Purification and characterization of a serine protease from Cucumis trigonus Roxburghi

Kachri fruit, Cucumis trigonus Roxburghi, contains high protease activity and has been used as meat tenderizer in the Indian subcontinent. A 67 kDa serine protease from Kachri fruit was purified by DEAE-Sepharose and CM-Sepharose chromatography, whose optimum activity was at pH 11 and 70 degrees C. Its activity was strongly inhibited by PMSF, but not by EDTA, pepstatin, or cysteine protease inhibitors. The substrate specificity of the purified protease towards synthetic peptides was comparable to cucumisin, the first characterized subtilisin class plant protease from the sarcocarp of melon fruit (Cucumis melo). These characteristics, along with the N-terminal amino acid sequence, indicated that the isolated protease from Cucumis trigonus Roxburghi is a cucumisin homologue, which belongs to the serine protease family.     

Purification and characterization of a keratinolytic serine protease from Purpureocillium lilacinum LPS # 876

A keratinolytic serine protease secreted by Purpureocillium lilacinum (formerly Paecilomyces lilacinus) upon culture in a basal medium containing 1% (w/v) hair waste as carbon and nitrogen source was purified and characterized. After purification the keratinase was resolved by SDS-PAGE as a homogeneus protein band of molecular mass 37.0 kDa. The extracellular keratinase of P. lilacinum was characterized by its appreciable stability over a broad pH range (from 4.0 to 9.0), and up to 65 °C, along with its strong inhibition by phenylmethylsulphonyl fluoride among the protease inhibitors tested (98.2% of inhibition), thus suggesting its nature as a serine protease. The enzyme was active and stable in the presence of organic solvents such as dimethylsulfoxide, methanol, and isopropanol; certain surfactants such as Triton X-100, sodium dodecylsulfate, and Tween 85; and bleaching agents such as hydrogen peroxide. These biochemical characteristics suggest the potential use of this enzyme in numerous industrial applications.     

Purification and characterization of a fibrinolytic subtilisin-like protease of Bacillus subtilis TP-6 from an Indonesian fermented soybean, Tempeh

We have isolated a bacterium (TP-6) from the Indonesian fermented soybean, Tempeh, which produces a strong fibrinolytic protease and was identified as Bacillus subtilis. The protease (TPase) was purified to homogeneity by ammonium sulfate fractionation and octyl sepharose and SP sepharose chromatography. The N-terminal amino acid sequence of the 27.5 kDa enzyme was determined, and the encoding gene was cloned and sequenced. The result demonstrates that TPase is a serine protease of the subtilisin family consisting of 275 amino acid residues in its mature form. Its apparent K _m and V _max for the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-pNA were 259 μM and 145 μmol mg⁻¹ min⁻¹, respectively. The fibrinogen degradation pattern generated by TPase as a function of time was similar to that obtained with plasmin. In addition, N-terminal amino acid sequence analysis of the fibrinogen degradation products demonstrated that TPase cleaves Glu (or Asp) near hydrophobic acids as a P1 site in the α- and β-chains of fibrinogen to generate fragments D′, E′, and D′ similar to those generated by plasmin. On plasminogen-rich fibrin plates, TPase did not seem to activate fibrin clot lysis. Moreover, the enzyme converted the active plasminogen activator inhibitor-1 to the latent form.Seong-Bo Kim and Dong-Woo Lee contributed equally to the work.     

Purification and characterization of a thermodynamic stable serine protease from Aspergillus fumigatus

A thermostable extracellular serine protease from Aspergillus fumigatus was purified 8.8-fold using a 4-step protocol. The enzyme was produced using a 36 h solid-state culture, had a molecular weight of 88 kDa and exhibited maximal enzyme activity at pH 7 and 60 °C. Structural analysis revealed that the protease is monomeric and non-glycosylated. Thermal inactivation of the pure enzyme followed first-order kinetics. The half-life (t_1/2) of the pure enzyme at 50, 60 and 70 °C was 65, 34 and 14 min, respectively. The denaturation and activation energies were 69 and 62 kJ mol⁻¹, respectively. Thermodynamic parameters (entropy and enthalpy) suggested that the protease was highly thermostable. This is the first report on the thermodynamic parameters of proteases produced by A. fumigatus.     

Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi

Bacillus subtilis DC33 producing a novel fibrinolytic enzyme was isolated from Ba-bao Douchi, a traditional soybean-fermented food in China. The strong fibrin-specific enzyme subtilisin FS33 was purified to electrophoretic homogeneity using the combination of various chromatographic steps. The optimum temperature, pH value, and pI of subtilisin FS33 were 55°C, 8.0, and 8.7, respectively. The molecular weight was 30 kDa measured by SDS–PAGE under both reducing and non-reducing conditions. The enzyme showed a level of fibrinolytic activity that was about six times higher than that of subtilisin Carlsberg. The first 15 amino acid residues of N-terminal sequence of the enzyme were A-Q-S-V-P-Y-G-I-P-Q-I-K-A-P-A, which are different from that of other known fibrinolytic enzymes. The amidolytic activities of subtilisin FS33 were inhibited completely by 5 mM phenylmethanesulfonyl fluoride (PMSF) and 1 mM soybean trypsin inhibitor (SBTI), but 1,4-dithiothreitol (DTT), β-mercaptoethanol, and p-hydroxymercuribenzoate (PHMB) did not affect the enzyme activity; serine and tryptophan are thus essential in the active site of the enzyme. The highest affinity of subtilisin FS33 was towards N-Succ-Ala-Ala-Pro-Phe-pNA. Therefore, the enzyme was considered to be a subtilisin-like serine protease. The fibrinolytic enzyme had a high degrading activity for the Bβ-chains and Aα-chain of fibrin(ogen), and also acted on thrombotic and fibrinolytic factors of blood, such as plasminogen, urokinase, thrombin, and kallikrein. So subtilisin FS33 was able to degrade fibrin clots in two ways, i.e., (a) by forming active plasmin from plasminogen and (b) by direct fibrinolysis.     

Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42

An extracellular serine alkaline protease of Bacillus clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3 days at pH 10.5 and 37°C. Highest alkaline protease activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate by DEAE-cellulose chromatography followed by (NH₄)₂SO₄ precipitation, with a yield of 58%. SDS-PAGE analysis revealed the molecular weight of the enzyme to be 26.50 kDa. The optimum temperature for enzyme activity was 60°C; however, it is shifted to 70°C after addition of 5 mM Ca²⁺ ions. The enzyme was stable between 30 and 40°C for 2 h at pH 10.5; only 14% activity loss was observed at 50°C. The optimal pH of the enzyme was 11.3. The enzyme was also stable in the pH 9.0–12.2 range for 24 h at 30°C; however, activity losses of 38% and 76% were observed at pH values of 12.7 and 13.0, respectively. The activation energy of Hammarsten casein hydrolysis by the purified enzyme was 10.59 kcal mol⁻¹ (44.30 kJ mol⁻¹). The enzyme was stable in the presence of the 1% (w/v) Tween-20, Tween-40,Tween-60, Tween-80, and 0.2% (w/v) SDS for 1 h at 30°C and pH 10.5. Only 10% activity loss was observed with 1% sodium perborate under the same conditions. The enzyme was not inhibited by iodoacetate, ethylacetimidate, phenylglyoxal, iodoacetimidate, n-ethylmaleimidate, n-bromosuccinimide, diethylpyrocarbonate or n-ethyl-5-phenyl-iso-xazolium-3′-sulfonate. Its complete inhibition by phenylmethanesulfonylfluoride and relatively high k _cat value for N-Suc-Ala-Ala-Pro-Phe-pNA hydrolysis indicates that the enzyme is a chymotrypsin-like serine protease. K _m and k _cat values were estimated at 0.655 μM N-Suc-Ala-Ala-Pro-Phe-pNA and 4.21×10³ min⁻¹, respectively.     

Purification and characterization of a salinity induced alkaline protease from isolated spinach chloroplasts

In cynobacteria and higher plants, salinity is known to inhibit the activity of several enzymes involved in photosynthesis and hence decreases the overall photosynthetic rate. This gave us an impetus to search for a protease, which may be involved in the turnover of non-functional enzymes produced under salinity stress. Taking the possible changes in pH gradient of the chloroplast under consideration, we have tried to identify a protease, which is induced under salinity and characterized it as an alkaline protease using spinach (Spinacia oleracea) leaves as a model system. The HIC-HPLC purified homogeneous alkaline serine protease from the isolated spinach chloroplasts had two subunits of molecular weight 63 and 32 kDa. The enzyme was maximally active at pH 8.5 and 50°C. The enzyme showed the property to hydrolyze the synthetic substrate like azocaesin and had sufficient proteolytic activity in gelatin bound native PAGE. The enzyme activity was also dependent upon the presence of divalent cations and reduced environment. The active site residues were identified and the homogeneous alkaline serine protease had cysteine, lysine and tryptophan residues at its active site.     

米曲霉耐热木聚糖酶纯化及酶学特性研究

对米曲霉原始发酵液中耐热木聚糖酶进行纯化和酶学特性研究,利用甘蔗渣为碳源培养米曲霉,通过超滤和阴离子交换柱两步纯化得到木聚糖酶XynH1,分子量35．402kDa,利用飞行时间质谱和SDS—PAGE分析,推断XynH1为XylanaseXynF1,分子量为35．402kDa。XynH1属于糖苷水解酶家族10,酶活为442．2IU／nag,最适pH和温度分别为pH6．0和65℃,80℃以下及pH4．0～10．5范围内较稳定。     

米曲霉酸性蛋白酶基因在毕赤酵母中的异源表达及酶学性质

酸性蛋白酶作为一类重要的天冬氨酸蛋白酶,被广泛应用于食品、医药和皮革等领域。为推动酸性蛋白酶的研究及应用,通过对发酵豆制品样品进行宏基因组测序,从中获得米曲霉酸性蛋白酶基因pepA,在毕赤酵母GS115中进行异源表达,并对重组酶PepA进行酶学性质分析。结果显示毕赤酵母发酵上清液中酸性蛋白酶的活性为50.62 U/mL。SDS-PAGE验证PepA的分子量约为50 kDa,且发酵上清液几乎无杂蛋白。PepA的最适pH值为4.5,最适温度为50℃,Mn~(2+)和Cu~(2+)对其具有激活作用,而Fe~(3+)、Fe~(2+)与Ca~(2+)则具有抑制作用。上述研究结果可为米曲霉酸性蛋白酶的异源表达及其相关工业应用提供指导。     

Purification and biochemical characterization of an extracellular serine peptidase from Aspergillus terreus

Peptidases are important because they play a central role in pharmaceutical, food, environmental, and other industrial processes. A serine peptidase from Aspergillus terreus was isolated after two chromatography steps that showed a yield of 15.5%. Its molecular mass was determined to be 43 kD, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This peptidase was active between pH 5.0 to 8.0 and had maximum activity at pH 7.0, at 45°C. When exposited with 1 M of urea, the enzyme maintained 100% activity and used azocasein as substrate. The N-terminal (first 15 residues) showed 33% identity with the serine peptidase of Aspergillus clavatus ES1. The kinetics assays showed that subsite S₂ did not bind polar basic amino acids (His and Arg) nonpolar acidic amino acids (Asp and Glu). The subsite S₁ showed higher catalytic efficiency than the S₂ and S₃ subsites.     

Purification and characterization of a novel fibrinolytic protease from <Emphasis Type="Italic">Fusarium</Emphasis> sp. CPCC 480097

A novel fibrinolytic enzyme from Fusarium sp. CPCC 480097, named Fu-P, was purified to electrophoretic homogeneity using ammonium sulfate precipitation and ion exchange and gel filtration chromatography. Fu-P, a single protein had a molecular weight of 28 kDa, which was determined by SDS-PAGE and gel filtration chromatography. The isoelectric point of Fu-P determined by isoelectric focusing electrophoresis (IEF) was 8.1, and the optimum temperature and pH value were 45°C and 8.5, respectively. Fu-P cleaved the α-chain of fibrin (ogen) with high efficiency, and the β-chain and γ-γ (γ-)-chain with lower efficiency. Fu-P activity was inhibited by EDTA and PMSF, and the enzyme exhibited a high specificity for the chymotrypsin substrate S-2586. Fu-P was therefore identified as a chymotrypsin-like serine metalloprotease. The first 15 amino acids of the N-terminal sequence of Fu-P were Q-A-S–S-G-T-P-A-T-I-R-V-L-V–V and showed no homology with that of other known fibrinolytic enzymes. This protease may have potential applications in thrombolytic therapy and in thrombosis prevention.     

Cloning and characterization of an extracellular temperature-labile serine protease gene from Aeromonas hydrophila

Aeromonas virulence is thought to depend on multigenic functions. The gene for an extracellular protease from Aeromonas hydrophila SO2/2 was cloned in Escherichia coli C600-1 by using pIJ860, bifunctional plasmid, as a vector. The gene encodes for a temperature-labile serine protease (P2) with a molecular mass of approx. 68 kDa which is highly inhibited by PMSF. The gene was expressed in Streptomyces lividans 1326 by transforming protoplasts with the original clone pPA2. We were also able to transfer and express the prt P2 gene in Pseudomonas putida by mating experiments. The protein P2 was secreted into the periplasms of both P. putida and E. coli C600-1 being identical in properties to one of the proteases secreted into the culture supernatant by A. hydrophila SO2/2.     

Purification and characterization of a biodegradable plastic-degrading enzyme from Aspergillus oryzae

We used biodegradable plastics as fermentation substrates for the filamentous fungus Aspergillus oryzae. This fungus could grow under culture conditions that contained emulsified poly-(butylene succinate) (PBS) and emulsified poly-(butylene succinate-co-adipate) (PBSA) as the sole carbon source, and could digest PBS and PBSA, as indicated by clearing of the culture supernatant. We purified the PBS-degrading enzyme from the culture supernatant, and its molecular mass was determined as 21.6 kDa. The enzyme was identified as cutinase based on internal amino acid sequences. Specific activities against PBS, PBSA and poly-(lactic acid) (PLA) were determined as 0.42 U/mg, 11 U/mg and 0.067 U/mg, respectively. To obtain a better understanding of how the enzyme recognizes and hydrolyzes PBS/PBSA, we investigated the environment of the catalytic pocket, which is divided into carboxylic acid and alcohol recognition sites. The affinities for different substrates depended on the carbon chain length of the carboxylic acid in the substrate. Competitive inhibition modes were exhibited by carboxylic acids and alcohols that consisted of C₄-C₆ and C₃-C₈ chain lengths, respectively. Determination of the affinities for different chemicals indicated that the most preferred substrate for the enzyme would consist of butyric acid and n-hexanol.This revised version was published online in February 2005 with corrections to Table 1.     

Purification and characterization of a prolyl endopeptidase isolated from Aspergillus oryzae

A new fungal strain that was isolated from our library was identified as an Aspergillus oryzae and noted to produce a novel proly endopeptidase. The enzyme was isolated, purified, and characterized. The molecular mass of the prolyl endopeptidase was estimated to be 60 kDa by using SDS-PAGE. Further biochemical characterization assays revealed that the enzyme attained optimal activity at pH 4.0 with acid pH stability from 3.0 to 5.0. Its optimum temperature was 30 °C and residual activity after 30 min incubation at 55 °C was higher than 80 %. The enzyme was activated and stabilized by Ca²⁺ but inhibited by EDTA (10 mM) and Cu²⁺. The K _m and k _cat values of the purified enzyme for different length substrates were also evaluated, and the results imply that the enzyme from A. oryzae possesses higher affinity for the larger substrates. Furthermore, this paper demonstrates for the first time that a prolyl endopeptidase purified from A. oryzae is able to hydrolyze intact casein.     

Purification and characterization of protease from a newly isolated Rhizopus oryzae

A newly isolated Rhizopus oryzae was found to exhibit some unusual phenomenon of secreting alkaline protease which was purified and characterized. The molecular weight was determined to be 28,600 dalton in gel electrophoresis. The enzyme is stable in the pH range from 3 to 11 and most active at pH 8. The temperature optimum of this thermostable biocatalyst is at 60 °C. The enzyme is sensitive to metal chelators, most of the metal ions (excepting a few monovalent cations) and inhibitor like PMSF. This indicates that the protease of isolated Rhizopus oryzae falls under alkaline serine group.     

Enzymatic properties and identification of a fibrinolytic serine protease purified from Bacillus subtilis DC33

A novel fibrinolytic enzyme subtilisin FS33 was purified from Bacillus subtilis DC33, isolated from a traditional flavour-rich food in China. The purified subtilisin FS33 was a single chain protein with a molecular mass of 30 kDa measured by SDS-PAGE. After activated SDS-PAGE, the enzyme band exhibited strong fibrinolytic activity on the fibrin plate. Subtilisin FS33 was temperature-stable below 60°C over the pH range 5–12, with a maximum activity at pH 8.0, but the activity completely disappeared after 10 min above 65°C. The NH₂-terminal amino acid sequence of the enzyme was different from that of other known fibrinolytic enzymes, such as NK, CK, SMCE, KA38, subtilisin E, subtilisin DFE and Katsuwokinase. The amidolytic activities of subtilisin FS33 were inhibited completely by phenylmethanesulfonyl fluoride (PMSF) and soybean trypsin inhibitor (SBTI). EDTA did not affect the enzyme activity, and none of the ions tested activated the activity. Therefore, the enzyme was thought to be a subtilisin-like serine protease. The enzyme degraded the Bβ-chains of fibrin(ogen) very rapidly and then degraded the Aα-chain and at least five fragments from fibrin(ogen) were obtained after hydrolysis. Subtilisin FS33 was also able to cleave blood clots in the absence of endogenous fibrinolytic factors.     

Purification and characterization of hevain,a serine protease from Hevea brasiliensis

A serine protease of MW 69000 has been isolated, in homogeneous form, from the latex of Hevea brasiliensis. The enzyme, named hevain, has only limited esterolytic and proteolytic abilities, a maximum activity in the pH range 6.5–7.5, and a pI of 4.3. Hevain has a notably high content of acidic amino acids, while the aromatic residues are present in relatively minor amounts.     

Purification and characterization of the two molecular forms of membrane acid protease from Aspergillus oryzae.

Two forms (M1 and M2) of the membrane-bound acid protease of Aspergillus oryzae have been purified by extraction with Triton X-100, washing with cold acetone, and repeated gel filtration on Bio-Gel A-15 m in the presence and absence of Triton X-100. The purified membrane enzymes, M1 and M2, moved as a single band in acrylamide gel electrophoresis and had apparent molecular weights of 150 000 and 60 000, respectively, as estimated by sodium dodecyl sulfate/acrylamide gel electrophoresis. These two membrane enzymes activated bovine pancreatic trypsinogen and had the same pH optima in the acid pH range. They immunologically cross-reacted with each other and with an extracellular acid protease from A. oryzae, and contained carbohydrate, ranging from 52.5 to 80.5% and comprising three hexoses, glucose, galactose, and mannose. While these catalytic, chemical and immunological properties are similar to those of the extracellular acid protease from A. oryzae, both membrane enzyme differed in their hydrophobic properties from external enzymes. Thus they are activated by the detergent Triton X-100 and some polar lipids.     

Purification and characterization of a milk clotting protease from Rhizomucor miehei

Benzamidine, an inhibitor of serine proteases, was used as an affinity ligand for the purification of aspartyl protease from culture filtrate of Rhizomucor miehei. The two step purification protocol (ion-exchange and affinity chromatography) resulted in a homogenous enzyme preparation with seven-fold purification and a final recovery of 22%. The purified enzyme was free of brown pigmentation, a factor inherently associated with the enzyme; it was stable and active at acidic pH (optimum pH 4.1 for proteolytic activity and 5.6 for milk clotting activity). The significant positive characteristic of the enzyme is its comparatively lower thermostability; the enzyme was comparable to calf rennet in its properties of thermostability, milk-clotting to proteolytic activity ratio and sensitivity to CaCl2. Limited protease digestion of the purified enzyme with proteinase K yielded a 20kDa fragment as shown by SDS–PAGE. Native gel electrophoresis of the digest showed an additional peak of activity corresponding to the 20kDa fragment on SDS–PAGE, this fragment retained both milk-clotting and proteolytic activities. It was also inhibited by pepstatin A and hence it is presumed that this fragment contained the active site of the enzyme.     

Purification and characterization of the two molecular forms of Aspergillus oryzae acid protease.

The isolation and partial characterization of the acid proteases A1 and A2 (EC3.4.23.6) from Aspergillus oryzae grown on solid bran culture are described. The purified preparations were essentially homogeneous by several criteria including sedimentation analysis and polyacrylamide gel electrophoresis. The physiochemical properties of the proteases A1 and A2 were as follows (in the order: A1, A2): molecular weight: 63 000 & 32 000; sedimentation coefficient s20, w: 3.93 and 3.16 S; diffusion constant D20, w, 5.63 - 10(-7) and 8.61 - 10(-7) CM2/S, partial specific volume, v: 0.73 ml/g for both; nitrogen content: 16.30 and 13.42%; E1% 1 cm at 280 nm: 5.9 and 11.1. The two enzymes had the same pH optima in the acid pH range, and both activated bovine pancreatic trypsinogen. The enzymes were essentially of the same amino acid composition and immunologically cross-reacted with each other. The protease A2 contained little or no carbohydrate, whereas the protease A1 was glycoprotein, containing 49% carbohydrate comprising glucose, mannose, and galactose. These results suggest that the protein portion of acid protease A1 is the same as that of acid protease A2.