Characterization of an Extracellular Protease from the Insect Pathogen Xenorhabdus luminescens

Xenorhabdus luminescens Hm cultured in gelatin broth produced a single extracellular protease. The protease was purified by a factor of 500 and characterized as a monomeric protein with an approximate molecular weight of 61,000. On the basis of inhibitor studies and its pH optimum, the protease was classified as an alkaline metalloprotease with a pH optimum near 8; the isoelectric point of the enzyme is 4.2 +/- 0.2. The protease may be a major factor in the ecology of X. luminescens, which is carried as a symbiom of some parasitic nematodes.     

The Fish Pathogen Yersinia ruckeri Produces Holomycin and Uses an RNA Methyltransferase for Self-resistance

Holomycin and its derivatives belong to a class of broad-spectrum antibacterial natural products containing a rare dithiolopyrrolone heterobicyclic scaffold. The antibacterial mechanism of dithiolopyrrolone compounds has been attributed to the inhibition of bacterial RNA polymerase activities, although the exact mode of action has not been established in vitro. Some dithiopyrrolone derivatives display potent anticancer activities. Recently the biosynthetic gene cluster of holomycin has been identified and characterized in Streptomyces clavuligerus. Here we report that the fish pathogen Yersinia ruckeri is a holomycin producer, as evidenced through genome mining, chemical isolation, and structural elucidation as well as genetic manipulation. We also identified a unique regulatory gene hom15 at one end of the gene cluster encoding a cold-shock-like protein that likely regulates the production of holomycin in low cultivation temperatures. Inactivation of hom15 resulted in a significant loss of holomycin production. Finally, gene disruption of an RNA methyltransferase gene hom12 resulted in the sensitivity of the mutant toward holomycin. A complementation experiment of hom12 restored the resistance against holomycin. Although the wild-type Escherichia coli BL21(DE3) Gold is susceptible to holomycin, the mutant harboring hom12 showed tolerance toward holomycin. High resolution liquid chromatography (LC)-ESI/MS analysis of digested RNA fragments demonstrated that the wild-type Y. ruckeri and E. coli harboring hom12 contain a methylated RNA fragment, whereas the mutated Y. ruckeri and the wild-type E. coli only contain normal non-methylated RNA fragments. Taken together, our results strongly suggest that this putative RNA methyltransferase Hom12 is the self-resistance protein that methylates the RNA of Y. ruckeri to reduce the cytotoxic effect of holomycin during holomycin production.     

Purification and Characterization of an Extracellular Protease from Penicillium chrysogenum Pg222 Active against Meat Proteins

An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60°C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45°C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products.     

Purification and Characterization of an Extracellular Alkaline Serine Protease from Aspergillus terreus (IJIRA 6.2)

An extracellular alkaline serine protease has been purified from Aspergillus terreus (IJIRA 6.2). The purification procedure involved chromatography on DEAE-Sephadex A25, phosphocellulose, hydroxyapatite, casein-Sepharose, gel filtration on Sephacryl-S-300 and by glycerol density gradient centrifugation. The enzyme was further purified to apparent homogeneity through a combination of electrophoresis in polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS) with or without protease substrate (gelatin) and subsequent regeneration of its activity in situ by removal of SDS. The active enzyme was visualized in a zymogram or on the basis of protease activity exhibited on an X-ray film. The protein in the unstained segment of the gel was electroeluted. The eluted protein with protease activity exhibited a molecular mass of 37,000-daltons on electrophoresis in SDS-polyacrylamide gel. A sedimentation coefficient of 3.2S was obtained by glycerol density gradient contrifugation. Maximum activity of protease was observed at pH 8.5 and at 37°C. Purified protease was active between pH 5.5 and 9.5 and was found to be stable up to 60°C. With Na-caseinate, the K _m of the purified protease was found to be 0.055 mM. Antipain, phenylmethane sulfonyl fluoride, and chymostatin served as non-competitive inhibitors. Substrate specificity was determined by using a synthetic chromogenic peptide containing N-P-Tosyl-Gly-Pro-Arg-p-nitroanilide. Results showed that the protease cleaved the peptide on the -COOH end of arginine residue. Received: 8 October 1999 / Accepted: 3 November 1999     

Purification, Characterization, and Functional Role of a Novel Extracellular Protease from Pleurotus ostreatus

A new extracellular protease (PoSl; Pleurotus ostreatus subtilisin-like protease) from P. ostreatus culture broth has been purified and characterized. PoSl is a monomeric glycoprotein with a molecular mass of 75 kDa, a pI of 4.5, and an optimum pH in the alkaline range. The inhibitory profile indicates that PoSl is a serine protease. The N-terminal and three tryptic peptide sequences of PoSl have been determined. The homology of one internal peptide with conserved sequence around the Asp residue of the catalytic triad in the subtilase family suggests that PoSl is a subtilisin-like protease. This hypothesis is further supported by the finding that PoSl hydrolysis sites of the insulin B chain match those of subtilisin. PoSl activity is positively affected by calcium. A 10-fold decrease in the K_m value in the presence of calcium ions can reflect an induced structural change in the substrate recognition site region. Furthermore, Ca²⁺ binding slows PoSl autolysis, triggering the protein to form a more compact structure. These effects have already been observed for subtilisin and other serine proteases. Moreover, PoSl protease seems to play a key role in the regulation of P. ostreatus laccase activity by degrading and/or activating different isoenzymes.     

Purification and Properties of Cold-active Metalloprotease from Curtobacterium luteum and Effect of Culture Conditions on Production

Curtobacterium luteum, a gram-positive psychrotrophic bacterium, secreting an extracellular protease was isolated from the soil of Gangotri glacier, Western Himalaya. The maximum enzyme production was achieved when isolate was grown in a pH-neutral medium containing skim milk at 15oC over 120 hour. The metal ions such as Zn2+ and Cr2+ enhanced enzyme production. The specific activity of purified enzyme was 8090 u/mg after 34.1 fold purification. The 115 kD enzyme was a metalloprotease (activity inhibited by EDTA and EGTA) and showed maximum activity at 20oC and pH 7. The enzyme was active over a broad pH range and retained 84% of its original activity between pH 6–8. There was no loss in enzyme activity when exposed for 3 hours at 4oC-20oC. However, lost 65% of activity at 30oC, and was almost inactivated at 50oC, but was resistant to repeated freezing and thawing. The enzyme activity was stimulated by manganese ions; however, it was inactivated by copper ions.     

Purification and characterization of an extracellular protease from the fish pathogen Yersinia ruckeri and effect of culture conditions on production.

A novel protease, hydrolyzing azocasein, was identified, purified, and characterized from the culture supernatant of the fish pathogen Yersinia ruckeri. Exoprotease production was detected at the end of the exponential growth phase and was temperature dependent. Activity was detected in peptone but not in Casamino Acid medium. Its synthesis appeared to be under catabolite repression and ammonium control. The protease was purified in a simple two-step procedure involving ammonium sulfate precipitation and ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified protein indicated an estimated molecular mass of 47 kDa. The protease had characteristics of a cold-adapted protein, i.e., it was more active in the range of 25 to 42 degrees C and had an optimum activity at 37 degrees C. The activation energy for the hydrolysis of azocasein was determined to be 15.53 kcal/mol, and the enzyme showed a rapid decrease in activity at 42 degrees C. The enzyme had an optimum pH of around 8. Characterization of the protease showed that it required certain cations such as Mg(2+) or Ca(2+) for maximal activity and was inhibited by EDTA, 1,10-phenanthroline, and EGTA but not by phenylmethylsulfonyl fluoride. Two N-methyl-N-nitro-N-nitrosoguanidine mutants were isolated and analyzed; one did not show caseinolytic activity and lacked the 47-kDa protein, while the other was hyperproteolytic and produced increased amounts of the 47-kDa protein. Azocasein activity, SDS-PAGE, immunoblotting by using polyclonal anti-47-kDa-protease serum, and zymogram analyses showed that protease activity was present in 8 of 14 strains tested and that two Y. ruckeri groups could be established based on the presence or absence of the 47-kDa protease.     

Biochemical and Genetic Characterization of an Extracellular Protease from Pseudomonas fluorescens CY091

Pseudomonas fluorescens CY091 cultures produce an extracellular protease with an estimated molecular mass of 50 kDa. Production of this enzyme (designated AprX) was observed in media containing CaCl₂ or SrCl₂ but not in media containing ZnCl₂, MgCl₂, or MnCl₂. The requirement of Ca²⁺ (or Sr²⁺) for enzyme production was concentration dependent, and the optimal concentration for production was determined to be 0.35 mM. Following ammonium sulfate precipitation and ion-exchange chromatography, the AprX in the culture supernatant was purified to near electrophoretic homogeneity. Over 20% of the enzyme activity was retained in the AprX sample which had been heated in boiling water for 10 min, indicating that the enzyme is highly resistant to heat inactivation. The enzyme activity was almost completely inhibited in the presence of 1 mM 1,10-phenanthroline, but only 30% of the activity was inhibited in the presence of 1 mM EGTA. The gene encoding AprX was cloned from the genome of P. fluorescens CY091 by isolating cosmid clones capable of restoring the protease production in a nonproteolytic mutant of strain CY091. The genomic region of strain CY091 containing the aprX gene was located within a 7.3-kb DNA fragment. Analysis of the complete nucleotide sequence of this 7.3-kb fragment revealed the presence of a cluster of genes required for the production of extracellular AprX in P. fluorescens and Escherichia coli. The AprX protein showed 50 to 60% identity in amino acid sequence to the related proteases produced by Pseudomonas aeruginosa and Erwinia chrysanthemi. Two conserved sequence domains possibly associated with Ca²⁺ and Zn²⁺ binding were identified. Immediately adjacent to the aprX structural gene, a gene (inh) encoding a putative protease inhibitor and three genes (aprD, aprE, and aprF), possibly required for the transport of AprX, were also identified. The organization of the gene cluster involved in the synthesis and secretion of AprX in P. fluorescens CY091 appears to be somewhat different from that previously demonstrated in P. aeruginosa and E. chrysanthemi.     

粗毛栓菌胞外锰过氧化物酶的纯化及性质

培养于麦草粉上的白腐担子菌粗毛栓菌分泌胞外木质纤维素降解酶(纤维素酶、木聚糖酶、漆酶、锰过氧化物酶和木质素过氧化物酶)。经过超滤、盐析、离子交换层析、凝胶过滤和活性聚丙烯酰胺凝胶电泳等步骤,获得了初步纯化的锰过氧化物酶组分。利用变性聚丙烯酰胺凝胶电泳和等电点聚焦技术所测定的锰过氧化物酶的相对分子质量和等电点分别为35.7 ku和pI 2.8。研究结果表明,所纯化的锰过氧化物酶在407nm处具有最大光吸收峰,该酶最适作用pH值和温度分别为pH 5.3和35℃。     

Purification and Characterization of an Extracellular Alkaline Phosphatase from Penicillium Chrysogenum

Abstract

An extracellular alkaline phosphatase from Penidllium chrysogenum was purified to homogeneity using DEAE ion-exchange chromatography and size exclusion chromatography. SDS-PAGE of the purified enzyme indicated a molecular weight of 58,000. The mobility of the native enzyme on a Superose 12 column suggests that the active form of the enzyme is a monomer. The enzyme catalyzes the hydrolysis of phosphate from a variety of substrates including p-Miitrophenyl phosphate, α-naphthyl phosphate and the anti-tumor compound etoposide phosphate. The apparent K_m for the substrate p-nitrophenyl phosphate is 1.3 mM and the enzyme is inhibited by inorganic phosphate. The pH optimum of the enzyme is 9.0 with a broad optimal temperature range between 40 and 50 °C. The isoelectric point of the enzyme is approximately 5.5. The enzyme is a glycoprotein; digestion with endoglycosidase H indicates that the protein consists primarily of N-inked carbohydrates. Enzymatic activity is enhanced by the addition of divalent cations such as Mg⁺⁺ and Mn⁺⁺ and inhibited by addition of a chelator such as EDTA suggesting a metal ion requirement. The enzyme was found to be an inexpensive catalyst for the conversion of etoposide phosphate to etoposide in the manufacture of this anti-tumor compound.     

Production,Rapid Purification and Catalytic Characterization of Extracellular Phytase from Aspergillus Ficuum

Abstract

A rapid purification scheme utilizing three chromatographic steps resulted in 6 fold purification of Aspergillus ficuum phytase (myo-inositol-hexakis-phosphate 3-phosphohydrolase, EC 3.1. 3.8). At pH 5.0 and 60°C the enzyme performed acceptably for 2.0 hr with only 30% diminished catalytic rate at the end. Substrate concentration exceeding 2nM was inhibitory. The inorganic orthophosphate, the product and a weak inhibitor, exhibited a Ki of 1.9 × 10^?3M. The extracellular phytase has the potential for industrial use since it can be over produced, easily purified, remain catalytically active for a longer period and is not subjected to severe product inhibition.     

Purification and Characterization of an Extracellular Proteinase from Brevibacterium linens ATCC 9174

An extracellular serine proteinase from Brevibacterium linens ATCC 9174 was purified to homogeneity. pH and temperature optima were 8.5 and 50(deg)C, respectively. The results for the molecular mass of the proteinase were 56 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 126 kDa by gel filtration, indicating that the native enzyme exists as a dimer. Mg(sup2+) and Ca(sup2+) activated the proteinase, as did NaCl; however, Hg(sup2+), Fe(sup2+), and Zn(sup2+) caused strong inhibition. The sequence of the first 20 N-terminal amino acids was NH(inf2)-Ala-Lys-Asn-Asp-Ala-Val-Gly-Gly-Met-Gly-Tyr-Leu-Ser-Met-Ile-Pro-Se r-Gln-Pro-Gly.     

Purification and Characterization of a Fibrinolytic Protease from a Culture Supernatant of Flammulina velutipes Mycelia

In this study we purified a fibrinolytic enzyme from the culture supernatant of Flammulina velutipes mycelia by ion exchange and gel filtration chromatographies, it was designated as F. velutipes protease (FVP-I). This purification protocol resulted in 18.52-fold purification of the enzyme at a final yield of 0.69%. The molecular mass of the purified enzyme was estimated to be 37 kDa by SDS–PAGE, fibrin-zymography and size exclusion by FPLC. This protease effectively hydrolyzed fibrin, preferentially digesting α-chain over β-and γ–γ chain. Optimal protease activity was found to occur at a pH of 6.0 and a temperature of 20 to 30 °C. The protease activity was inhibited by Cu²⁺, Fe²⁺ and Fe³⁺ ions, but was found to be enhanced by Mn²⁺ and Mg²⁺ ions. Furthermore, FVP-I activity was potently inhibited by EDTA and EGTA, and it was found to exhibit a higher specificity for chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The first 20 amino acid residues of the N-terminal sequence of FVP-I were LTYRVIPITKQAVTEGTELL. They had a high degree of homology with hypothetical protein CC1G_11771, GeneBank Accession no. EAU86463.     

Purification and Properties of an Extracellular Protease Produced by the Entomopathogenic Fungus Beauveria bassiana

Beauveria bassiana GK2016 grown in a medium with gelatin as the sole carbon and nitrogen source produced an extracellular protease. The protease production was highest when the fungus was grown on a semiliquid medium and was purified about 18-fold, with a recovery of 21%. The protease molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be about 35,000. It had an optimum activity at pH 8.5 and 37 degrees C and was rapidly inactivated at 50 degrees C. Its enzymatic activity was that of an endopeptidase which hydrolyzed elastin, casein, and gelatin but was much less active on bovine serum albumin and collagen. No trypsinlike activity was detected on N-alpha-benzoyl-dl-arginine-p-nitroanilide. It was, however, inhibited by phenylmethylsulfonyl fluoride, indicating that a serine residue is present in the active site. The protease was unaffected by metal-chelating agents, sulfhydryl reagents, trypsin inhibitor, and chymotrypsin inhibitor.     

Purification and Characterization of an Extracellular Acid Proteinase from the Ectomycorrhizal Fungus Hebeloma crustuliniforme

Hebeloma crustuliniforme produced an extracellular acid proteinase in a liquid medium containing bovine serum albumin as the sole nitrogen source. The proteinase was purified 26-fold with 20% activity recovery and was shown to have a molecular weight of 37,800 (as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and an isoelectric point of 4.8 +/- 0.2. The enzyme was most active at 50 degrees C and pH 2.5 against bovine serum albumin and was stable in the absence of substrates at temperatures up to 45 degrees C and pHs between 2.0 and 5.0. Pepstatin A, diazoacetyl-dl-norleucine methylester, metallic ions Fe and Fe, and phenolic acids severely inhibited the enzyme activity, while antipain, leupeptin, N-alpha-p-tosyl-l-lysine chloromethyl ketone, and trypsin inhibitor inhibited the activity moderately. The proteinase hydrolyzed bovine serum albumin and cytochrome c rapidly compared with casein and azocasein but failed to hydrolyze any of the low-molecular-weight peptide derivatives tested.     

Purification and Characterization of an Extracellular β-Glucosidase from the Wood-Grown Fungus Xylaria regalis

Xylaria regalis, a wood-grown ascomycete isolated in Taiwan, produces β-glucosidase (EC 3.2.1.21) extracellularly. The β-glucosidase was purified to homogeneity by ammonium sulfate precipitation, ion-exchange, and gel filtration chromatography. The molecular mass of the purified enzyme was estimated to be 85 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. With p-nitrophenyl β-D-glucopyranoside (PNPG) as the substrate at pH 5.0 and 50°C, the K _m was 1.72 mM and V _max was 326 μmol/min/mg. Optimal activity with PNPG as the substrate was at pH 5.0 and 50°C. The enzyme was stable at pH 5.0 at temperatures up to 50°C. The purified β-glucosidase was active against PNPG, cellobiose, sophorose, and gentiobiose, but did not hydrolyze lactose, sucrose, Avicel, and o-nitrophenyl β-D-galactopyranoside. The activity of β-glucosidase was stimulated by Ca²⁺, Mg²⁺, Mn²⁺, Cd²⁺ and β-mercaptoethanol, and inhibited by Ag⁺, Hg²⁺, SDS, and p-chloromercuribenzoate (PCMB). Received: 30 March 1996 / Accepted: 3 May 1996     

Comparison Study of Synthesized Red (or Blood) Orange Peels and Juice Extract-Nanoflowers and Their Antimicrobial Properties on Fish Pathogen (Yersinia ruckeri)

AbstractIn this work, we synthesized blood orange peel extract-copper (II) (Cu²⁺) ions nanoflower (NFs) and blood orange juice extract-copper (II) (Cu²⁺) ions nanoflower examine their antimicrobial properties on the fish pathogen (Yersinia ruckeri). The main compounds of the blood orange peel extract and the blood orange juice extract were organic components, and the copper (II) (Cu^{2 +}) ions were inorganic components. BOPE-Cu^{2 +} nanoflowers are quite compact, porous, and uniform as compared to BOJE-Cu²⁺ nanoflowers. Scanning Electron Microscopy, Fourier Transform Infrared spectrometry, and Energy-Dispersive X-ray spectroscopy were used to observe the structures of the NFs. The findings of FT-IR show Cu–O and Cu–N bonds in NF, which may be an indicator of the development of NFs. Although the antimicrobial actions of BOPE-hNFs and BOJE-hNFs against Yersinia ruckeri (NCTC 12,268) have been confirmed.Graphic Abstract      

发状念珠藻胞外多糖的纯化与性质分析

采用DEAE阴离子交换层析和Sephadex G100凝胶层析对液体悬浮培养发状念珠藻胞外多糖进行纯化, 得到两个组分NFPS1和NFPS2。对组分NFPS2进行理化性质分析, 并与野生发状念珠藻多糖NFPS0的性质进行对比。结果表明二者具有相似的单糖组成, 均为葡萄糖、木糖、半乳糖、甘露糖; 表观分子量分别为2.79×105、2.26×105; 均不含核酸、蛋白质等物质, 是非硫酸化多糖; 有较高的热稳定性, 其降解温度在245oC左右。但在微观结构上, 两者存在一定差别。     

Purification and Characterization of Extracellular Pectinesterases from Phytophthora infestans

Constitutively produced extracellular pectinesterases from culture filtrates of the potato late blight fungus Phytophthora infestans were purified and characterized. One enzyme (PE II) was purified to homogeneity. Sodium dodecyl sulfate electrophoresis of the second enzyme (PE I) revealed two protein bands; there are indications that both proteins are pectinesterases, which were not separable by a number of different techniques. Thus, P. infestans might produce three pectinesterases in vitro. Enzyme activities were optimal in the neutral pH range and were largely dependent on the presence of NaCl or CaCl₂ in the reaction medium. The molecular weight of the PE I-complex was between 45 and 48 kilodaltons, and the one of PE II was between 35 and 40 kilodaltons. Further investigations will help us to clarify the role of these enzymes during pathogenesis.     

发状念珠藻胞外多糖的纯化与性质分析

采用DEAE阴离子交换层析和Sephadex G100凝胶层析对液体悬浮培养发状念珠藻胞外多糖进行纯化, 得到两个组分NFPS1和NFPS2。对组分NFPS2进行理化性质分析, 并与野生发状念珠藻多糖NFPS0的性质进行对比。结果表明二者具有相似的单糖组成, 均为葡萄糖、木糖、半乳糖、甘露糖; 表观分子量分别为2.79×105、2.26×105; 均不含核酸、蛋白质等物质, 是非硫酸化多糖; 有较高的热稳定性, 其降解温度在245oC左右。但在微观结构上, 两者存在一定差别。