Isolation and Characterization of Two Momordins,Ribosome-Inactivating Proteins from the Seeds of Bitter Gourd (Momordica charantia)

Cathepsins L and L-like (58 kDa) proteinase from mackerel were purified to electrophoretical homogeneity by Concanavalin A-Sepharose and Econo-Pac S chromatographies. The molecular weights of cathepsins L and L-like proteinase were 30,000 and 58,000, and the optimal pH for the hydrolysis of Z-Phe-Arg-MCA (benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-[4-methyl] coumarylamide) were 5.0 and 5.5, respectively. The stability of both purified proteinases at various pHs was low, when the pH was above 7.0. According to the substrate specificity analysis, these proteinases hydrolyzed Z-Phe-Arg-MCA and Z-Arg-Arg-MCA, but did not hydrolyze Z-Arg-MCA and L-Arg-MCA. The activities of these two proteinases were effectively activated by cysteine and dithiothreitol. Their thiol-dependent proteolytic activity against Z-Phe-Arg-MCA was strongly inhibited by E-64 (trans-epoxysuccinyl-L-leucylamido[4-guanidino]butane), antipain, chymostatin, iodoacetic acid, and leupeptin, but not inhibited by pepstatin or phenylmethane sulfonyl floride. The inactivation rate constants (K_D) of cathepsins L and L-like proteinases at 50°C were 5.1 × 10^?5 and 6.9 × 10^?4 s^?1, respectively. K⁺, Na⁺, Mg⁺, and Sr⁺ did not affect them, while Zn²⁺, Cd²⁺, Co²⁺, Ni²⁺, Cu²⁺, Hg²⁺, Fe²⁺, and Fe³⁺ inhibited the activity of the purified cathepsins L and L-like proteinase.     

Miltpain, a cysteine proteinase, from milt of Pacific cod (Gadus macrocephalus): purification and characterization

Miltpain (EC.3.4.22.-) is a cysteine proteinase that preferentially hydrolyzes basic proteins, previously found in the milt of chum salmon. Here we report a similar cysteine proteinase in the milt of the marine Pacific cod. The enzyme was isolated and purified 6900-fold and with an estimated mass of 63 kDa by gel filtration chromatography and 72 kDa by SDS/PAGE. Cod miltpain has an optimum pH of 6.0 for Z-Arg-Arg-MCA hydrolysis, and K_m of 11.5 μM and k_cat of 19.0 s⁻¹ with Z-Arg-Arg-MCA. It requires a thiol-inducing reagent for activation and is inhibited by E-64, iodoacetamide, CA-074, PCMB, NEM, TLCK, TPCK, ZPCK and o-phenanthroline. This proteinase strongly hydrolyzes basic proteins such as salmine, clupeine and histone, and exhibits unique substrate specificity toward paired basic residues such as Lys-Arg, Arg-Arg on the substrates of P2-P1. The isoelectric point is 5.2 by isoelectric focusing. N-Terminal sequencing gave a sequence of <EVPVEVVRXYVTSAPEK. The cysteine proteinase from Pacific cod very closely matches the previously reported miltpain from chum salmon.     

Simultaneous isolation of human kidney cathepsins B, H, L and C and their characterisation

A procedure for the simultaneous isolation of four cysteine proteinases, cathepsins B, H, L and C, from human kidney is described. The method includes concentration of the acidified homogenate by ammonium sulphate precipitation. The resuspended and dialysed precipitate was chromatographed on DEAE-cellulose DE-32, to allow separation of cathepsins H and C from cathepsins B and L. The main isoform of cathepsin H was separated from cathepsin C by cation-exchange chromatography on CM-Sephadex C-50. These two enzymes were further purified by covalent chromatography on thiopropyl Sepharose and gel permeation on Sephacryl S-200. The last step allowed separation of cathepsin C and the minor isoform of cathepsin H. Purification of the other two enzymes, cathepsins B and L, was carried out on thiol Sepharose, followed by chromatography on CM-Sepharose C-50. In this step, pure cathepsin L was obtained, while two isoforms of cathepsin B had to be finally purified on Sephacryl S-200 columns. The purity of each enzyme was analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis, isoelectric focusing on polyacrylamide gels and N-terminal sequencing. The activities of the purified cathepsins B, H and L were determined in terms of k_cat/K_M for three substrates, Z-Phe-Arg-MCA, Z-Arg-Arg-MCA and Arg-MCA. The method produced 25 mg of cathepsin B, 6.5 mg of cathepsin H, 1.5 mg of cathepsin L and 3.8 mg of cathepsin C from 3.5 kg of human kidney.     

Studies on the <Emphasis Type="Italic">Pycnoporus sanguineus</Emphasis> CCT-4518 laccase purified by hydrophobic interaction chromatography

A laccase from Pycnoporus sanguineus was purified by two steps using phenyl-Sepharose columm. A typical procedure provided 54.1-fold purification, with a yield of 8.37%, using syringaldazine as substrate. The molecular weight of the purified laccase was 69 and 68 kDa as estimated by 12% (w/v) SDS-PAGE gel and by gel filtration, respectively. The K _m values for the substrates ABTS, syringaldazine, and guaiacol were 58, 8.3, and 370 μM, respectively. The enzyme’s pH optimum for syringaldazine was 4.2 and optimal activity was 50°C. The enzyme showed to be thermostable because when kept at 50°C for 24 and 48 h it retained 93 and 76% activity. This laccase was inhibited by l-cysteine, β-mercaptoethanol, NaN₃, NaF, and HgCl₂.     

Hemoglobinase activity of a cysteine protease from the ixodid tick Haemaphysalis longicornis

We report here the molecular characterization and possible function of a cysteine protease (termed HlCPL-A) identified in the midgut of the hard tick Haemaphysalis longicornis. HlCPL-A is a 333 amino acid protein belonging to the papain family of the cysteine protease. A construct encoding proHlCPL-A was expressed in Escherichia coli and purified as both procathepsin L and active processed cathepsin L forms. The HlCPL-A gene expression was up-regulated by blood-feeding process. HlCPL-A exhibited substrate specificity against synthetic peptidyl substrates (Z-Phe-Arg-MCA and Z-Arg-Arg-MCA; k_cat / K_m = 0.19 and 0.0023 M^− 1 S^− 1, respectively). The proteolytic activity of HlCPL-A was inhibited by leupeptin, antipain and E-64 but was unaffected by pepstatin. HlCPL-A was capable of degrading bovine hemoglobin at pH 3.2 to 5.6. These results suggest that HlCPL-A may play important roles in the digestion of host hemoglobin in ticks.     

Purification and characterization of a new type of serine carboxypeptidase from<Emphasis Type="Italic"> Monascus purpureus</Emphasis>

Carboxypeptidase produced by Monascus purpureus IFO 4478 was purified to homogeneity. The purified enzyme is a heterodimer with a molecular mass of 132 kDa and consists of two subunits of 64 and 67 kDa. It is an acidic glycoprotein with an isoelectric point of 3.67 and 17.0% carbohydrate content. The optimum pH and temperature were 4.0 and 40 °C, respectively. The enzyme was stable between pH 2.0 and 8.0 at 37 °C for 1 h, and up to 50 °C at pH 5.0 for 15 min. The enzyme was strongly inhibited by piperastatin A, diisopropylfluoride phosphate (DFP), phenylmethylsulfonylfluoride (PMSF), and chymostatin, suggesting that it is a chymotrypsin-like serine carboxypeptidase. Monascus purpureus carboxypeptidase was also strongly inhibited by p-chloromercuribenzoic acid (PCMB) but not by ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline, indicating that it requires cysteine residue but not metal ions for activity. Benzyloxycarbonyl-l-tyrosyl-l-glutamic acid (Z-Tyr-Glu), among the substrates tested, was the best substrate of the enzyme. The K_m, V_max, K_cat, and K_cat/K_m values of the enzyme for Z-Tyr-Glu at pH 4.0 and 37 °C were 0.86 mM, 0.917 mM min^–1, 291 s^–1, and 339 mM^–1 s^–1, respectively.     

Digestive physiology and characterization of digestive cathepsin L-like proteinase from the sugarcane weevil Sphenophorus levis

Sugarcane is an important crop that has recently become subject to attacks from the weevil Sphenophorus levis, which is not efficiently controlled with chemical insecticides. This demands the development of new control devices for which digestive physiology data are needed. In the present study, ion-exchange chromatography of S. levis whole midgut homogenates, together with enzyme assays with natural and synthetic substrates and specific inhibitors, demonstrated that a cysteine proteinase is a major proteinase, trypsin is a minor one and chymotrypsin is probably negligible. Amylase, maltase and the cysteine proteinase occur in the gut contents and decrease throughout the midgut; trypsin is constant in the entire midgut, whereas a membrane-bound aminopeptidase predominates in the posterior midgut. The cysteine proteinase was purified to homogeneity through ion-exchange chromatography. The purified enzyme had a mass of 37 kDa and was able to hydrolyze Z-Phe-Arg-MCA and Z-Leu-Arg-MCA with k_cat/K_m values of 20.0 ± 1.1 μM⁻¹ s⁻¹ and 30.0 ± 0.5 μM⁻¹ s⁻¹, respectively, but not Z-Arg-Arg-MCA. The combined results suggest that protein digestion starts in the anterior midgut under the action of a cathepsin L-like proteinase and ends on the surface of posterior midgut cells. All starch digestion takes place in anterior midgut. These data will be instrumental to developing S. levis-resistant sugarcane.     

Stachyose synthesis in leaves of Cucurbita pepo

An enzyme synthesizing stachyose, galactinol-raffinose galactosyltransferase (EC2.4.1.67), has been purified ca 40-fold from mature leaves of Cucurbita pepo using ammonium sulphate precipitation, Sephadex gel filtration and DEAE-Sephadex gel chromatography. The purified enzyme fraction was separated from all but 2 % of the total,α-galactosidase activity extracted from the tissue. The enzyme was optimally active at pH 6.9 and was stable for at least a month at 4° in the presence of 20 mM 2-mercaptoethanol. The enzyme displayed high specificity for the donor galactinol (K_m 7.7 mM) and the acceptor raffinose (K_m 4.6 mM) and was unable to effect synthesis of any other member of the raffinose series of galactosyl-sucrose oligosaccharides. Co²⁺, Hg²⁺, Mn²⁺ and Ni²⁺ ions were particularly inhibitory; no metal ion promotion was observed and 5 mM EDTA was ineffective. Myo-inositol was strongly inhibitory (K_i 2 mM), melibiose weakly so. Tris buffer (0. 1 M) was also inhibitory. Galactinol hydrolysis occurred in the absence of the acceptor raffinose but there was no hydrolysis of either raffinose or stachyose in the absence of the donor galactinol. The reaction was readily reversible and exchange reactions were detected between substrates and products. It is proposed that the synthesis of stachyose in mature leaves ofC. pepo proceeds via this galactosyltransferase and not via α-galactosidase.     

Echinococcus multilocularis: identification and functional characterization of cathepsin B-like peptidases from metacestode

Cysteine peptidases have potent activities in the pathogenesis of various parasitic infections, and are considered as targets for chemotherapy and antigens for vaccine. In this study, two cathepsin B-like cysteine peptidases (EmCBP1 and EmCBP2) from Echinococcus multilocularis metacestodes were identified and characterized. Immunoblot analyses demonstrated that EmCBP1 and EmCBP2 were present in excretory/secretory products and extracts of E. multilocularis metacestodes. By immunohistochemistry, EmCBP1 and EmCBP2 were shown to localize to the germinal layer, the brood capsule and the protoscolex. Recombinant EmCBP1 and EmCBP2 expressed in Pichia pastoris, at optimum pH 5.5, exhibited substrate preferences for Z-Phe-Arg-MCA, Z-Val-Val-Arg-MCA, and Z-Leu-Arg-MCA, and low levels of hydrolysis of Z-Arg-Arg-MCA. Furthermore, recombinant enzymes degraded IgG, albumin, type I and IV collagens, and fibronectin. These results suggested that EmCBP1 and EmCBP2 may play key roles in protein digestion for parasites’ nutrition and in parasite–host interactions.     

Characterization of a peptidase fromDiplococcus pneumoniae

Some properties of a purified peptidase fromDiplococcus pneumoniae have been studied. The enzyme has a broad pH optimum between 6 and 8 and a K_m (onl-leucylglycylglycine) of 2.8mm. It is activated by low levels of Hg⁺⁺ and is inhibited by Mn⁺⁺, Co⁺⁺, β-mercaptoethanol and EDTA. Substrate specificity studies show that the enzyme is an exopeptidase of the aminopeptidase type, most active on tripeptide substrates bearing bulky substituents at the NH₂ ⁻ terminal end.     

Purification and characterization of heat-stable alkaline proteinase from bigeye snapper (Priacanthus macracanthus) muscle

Heat-stable alkaline proteinase was purified from bigeye snapper (Priacanthus macracanthus) ordinary muscle by heat-treatment and a series of chromatographies including Phenyl-Sepharose 6 Fast Flow, Source 15Q and Superose 12 HR 10/30. It was purified to 5180-fold with a yield of 0.8%. The molecular weight of purified proteinase was estimated to be 72 kDa by gel filtration. The proteinase appeared as two proteinase activity bands with molecular weights of 66 and 13.7 kDa on non-reducing SDS-substrate gel. Accordingly, it was found to consist of two different subunits. The optimum pH and temperature for casein hydrolysis were 8.5 and 60 °C, respectively. The proteolytic activity was strongly inhibited by soybean trypsin inhibitor and partially inhibited by ethylenediaminetetraacetic acid, while pepstatin A and E-64 showed no inhibition. Purified proteinase was able to hydrolyze Boc-Phe-Ser-Arg-MCA, but rarely hydrolyzed Z-Phe-Arg-MCA and Z-Arg-Arg-MCA. In addition, it mainly degraded myosin heavy chain, not actin. These results suggest that purified proteinase was serine proteinase, which is probably involved in gel weakening of bigeye snapper surimi.     

Purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of horn fly, Haematobia irritans irritans (Diptera: Muscidae)

This work describes the purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of Haematobia irritans irritans (Diptera: Muscidae). The enzyme was purified using a one-step process, consisting of affinity chromatography on SBTI-Sepharose. The purified protease showed one major active proteinase band on reverse zymography with 0.15% gelatin, corresponding to a molecular mass of 25.5 kDa, with maximum activity at pH 9.0. The purified trypsin-like enzyme preferentially hydrolyzed synthetic substrates with arginine residue at the P1 position. The K _m values determined for three different substrates were 1.88 × 10^–4, 1.28 × 10^–4, and 1.40 × 10^–4 M for H--benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222), dl-Ile-Pro-Arg-p-nitroanilide (S2288), and DL-Phe-Pip-Arg-p-nitroanilide (S2238), respectively. The enzyme was strongly inhibited by typical serine proteinase inhibitors such as SBTI (soybean trypsin inhibitor, K _i = 0.19 nM) and BuXI (Bauhinia ungulata factor Xa inhibitor, K _i = 0.48 nM), and less inhibited by LDTI (leech-derived tryptase inhibitor, K _i = 1.5 nM) and its variants LDTI 2T and 5T (0.8 and 1.5 nM, respectively). The most effective inhibitor for this protease was r-aprotinin (r-BPTI) with a K _i value of 39 pM. Synthetic serine protease inhibitors presented only weak inhibition, e.g., benzamidine with K _i = 3.0 × 10^–4 M and phenylmethylsulfonyl fluoride (PMSF) showed traces of inhibition. The purified trypsin-like enzyme also digested natural substrates such as fibrinogen and fibrin net. The protease showed higher activity against fibrinogen and fibrin than did bovine trypsin. These data suggest that the proteolytic enzyme of H. irritans irritans is more specific to proteins from blood than are the vertebrate digestive enzymes. This enzyme's characteristics may be an adaptation resulting from the feeding behavior of this hematophagous insect.     

Purification and characterization of an iron-containing alcohol dehydrogenase in extremely thermophilic bacterium <Emphasis Type="Italic">Thermotoga hypogea</Emphasis>

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90°C. It uses carbohydrates and peptides as carbon and energy sources to produce acetate, CO₂, H₂, l-alanine and ethanol as end products. Alcohol dehydrogenase activity was found to be present in the soluble fraction of T. hypogea. The alcohol dehydrogenase was purified to homogeneity, which appeared to be a homodimer with a subunit molecular mass of 40 ± 1 kDa revealed by SDS-PAGE analyses. A fully active enzyme contained iron of 1.02 ± 0.06 g-atoms/subunit. It was oxygen sensitive; however, loss of enzyme activity by exposure to oxygen could be recovered by incubation with dithiothreitol and Fe²⁺. The enzyme was thermostable with a half-life of about 10 h at 70°C, and its catalytic activity increased along with the rise of temperature up to 95°C. Optimal pH values for production and oxidation of alcohol were 8.0 and 11.0, respectively. The enzyme had a broad specificity to use primary alcohols and aldehydes as substrates. Apparent K _m values for ethanol and 1-butanol were much higher than that of acetaldehyde and butyraldehyde. It was concluded that the physiological role of this enzyme is likely to catalyze the reduction of aldehydes to alcohols.     

Purification and characterization of a high molecular mass serine carboxypeptidase from <Emphasis Type="Italic">Monascus pilosus</Emphasis>

Two serine carboxypeptidases, MpiCP-1 and MpiCP-2, were purified to homogeneity from Monascus pilosus IFO 4480. MpiCP-1 is a homodimer with a native molecular mass of 125 kDa composed of two identical subunits of 61 kDa, while MpiCP-2 is a high mass homooligomer with a native molecular mass of 2,263 kDa composed of about 38 identical subunits of 59 kDa. This is unique among carboxypeptidases and distinguishes MpiCP-2 as the largest known carboxypeptidase. The two purified enzymes were both acidic glycoproteins. MpiCP-1 has an isoelectric point of 3.7 and a carbohydrate content of 11%, while for MpiCP-2 these values were 4.0 and 33%, respectively. The optimum pH and temperature were around 4.0 and 50°C for MpiCP-1, and 3.5 and 50°C for MpiCP-2. MpiCP-1 was stable over a broad range of pH between 2.0 and 8.0 at 37°C for 1 h, and up to 55°C for 15 min at pH 6.0, but MpiCP-2 was stable in a narrow range of pH between 5.5 and 6.5, and up to 50°C for 15 min at pH 6.0. Phenylmethylsulfonylfluoride strongly inhibited MpiCP-1 and completely inhibited MpiCP-2, suggesting that they are both serine carboxypeptidases. Of the substrates tested, benzyloxycarbonyl-l-tyrosyl-l-glutamic acid (Z-Tyr-Glu) was the best for both enzymes. The K_m, V_max, K_cat and K_cat/K_m values of MpiCP-1 for Z-Tyr-Glu at pH 4.0 and 37°C were 1.33 mM, 1.49 mM min^–1, 723 s^–1 and 545 mM^–1 s^–1, and those of MpiCP-2 at pH 3.5 and 37°C were 1.55 mM, 1.54 mM min^–1, 2,039 s^–1 and 1,318 mM^–1 s^–1, respectively.     

Novel halophilic 2-aminobutyrate dehydrogenase from Halobacterium saccahrovorum DSM 1137

A halophilic NAD⁺-dependent 2-aminobutyrate dehydrogenase (EC1.4.1.1) was purified to homogeneity from a crude extract of an extreme halophile, Halobacterium saccharovorum DSM 1137, with a 30% yield. The enzyme had a molecular mass of about 160 kDa and consisted of four identical subunits. It retained more than 70% of the activity after heating at 60 °C for 1 h and kept it at 30 °C for 8 months in the presence of 2 M NaCl. The enzyme showed maximum activity in the presence of 2 M RbCl or KCl. The enzyme required NAD⁺ as a coenzyme and used -2-aminobutyrate, -alanine, and -norvaline as substrates. The best substrate was -2-aminobutyrate. The optimum pH was 9.3 for the oxidative deamination of -2-aminobutyrate and 8.6 for the reductive amination of 2-ketobutyrate. The Michaelis constants were 1.2 mM for -2-aminobutyrate, 0.16 mM for NAD⁺, 0.012 mM for NADH, 0.78 mM for 2-ketobutyrate, and 500 mM for ammonia in the presence of 2 M KCl. The K_m values for the substrates depended on the concentration of KCl, and the K_m values decreased under high salt conditions.     

Purification and characterization of two cold-adapted extracellular tannin acyl hydrolases from an Antarctic strain <Emphasis Type="Italic">Verticillium</Emphasis> sp. P9

Two extracellular tannin acyl hydrolases (TAH I and TAH II) produced by an Antarctic filamentous fungus Verticillium sp. P9 were purified to homogeneity (7.9- and 10.5-fold with a yield of 1.6 and 0.9%, respectively) and characterized. TAH I and TAH II are multimeric (each consisting of approximately 40 and 46 kDa sub-units) glycoproteins containing 11 and 26% carbohydrates, respectively, and their molecular mass is approximately 155 kDa. TAH I and TAH II are optimally active at pH of 5.5 and 25 and 20°C, respectively. Both the enzymes were activated by Mg²⁺and Br⁻ ions and 0.5–2.0 M urea and inhibited by other metal ions (Zn²⁺, Cu²⁺, K⁺, Cd²⁺, Ag⁺, Fe³⁺, Mn²⁺, Co²⁺, Hg²⁺, Pb²⁺ and Sn²⁺), anions, Tween 20, Tween 60, Tween 80, Triton X-100, sodium dodecyl sulphate, β-mercaptoethanol, α-glutathione and 4-chloromercuribenzoate. Both tannases more efficiently hydrolyzed tannic acid than methyl gallate. E _a of these reactions and temperature dependence (at 0–30°C) of k _cat, k _cat/K _m, ΔG*, ΔH* and ΔS* for both the enzymes and substrates were determined. The k _cat and k _cat/K _m values (for both the substrates) were considerably higher for the combined preparation of TAH I and TAH II.     

Purification and Characterization of Invertase from Lactobacillus reuteri CRL 1100

The invertase of Lactobacillus reuteri CRL 1100 is a glycoprotein composed by a single subunit with a molecular weight of 58 kDa. The enzyme was stable below 45°C over a wide pH range (4.5–7.0) with maximum activity at pH 6.0 and 37°C. The invertase activity was significantly inhibited by bivalent metal ions (Ca⁺⁺, Cu⁺⁺, Cd⁺⁺, and Hg⁺⁺), β-mercaptoethanol, and dithiothreitol and partially improved by ethylenediaminetetraacetic acid. The enzyme was purified 32 times over the crude extract by gel filtration and ion-exchange chromatography with a recovery of 17%. The K _m and V_max values for sucrose were 6.66 mM and 0.028 μmol/min, respectively. An invertase is purified and characterized for the first time in Lactobacillus, and it proved to be a β-fructofuranosidase. Received: 13 August 1999 / Accepted: 15 September 1999     

Purification and characterization of a thermostable uricase from Microbacterium sp. strain ZZJ4-1

In order to study the properties of a thermostable uricase produced by Microbacterium sp. strain ZZJ4-1, the enzyme was purified by ammonium sulfate precipitation and DEAE-cellulose ion exchange, hydrophobic and molecular sieve chromatography. The molecular mass of the purified enzyme was estimated to be 34 kDa by SDS-PAGE. The enzyme was stable between pH 7.0 and 10.00. The optimal reaction temperature of the enzyme was 30 °C at pH 8.5. The K _m and K _cat of the enzyme were 0.31 mM and 3.01 s⁻¹, respectively. Fe³⁺ could enhance the enzyme activity, whereas Ag⁺, Hg²⁺, o-phenanthroline and SDS inhibited the activity of the enzyme considerably. After purification, the enzyme was purified 19.7-fold with 31% yield. As compared with uricases from other microbial sources, the purified enzyme showed excellent thermostability and other unique characteristics. The results of this work showed that strains of Microbacterium could be candidates for the production of a thermostable uricase, which has the potential clinical application in measurement of uric acid.     

Purification and Some Properties of Trametes sanguinea Rhodanese

A fungal rhodanese from the spray-dried powder of a culture filtrate of Trametes sanguinea was purified to 142-fold by ammonium sulfate precipitation and DEAE-cellulose and Sephadex G–100 column chromatography. The purified rhodanese (pI 5.10) showed a single band on disc electrophoresis, and its molecular weight was estimated to be 51,700 by gel filtration technique. The enzyme had a broad pH optimum between 7.5 and 8.5 and was stable at pH values from 4 to 8 at 30°C for 44 hr. Its activity was inhibited by p-chloromercuribenzoate at pH 9.5, but not at pH 8.0, and was inhibited by cysteine, β-mercaptoethanol and sodium borohydride at pH 8.0. Both thiosulfate and cyanide showed substrate inhibition at high concentrations. Dihydrolipoate and benzenethiosulfonate were also good substrates.     

Multisubstrate specifics amylase from mushroomTermitomyces clypeatus

An amylase was purified from the culture filtrate ofTermitomyces clypeatus by ammonium sulphate precipitation, DEAE-Sephadex chromatography and gel filtration on Bio-Gel P-200 column. The electrophoretically homogeneous preparation also exhibited hydrolytic activity (in a decreasing order) on amylose, xylan, amylopectin, glycogen, arabinogalactan and arabinoxylan. The enzyme had characteristically endo-hydrolytic activity on all the substrates tested and no xylose, glucose, arabinose or glucuronic acid could be detected even after prolonged enzymatic digestion of the polysaccharides. Interestingly the enzyme had similar pH optima (5.5), temperature optima (55°C), pH stability (pH 3–10) and thermal denaturation kinetics when acted on both starch and xylan (larch wood) .K _m values were found to be 2.63 mg/ml for amylase and 6.25 mg/ml for xylanase activity. Hill’s plot also indicated that the enzyme contained a single active site for both activities. Hg²⁺ was found to be most potent inhibitor. Ca²⁺, a common activator for amylase activity, appeared to be an inhibitor for this enzyme. Thus it appeared that the enzyme had multisubstrate specificity acting as α-amylase on starch and also acting as xylanase on side chain oligosaccharides of xylan containing α-linked sugars.