Proteases of Senescing Oat Leaves: II. Reaction to Substrates and Inhibitors

Two proteases isolated from senescent oat (Avena sativa) leaves have been subjected to further study. One of these, an acid protease active at pH 4.2, is inhibited by phenylmethylsulfonyl fluoride (PMSF) but not by iodoacetamide (IAc). The other, active at pH 6.6, is inhibited by both PMSF and IAc. These results, together with previously reported evidence that mercaptoethanol stimulates the activity of only the neutral protease, are taken to indicate that the acid protease is probably of the serine type, whereas the neutral enzyme is of the sulfhydryl type. Both enzymes are inhibited by irradiation in the presence of rose bengal, a selective histidine modification reagent. The acid protease was completely unaffected by chelators, but data on the neutral protease were equivocal.

All protein substrates tested were attacked by both enzymes, though at strikingly different rates. Characterization of the digestion products, with denatured hemoglobin as substrate, indicated that the acidic enzyme is an endoprotease, while the neutral one is an exoprotease. Evidence is presented that these proteases undergo autolysis in vitro.

     

Proteolysis of peripheral nerve myelin in acute experimental allergic neuritis

Proteolysis of peripheral nerve myelin was studied in rats with experimental allergic neuritis (EAN). In vitro measurements using rat sciatic nerve homogenate and denatured bovine myelin as a substrate showed two myelin specific enzyme activities at pH 3.8 (inhibited by pepstatin) and pH 5.8 (inhibited by PMSF) in the normal rat and newly appearing activities at pH 2.8 (inhibited by pepstatin) and pH 5.0 (not characterized) in the EAN rat. In EAN the proteolytic activity was not restricted to myelin substrate but degraded total sciatic nerve protein as well. Endogenous sciatic nerve protease at pH 5.8 did not significantly change in activity during the course of disease. On the contrary, activity of acid protease at pH 2.8 corresponded well to the disease. Myelin degradation in EAN, therefore, appears to be mainly due to exogenous non-tissue protease.Abbreviations EAN experimental allergic neuritis - EDTA ethylenediaminetetraacetic acid - HBM hydroxymercuro benzoate - PLP proteolipid protein - PMSF phenylmethylsulfonyl fluoride - PNS peripheral nervous system - SDS sodium dodecylsulfate - TCA trichloroacetic acid This work is part of the M.D. thesis of R. B.     

Detection and partial characterization of lymphoid cell surface proteases

Cultures of viable thymocytes and lymph node cells (LNC) were found to exhibit neutral protease activity toward radiolabeled protein substrates. Proteases were not actively secreted in serum-free culture. Thymocyte surface proteases were not affected by incubation of the cells in 1 mM ethylenediaminetetraacetic acid (EDTA) or 1 mM ethylene glycol bis(aminoethyl ether) N, N'-tetraacetic acid (EGTA); however, approximately 25% of lymph node cell surface protease activity was released from the cells by EDTA. It was concluded that the majority of protease activity displayed by both cell types was tightly associated with the cell surface. The inhibitor sensitivity of the cell surface proteases detected on hamster thymocytes and LNC and rat thymocytes was very similar. Cell surface protease activity was inhibited (85%) by the serine protease inhibitors diisopropylfluorophosphate (DFP) and phenylmethylsulfonylfluoride (PMSF) and was partially inhibited by l-1-tosylamide-2-phenylethylchloromethyl ketone(TPCK) and soybean trypsin inhibitor (SBTI), but not by N-α-p-tosyl-l-lysine-chloromethyl ketone (TLCK) or ?-aminocaproic acid (EACA). The bacterial protease inhibitor antipain was strongly inhibitory whereas leupeptin was less effective and elastinal did not inhibit cell surface protease activity. Thymocyte surface proteases were also inhibited (65%) by ZnCl₂, but not be several other divalent cations. In LNC, both ZnCl₂ and NiCl₂ were inhibitory to a lesser extent (32% inhibition). At least one surface protease in both thymocytes and LNC could function as a plasminogen activator.     

球形芽孢杆菌C_3—41菌株胞外蛋白酶特性

球形芽孢杆菌能够合成具杀蚊活性的蛋白晶体,该晶体在蚊中肠碱性条件下降解产生毒性,尽管球形芽孢杆菌蛋白酶与杀蚊毒素的降解无关,但它在球形芽孢杆菌杀蚊制剂的产生中有重要意义。同时球形芽孢杆菌产生的碱性蛋白酶具有潜在的医疗价值。 我们以本实验室分离的高效杀蚊菌C_3—41菌株为材料,研究了球形芽孢杆菌蛋白酶的产生特性及其理化性质,在国内尚属首次报道。     

Angiotensin I [Phe8-His9] hydrolase and bradykininase from human lung.

Atypical angiotensin I “converting enzyme” (angiotensin I [Phe⁸-His⁹] hydrolase or APHH) was purified from human lung tissue. Two enzyme preparations from different lungs were found to fragment and inactive bradykinin. Fragmentation was demonstrated by electrophoretic techniques and biological inactivation was demonstrated by bioassay. Bradykininase activity was inhibited by low concentrations of 2,3-dimercaptol-propanol (BAL) and ethylenediaminetetraacetic acid (EDTA), but not by phenylmethylsulfonyl fluoride (PMSF) or p-chloromercuriphenyl sulfonic acid (CMPSA). Conversion of angiotensin I was inhibited by BAL, PMSF, and CMPSA but not by EDTA. APHH from a third lung preparation was free of significant bradykininase activity as determined by bioassay. It is concluded that these two enzymatic activities are probably associated with separate enzymes.     

Digestive enzyme patterns and evaluation of protease classes in Catla catla (family: Cyprinidae) during early developmental stages

Digestive enzymes of Catla catla were studied during ontogenic development. Specific amylase activity was 0.12+/-0.01 mg maltose mg protein(-1) h(-1) in fish 4 days after hatching (DAH) and reached a maximum on (0.41+/-0.12 mg maltose mg protein(-1) h(-1)) 34 DAH. Total protease activity was minimum (123.2+/-16.5 mU mg protein(-1) min(-1)) on day-8 and reached its highest level (2713+/-147.2 mU mg protein(-1) min(-1)) on day-32. Trypsin activity showed constant increasing trend from day-16 onwards and was maximum on day-34 (118.1+/-7.09 mU mg protein(-1) min(-1)). Highest chymotrypsin activity was found on day-32 (1789.0+/-111.7 mU mg protein(-1) min(-1)). Lipase activity was detected in 4 DAH catla. Lipase activity increased steadily from day-22 onwards. SDS-PAGE of crude enzyme extracts showed that high molecular mass bands (41.8-127.8 kDa) appeared during the early stages followed by low molecular mass bands (17.8-37.2 kDa). The number of protease activity bands in substrate SDS-PAGE increased with age of fish. During ontogenesis of carp, soybean trypsin inhibitor (SBTI), PMSF and TLCK inhibited 75.5+/-1.19% to 92.8+/-0.85%, 53.3+/-9.47% to 90.5+/-2.6% and 39.8+/-3.8% to 84.7+/-1.54% of total protease activity, respectively. There was only 2.58+/-0.66% to 10.21+/-0.09% inhibition of protease activity with EDTA. SBTI and PMSF inhibited 8 and 4 activity bands, respectively. TLCK, a specific trypsin inhibitor, inhibited four trypsin-like enzymes in carp during ontogenesis.     

Biochemical and functional properties of serine esterases in acidic cytoplasmic granules of cytotoxic T lymphocytes

Percoll gradient fractions of homogenates of murine cloned cytotoxic T lymphocytes (CTL) were analyzed for the trypsin-like enzyme alpha-N-benzyloxy-carbonyl-L-lysinethiobenzyl ester (BLT) esterase recently described in CTL homogenates. Enzymatic activity was found in three areas of the gradient: the dense cytolysin containing granules; a light granule fraction; and a variable amount in the soluble fraction at the top of the gradient. Gel filtration columns showed a major peak of BLT esterase activity eluted at the position of a 60-kDa protein, and an additional, minor BLT esterase peak eluting at about 27 kDa. The separated enzymes were both significantly inhibited by the serine protease inhibitors diisopropylfluorophosphate and phenylmethyl sulfonyl fluoride (PMSF), indicating they are both serine proteases, but showed different patterns of inhibition by a series of inhibitors, suggesting the larger enzyme is not a simple dimer of the smaller. pH activity profiles of both CTL BLT esterases showed an optimum at about pH 8. PMSF inactivation of BLT esterase in detergent extracts of CTL diminished sharply as the pH was dropped below 7. Agents which raise the pH of acidic intracellular compartments were found to markedly enhance the PMSF inactivation of BLT esterase in intact CTL, showing that the granules have a low internal pH. Similarly, [3H]diisopropylfluorophosphate labeling of intact CTL gave four protein bands on non-reduced gels, of which two were labeled threefold more effectively in the presence of chloroquine. In parallel studies of inactivation of CTL lytic activity, PMSF pretreatment caused a 50% reduction of the lytic activity under conditions where greater than 90% of the BLT esterase activity was inactivated. Addition of agents raising the intragranular pH dramatically enhanced the BLT esterase inactivation but did not concomitantly reduce CTL lytic activity. These results indicate that inactivation of lytic function by PMSF is unlikely to be due to its reaction with protease in acidic granules, and suggest that the activity of these enzymes may not be required for cytotoxicity.     

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.     

Matrix metalloproteinases and their tissue inhibitors in the developing neonatal mouse uterus

Postnatal development of the mouse uterus involves differentiation and development of the endometrial glands as well as the myometrium. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in extracellular matrix breakdown and morphogenesis of many epitheliomesenchymal organs. As a first step to understanding their roles in postnatal mouse uterine development, MMPs and TIMPs found to be expressed in the neonatal mouse uterus by microarray analysis were localized by in situ hybridization. The MMP-2 mRNA was detected only in the uterine stroma, whereas the MMP-10 mRNA was present only in the uterine epithelium from Postnatal Day (PND) 3 to PND 9. All other MMPs (MMP-11, MMP-14, and MMP-23) as well as TIMP-1, TIMP-2, and TIMP-3 were detected in both epithelial and stromal cells of the endometrium, but not in the myometrium. Uterine extracts were then analyzed by gelatin and casein gel zymography to detect active gelatinases and stromelysins, respectively. Five major gelatinase bands of activity were detected and inhibited by the MMP inhibitors, EDTA or 1,10-phenanthroline, but not by PMSF, a serine protease inhibitor. Western blot analysis confirmed the presence of MMP-2 and MMP-9 proteins in the uterus. Immunoreactive MMP-9 protein was detected only in the endometrial stroma, whereas immunoreactive MMP-2 protein was detected in both the stroma and epithelium of the uterus. Casein zymography detected three major bands of activity ( approximately 54, 63, and 80 kDa) that were inhibited by the serine protease inhibitor, PMSF, but not by the MMP inhibitors, EDTA or 1,10-phenanthroline, suggesting that they were serine proteases. These results support the hypothesis that MMPs and TIMPs regulate postnatal development of the mouse uterus.     

Aspartic proteinase in Dugesia tigrina (Girard) planaria

A proteolytic activity was identified in Dugesia tigrina planaria using the chromogenic substrate Phe-Ala-Ala-Phe (4-NO2)-Phe-Val-Leu-O4MP. The activity of the enzyme increased four times during the regeneration and presented a maximum at 120 hr being higher in tail than head regenerating segments. The protease that displays this activity was purified from worms by a single step on pepstatin-agarose followed by gel-filtration high performance liquid chromatography. The purification resulted in a 34-fold increase in specific activity and the final yield was 10%. The active D. tigrina hydrolase appears to be a dimeric protein composed of identical subunits with 34 kDa associated by disulphide bridges similar to vertebrate cathepsin D. By SDS-PAGE several bands were detected but upon gel filtration HPLC one proteolytically active component, termed Asp-68, was detected and isolated. The maximal activity was observed in a range between pH 3.5-5.0 and the enzyme became inactivated at a pH value above 7.2. The purified enzyme was not inhibited by inhibitors from serine (aprotinin, TPCK, PMSF and TLCK), metallo (EDTA) and cysteine proteinase (E-64) classes. In contrast, inhibitors such as pepstatin, EPNP, and 4-beta-PMA efficiently inhibited the activity of the 68-kDa protease.     

Purification and characterization of two thermostable proteases from the thermophilic fungus Chaetomium thermophilum

Thermostable protease is very effective to improve the industrial processes in many fields. Two thermostable extracellular proteases from the culture supernatant of the thermophilic fungus Chaetomium thermophilum were purified to homogeneity by fractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sepharose, and PhenylSepharose hydrophobic interaction chromatography. By SDS-PAGE, the molecular mass of the two purified enzymes was estimated to be 33 kDa and 63 kDa, respectively. The two proteases were found to be inhibited by PMSF, but not by iodoacetamide and EDTA. The 33 kDa protease (PRO33) exhibited maximal activity at pH 10.0 and the 63 kDa protease (PRO63) at pH 5.0. The optimum temperature for the two proteases was 65 degrees C. The PRO33 had a K(m) value of 6.6 mM and a V(max) value of 10.31 micromol/l/min, and PRO63 17.6 mM and 9.08 micromol/l/min, with casein as substrate. They were thermostable at 60 degrees C. The protease activity of PRO33 and PRO63 remained at 67.2% and 17.31%, respectively, after incubation at 70 degrees C for 1 h. The thermal stability of the two enzymes was significantly enhanced by Ca2+. The residual activity of PRO33 and PRO63 at 70 degrees C after 60 min was approximately 88.59% and 39.2%, respectively, when kept in the buffer containing Ca2+. These properties make them applicable for many biotechnological purposes.     

Purification and partial characterization of a toxic serine protease produced by pathogenic Vibrio alginolyticus

An extracellular lethal toxin produced by Vibrio alginolyticus strain Swy originally isolated from diseased kuruma prawn (Penaeus japonicus) was purified using the AKTA purifier system with hydrophobic interaction chromatography, anion exchange and gel filtration columns. The toxin is an alkaline serine protease, inhibited by phenyl methylsulphonyl fluoride (PMSF), antipain and shows maximal activity at pH 8 to 11, having a pI of 4.3 and a molecular weight of approximately 33 kD. The toxin was completely inhibited by FeCl2 but partially inhibited by 3,4-dichloroisocoumarin (3,4-DCI), ethylenediamine tetraacetic acid (EDTA), ethylene glycol-bis(beta-amino-ethyl ether) N,N,N',N'-tetraacetic acid (EGTA), CuCl2 and ZnCl2. The purified protease was lethal for kuruma prawn at an LD50 of 0.29 microgram protein/g body weight. The haemolymph withdrawn from the moribund prawns injected with the toxic protease was unable to clot. The coagulogen in the kuruma prawn plasma showed an increased migration rate after incubation with this serine protease, and a plasma colour change from blue to pink was recorded. The addition of PMSF completely inhibited the lethal toxicity of the purified protease, indicating that this serine protease was a lethal toxin produced by the bacterium. The 33 kD protease was therefore a toxic protease produced by V. alginolyticus strain Swy.     

Characterization of a metalloenzyme from a wild mushroom, Tricholoma saponaceum

Two kinds of metalloendopeptidases from the fruiting bodies of Tricholoma saponaceum (TSMEP1 and TSMEP2) have been purified, and TSMEP1 has been characterized based on their fibrinolytic activity. The enzymes have the same N-terminal amino acid sequence, Ala-Leu-Tyr-Val-Gly-X-Ser-Pro-X-Gln-Gln-Ser-Leu-Leu-Val, but slightly different molecular weights of 18,147 and 17,947, as measured by matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. The N-terminal sequence do not match with any known protein or open reading frame. TSMEP1 hydrolyzes fibrinogen as well as fibrin, but does not show any proteolytic activity for other blood proteins such as thrombin, human albumin, human IgG, hemoglobin, or urokinase. The enzyme hydrolyzes both A alpha and B beta subunits of human fibrinogen with equal efficiency but didn't show any reactivity for the gamma form of human fibrinogen. The enzymatic activity is strongly inhibited by EDTA and 1,10-phenanthroline, indicating that the enzymes are metalloproteases. No inhibition was found with phenylmethylsulfonyl fluoride (PMSF), L-trans-epoxysuccinyl leucylamido-(4-guanidino)-butane (E-64), pepstatin and 2-mercaptoethanol. The activity of the purified enzyme was increased by Mg2+, Fe2+, Zn2+, and Co2+, and slightly decreased by Ca2+, but the enzyme activity was dramatically decreased by Cu2+, and totally inhibited by Hg2+. It has broad substrate specificity for synthetic peptides, and keep the high activity from pH 7.5 to 9, suggesting that the purified enzyme was a basic protease. The enzyme was stable up to 30 degrees C and the maximum fibrinolytic activity was at 55 degrees C.     

Production and properties of an alkaline protease by Aureobasidium pullulans

A strain of the yeast-like fungus Aureobasidium pullulans was grown on whey to produce an extracellular protease. The protease was totally inhibited by the serine inhibitor, phenyl methyl sulphonyl fluoride (PMSF), and partially inhibited by the chelating agent EDTA. The enzyme showed maximal activity in the alkaline range with an optimum pH of 9·5–10·5. The optimum temperature for protease activity was 41C. As well as being active against the non-specific proteolytic substrate Azocoll, the protease readily degraded purified α-casein. A molecular weight of 27000 ± 350 was determined for the protease using gel filtration chromatography.     

Purification and characterization of the cuticle-degrading protease produced by the entomopathogenic fungus,Beauveria bassiana in the presence of Sunn pest,Eurygaster integriceps (Hemiptera: Scutelleridae) cuticle

The extracellular protease from the entomopathogenic fungus, Beauveria bassiana in the presence of Eurygaster integriceps cuticle was isolated, purified and characterized. Isolate B1 of B. bassiana that shows high virulence against E. integriceps was examined for the production of the cuticle-degrading proteases. Results showed that both subtilisin-like (Pr1) and trypsin-like (Pr2) cuticle-degrading proteases were produced and the enzyme kinetic properties showed better activity of Pr1 in comparison with Pr2. The proteases were purified using acetone precipitation, Sephadex G-100 gel filtration and CM-Sepharose ion exchange chromatography, with a 5.09-fold increase in specific activity and 21.86% recovery. The enzyme molecular weight was estimated to be 47 kDa and the optimal pH and temperature were 8 and 45°C, respectively. The purified protease was activated by divalent cations, Ca^{2 +} and Mg^{2 +}, and inhibited by NaCl, KCl and determined as a serine protease by inhibition of its activity due to using PMSF, EDTA, mercaptoethanol and SDS. Studies on the timing of the protease secretion in the presence of cuticular substrates could provide information about the role of the accumulated hydrolytic enzymes during pathogenesis to better understand these processes.     

Intracellular proteolytic activity during sporulation ofBacillus megaterium

Intracellular proteolytic activity increased during incubation of the sporogenic strain ofBacillus megaterium KM in a sporulation medium together with excretion of an extracellular metalloprotease. The exocellular protease activity in a constant volume of the medium reached a 100-fold value with respeot to the intracellular activity. Maximal values of the activity of both the extracellular and intracellular enzyme were reached after 3 – 5 h of incubation. After 7 h 20 – 50% cells formed refractile spores. The intracellular proteolytic system hydrolyzed denatured proteinsin vitro at a rate up to 150 μg mg_-1 h_-1 and native proteins at a rate up to 70 μg mg^-1 h^-1. Degradation of proteinsin vivo proceeded from the beginning of transfer to the sporulation medium at a constant rate of 40 μg mg^-1 h^-1 and the inactivation of beta-galactosidase at a rate of 70 μg mg^-1 h^-1. The intracellular proteolytic activity was inhibited to 65 – 88% by EDTA, to 23 – 76% by PMSF. Proteolysis of denatured proteins was inhibited both by EDTA and PMSF more pronouncedly than proteolysis of native proteins; 50 – 65% of the activity were localized in protoplasts. Another strain ofBacillus megaterium (J) characterized by a high (up to 90%) and synchronous sporulation activity was found to behave in a similar way, but the rate of protein turnover in this strain was almost twice as high. The asporogenic strain ofBacillus megaterium KM synthesized the exocellular protease in the sporulation medium, but its protein turnover was found to decrease substantially after 3 – 4 h. The intraeellular proteolytic system of the sporogenic strain J and the asporogenic strain KM were also inhibited by EDTA and PMSF.     

Purification and characterization of a novel salt-tolerant protease from Aspergillus sp. FC-10, a soy sauce koji mold

A novel salt-tolerant protease produced by Aspergillus sp. FC-10 was purified to homogeneity through anion-exchange chromatography, preparative isoelectric-focusing electrophoresis, and gel filtration chromatography, with an overall recovery of 12.7%. This protease demonstrated an optimum pH range of 7.0-9.0 for activity, with a stable pH range of 5.0-9.0. The optimum process temperature at pH 7.0 was 65 degrees C. The enzyme has a molecular mass of 28 kDa and was deduced as a monomer with an isoelectric point of 3.75. Enzyme activity was strongly inhibited by 5 mM of HgCl(2) and FeCl(3), and significantly inhibited by 5 mM of CuSO(4), FeSO(4), and MnCl(2). The activity of this purified protease was inhibited by Na(2).EDTA; however, leupeptin, pepstatin A, PMSF, and E-64 did not affect the activity. Based on the N-terminal amino acid sequence and amino acid composition, this purified protease should be classified as a member of the deuterolysin family.     

Proteolytic activity of a rumen anaerobic fungus

Abstract A strain of the anaerobic phycomycetous fungus Neocallimastix frontalis isolated from the rumen of a sheep had a high proteolytic activity which became predominantly extracellular during growth. Proteolytic activity appeared to be due to a metalloprotease, as it was inhibited by 1,10-phenanthroline, EDTA and other chelators but not by phenylmethylsulphonyl fluoride (PMSF). Inhibition by EDTA was fully reversed by the addition of Zn²⁺, Ca²⁺ or Co²⁺, whereas addition of metal ions in the presence of 1,10-phenanthroline restored only a little activity. p -Chloromercuribenzoate (PCMB) was also inhibitory in dialysed supernatant fluid. N-α-p-Tosyl- l -lysine chloromethylketone (TLCK) inhibited proteolysis, suggesting that the protease(s) has a trypsin-like specificity, but benzoylarginine p -nitroanilide was not hydrolysed. Protease activity has a broad pH profile with a maximum at pH 7.5. Gel fractionation indicated that most of the activity was in a high- M _r form.     

Proteases and Their Involvement in the Infection and Immobilization of Nematodes by the Nematophagous Fungus Arthrobotrys oligospora

The nematophagous fungus Arthrobotrys oligospora produced extracellular proteases when grown in a liquid culture, as revealed by measuring the hydrolysis of the chromogenic substrate Azocoll. The extracellular protease activity was inhibited by phenylmethylsulfonyl fluoride (PMSF) and other serine protease inhibitors and partly inhibited by the aspartate protease inhibitor pepstatin and by a cysteine protease inhibitor [l-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane, or E-64]. Substrate gel electrophoresis showed that the fungus produced several different proteases, including multiple serine proteases. The function of proteases in the infection of nematodes was examined by treating the fungus with various protease inhibitors. None of the inhibitors tested affected the adhesion of nematodes to the traps, but incubating trap-bearing mycelium with a serine protease inhibitor, PMSF, antipain, or chymostatin, or the metalloprotease inhibitor phenanthroline significantly decreased the immobilization of nematodes captured by the fungus. Inhibitors of cysteine or aspartic proteases did not affect the immobilization of captured nematodes. The effects of PMSF on the immobilization of nematodes were probably due to serine proteases produced by the fungus, since the effects were observed when unbound inhibitor was washed away from the fungus before the nematodes were added to the system. No effects were observed when the nematodes only were pretreated with PMSF.     

A novel alkaline protease from wild edible mushroom Termitomyces albuminosus

A protease with a molecular mass of 30 kDa and the N-terminal sequence of GLQTNAPWGLARSS, was isolated from fresh fruiting bodies of the wild edible mushroom Termitomyces albuminosus. The purification protocol included ion exchange chromatography on DEAE-cellulose, Q-Sepharose, SP-Sepharose and FPLC-gel filtration on Superdex 75. The protein was unadsorbed on DEAE-cellulose and Q-Sepharose, but adsorbed on SP-Sepharose. The optimal pH and temperature of the purified enzyme were 10.6 and 60 °C, respectively. The enzyme was stable in the presence of 2 % (v/v) Tween 80 and 4 M urea. More than 80 % of the enzyme activity was retained in 2 % (v/v) Triton X 100, 54 % in 10 mM EDTA and 31 % in 2 % (w/v) SDS. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), but not inhibited by dithiothreitol (DTT), pepstatin or lima bean trypsin inhibitor suggesting that it was a serine protease but not a trypsin-like one. The protease was inhibited by Hg(2+), Cu(2+), and Fe(3+) ions. The K(m) and V(max) values of the purified enzyme for casein were 8.26 mg ? ml(-1) and 0.668 mg ? ml(-1) ? min(-1), respectively.