL/L. With this composition, the Lvs production was 157 mg/L after 10 d of cultivation. In comparison with glycerol and glucose, the rice powder becomes a more suitable carbon source and represents a great potential for the production of Lvs.     

Cosecretion of protease inhibitor stabilizes antibodies produced by plant roots

A plant-based system for continuous production of monoclonal antibodies based on the secretion of immunoglobulin complexes from plant roots into a hydroponic medium (rhizosecretion) was engineered to produce high levels of single-chain and full-size immunoglobulins. Replacing the original signal peptides of monoclonal antibodies with a plant-derived calreticulin signal increased the levels of antibody yield 2-fold. Cosecretion of Bowman-Birk Ser protease inhibitor reduced degradation of the immunoglobulin complexes in the default secretion pathway and further increased antibody production to 36.4 microg/g root dry weight per day for single-chain IgG1 and 21.8 microg/g root dry weight per day for full-size IgG4 antibodies. These results suggest that constitutive cosecretion of a protease inhibitor combined with the use of the plant signal peptide and the antibiotic marker-free transformation system offers a novel strategy to achieve high yields of complex therapeutic proteins secreted from plant roots.     

Purification and characterization of three novel keratinolytic metalloproteases produced by Chryseobacterium indologenes TKU014 in a shrimp shell powder medium

A protease-producing bacterium was isolated and identified as Chryseobacterium indologenes TKU014. The optimized condition for protease production was found when the culture was shaken at 30 °C for one day in 50 mL of medium containing 0.5% shrimp shell powder (w/v), 0.1% K₂HPO₄, and 0.05% MgSO₄ · 7H₂O. Three extracellular proteases (P1, P2, and P3) were purified from culture by DEAE-Sepharose and Phenyl Sepharose chromatography. Three enzymes all showed activities of keratinase and elastase with molecular weights of 56, 40, 40 kDa, respectively. The inhibitory effect of metal chelator EDTA and Zn-specific chelator 1,10-phenanthroline characterized three enzymes as Zn-metalloproteases. Peptide mass fingerprints of P1, P2, and P3 were determined by using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Similarity search in the NCBI non-redundant protein sequence database revealed that three enzymes exhibited no significant homology to any other reported microbial peptides. Therefore, P1, P2, and P3 are most likely novel proteins.     

Biochemical and physiological properties of an extracellular protease produced by Bacillus megaterium

A protease, excreted by a sporogeneous strain of B. megaterium, growing exponentially in a minimum glucose ammonium medium, was isolated. It is a neutral endopeptidase, stabilized by Ca⁺⁺, inhibited by o-phenanthroline, but not by di-isopropylfluorophosphate. The specificity, studied on insulin B-chain, glucagon, cytochrome c, and dipeptides substrates, indicated the need for a dipeptide backbone with both substituted amino and carboxyl groups. A requirement was observed for a nonpolar lateral chain in the amino acid whose amino group was involved in the peptide bond (Leu, Phe, Ala, He, Val). Rates of hydrolysis varied also with the amino acid whose carboxyl group was involved (e.g., His > Ser > Ala > Gly). In complex medium, supplemented with Yeast Extract, the biosynthesis of the protease was repressed during growth, but the same enzyme was excreted during sporulation. The repression was apparently of the same nature as that controlling sporulation during and after growth (e.g., repression by a mixture of amino acids or high concentration of glucose). An asporogeneous mutant showed a normal product ion of protease under all conditions, and a low intracellular protease turnover after growth. A mutant unable to produce protease showed a normal sporulation and a high protein turnover. This protease, here termed megapeptidase, seems to be a typical growth enzyme, not related to either the sporulation process or to the protein turnover after growth.     

Purification and properties of a new type of protease produced by Microbacterium liquefaciens

A bacterium, identified as Microbacterium liquefaciens MIM-CG-9535-I, was isolated from a soil sample taken from the industrial site of a gelatin manufacturer. A new type of protease, which restrictively decomposes gelatin at one or two positions, was purified from the bacterial culture. The molecular mass of the purified enzyme was 21 kDa by SDS-polyacrylamide gel electrophoresis. The purified enzyme specifically degraded the alpha-chain of gelatin with a molecular weight of 100 kDa into two peptides of 60 kDa and 40 kDa. Native collagen was not a substrate for the enzyme.     

Characterization of two forms of hemagglutinin/protease produced by Vibrio cholerae non-O1

Two forms (34 kDa and 32 kDa) of hemagglutinin/protease produced by Vibrio cholerae non-O1 were characterized. The hemagglutinin/protease purified by immunoaffinity column chromatography using a monoclonal antibody was essentially a 34-kDa form. By incubation of the purified 34-kDa form at 37 degrees C, it was processed (autodigested) to the 32-kDa form. The N-terminal 20 amino acid sequences of both the 34- and 32-kDa forms were identical, suggesting that proteolytic processing at the C-terminal region of the 34-kDa hemagglutinin/protease resulted in the 32-kDa form. With this shift, protease activity increased, but hemagglutinating activity decreased, suggesting that the C-terminal region of the hemagglutinin/protease is related to hemagglutinating activity.