Isolation, characterization and antigenic cross-reactivities of the major hemorrhagin from Cryptelytrops purpureomaculatus venom.

The major hemorrhagin from C. purpureomaculatus (mangrove pit viper) venom was purified to homogeneity and termed Maculatoxin. Maculatoxin has a molecular weight of 38 kDa as determined by SDS-PAGE. It is an acidic protein (pI= 4.2) and exhibited proteolytic and hemorrhagic activities (MHD10 = 0.84 μg in mice) but was not lethal to mice at a dose of 1 μg/g. The hemorrhagic activity of Maculatoxin was completely inactivated by EDTA and partially inhibited by ATP and citrate. The N-terminal sequence of Maculatoxin (TPEQQRFPPTYIDLGIFVDHGMYAT) shares a significant degree of homology with the metalloprotease domain of other venom hemorrhagins. Indirect ELISA showed anti-Maculatoxin cross reacted with protein components of many snake venoms. In the double-sandwich ELISA, however, anti-Maculatoxin cross-reacted only with venoms of certain species of the Trimeresurus (Asia lance-head viper) complex, and the results support the recent proposed taxonomy changes concerning the Trimeresurus complex

Effect of laundry detergent formulation on the performance of alkaline phytoproteases

Background: Proteases constitute the largest product segment in the global industrial enzymes market; they are used in food, pharmaceutical, leather, textile, wood and detergent industries. Alkaline proteases improve the cleaning efficiency of detergents and represent one of the most successful applications of modern industrial biotechnology. The aim of this work was to study the performance of two alkaline phytoproteases, araujiain (Araujia hortorum Fourn.) and asclepain (Asclepias curassavica L.), for their potential application as additive in laundry detergent formulations. Results: The effect of pure non-ionic and ionic surfactants on proteolytic activity of araujiain and asclepain was analyzed measuring the remaining activity after 1 hr of incubation of those enzymes in aqueous solutions of surfactants at different concentrations (0.1, 0.4 and 1% v/v) and temperatures (25, 40 and 60ºC). Besides, the compatibility of the enzymes with six commercial laundry detergents was also studied measuring the remaining proteolytic activity at 37ºC after 1 hr. Commercial detergent components influenced in different ways on araujiain and asclepain, in spite of the similar behaviour of the two enzymes in buffer. In commercial detergent solutions, araujiain expressed between 60% and 140% of its remaining proteolytic activity in buffer (pH 8.5) at 37ºC after 1 hr, while asclepain, was practically inactivate in most of them at the same conditions. Conclusions: Proteolytic extract of Araujia hortorum fulfilled all the requirements for its application as additive for laundry detergents: high stability in a broad temperature range (25-70ºC), high activity in alkaline pH (7.5-9.5) and very good compatibility with the commercial detergent additives. Nevertheless, in spite of its high stability and activity in buffer, the proteolytic extract of Asclepias curassavica did not show the same performance than araujiain.

Isolation and characterization of a metal ion-dependent alkaline protease from a halotolerant Bacillus aquimaris VITP4.

A halotolerant bacterium Bacillus acquimaris VITP4 was used for the production of extracellular protease. Fractional precipitation using ammonium chloride was used to obtain the enzyme. The protease exhibited optimum activity at pH 8.0 and 40°C and retained 50% of its optimal proteolytic activity even in the presence of 4 M NaCl, suggesting that it is halotolerant. The molecular mass of protease, as revealed by SDS-PAGE was found to be 34 kDa and the homogeneity of the enzyme was confirmed by gelatin zymography and reverse-phase HPLC. Upon purification, the specific activity of th enzyme increased from 533 U/mg to 1719 U/mg. Protease inhibitors like phenyl methane sulphonyl fluoride and 2-mercaptoethanol did not affect the activity of the enzyme, but EDTA inhibited the activity, indicating the requirement of metal ions for activity. Cu­­­2+, Ni2+ and Mn2+ enhanced the enzyme activity, but Zn2+, Hg2+ and Fe2+ decreased the activity, while Mg2+, Ca2+ and K+ had no effect on the enzyme activity. The protease was quite stable in the presence of cationic (CTAB), anionic (SDS) and neutral detergents (Triton X-100 and Tween-20) and exhibited antimicrobial activity against selected bacterial and fungal strains. The stability characteristics and broad spectrum antimicrobial activity indicated the potential use of this protease in industrial applications.

Endopeptidases of Bacillus subtilis IBTC-3 and B. alcalophilus PB92 in synthesis of precursors of biologically active peptides.

Two endopeptidases (from Bacillus subtilis IBTC-3 and from B. alcalophilus PB92-commercial preparation) efficiently synthesized amino acid esters (NAc-Tyr-OEt and NAc-Phe-OEt) and dipeptides (NAc-Tyr-Gly-NH2 and NAc-Tyr-Arg-NH2) in organic solvent/water systems. The rate of NAc-Tyr-OEt synthesis mediated by the native subtilisin IBTC-3 was maximum (0.23 Umg-1) in ethanol/5-7% w/v water system, while the highest activity of the freeze-dried enzyme (0.18 Umg-1) was achieved, when water content was 9-10% w/v. The preferred system for dipeptide synthesis (using NAc-Tyr-OEt as acyl donor) by both the enzymes was acetonitrile/4% w/v water. In this system, the maximum yield of NAc-Tyr-GlyNH2 was 71 and 80% and that of NAc-Tyr-Arg-NH2 was 53 and 40% for subtilisin IBTC-3 and peptidase PB92, respectively. In contrast to the peptidase PB92, the subtilisin efficiently catalyzed esterification of NAc-Tyr with 1-butanol and isopropanol.

Production and purification of a protease from an alkalophilic Bacillus sp. 2-5 strain isolated from soil

This research has focused on isolation and characterization of a strain of Bacillus sp. from alkaline soil, which was able to produce extracellular alkaline protease at pHs ranging from 8 to 11 and temperatures of 20 to 50§C. Also the impact of different carbon and nitrogen sources were investigated. The yield and fold of enzyme purification was 24% and 50 times, respectively. Molecular weight of purified enzyme was measured by SDS-PAGE as 24.7 kDa. The alkaline protease produced by Bacillus sp. 2 - 5 showed the most caseinolytic activity [without any gelatinolytic activity] at pH>10

Isolation and purification of an extracellular protease from a new strain of Bacillus subtilis, viz.NCIM 2711.

A new extracellular protease having a prospective application in the food industry was isolated from Bacillus sUbtilis NCIM 2711 by (NH4)2SO4 precipitation from the cell broth. It was purified using DEAE-Cellulose and CM-Sephadex C-50 ion-exchange chromatography. With casein as a substrate, the proteolytic activity of the purified protease was found to be optimal at pH 7.0 and temperature 55 degrees C with Km 1.06 mg/ml. The enzyme was stable over a pH range 6.5-8.0 at 30 degrees C for 1 hr in presence of CaCl2 x 2H2O. At 55 degrees C, the enzyme retained 60% activity up to 15 min in presence of CaCl2 x 2H2O. EDTA and o-phenanthroline (OP) completely inhibited the enzyme activity while DFP, PMSF and iodoacetamide were ineffective. The enzyme was completely inhibited by Hg2+ and partially by Cd2+, Cu2+, Ni2+, Pb2+ and Fe2+. The OP inhibited enzyme could be reactivated by Zn2+ and Co2+ up to 75% and 69% respectively. It is a neutral metalloprotease showing a single band of 43 kDa on SDS-PAGE.

Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis

The protease ZapA, secreted by Proteus mirabilis, has been considered to be a virulence factor of this opportunistic bacterium. The control of its expression requires the use of an appropriate methodology, which until now has not been developed. The present study focused on the replacement of azocasein with fluorogenic substrates, and on the definition of enzyme specificity. Eight fluorogenic substrates were tested, and the peptide Abz-Ala-Phe-Arg-Ser-Ala-Ala-Gln-EDDnp was found to be the most convenient for use as an operational substrate for ZapA. A single peptide bond (Arg-Ser) was cleaved with a Km of 4.6 µM, a k cat of 1.73 s-1, and a catalytic efficiency of 376 (mM s)-1. Another good substrate for ZapA was peptide 6 (Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Gln-EDDnp) which was cleaved at a single bond (Phe-Ser) with a Km of 13.6 µM, a k cat of 3.96 s-1 and a catalytic efficiency of 291 (mM s)-1. The properties of the amino acids flanking the scissile bonds were also evaluated, and no clear requirement for the amino acid residue at P1 was found, although the enzyme seems to have a preference for a hydrophobic residue at P2.

Protease from Sporosarcina sp. RRLJ 1.

Protease was isolated from Sporosarcina RRLJ1 which was collected from acid tea (Camellia sinensis) plantations. It showed potential for production of the enzyme for commercial purposes. The study revealed that optimum pH for growth of the organism was 6.5-7 and supplement of casein (1%) in the medium was required for production of protease. Enzyme production and enzyme activity was maximum in 72 hr old broth culture. Maximum activity of the enzyme was found at pH 6.5.

High and low molecular weight proteolytic activities in prawn (Metapenaeus brevicornis) muscle.

Muscle extract of prawn (Metapenaeus brevicornis) expressed high azocoll lytic activity compared to extracts of many other prawn varieties; the activity was also inhibited to a small extent by dithiothreitol. Ammonium sulphate precipitation, subsequent extraction at pH 5.6 and chromatography revealed the occurrence of two types of azocoll lytic activities: one, high molecular weight (630 kDa) and the other low molecular weight (< 30 kDa) enzyme. The former was stimulated by dithiothreitol whereas the latter was inhibited. SDS PAGE of high molecular weight preparation did not show homogeneity but the profile was similar to that of the low molecular weight fraction. Gel filtration of high molecular weight enzyme following incubation at high pH revealed the formation of low molecular weight fractions having activity towards azocoll. Chymotrypsin-like activity associated with high molecular weight enzyme was also susceptible to dissociation by high pH. Azocoll lytic activity of both enzymes was strongly inhibited by 1,10-phenanthroline.

Partial characterization of affinity purified excretory-secretory protease from Setaria digitata.

Excretory-secretory protease of S. digitata released along with the microfilariae (mf) during hatching has been purified by affinity chromatography. No other activity could be detected in the affinity purified material. Homogeneity is checked by native PAGE. It has a pH optimum of 5.4 and a molecular weight of 70 kD. The purified material showed positivity against antibodies raised against ES material.

Purification and characterization of an alkaline proteinase from kidney cortex lysosomes.

An alkaline proteinase was purified to apparent homogeneity from buffalo (Bubalus bubalis) kidney cortex lysosomes by affinity chromatography on STI sepharose 4B and gel filtration over Sephadex G-100. The molecular weight of the enzyme was 17,000 and 21,000 by gel filtration and SDS/PAGE respectively. The purified enzyme was optimally active at pH 8.5-9.0 at 50 degrees C and hydrolysed synthetic substrates of chymotrypsin but not those of elastase or trypsin. It was inhibited by serine proteinase inhibitors like soybean trypsin inhibitor, limabean trypsin inhibitor and phenylmethyl sulphonyl fluoride. Immunologically, the enzyme was similar to chymotrypsin. The amino acid composition showed high content of acidic amino acids. This protein was detected in kidney, liver, spleen, pancreas and heart.

Rapid purification of secretory acid proteinase from Candida albicans and its characterization.

Secretory acid proteinase from C. albicans was purified from culture supernatant to apparent homogeneity by ion-exchange chromatography. Two isozymes of the proteinase were resolved using a novel chromatofocusing method. The enzyme, which appears to be a glycoprotein, consists of a single polypeptide chain with glutamine at the N-terminus. Its molecular weight is about 45,000 and isoelectric point is pH 4.6. At pH 5, the proteinase is stable at 45 degrees C for at least 15 min. It has a broad substrate specificity. With BSA as a substrate, Km was determined to be 1.6 x 10(-4) M. The enzyme is inhibited by pepstatin and thus is a carboxyl proteinase. It undergoes autocatalytic digestion at or below pH 5.0. The kinetics of induction of proteinase by various proteins are also reported.

Aislamiemto y algunas propiedades de la proteinasa I del veneno de la serpiente peruana Lachesis muta / Isolation and various properties of proteinase I from the venom of the Peruvian snake Lachesis muta

Se ha purificado una enzima proteolítica con elevada actividad sobre caseína a partir del veneno de la serpiente Lachesis muta. Esta proteína denominada "Proteinasa I" fue obtenida mediante una cromatografía de filtración sobre sephadex G-100 a pH 6.5 con buffer acetato de amonio 0.1 seguida de una cromatografía de intercambio iónico con DEAE-celulosa a pH 7.5 y una recromatografía en DEAE-celulosa a pH 9.0 y 7.8. La proteína aislada mostró un banda homogenea en electroforeis en gel de poliacrilamida. El peso molecular calculado por filtración en gel fue 26,100 y su pH óptimo 8.4. La ctividad enzimática no fue alterada depués de un calentamiento a 45-C por 10 minutos, mientras que a 70- sólo se registró un 4%de actividad. La enzima no ataca ésteres sintético como TAME y BAEE, no es afectada por lo iones de calcio y carecen de actividad hemorrágica

Isolation and partial characterization of Lachesis muta melanocephala coagulant proteinase: biochemical parameters of the venom

The coagulant proteinase of L. m. melanocephala was purified by DEAE Sephadex A-50 followed by agmatine CH- Sepharose and gel filtration on Sephadex G-100. The enzyme exhibited m,any of the properties adscribed to -mudasa-, the coagulant proteinase from L. m. stenophirs venom. Its molecular weight by gel filtration was 35 kDa and its specific clotting activity was 702 NIH/mg of protein. A 32 fold increase in the clotting was obtained by purification. The coagulant proteinase exibited esterolytic activities toward lysine and arginine esters as well as amidolytic. Significant differences are observed when compared with the activities of -mudasa-, the former is less esterolytic, although its activity toward TLEME is higher. Significant differences in the activities are also observed when the venom from the Pacific and Atlantic L. muta populations (corresponding to the subspecies L.m. melanocefala and L.m. Stenophyrs) are compared toward the same substrates. The Pacific type is less amidolytic and more esterolytic toward BAME and BAEE, although toward the lysine and tyrosine esters no significant differences are observed. The venon from the Pacific populations is more coagulant and less proteolytic than the venom from the Atlantic population. Analytical isoelectric focusing of both populations of venom revealed important differences in the number and intensity of the protein bands. The results here given further substantiate the taxonomical differentiation already given to the Pacific and Atlantic Costa Rican population of L. muta.