Purification, Characterization, and Specificity Determination of a New Serine Protease Secreted by Penicillium waksmanii

The purpose of this work was to purify a protease from Penicillium waksmanii and to determine its biochemical characteristics and specificity. The extracellular protease isolated that was produced by P. waksmanii is a serine protease that is essential for the reproduction and growth of the fungus. The protease isolated showed 32 kDa, and has optimal activity at pH 8.0 and 35 °C towards the substrate Abz-KLRSSKQ-EDDnp. The protease is active in the presence of CaCl₂, KCl, and BaCl, and partially inhibited by CuCl₂, CoCl₂ and totally inhibited by AlCl₃ and LiCl. In the presence of 1 M urea, the protease remains 50 % active. The activity of the protease increases 60 % when it is exposed to 0.4 % nonionic surfactant-Triton X-100 and loses 10 % activity in the presence of 0.4 % Tween-80. Using fluorescence resonance energy transfer analysis, the protease showed the most specificity for the peptide Abz-KIRSSKQ-EDDnp with k _cat/K _m of 10,666 mM^?1?s^?1, followed by the peptide Abz-GLRSSKQ-EDDnp with a k _cat/K _m of 7,500 mM^?1?s^?1. Basic and acidic side chain-containing amino acids performed best at subsite S₁. Subsites S₂, S₃, S^′ ₂, and S^′ ₁, S^′ ₃ showed a preference for binding for amino acids with hydrophobic and basic amino acid side chain, respectively. High values of k _cat/K _m were observed for the subsites S₂, S₃, and S^′ _2. The sequence of the N-terminus (ANVVQSNVPSWGLARLSSKKTGTTDYTYD) showed high similarity to the fungi Penicillium citrinum and Penicillium chrysogenum, with 89 % of identity at the amino acid level.     

Purification and Biochemical Characterization of a New Protease Inhibitor from Conyza dioscoridis with Antimicrobial,Antifungal and Cytotoxic Effects

The main objective of the current study was the extraction, purification, and biochemical characterization of a protein protease inhibitor from Conyza dioscoridis. Antimicrobial potential and cytotoxic effects were also examined. The protease inhibitor was extracted in 0.1 M phosphate buffer (pH 6–7). Then, the protease inhibitor, named PDInhibitor, was purified using ammonium sulfate precipitation followed by filtration through a Sephadex G-50 column and had an apparent molecular weight of 25 kDa. The N-terminal sequence of PDInhibitor showed a high level of identity with those of the Kunitz family. PDInhibitor was found to be active at pH values ranging from 5.0 to 11.0, with maximal activity at pH 9.0. It was also fully active at 50 °C and maintained 90% of its stability at over 55 °C. The thermostability of the PDInhibitor was clearly enhanced by CaCl₂ and sorbitol, whereas the presence of Ca²⁺ and Zn²⁺ ions, Sodium taurodeoxycholate (NaTDC), Sodium dodecyl sulfate (SDS), Dithiothreitol (DTT), and β-ME dramatically improved the inhibitory activity. A remarkable affinity of the protease inhibitor with available important therapeutic proteases (elastase and trypsin) was observed. PDInhibitor also acted as a potent inhibitor of commercial proteases from Aspergillus oryzae and of Proteinase K. The inhibitor displayed potent antimicrobial activity against gram+ and gram- bacteria and against fungal strains. Interestingly, PDInhibitor affected several human cancer cell lines, namely HCT-116, MDA-MB-231, and Lovo. Thus, it can be considered a potentially powerful therapeutic agent.     

Neutral Serine Protease from Penicillium italicum. Purification,Biochemical Characterization,and Use for Antioxidative Peptide Preparation from Scorpaena notata Muscle

In the present study, purification and properties of an extracellular neutral serine protease from the fungus Penicillium italicum and its potential application as an antioxidant peptides producer are reported. The protease was purified to homogeneity using ammonium sulfate precipitation, Sephacryl S-200 gel filtration, diethylaminoethanol (DEAE)-Sepharose ion exchange chromatography, and TSK-HPLC gel filtration with a 10.2-fold increase in specific activity and 25.8 % recovery. The purified enzyme appeared as single protein band with a molecular mass of 24 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature for the proteolytic activity were pH 7.0 and 50 °C, respectively. The enzyme was stable in the pH range of 6.0–9.0. The protease was activated by divalent cations such as Ca²⁺ and Mg²⁺. Complete inhibition of the purified enzyme by phenylmethylsulfonyl fluoride confirmed that the protease was of serine-type. The purified enzyme revealed high stability and relatively broad specificity. Scorpaena notata muscle protein hydrolysates prepared using purified serine protease (protease from P. italicum (Prot-Pen)) showed good in vitro antioxidative activities. The antioxidant activities of Scorpaena muscle protein hydrolyzed by Prot-Pen (SMPH-PP) were evaluated using various antioxidant assays: 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, reducing power, ferrous chelating activity, and DNA nicking assay. SMPH-PP showed varying degrees of antioxidant activity and almost the same strongest protection against hydroxyl radical induced DNA breakage.     

Purification and Characterization of a New Metallo-Neutral Protease for Beer Brewing from Bacillus amyloliquefaciens SYB-001

The increased additive amount of adjuncts in the raw materials of Chinese beer requires the usage of protease to release more water-soluble proteins. Here, a metallo-neutral protease suited for brewing industry was purified from Bacillus amyloliquefaciens SYB-001. A 5.6-fold purification of the neutral protease was achieved with a 4-step procedure including ammonium sulfate precipitation, ion-exchange, hydrophobic interaction, and gel-filtration chromatography. The molecular mass of the enzyme was estimated to be 36.8 kDa. The protease was active and stable at a wide range of pH from 6.0–10.0 with an optimum at pH 7.0. The highest activity of the purified enzyme was found at 50 °C. The existence of manganese ion would specifically enhance the protease activity. Comparing with other commercial neutral proteases in China, adding the new neutral protease during mashing process would release more amino acids from wort such as aspartic acid, arginine, methione, and histidine, resulting in a better amino acid profile in wort. Moreover, the wort processed with the new neutral protease had a higher α-amino nitrogen concentration, which would ensure a vigorous yeast growth and better flavor. The study of the enzyme could lay a foundation for its industrial application and further research.     

Physicochemical Properties and Elimination of the Activity of Anti-Nutritional Serine Protease Inhibitors from Mulberry Leaves

Mulberry leaf is an excellent protein resource that can be used as feed additive for livestock and poultry. Nevertheless, the use of mulberry leaves in animal diets is limited by its protease inhibitors, tannic acid and other anti-nutritional factors. This study systematically analyzed the type and activity of serine protease inhibitors (SPIs) from the leaves of 34 mulberry varieties, aiming to reveal the physicochemical properties and inactivation mechanism of SPIs. The types and activities of trypsin inhibitors (TIs) and chymotrypsin inhibitors (CIs) exhibited polymorphisms among different mulberry varieties. The highest number of types of inhibitors was detected in Jinshi, with six TIs (TI-1~TI-6) and six CIs (CI-1~CI-6). TIs and CIs exhibited strong thermal and acid–base stability. High-temperature and high-pressure treatment could reduce the activities of TIs and CIs to a certain extent. β-mercaptoethanol treatment could completely abolish TIs and CIs, suggesting that the disulfide bridges were critical for their inhibitory activities. The Maillard reaction could effectively eliminate the inhibitory activities of TI-1~TI-4 and CI-1~CI-4. This study reveals the physicochemical properties and inactivation mechanisms of the anti-nutritional SPIs from mulberry leaves, which is helpful to exploit mulberry-leaf food with low-activity SPIs, promote the development and utilization of mulberry-leaf resources in animal feed and provide reference for mulberry breeding with different functions.     

New Bacteriocin from Bacillus clausii StrainGM17: Purification, Characterization, and Biological Activity

A bacteriocin-producing strain (9,000 AU/ml) was isolated from the rhizosphere of Algerian healthy plants Ononis angustissima Lam. and identified as Bacillus clausii strain GM17. The bacteriocin, called Bac-GM17, was purified from the culture supernatant after heat treatment, ammonium sulfate precipitation, Sephadex G-50 chromatography and Mono Q fast-performance liquid chromatography (FPLC). Based on matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, the purified Bac-GM17 is a monomer protein with a molecular mass of 5,158.11 Da. The N-terminal sequencing allowed for the straightforward identification of its first 20 residues, which were of pure bacteriocin. It also revealed that this bacteriocin contained a unique sequence, namely DWTCSKWSCLVCDDCSVELT, which suggests the identification of a novel compound. Bac-GM17 was extremely heat stable (20 min at 120 °C) and was stable within the pH range (3–9). It was found to be resistant to the proteolytic action of trypsin, pepsin, papain, pronase E, and proteinase K. It was also noted to display a bactericidal mode of action against Agrobacterium tumefaciens C58 and a fungistatic mode of action against Candida tropicalis R2 CIP203.     

Production, Purification, and Biochemical Characterization of a Fibrinolytic Enzyme from Thermophilic Streptomyces sp. MCMB-379

Production of the fibrinolytic enzyme was carried out using 2.5-L glass fermentor, culture of thermophilic Streptomyces sp., and glucose yeast extract peptone medium of pH 8.0. Five successive batches were carried out under controlled fermentation conditions viz., agitation 140 rpm, aeration 0.5 vvm, 55 °C, and 18 h. The total protein extracellularly produced in the cell-free broth was ~300-500 mg/L. The enzyme belongs to serine endopeptidase type. Studies on the fibrin degradation indicate that the enzyme degrades the fibrin into small molecular weight products as seen from HPLC profile. Phase-contrast microscopic structure of fibrin showed that enzyme cleaves the fibrin filaments. The ex vivo activity of the actinokinase was compared with 500 IU of urokinase and 350 IU of streptokinase. The ex vivo clot lysis was found to be faster as compared to the commercial available enzymes.     

Purification and Characterization of a Milk Clotting Protease fromMucor bacilliformis

An acid protease having milk clotting activity has been isolated fromMucor bacilliformis cultures. The enzyme was basically purified by ionic exchange chromatography. An average yield of 29 mg purified product was obtained from 100 mL crude extract. As purity criteria, SDS-PAGE, reverse-phase HPLC, and N-terminal analysis were performed. The protease is a protein composed of a single polypeptide chain with glycine at the N-terminus. The mol wt is approx 32,000, and its amino acid composition is very similar to those of other fungal proteases. As expected, its clotting activity was drastically inhibited by pepstatin A action. On the other hand, its instability against heat treatment and its clotting/proteolytic activity ratio indicate that it may be considered as a potential substitute for bovine chymosin. Index Entries:Mucor bacilliformis protease; milk clotting enzyme; acid protease; fungal protease; aspartyl protease.     

Depsipeptides from a Guamanian Marine Cyanobacterium, Lyngbya bouillonii, with Selective Inhibition of Serine Proteases

Bouillomides A (1) and B (2) are two depsipeptide analogues of dolastatin 13. Isolated from a Guamanian sample of Lyngbya bouillonii, the planar structures were elucidated on the basis of HR-ESI-MS and NMR data, while the absolute configurations were determined by employing functional group conversions, modified Marfey’s analysis, and detailed analyses of ROESY correlations. Compounds 1 and 2 selectively inhibited serine proteases elastase (IC₅₀ = 1.9 μM for both) and chymotrypsin (IC₅₀ = 0.17 and 9.3 μM, respectively) while showing no inhibition of trypsin (IC₅₀ >100 μM).     

Purification and Characterization of Bowman‐Birk Protease Inhibitor from Rice Coleoptiles

A 15 kDa rice Bowman‐Birk inhibitor from fast elongating coleoptiles has been purified and identified using partial N‐terminal sequence, LC‐MS, and MALDI‐TOF MS as a 133 amino acid polypeptide (BBIrc 1). The kinetic study shows this protease inhibitor displays competitive inhibition toward trypsin with Ki of 4.0 × 10^?7 M and non‐competitive inhibition toward α‐chymotrypsin with Ki of 9.3 × 10^?6 M. The Western blotting results of the anti‐sera raised against this 15 kDa protein showed that this anti‐serum recognized two BBI proteins with molecular size around 15 kDa (BBIrc 1) and 25 kDa (BBIrc2) and the quantity of the expression of 15 kDa was nearly constant under both aerobic and hypoxia conditions; however, the 25 kDa expression was greatly up‐regulated when the fast elongating coleoptiles were transferred from hypoxia conditions to the aerobic conditions. The results indicate that the expression pattern of BBIs proteins correlated to the developmental stage in terms of morphological changes. The partial N‐terminal sequence of the first 9 amino acids of 25 kDa was AEAPPRPPK, which is the same as the amino acid sequence of 37th to 45th of RBBI3‐1 and LC‐MS study shows that several mass fragments fit to RBBI3‐1. The 25 kDa protein also shows specific binding to bovine trypsin. This expression pattern demonstrates for the first time that environmental factor, oxygen, can select and enhance specific BBI gene expression. The results of this study suggest BBI proteins might play multiple biological functions inside rice coleoptiles.     

Characterization of Thermostable Serine Alkaline Protease from an Alkaliphilic Strain Bacillus pumilus MCAS8 and Its Applications

This study describes the characterization and optimization of medium components for an extracellular detergent, surfactant, organic solvent and thermostable serine alkaline protease produced by alkaliphilic Bacillus pumilus MCAS8 strain isolated from Pulicat lake sediments, Tamil Nadu, India. The strain yielded maximum protease (2,214?U/ml) under optimized conditions: carbon source, citric acid??1.5?% (w/w); inducer, soyabean meal??2?% (w/w); pH?11.0; shaking condition 37?°C for 48?h. The enzyme had pH and temperature optima of 9.0 and 60?°C, respectively. The enzyme displayed the molecular mass of 36?kDa in sodium dodecyl sulphate?Cpolyacrylamide gel electrophoresis study and exhibited activity at a wide range of pH (6.0?C11.0) and thermostability (20?C70?°C). More than 70?% residual activity was observed when the enzyme was incubated with dithiothreitol, ethylenediaminetetraacetic acid, ethylene glycol tetraacetic acid and H₂O₂ for 30?min. The protease activity was also enhanced by divalent cations such as Ba²⁺, Ca²⁺ and Mg²⁺ and was strongly inhibited by Fe²⁺, Zn²⁺, Sr²⁺, Hg²⁺ and urea. The enzyme retained more than 50?% of its initial activity after pre-incubation for 1?h in the presence of 5?% (v/v) organic solvents such as dimethyl sulphoxide and acetone. The protease could hydrolyse various native proteinaceous substrates (1?%?w/v) such as bovine serum albumin, casein, skim milk, gelatine, azocasein and haemoglobin. Wash performance analysis of enzyme revealed that it could effectively remove blood stains from the cotton fabric, thus making it suitable to use as an effective detergent additive. The protease enzyme also exhibited promising result in the dehairing of goat skin. The potency of the eco-friendly enzyme without using any chemicals against washing and dehairing showed that the enzyme could be used for various industrial applications.     

Separation,Purification, Structural Characterization,and Anticancer Activity of a Novel Exopolysaccharide from Mucor sp.

Mucor sp. has a wide range of applications in the food fermentation industry. In this study, a novel exopolysaccharide, labeled MSEPS, was separated from Mucor sp. fermentation broth through ethanol precipitation and was purified by ion-exchange chromatography, as well as gel filtration column chromatography. MSEPS was composed mostly of mannose, galactose, fucose, arabinose, and glucose with a molar ratio of 0.466:0.169:0.139:0.126:0.015 and had a molecular weight of 7.78 × 10⁴ Da. The analysis of methylation and nuclear magnetic resonance results indicated that MSEPS mainly consisted of a backbone of →3,6)-α-d-Manp-(1→3,6)-β-d-Galp-(1→, with substitution at O-3 of →6)-α-d-Manp-(1→ and →6)-β-d-Galp-(1→ by terminal α-l-Araf residues. MTT assays showed that MSEPS was nontoxic in normal cells (HK-2 cells) and inhibited the proliferation of carcinoma cells (SGC-7901 cells). Additionally, morphological analysis and flow cytometry experiments indicated that MSEPS promoted SGC-7901 cell death via apoptosis. Therefore, MSEPS from Mucor sp. can be developed as a potential antitumor agent.     

Zingipain, a Ginger Protease with Acetylcholinesterase Inhibitory Activity

In order to search for new acetylcholinesterase inhibitors (AChEIs), 15 Zingiberaceae plants were tested for AChEI activity in rhizome extracts. The crude homogenate and ammonium sulfate cut fraction of Zingiber officinale contained a significant AChEI activity. Eighty percent saturation ammonium sulfate precipitation and diethylaminoethyl cellulose ion exchange chromatography (unbound fraction) enriched the protein to a single band on nondenaturing and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (approximately 33.5 kDa). Gelatin-degrading zymography showed that the AChEI-containing band also contained cysteine protease activity. The AChEI activity was largely stable between ?20 and 60 °C (at least over 120 min) and over a broad pH range (2–12). The AChEI activity was stimulated strongly by Mn²⁺ and Cu²⁺ at 1–10 mM and weakly by Ca²⁺, Fe²⁺, Mg²⁺, and Zn²⁺ at 1 mM, but was inhibited at 10 mM. In contrast, Hg²⁺ and ethylenediaminetetraacetic acid were very and moderately strongly inhibitory, respectively. In-gel tryptic digestion with liquid chromatography–tandem mass spectroscopy resolution revealed two heterogeneous peptides, a 16-amino-acid-long fragment with 100 % similarity to zingipain-1, which is a cysteine protease from Z. officinale, and a 9-amino-acid-long fragment that was 100 % identical to actinidin Act 2a, suggesting that the preparation was heterogeneous. AChEI exhibited noncompetitive inhibition of AChE for the hydrolysis of acetylthiocholine iodide with a K _i value of 9.31 mg/ml.     

Expression,Purification and Characterization of a Novel Hybrid Peptide CLP with Excellent Antibacterial Activity

CLP is a novel hybrid peptide derived from CM4, LL37 and TP5, with significantly reduced hemolytic activity and increased antibacterial activity than parental antimicrobial peptides. To avoid host toxicity and obtain high-level bio-production of CLP, we established a His-tagged SUMO fusion expression system in Escherichia coli. The fusion protein can be purified using a Nickel column, cleaved by TEV protease, and further purified in flow-through of the Nickel column. As a result, the recombinant CLP with a yield of 27.56 mg/L and a purity of 93.6% was obtained. The purified CLP exhibits potent antimicrobial activity against gram+ and gram- bacteria. Furthermore, the result of propidium iodide staining and scanning electron microscopy (SEM) showed that CLP can induce the membrane permeabilization and cell death of Enterotoxigenic Escherichia coli (ETEC) K88. The analysis of thermal stability results showed that the antibacterial activity of CLP decreases slightly below 70 °C for 30 min. However, when the temperature was above 70 °C, the antibacterial activity was significantly decreased. In addition, the antibacterial activity of CLP was stable in the pH range from 4.0 to 9.0; however, when pH was below 4.0 and over 9.0, the activity of CLP decreased significantly. In the presence of various proteases, such as pepsin, papain, trypsin and proteinase K, the antibacterial activity of CLP remained above 46.2%. In summary, this study not only provides an effective strategy for high-level production of antimicrobial peptides and evaluates the interference factors that affect the biological activity of hybrid peptide CLP, but also paves the way for further exploration of the treatment of multidrug-resistant bacterial infections.     

Purification and Characterization of a Novel Tetradecapeptide from Ginseng Polypeptides with Enhancing Memory Activity for Mice

Aiming at isolating and investigating the active ingredients of the aqueous extract from Panax ginseng which showed enhancing memory activity, the authors characterized one ingredient. To separate the oligosaccharides and polypeptides, a DEAE-Sephadex A-50 colum was used. The enhanced memory activity in mice was studied by Mirros water maze tesk in mice. The dose of oligosacchrides, polypeptides or Piracetam was 30 mg/kg per day with intraperitoneal administration. The oligosaccharides did not show enhancing memory effect, but polypeptides did show. This result demonstrates that the active ingredients of the aqueous extract from Panax ginseng which showed enhancing memory effect was polypeptides. The purification of the polypeptides was performed on a Sephadex G-25 column. A novel tetradecapeptide was purified from the polypeptides and its structure was determined by liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry(LC-ESI-Q-TOF-MS) with the amino acid sequence of Lys-Ser-Leu-Thr-Leu-Thr-Ser-Ser-Leu-Ser-Tyr-Thr-Asp-Ser.     

Purification and Biochemical Characterization of a Novel Alkaline Protease Produced by Penicillium nalgiovense

Penicillium nalgiovense PNA9 produces an extracellular protease during fermentation with characteristics of growth-associated product. Enzyme purification involved ammonium sulfate precipitation, dialysis, and ultrafiltration, resulting in 12.1-fold increase of specific activity (19.5 U/mg). The protein was isolated through a series of BN-PAGE and native PAGE runs. ESI-MS analysis confirmed the molecular mass of 45.2 kDa. N-Terminal sequencing (MGFLKLLKGSLATLAVVNAGKLLTANDGDE) revealed 93 % similarity to a Penicillium chrysogenum protease, identified as major allergen. The protease exhibits simple Michaelis-Menten kinetics and K _m (1.152 mg/ml), V _max (0.827 mg/ml/min), and k _cat (3.2?×?10²) (1/s) values against azocasein show that it possesses high substrate affinity and catalytic efficiency. The protease is active within 10–45 °C, pH 4.0–10.0, and 0–3 M NaCl, while maximum activity was observed at 35 °C, pH 8.0, and 0.25 M NaCl. It is active against the muscle proteins actin and myosin and inactive against myoglobin. It is highly stable in the presence of non-ionic surfactants, hydrogen peroxide, BTNB, and EDTA. Activity was inhibited by SDS, Mn²⁺ and Zn²⁺, and by the serine protease inhibitor PMSF, indicating the serine protease nature of the enzyme. These properties make the novel protease a suitable candidate enzyme in meat ripening and other biotechnological applications.     

Herinase: A Novel Bi-functional Fibrinolytic Protease from the Monkey Head Mushroom, Hericium erinaceum

Herinase, a new bi-functional fibrinolytic metalloprotease, was purified from a medicinal and edible mushroom Hericium erinaceum. The enzyme was monomeric with a molecular mass of 51 kDa. Analysis of fibrin zymography showed an active band with a similar molecular mass. The N-terminal sequence of herinase VPSSFRTTITDAQLRG was highly distinguished from known fibrinolytic enzymes. Moreover, the enzyme activity was strongly inhibited by EDTA and EGTA, indicating that herinase is a metalloprotease. Herinase exhibited high specificity for the substrate t-PA followed by plasmin. The K _m and V _max values for H-D-Ile-Pro-Arg-PNA were found to be 4.7 mg and 26.7 U/ml respectively. Similarly, fibrin plate assays revealed that it was able to degrade fibrin clot directly and also able to activate plasminogen. Herinase provoked a rapid degradation of fibrin and fibrinogen α chains and slower degradation of γ chains. It had no activity on the β chains of fibrin and fibrinogen. This result suggests that herinase could possibly contain higher amount of α-fibrinogenase. The activity of herinase was stimulated by metal ions such as Ca²⁺, Mg²⁺, and Mn²⁺, but inhibited by Cu²⁺, Fe²⁺, and Zn²⁺. Herinase exhibited maximum activity at 30 °C and pH 7.0. These results demonstrate that herinase could be a novel fibrinolytic enzyme.     

新型水稻黄单胞菌Harpin蛋白的纯化及其特性研究

报道水稻黄单胞(Xanthomonas oryzae)的两个致病变种(pv.oryzae和 pv.oryzicola)所产生的Harpin蛋白. 结果表明， 表达菌株HRF1和HRF2分别携带两个致病变种编码Harpin的hrf1和hrf2基因. 在IPTG诱导下, 两个表达菌株的无细胞破碎液均具有激发烟草叶片过敏反应(HR)的活性. 采用(NH4)2SO4沉淀、 阴离子交换层析、 Native-PAGE微量制备等方法, 分别纯化出分子量为15 600和15 300, pI皆为4.5左右的单一条带; 这两个单一组分符合典型Harpin蛋白的特征: 可激发烟草HR， 诱导烟草抗TMV， 对蛋白酶K敏感、 对热稳定; 放线菌素D、 环己酰亚胺和氯化镧等真核生物代谢抑制剂可消除它们的生物活性; 琼脂双扩散试验(ODD)血清反应表明， 两个Harpin蛋白有交叉反应.