Comprehensive evaluation of chitosan nanoparticle based phage lysin delivery system; a novel approach to counter S. pneumoniae infections.

Cpl-1, an endolysin derived from Cp-1 phage has been found to be effective in a number of in-vitro and in-vivo pneumococcal infection models. However its lower bioavailability under in-vivo conditions limits its applicability as therapeutic agent. In this study, Cpl-1 loaded chitosan nanoparticles were set up in order to develop a novel therapeutic delivery system to counter antibiotic resistant S. pneumoniae infections. Interactions of chitosan and Cpl-1 were studied by in-silico docking analysis. Chitosan nanoparticles and Cpl-1 loaded chitosan nanoparticles were prepared by using ionic gelation method and the process was optimized by varying chitosan:TPP ratio, pH, stirring time, stirring rate and Cpl-1 concentration. Chitosan nanoparticles and Cpl-1 loaded chitosan nanoparticles were characterized to ascertain successful formation of nanoparticles and entrapment of Cpl-1 into nanoparticles. Chitosan nanoparticles and Cpl-1 loaded nanoparticles were also evaluated for nanoparticle yield, entrapment efficiency, in-vitro release, stability, structural integrity of Cpl-1, in-vitro bioassay, swelling studies, in-vitro biodegradation and heamolysis studies. Mucoadhesion behavior of chitosan nanoparticles and Cpl-1 loaded nanoparticles was explored using mucous glycoprotein assay and ex-vivo mucoadhesion assay, both preparations exhibited their mucoadhesive nature. Cellular cytotoxicity and immune stimulation studies revealed biocompatible nature of nanoparticles. The results of this study confirm that chitosan nanoparticles are a promising biocompatible candidate for Cpl-1 delivery with a significant potential to increase bioavailability of enzyme that in turn can increase its in-vivo half life to treat S. pneumoniae infections.

Production, purification and biochemical characterization of a novel detergent-stable serine alkaline protease from Bacillus safensis strain RH12.

A novel extracellular protease (SAPRH) was hyper-produced (9000 U/mL) from Bacillus safensis RH12, a newly isolated enzyme from a Tunisian offshore oil field. The enzyme was purified to homogeneity, using salt-precipitation, heat-treatment and FPLC anion-exchange chromatography. The purified enzyme was a monomer of molecular mass of ~28 kDa. The NH2-terminal 23 amino-acid sequence of SAPRH showed high homology with those of Bacillus-proteases. SAPRH displayed optimal activity at pH 9 and 60 °C. It was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), indicating that it belongs to the serine-proteases family. Moreover, SAPRH was extremely stable at a broad range of temperature and pH retaining 85% of its activity at 50 °C and 75% at pH 11. The enzyme exhibited excellent stability and compatibility with surfactants and commercial detergents, revealing 90% stability with SDS and 100% stability with Class commercial laundry detergent. One of the most distinctive properties is its catalytic efficiency, which is higher than that of Alcalase 2.5 L, typeDX (commercial enzyme) and SAPB from B. pumilus CBS. Interestingly, the results of the wash performance analysis demonstrated considerably good de-staining at 40 °C for 30 min with low supplementation (500 U/mL). Accordingly, such a protease could be considered as a good detergent-additive in detergent industry.

Synergistic Removal of Static and Dynamic Staphylococcus aureus Biofilms by Combined Treatment with a Bacteriophage Endolysin and a Polysaccharide Depolymerase.

Staphylococcus aureus is an important pathogen and biofilm former. Biofilms cause problems in clinics and food production and are highly recalcitrant to antibiotics and sanitizers. Bacteriophage endolysins kill bacteria by degrading their cell wall and are therefore deemed promising antimicrobials and anti-biofilm agents. Depolymerases targeting polysaccharides in the extracellular matrix have been suggested as parts of a multi-enzyme approach to eradicate biofilms. The efficacy of endolysins and depolymerases against S. aureus biofilms in static models has been demonstrated. However, there is a lack of studies evaluating their activity against biofilms grown under more realistic conditions. Here, we investigated the efficacy of the endolysin LysK and the poly-N-acetylglucosamine depolymerase DA7 against staphylococcal biofilms in static and dynamic (flow cell-based) models. LysK showed activity against multiple S. aureus strains, and both LysK and DA7 removed static and dynamic biofilms from polystyrene and glass surfaces at low micromolar and nanomolar concentrations, respectively. When combined, the enzymes acted synergistically, as demonstrated by crystal violet staining of static biofilms, significantly reducing viable cell counts compared to individual enzyme treatment in the dynamic model, and confocal laser scanning microscopy. Overall, our results suggest that LysK and DA7 are potent anti-biofilm agents, alone and in combination.

Development of Phage Lysins as Novel Therapeutics: A Historical Perspective.

Bacteriophage lysins and related bacteriolytic enzymes are now considered among the top antibiotic alternatives for solving the mounting resistance problem. Over the past 17 years, lysins have been widely developed against Gram-positive and recently Gram-negative pathogens, and successfully tested in a variety of animal models to demonstrate their efficacy. A lysin (CF-301) directed to methicillin resistant Staphylococcus aureus (MRSA) has effectively completed phase 1 human clinical trials, showing safety in this novel therapeutic class. To validate efficacy, CF-301 is currently the first lysin to enter phase 2 human trials to treat hospitalized patients with MRSA bacteremia or endocarditis. If successful, it could be the defining moment leading to the acceptance of lysins as an alternative to small molecule antibiotics. This article is a detailed account of events leading to the first therapeutic use and ultimate development of phage-encoded lysins as novel anti-infectives.

Purification and characterization of a novel high molecular weight alkaline protease produced by an endophytic Bacillus halotolerans strain CT2.

A protease-producing strain CT2 isolated from Tunisian potatoes, exhibiting a potent protease activity (prot CT2), was identified as Bacillus halotolerans according to 16S ribosomal DNA sequence analysis. Maximum prot CT2 production was obtained in medium supplemented with bean seed proteins. Proteolytic activity was purified by ammonium sulphate precipitation, Sephacryl S-200 gel filtration and SP-sepharose cation-exchange chromatography. Optimal enzyme activity was reached at pH 9 and temperature of 50 °C. Proteolytic activity was enhanced by Ca2+ and Mn2+ ions, completely inhibited by PMSF suggesting a serine protease nature and exhibited high stability in the presence of commercial detergents. Prot CT2 showed broad substrate specificity towards both synthetic and natural substrates, with a high capacity to hydrolyze legume seed proteins. Using electrophoretic analysis, its molecular weight was around 250 kDa with two major subunit showing important homologies with serine proteases belonging to the subtilisin-like serine proteases. Based on the Lineweaver-Burk plots Km and Vmax values were 10 mg/ml and 50,000 U/mg respectively. This newly described prot CT2 displays relevant properties which highlight its potential use in various industrial and biotechnological applications.

Defense-related Proteins from Chelidonium majus L. as Important Components of its Latex.

The aim of this review is to cover most recent research on plant pathogenesis- and defenserelated proteins from latex-bearing medicinal plant Chelidonium majus (Papaveraceae) in the context of its importance for latex activity, function, pharmacological activities, and antiviral medicinal use. These results are compared with other latex-bearing plant species and recent research on proteins and chemical compounds contained in their latex. This is the first review, which clearly summarizes pathogenesisrelated (PR) protein families in latex-bearing plants pointing into their possible functions. The possible antiviral function of the latex by naming the abundant proteins present therein is also emphasized. Finally latex-borne defense system is hypothesized to constitute a novel type of preformed immediate defense response against viral, but also non-viral pathogens, and herbivores.

Destabilase-lysozyme-2 - original recombinant thrombolytic preparation of medicinal leech inhibits horse platelets aggregation.

The purpose. Identifying the capacity of the medicinal leech novel original recombinant thrombolytic preparation Destabilase-Lysozyme-2 to inhibit the blood platelet aggregation. Methods: Gene of destabilase-lysozyme. ds2 (mlDL-Ds2 ), was cloned in E.coli cells. Recombinant protein was isolated in denaturing conditions using metal-chelate chromatography followed by denaturation of the polypeptide by rapid dilution in exact accordance with the procedure described by Kurdyumov A.S. et al. ( 2016, Russian Journal of Bioorganic Chemistry, v.42, s. 42-52). Blood was collected from the jugular vein of 18 horses. The functional status of platelets in the presence of different destabilase-lysozyme concentrations were evaluated for their aggregation in Platelet Rich Plasma ( PRP) and in Washed Platelet suspension (WP) using aggregometers Chrono-Log-700 and Сhrono-Log-560, USA560, США. As used aggregation inducers of ADP, collagen type III and human thrombin. Results: First demonstrated the ability of newly synthesized (Kurdyumov A.S. et al. 2016, Russian Journal of Bioorganic Chemistry, v42, s. 42-52) thrombolytic recombinant enzyme destabilase-lyzosyme to inhibit more than 40% of ADP-stimulated PRP aggregation and ADP- stimulated aggregation of horse blood washed platelets. Conclusion: The ability of destabilase-lyzosyme -2 to inhibit platelets aggregation extends biological properties of recombinant thrombolytic enzyme, pre-clinical trials which resulted in the end of 2015.

A comparison of the enzymatic properties of three recombinant isoforms of thrombolytic and antibacterial protein--Destabilase-Lysozyme from medicinal leech.

BACKGROUND: Destabilase-Lysozyme (mlDL) is a multifunctional i-type enzyme that has been found in the secretions from the salivary glands of medicinal leeches. mlDL has been shown to exhibit isopeptidase, muramidase and antibacterial activity. This enzyme attracts interest because it expresses thrombolytic activity through isopeptidolysis of the ε-(γ-Glu)-Lys bonds that cross-link polypeptide chains in stabilised fibrin. To date, three isoforms of mlDL have been identified. The enzymatic properties of pure mlDL isoforms have not yet been described because only destabilase complexes containing other proteins could be isolated from the salivary gland secretion and because low product yield from the generation of recombinant proteins has made comprehensive testing difficult. RESULTS: In the present study, we optimised the procedures related to the expression, isolation and purification of active mlDL isoforms (mlDL-Ds1, mlDL-Ds2, mlDL-Ds3) using an Escherichia coli expression system, and we detected and compared their muramidase, lytic, isopeptidase and antimicrobial activities. After optimisation, the product yield was 30 mg per litre of culture. The data obtained in our study led to the suggestion that the recombinant mlDL isoforms isolated from inclusion bodies form stable oligomeric complexes. Analyses of the tested activities revealed that all isoforms exhibited almost identical patterns of pH and ionic strength effects on the activities. We determined that mlDL-Ds1, 2, 3 possessed non-enzymatic antibacterial activity independent of their muramidase activity. For the first time, we demonstrated the fibrinolytic activity of the recombinant mlDL and showed that only intact proteins possessed this activity, suggesting their enzymatic nature. CONCLUSIONS: The recombinant Destabilase-Lysozyme isoforms obtained in our study may be considered potential thrombolytic agents that act through a mechanism different from that of common thrombolytics.

A novel endolysin disrupts Streptococcus suis with high efficiency.

Streptococcus suis serotype 2 (S. suis 2) is a zoonotic pathogen that exhibits high-level resistance and multi-drug resistance to classic antibiotics and causes serious human casualties and heavy economic losses in the swine industry worldwide. Therefore, alternative therapies or novel antibacterial agents need to be developed to combat this pathogen. A novel endolysin derived from the S. suis temperate phage phi7917, termed Ly7917, was identified, which had broad lytic activity against S. suis type 1, 2, 7 and 9. Ly7917 consisted of an N-terminal cysteine, histidine-dependent amidohydrolases/peptidase catalytic domain and C-terminal SH3b cell wall binding domain. The endolysin maintained activity at high pH and its catalytic activity could be improved by addition of 10 µM 1.5 mM Ca(2+). In animal studies, 90% of BALB/c mice challenged with typical virulent strain HA9801 of S. suis 2 were protected by Ly7917 treatment. The bacterial load in the blood of HA9801-challenged mice was efficiently reduced almost 50% by Ly7917 while that of penicillin-G-treated mice kept almost unchanged. Our data suggest that Ly7917 may be an alternative therapeutic agent for infections caused by virulent S. suis strains.

The First Paenibacillus larvae Bacteriophage Endolysin (PlyPl23) with High Potential to Control American Foulbrood.

Endolysins, which are peptidoglycan-degrading enzymes expressed during the terminal stage of the reproduction cycle of bacteriophages, have great potential to control Gram-positive pathogens. This work describes the characterization of a novel endolysin (PlyPl23) encoded on the genome of Paenibacillus larvae phage phiIBB_Pl23 with high potential to control American foulbrood. This bacterial disease, caused by P. larvae, is widespread in North America and Europe and causes important economic losses in apiculture. The restriction to antibiotic residues in honey imposed by the EU legislation hinders its therapeutic use to combat American foulbrood and enforces the development of alternative antimicrobial methods. The new endolysin described herein has an N-acetylmuramoyl-L-alanine amidase catalytic domain and exhibits a broad-spectrum activity against common P. larvae genotypes. Moreover, the enzyme displays high antimicrobial activity in a range of pH that matches environmental conditions (pH between 5.0 and 7.0), showing its feasible application in the field. At pH 7.0, a concentration of 0.2 µM of enzyme was enough to lyse 104 CFU.mL-1 of P. larvae in no more than 2 h. The presence of sucrose and of the substances present in the larvae gut content did not affect the enzyme activity. Interestingly, an increase of activity was observed when PlyPl23 was previously incubated in royal jelly. Furthermore, in vivo safety evaluation assays demonstrated that this enzyme is not toxic to the bee larvae. The present work describes for the first time an endolysin encoded in a P. larvae phage that presents high potential to integrate a commercial product to control the problematic American foulbrood.

Improved peripheral nerve regeneration in streptozotocin-induced diabetic rats by oral lumbrokinase.

We assessed the therapeutic effects of lumbrokinase, a group of enzymes extracted from the earthworm, on peripheral-nerve regeneration using well-defined sciatic nerve lesion paradigms in diabetic rats induced by the injection of streptozotocin (STZ). We found that lumbrokinase therapy could improve the rats' circulatory blood flow and promote the regeneration of axons in a silicone rubber conduit after nerve transection. Lumbrokinase treatment could also improve the neuromuscular functions with better nerve conductive performances. Immunohistochemical staining showed that lumbrokinase could dramatically promote calcitonin gene-related peptide (CGRP) expression in the lamina I-II regions in the dorsal horn ipsilateral to the injury and cause a marked increase in the number of macrophages recruited within the distal nerve stumps. In addition, the lumbrokinase could stimulate the secretion of interleukin-1 (IL-1), nerve growth factor (NGF), platelet-derived growth factor (PDGF), and transforming growth factor-ß (TGF-ß) in dissected diabetic sciatic nerve segments. In conclusion, the administration of lumbrokinase after nerve repair surgery in diabetic rats was found to have remarkable effects on promoting peripheral nerve regeneration and functional recovery.

Dilong prevents the high-KCl cardioplegic solution administration-induced apoptosis in H9c2 cardiomyoblast cells mediated by MEK.

Infusion of high-KCl cardioplegic solution (High-KCS) is the most common method used to induce asystole before cardiac surgery. However, our previous study showed the High-KCS can cause the apoptosis of cardiomyocytes in patients who were administered High-KCS prior to undergoing coronary artery bypass graft (CABG) to treat coronary artery disease (CAD). Therefore, it is urgent today to find a complementary medicine to reduce this damage. Dilong (earthworm) has been used as a traditional medicine in China for several thousand years, and extract from the dilong has been empirically used in Asia for the treatment of vascular disorders. In this study, we applied dilong extract to reduce myocardial cell damage from High-KCS infusion and further investigated the mechanisms. H9c2 cardiomyoblast cells were cultured in serum-free medium for 4 h and then treated with dilong at 31.25, 62.5, 125, and 250 mg/mL for 24 h, which was then followed by High-KCS treatment for 3 h to detect the protective mechanisms of dilong behind cardiomyocyte apoptosis and cardiac fibrosis. Cells were harvested for MTT assay, TUNEL assay, and western blot analysis. We found that High-KCS-induced cardiomyocyte apoptosis, enhanced the protein level of pro-apoptotic Bad, released cytochrome c, and activated caspase-3 in H9c2 cells. The IGF-I/IGF-IR/ERK pathway involved in non-cardiomyocyte proliferation, and the expression/activation of uPA, Sp-1 and CTGF, which are implicated in the development of cardiac fibrosis were up-regulated, but the Akt for cardiomyocyte survival was greatly deactivated in postcardioplegic H9c2 cardiomyoblast cells. However, dilong was highly protective and totally reversed the apoptosis and cardiac fibrosis effects induced by High-KCS. Chemical inhibitors P38 (SB203580), JNK (SP600125), MEK (U0126), IGF-1 (AG1024), and PI3K (LY294002) were applied to investigate which is the mediator for dilong attenuated High-KCS stimulated caspase 3 activation. MEK (U0126) inhibitor completely blocked dilong inhibited caspase 3 activation in High-KCS treated H9c2 cells. The MEK siRNA was further applied to knockdown MEK to confirm our finding. We found dilong worked through MEK to inhibit caspase 3 activity induced by High-KCS in H9c2 cells. Furthermore, we used the pure component of dilong, Lumbrokinase, to block the High-KCS effect. Using the microscope to observe the cell viability, we found Lumbrokinase could reverse the High-KCS effect. Lumbrokinase could also reduce the protein levels of caspase 8, caspase 9, and caspase 3, and enhance the survival related proteins PI3K/Akt and Bcl2. These results demonstrate that dilong could be used as a potential agent to block the side effects caused by High-KCS in CABG surgery patients.

[Highly active fractions of the medicinal leech recombinant destabilase-lysozyme].

From the highly purified but lowly active recombinant protein Destabilas-Lysozyme (Dest-Lys) by use cation-exchange column TSK CM 3-SW chromatography, it was separated non-active fraction IV, contained 90% of protein. Fractions I, II and III, represented proteins with lysozyme and isopeptidase activities. Their lysozyme activity correlates with the activity of natural Des-Lys. The ratio of the activities in fractions I - III is such, that maximal lysozyme activity is concentrated in fraction III, isopeptidase - in fraction I. It is discussed the possibility of Dest-Lys different functions regulation is depended on the formation of protein complex forms.

Novel thrombolytic protease from edible and medicinal plant Aster yomena (Kitam.) Honda with anticoagulant activity: purification and partial characterization.

A thrombolytic protease named kitamase possessing anticoagulant property was purified from edible and medicinal plant Aster yomena (Kitam.) Honda. Kitamase showed a molecular weight of 50 kDa by SDS-PAGE and displayed a strong fibrin zymogram lysis band corresponding to the similar molecular mass. The enzyme was active at high temperatures (50°C). The fibrinolytic activity of kitamase was strongly inhibited by EDTA, EGTA, TPCK and PMSF, inhibited by Zn(2+). The Km and Vmax values for substrate S-2251 were determined as 4.31 mM and 23.81 mM/mg respectively. It dissolved fibrin clot directly and specifically cleaved the α, Aα and γ-γ chains of fibrin and fibrinogen. In addition, kitamase delayed the coagulation time and increased activated partial thromboplastin time and prothrombin time. Kitamase exerted a significant protective effect against collagen and epinephrine induced pulmonary thromboembolism in mice. These results suggest that kitamase may have the property of metallo-protease like enzyme, novel fibrino(geno)lytic enzyme and a potential to be a therapeutic agent for thrombosis.

Purification, characterization, and properties of an alkaline protease produced by Serratia marcescens S3-R1 inhabiting Korean ginseng rhizosphere.

BACKGROUND: An alkaline protease produced by the Serratia marcescens S3-R1 which inhabits in the Korean ginseng rhizosphere was investigated. The purposes of this study were to characterize and purify the bacterial enzyme by four different purification steps: precipitation of enzyme fraction by ammonium sulfate, loading the enzyme pellets on a DEAE-Sepharose anion-exchange chromatograph, separation of the fraction containing enzyme activity by fast protein liquid Mono Q chromatography and identification of the single-band fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then quantification of the single-band fraction by reverse-phase high-performance liquid chromatography. RESULTS: The molecular weight of the purified protease was estimated as 50 308 Da by matrix-assisted laser desorption ionization time-of-flight analysis. The N-terminal amino acid sequence of the protease was identified as Ala-Val-Thr-Ile-Glu-Asp-Ala-Val-Asp-Asp, and the enzyme belongs to the metalloprotease family. The optimal activities of the protease occurred at pH 7-9 and a temperature 40 °C. The ranges of pH and thermal stability of the enzyme were at 7-10 and 30-40 °C, respectively. CONCLUSION: The alkaline protease was successfully purified and characterized from the bacterium Serratia marcescens S3-R1, which has potential for industrial application, including milk protein hydrolysates.

Multiple forms of medicinal leech destabilase-lysozyme.

Earlier, three genes Ds1, Ds2, and Ds3 encoding corresponding destabilase-lysozyme isoforms were identified. However only one form of the enzyme encoded by Ds3 gene coincided with the protein CNBr fragments [Mol. Gen. Genet. 253 (1996) 20]. In this work we found by ESI-TOF mass spectrometry that the enzyme preparation consists of at least three forms with molecular masses of 12677.6, 12839.7, and 12938.2Da, each of which contains seven disulfide bridges. Only one mass (12839.7Da) fits to the calculated mass for the protein encoded by Ds3 gene. Further analysis of the CNBr fragments of the enzyme showed the heterogeneity of large 5.5 kDa peptide at positions 64 (threonine or arginine) and 67 (histidine or arginine) in the wild-type amino acid sequence. One CNBr peptide, with Arg and His at positions 64 and 67, respectively, correlates in the molecular mass with the protein encoded by Ds3. In addition, we have found a new acid form of destabilase-lysozyme, P-Ac, which differs from all known destabilase-lysozyme structures by its N-terminal amino acid sequence.

Isolation and partial characterization of rennet-like proteases from Australian cardoon (Cynara cardunculus L.).

The yield, protein content, proteolytic activity, and substrate specificity of crude and partially purified extracts from dried and fresh Australian cardoon (Cynara cardunculus L.) flowers were determined. Crude water extracts had high yield but low protein content and proteolytic activity, whereas citric acid extracts had low yield but high protein content and proteolytic activity. Fresh flower extracts gave higher yield and proteolytic activity but lower protein content in comparison with dried flower extracts. Purification with ammonium sulfate resulted in significantly increased proteolytic activity for water extracts from both fresh and dried cardoon flowers, whereas the proteolytic activity of citric acid extracts did not change significantly after purification. Irrespective of extraction method, all extracts had higher proteolytic activity against ovine whole and kappa-caseins compared to their bovine counterparts, showing optimal activity at 37 degrees C and pH 6.0. Separation of purified extracts by ion-exchange liquid chromatography yielded three active fractions, each of which when assayed with sodium dodecyl sulfate capillary electrophoresis revealed two subunits with molecular masses of 15.5 and 33.1 kDa, respectively.

Characterization and cDNA cloning of halyxin, a heterogeneous three-chain anticoagulant protein from the venom of Agkistrodon halys brevicaudus.

We report upon the isolation, characterization, and cDNA cloning of an anticoagulant protein, halyxin from Agkistrodon halys brevicaudus venom. The protein exists as a 29kDa protein, and is separated into three chains on SDS-PAGE under reducing conditions. However, we cloned only two cDNAs encoding halyxin from the cDNA library of the snake venom gland, on the basis of the determined amino acid sequences. The complete amino acid sequences were deduced from their nucleotide sequences and named halyxin A (129 amino acid residues) and B chain (123 amino acid residues). The deduced amino acid sequence of halyxin A chain corresponds to the two smaller chains. Thus, it is considered that halyxin A chain could be synthesized as a single-chain protein that is subsequently cleaved to yield the mature two-chain protein. The amino acid sequence of halyxin is similar to that of other snake venom proteins of the C-type lectin superfamily, and prolongs plasma-clotting time. In the presence of Ca(2+) ions, halyxin binds to coagulation factors IX, X, IXa, and Xa, but not to other vitamin K-dependent coagulation factors. It also inhibits factor Xa in a non-competitive manner but does not affect other activated coagulation factors.

[Factors influencing the activity of fibrinolytic enzymes from earthworm, Eeisenia foetida].

OBJECTIVE: To study the factors influencing the activity of fibrinolytic enzymes from earthworm and to obtain the better way to extract fibrinolytic enzymes as well as keep its optimum activity. METHOD: 75% alcohol, 0.9% NaCl and 10% saccharose was used to extract the crude fibrinolytic enzymes from earthworm, the method of urokinase gelose-fibrin plate was used to measure the activity of fibrinolytic enzymes from earthworm. and the method of 3,3'-diaminobezidine tetrahydrochloride colorimetry to was used measure the content of selenium. The method use ts of measuring the content of arsenic was silver diethyldithiocarbamate colorimetry. RESULT: The fibrinolytin of earthworms reared with cattle soils had higher activity than that reared with garbage. The arsenic in the earthworm's body could improve the activity of earthworm's fibrinolytin. However, the selenium had litter influence on it. Among the three methods of extraction, the 75% alcohol one was the most efficient, the 0.9% NaCl was next, and the 10% saccharose was the lowest. The influence of dialysis on the activity of fibrinolytin was less than that of ultrafiltration, when the earthworm's fibrinolytin enzyme was further sublimated. CONCLUSION: The activity of the earthworm's fibrinolytin will be increased earthworm is reared with the fitting baits and when appropriate methods, of extraction and purification are used.

[Purification and immobilization of the proteinase from mung bean burgeon inactivating soybean trypsin inhibitor].

By 30%-60% s(NH4)2SO4 fractional precipitation, anion-exchange chromatographs on DEAE-Sepharose CL-6B, gel filtration on Sephacryl S-200 and anion-exchange chromatographs on Waters AP-1 column(ProteinTm-Pak DEAE 15HR), a proteinase which can inactivated STI was purified from mung bean(Phaseolus aureus) burgeon. It was stable at temperatures lower than 50 degrees C and pH7.5-8.5, and the Km and Vmax of the proteinase for STI was 769.2 alpha-N-benzoyl-L-arginine ethyl ester(BAEE)/mL and 115.3BAEE/min/mL respectively. The molecular weight of the proteinase was estimated to be 29.8kD by SDS-PAGE. The proteinase immobilized by polyacrylamide was stable at temperatures lower than 60 degrees C and pH7.0-9.0, and the apparent Km* and Vmax* of the immobilized proteinase for STI was 1303.8 (BAEE)/mL and 94.34(BAEE)/min/mL respectively. The half-life of the immobilized proteinase was about 12 days at 4 degrees C.