Trypsin-like Enzymes Research Articles

Bowman-Birk Protease Inhibitor from Vigna unguiculata Seeds Enhances the Action of Bradykinin-Related Peptides

The hydrolysis of bradykinin (Bk) by different classes of proteases in plasma and tissues leads to a decrease in its half-life. Here, Bk actions on smooth muscle and in vivo cardiovascular assays in association with a protease inhibitor, Black eyed-pea trypsin and chymotrypsin inhibitor (BTCI) and also under the effect of trypsin and chymotrypsin were evaluated. Two synthetic Bk-related peptides, Bk1 and Bk2, were used to investigate the importance of additional C-terminal amino acid residues on serine protease activity. BTCI forms complexes with Bk and analogues at pH 5.0, 7.4 and 9.0, presenting binding constants ranging from 103 to 104 M−1. Formation of BTCI-Bk complexes is probably driven by hydrophobic forces, coupled with slight conformational changes in BTCI. In vitro assays using guinea pig (Cavia porcellus) ileum showed that Bk retains the ability to induce smooth muscle contraction in the presence of BTCI. Moreover, no alteration in the inhibitory activity of BTCI in complex with Bk and analogous was observed. When the BTCI and BTCI-Bk complexes were tested in vivo, a decrease of vascular resistance and consequent hypotension and potentiating renal and aortic vasodilatation induced by Bk and Bk2 infusions was observed. These results indicate that BTCI-Bk complexes may be a reliable strategy to act as a carrier and protective approach for Bk-related peptides against plasma serine proteases cleavage, leading to an increase in their half-life. These findings also indicate that BTCI could remain stable in some tissues to inhibit chymotrypsin or trypsin-like enzymes that cleave and inactivate bradykinin in situ.

Effects of Fraxinellone on the midgut enzyme activities of the 5th Instar Larvae of Oriental Armyworm, Mythimna separata walker.

Isolated from Dictamnus dasycarpus Turcz., fraxinellone exhibited multiple bioactivities against insects. In the present paper, the changes of digestive enzymes and detoxification enzymes of Mythimna separata Walker (5th instar larvae), treated with fraxinellone, were investigated. Compared with those of the control, the α-amylase activity of the fraxinellone-treated 5th instar larvae was inhibited, whereas the level of their protease activity was increased. Based upon further studies on the specific proteases, the levels of the active alkaline trypsin-like enzyme (BApNA as the substrate) and the chymotrypsin-like enzyme (BTEE as the substrate) activities of the treated larvae were declined; however, the level of activity of the weak alkaline trypsin-like enzyme (TAME as the substrate) of the treated ones was increased. Meanwhile, the activities of two detoxification enzymes, such as carboxylesterase (CarE) and glutathione S-transferase (GST), of the treated larvae were increased to some extent, but the activities of NADPH-P450 reductase and O-demethylase of the treated ones declined. Therefore, protease (especially the weak alkaline trypsin-like enzyme), CarE and GST played important roles in the metabolism of fraxinellone in the midgut of Mythimna separata (M. separata).

Effects of Streptococcus thermophilus on volatile sulfur compounds produced by Porphyromonas gingivalis

Halitosis as oral malodour is an unpleasant odour caused by volatile sulfur compounds (VSCs). VSCs are produced primarily by anaerobic bacteria that abundantly produce proteinase as trypsin-like enzyme. General therapies, such as mouthwash and plaque control, do not provide a continuous effect on oral halitosis. Streptococcus thermophilus is a probiotic bacterium that is beneficial for human health. The aim of this study was to evaluate the effect of S. thermophilus on Porphyromonas gingivalis-producing VSCs and to analyze the inhibitory mechanism of halitosis. P. gingivalis was cultured with or without S. thermophilus, and the emission of VSCs from the spent culture medium was measured by gas chromatography. In order to analyze the inhibitory effect, the antibacterial activity of S. thermophilus against P. gingivalis was assessed. After the spent culture medium or whole bacterial of S. thermophilus was mixed with the spent culture medium of P. gingivalis, VSCs were again measured by gas chromatograph. When S. thermophilus and P. gingivalis were co-cultivated, VSCs were present at a lower level than those of single-cultured P. gingivalis. S. thermophilus inhibited growth of P. gingivalis, and the whole bacteria and the spent culture medium of S. thermophilus reduced emission of VSCs gas. S. thermophilus may reduce oral malodour by inhibition of P. gingivalis growth and neutralizing VSCs with their metabolites or themselves.

Correlation between Pr1 and Pr2 gene content and virulence in Metarhizium anisopliae strains.

Metarhizium anisopliae is a widely studied model to understand the virulence factors that participate in pathogenicity. Proteases such as subtilisin-like enzymes (Pr1) and trypsin-like enzymes (Pr2) are considered important factors for insect cuticle degradation. In four M. anisopliae strains (798, 6342, 6345, and 6347), the presence of pr1 and pr2 genes, as well as the enzymatic activity of these genes, was correlated with their virulence against two different insect pests. The 11 pr1 genes (A, B, C, D, E, F, G, H, I, J, and K) and pr2 gene were found in all strains. The activity of individual Pr1 and Pr2 proteases exhibited variation in time (24, 48, 72, and 96 h) and in the presence or absence of chitin as the inductor. The highest Pr1 enzymatic activity was shown by strain 798 at 48 h with chitin. The highest Pr2 enzymatic activity was exhibited by the 6342 and 6347 strains, both grown with chitin at 24 and 48 h, respectively. Highest mortality on S. exigua was caused by strain 6342 at 48 h, and strains 6342, 6345, and 6347 caused the highest mortality 7 days later. Mortality on Prosapia reached 30% without variation. The presence of subtilisin and trypsin genes and the activity of these proteases in M. anisopliae strains cannot be associated with the virulence against the two insect pests. Probably, subtilisin and trypsin enzyme production is not a vital factor for pathogenicity, but its contribution is important to the pathogenicity process.

Spatial characterization of proteolytic enzyme activity in the foregut region of the adult necrophagous fly, Protophormia terraenovae

The spatial distribution of proteolytic enzymes in the adult foregut of Protophormia terraenovae was studied in the context of protein digestion and regurgitation. Based on substrate specificity, pH optima, and use of specific protease inhibitors, all adults tested displayed enzyme activity in the foregut consistent with pepsin, trypsin and chymotrypsin. Chymotrypsin-like and trypsin-like enzyme activity were detected in all gut fluids and tissues tested, with chymotrypsin displaying the highest activity in saliva and salivary gland tissue, whereas maximal trypsin activity was evident in the crop. Pepsin-like activity was only evident in crop fluids and tissues. The activity of all three enzymes was low or undetectable (pepsin) in the fluids and tissue homogenates derived from the esophagus and cardia of any of the adults assayed. Fed adult females displayed higher enzyme activities than fed males, and the activity of all three enzymes were much more prevalent in fed adults than starved. The pH optimum of the trypsin-like enzyme was between pH 7.0 and 8.0; chymotrypsin was near pH 8.0; and maximal pepsin-like activity occurred between pH 1.0 and 2.0. Regurgitate from fed adult females displayed enzyme activity consistent with the proteolytic enzymes detected in crop gut fluids. Enzymes in regurgitate were not derived from food sources based on assays of bovine liver samples. These latter observations suggest that adult flies release fluids from foregut when encountering dry foods, potentially as a means to initiate extra-oral digestion.

Stylet Bundle Morphology and Trophically Related Enzymes of the Hemlock Woolly Adelgid (Hemiptera: Adelgidae)

Abstract The hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), is a pest of eastern and Carolina hemlocks (Tsuga canadensis (L.) Carrière and Tsuga caroliniana Engelmann, respectively) in the eastern United States and has already caused catastrophic changes to eastern forests. As one of the significant exotic forest pests, it is imperative that the basic biology of hemlock woolly adelgid be understood for use in novel and improved management techniques. Scanning and transmission electron microscopy and enzyme assays were used to elucidate the feeding biology of hemlock woolly adelgid and are discussed in the context of the pest—plant interactions and the implications for host plant resistance. Morphological characters indicate that hemlock woolly adelgids may use labial sensilla and neural canals within the mandibular stylets to guide their stylets through close-range host acceptance processes. Stylet bundle insertion is likely assisted by external sheath material that secures the stylet bundle to the plant surface and mandibular dentitions that may assist entry into or within plant tissues. In addition, results support the theory that extra-oral digestion is likely used by hemlock woolly adelgid, suggested by both a narrow food canal and the presence of four trophically related enzymes (a trypsin-like enzyme, an amylase-like enzyme, peroxidase, and polyphenol oxidase). The presence of these enzymes also has implications for causing a systemic response in host trees.

Functional and Structural Characterization of Vibrio cholerae Extracellular Serine Protease B, VesB

The chymotrypsin subfamily A of serine proteases consists primarily of eukaryotic proteases, including only a few proteases of bacterial origin. VesB, a newly identified serine protease that is secreted by the type II secretion system in Vibrio cholerae, belongs to this subfamily. VesB is likely produced as a zymogen because sequence alignment with trypsinogen identified a putative cleavage site for activation and a catalytic triad, His-Asp-Ser. Using synthetic peptides, VesB efficiently cleaved a trypsin substrate, but not chymotrypsin and elastase substrates. The reversible serine protease inhibitor, benzamidine, inhibited VesB and served as an immobilized ligand for VesB affinity purification, further indicating its relationship with trypsin-like enzymes. Consistent with this family of serine proteases, N-terminal sequencing implied that the propeptide is removed in the secreted form of VesB. Separate mutagenesis of the activation site and catalytic serine rendered VesB inactive, confirming the importance of these features for activity, but not for secretion. Similar to trypsin but, in contrast to thrombin and other coagulation factors, Na(+) did not stimulate the activity of VesB, despite containing the Tyr(250) signature. The crystal structure of catalytically inactive pro-VesB revealed that the protease domain is structurally similar to trypsinogen. The C-terminal domain of VesB was found to adopt an immunoglobulin (Ig)-fold that is structurally homologous to Ig-folds of other extracellular Vibrio proteins. Possible roles of the Ig-fold domain in stability, substrate specificity, cell surface association, and type II secretion of VesB, the first bacterial multidomain trypsin-like protease with known structure, are discussed.

Purification of a Trypsin-Like Enzyme and Cloning of Its Gene from Chinese Ground Beetle (E upolyphaga sinensis )

An insect trypsin-like enzyme with similar biochemical properties to psychrophilic analogs was purified from Chinese ground beetle (Eupolyphaga sinensis) for the first time. The purified trypsin-like enzyme was designated as E. sinensis trypsin-like enzyme (ESTL) with molecular weight of 22.8 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The maximal activity of ESTL was observed at pH 9.5, and the temperature optimum of ESTL was observed at 45C by its amidolytic effect on the substrate benzoyl-L-Arg-p-nitroanilide. The analysis of inhibitors showed that specific inhibitor of serine proteases (phenylmethanesulfonyl fluoride) and trypsin inhibitors (tosyl–lysine chloromethyl ketone, bovine pancreatic trypsin inhibitor and benzamidine) inactivated ESTL almost completely. Over the range of tested temperature of 10–30C, the catalytic efficiency (kcat/Km) of the ESTL was about five times than that of bovine trypsin. The N-terminal sequencing of ESTL revealed the following sequence: I1VGGSTTTIQ10NFPYQVSL, and the complementary DNA (cDNA) of ESTL was cloned by rapid amplification of cDNA ends. Practical Applications In recent years, increasing attention has been drawn to the psychrophilic (cold-adapted) enzymes. Psychrophilic trypsins with high activity may be interesting for several industrial applications of enzymes, such as in certain food-processing operations that require low-processing temperatures. For example, cod trypsin is already used in food production and cosmetics. In this article, a trypsin-like enzyme [Eupolyphaga sinensis trypsin-like enzyme (ESTL) ] was purified from Chinese ground beetle by a relatively simple method, and the complementary DNA (cDNA) of this enzyme was cloned by rapid amplification of cDNA ends. This enzyme was characterized for molecular and enzymatic properties, as demonstrated that it had high activity at low and moderate temperature compared with the mammalian trypsins, and maintained good thermal stability compared with psychrophilic trypsins. ESTL has potential interest in food industry.

Metal-sensitive and thermostable trypsin from the crevalle jack (Caranx hippos) pyloric caeca: purification and characterization.

BackgroundOver the past decades, the economic development and world population growth has led to increased for food demand. Increasing the fish production is considered one of the alternatives to meet the increased food demand, but the processing of fish leads to by-products such as skin, bones and viscera, a source of environmental contamination. Fish viscera have been reported as an important source of digestive proteases with interesting characteristics for biotechnological processes. Thus, the aim of this study was to purify and to characterize a trypsin from the processing by-products of crevalle jack (Caranx hippos) fish.ResultsA 27.5 kDa trypsin with N-terminal amino acid sequence IVGGFECTPHVFAYQ was easily purified from the pyloric caeca of the crevalle jack. Its physicochemical and kinetic properties were evaluated using N-α-benzoyl-DL-arginine-p-nitroanilide (BApNA) as substrate. In addition, the effects of various metal ions and specific protease inhibitors on trypsin activity were determined. Optimum pH and temperature were 8.0 and 50°C, respectively. After incubation at 50°C for 30 min the enzyme lost only 20% of its activity. K m , k cat, and k cat /K m values using BApNA as substrate were 0.689 mM, 6.9 s-1, and 10 s-1 mM-1, respectively. High inhibition of trypsin activity was observed after incubation with Cd2+, Al3+, Zn2+, Cu2+, Pb2+, and Hg2+ at 1 mM, revealing high sensitivity of the enzyme to metal ions.ConclusionsExtraction of a thermostable trypsin from by-products of the fishery industry confirms the potential of these materials as an alternative source of these biomolecules. Furthermore, the results suggest that this trypsin-like enzyme presents interesting biotechnological properties for industrial applications.

Kinetics of inhibition of trypsin-like enzyme from Euphausia superba by STI

To study the kinetics of inhibition of trypsin-like protease from Euphausia superba by soybean trypsin inhibitor(STI),the kinetic theory by Tsou of irreversible inactivation was applied to measure its microscopic rate constants.The values of Km and Vmax were 0.155 mmol/L and 8.44 μmol/(L·min),respectively.As the concentra-tion of STI was increased,the activity decreased with IC50 of 2.8 μg/mL.STI slowly,reversibly and competitively inhibited the enzyme activity at low concentration.The rate constant of the forward inactivation(k+0) was 0.184 min-1,which was about five times higher than the value of 0.039 4 min-1 for the reverse reactivation(k-0).There-fore,at sufficiently high STI concentrations,the enzyme would be completely inactivated.This study provides experimental data for further research into the inhibition technology of the trypsin-like protease from E.superba.

Effects of an artificial diet on development, reproduction and digestive physiology of Chrysopa septempunctata

The effects of an artificial diet on growth, development, reproduction and digestive physiology of Chrysopa septempunctata Wesmael were determined. The results showed that the reproduction was highly correlated with weights of females and activities of trypsin-like and chymotrypsin-like enzymes of C. septempunctata. The influences of diet on the digestive physiology such as digestive enzyme activity of a predator may be used to assess the reproductive potential of a predator, and therefore to provide a way to speed up the development and screening of artificial diets.

Serine Proteinases Secreted by Two Isolates of the Fungus Alternaria solani

It is well-known Alternaria solani Sorauer is the causative agent of alternariosis. In this paper, serine proteinases secretion by two genetically related isolates of the fungus, collected from potato and tomato plants grown in central Russia have been studied. The data clarify functions of these enzymes in the process of pathogenesis in which they can play a pivotal role. Also, the data should allow classifying Alternaria’s strains more precisely. It was found that the two isolates produced trypsin-like and subtilisin-like proteinases during growth both in synthetic culture medium and in medium containing heat-stable vegetable proteins. There were significant differences in the influence of the environment on the serine proteinase secretion by the potato and tomato isolates of A. solani. The proportion of such serine proteinases as trypsin-like and subtilisin-like enzymes depends on the composition of the growth medium, especially on the available organic nitrogen form, as well as features both of the pathogenic fungus and of the host plant. So, the tomato isolate demonstrated weak growth and low level or absence of serine proteinase excretion on cultivation with the medium containing proteins extracted from potato tubers and pea seeds. The potato isolate secreted many more serine proteinases, among which the trypsin-like enzymes dominated. Our data suggest that the tomato isolate, when grown on medium with proteins extracted from potato tubers, lost pathogenicity and became to behave as a saprophyte, while the potato isolate retained its pathogenic properties on growth on any tested medium.

Методические подходы к получению фракции трипсиноподобных ферментов из морских гидробионтов (на примере антарктического криля)

The rapid development of biotechnology and pharmacology of the our days, one way or another, related to the targeted search of biologically active substances in the tissues of a wide variety of organisms, including the antarctic marine organisms. This group of organisms has attracted the attention of researchers due to its scarcity, as well as the presence of various protein composition. A promising direction is the creation of pharmaceutical products, based on proteins extracted from tissues of antarctic marine animals. Of particular interest is the possibility of molecules with directed action – enzymes with different specificity. In our work submitted the development methodological approaches for the isolation and testing fraction of trypsin-like enzymes from the tissues of the antarctic krill (Euphausia superba).

Selective inhibitors of digestive enzymes from Aedes aegypti larvae identified by phage display

Dengue is a serious disease transmitted by the mosquito Aedes aegypti during blood meal feeding. It is estimated that the dengue virus is transmitted to millions of individuals each year in tropical and subtropical areas. Dengue control strategies have been based on controlling the vector, Ae. aegypti, using insecticide, but the emergence of resistance poses new challenges. The aim of this study was the identification of specific protease inhibitors of the digestive enzymes from Ae. aegypti larvae, which may serve as a prospective alternative biocontrol method. High affinity protein inhibitors were selected by all of the digestive serine proteases of the 4th instar larval midgut, and the specificity of these inhibitors was characterized. These inhibitors were obtained from a phage library displaying variants of HiTI, a trypsin inhibitor from Haematobia irritans, that are mutated in the reactive loop (P1–P4′). Based on the selected amino acid sequence pattern, seven HiTI inhibitor variants were cloned, expressed and purified. The results indicate that the HiTI variants named T6 (RGGAV) and T128 (WNEGL) were selected by larval trypsin-like (IC50 of 1.1 nM) and chymotrypsin-like enzymes (IC50 of 11.6 nM), respectively. The variants T23 (LLGGL) and T149 (GGVWR) inhibited both larval chymotrypsin-like (IC50 of 4.2 nM and 29.0 nM, respectively) and elastase-like enzymes (IC50 of 1.2 nM for both). Specific inhibitors were successfully obtained for the digestive enzymes of Ae. aegypti larvae by phage display. Our data also strongly suggest the presence of elastase-like enzymes in Ae. aegypti larvae. The HiTI variants T6 and T23 are good candidates for the development as a larvicide to control the vector.

Elevated CO2 Reduces the Resistance and Tolerance of Tomato Plants to Helicoverpa armigera by Suppressing the JA Signaling Pathway

Both resistance and tolerance, which are two strategies that plants use to limit biotic stress, are affected by the abiotic environment including atmospheric CO2 levels. We tested the hypothesis that elevated CO2 would reduce resistance (i.e., the ability to prevent damage) but enhance tolerance (i.e., the ability to regrow and compensate for damage after the damage has occurred) of tomato plants to the cotton bollworm, Helicoverpa armigera. The results showed that elevated CO2 reduced resistance by decreasing the jasmonic acid (JA) level and activities of lipoxygenase, proteinase inhibitors, and polyphenol oxidase in wild-type (WT) plants infested with H. armigera. Consequently, the activities of total protease, trypsin-like enzymes, and weak and active alkaline trypsin-like enzymes increased in the midgut of H. armigera when fed on WT plants grown under elevated CO2. Unexpectedly, the tolerance of the WT to H. armigera (in terms of photosynthetic rate, activity of sucrose phosphate synthases, flower number, and plant biomass and height) was also reduced by elevated CO2. Under ambient CO2, the expression of resistance and tolerance to H. armigera was much greater in wild type than in spr2 (a JA-deficient genotype) plants, but elevated CO2 reduced these differences of the resistance and tolerance between WT and spr2 plants. The results suggest that the JA signaling pathway contributes to both plant resistance and tolerance to herbivorous insects and that by suppressing the JA signaling pathway, elevated CO2 will simultaneously reduce the resistance and tolerance of tomato plants.

Purification of a membrane-bound trypsin-like enzyme from the gut of the velvetbean caterpillar (Anticarsia gemmatalis Hübner)

Disruption of protein digestion in insects by specific endoprotease inhibitors is being regarded as an alternative to conventional insecticides for pest control. To optimize the effectiveness of this strategy, the understanding of the endoprotease diversity of the target insect is crucial. In this sense, a membrane-bound trypsin-like enzyme from the gut of Anticarsia gemmatalis fifth-instar larvae was purified. Non-soluble fraction of the gut extract was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and subjected to a p-aminobenzamidine affinity chromatography followed by anion-exchange chromatography. The yield of the purified enzyme was 11% with a purification factor of 143 and a final specific activity of 18.6 µM min.-1 mg-1 protein using N-α-benzoyl-L- Arg-p-nitroanilide (L-BApNA) as substrate. The purified sample showed a single band with proteolytic activity active and apparent molecular mass of 25 kDa on SDS-PAGE. Molecular mass determined by MALDI-TOF mass spectrometry was 28,632 ± 26 Da. Although the low recovery and the difficulties in purifying large enzyme amounts limited its further characterization, the results contribute for the understanding of the proteases present on A. gemmatalis gut, which are potential targets for natural or specifically designed protease inhibitors.

Proteinase-Activated Receptor-4 Plays a Major Role in the Recruitment of Neutrophils Induced by Trypsin or Carrageenan during Pleurisy in Mice

Background/Aims: The activation of proteinase-activated receptors (PARs) has been implicated in the development of important hallmarks of inflammation, including in vivo leukocyte recruitment. Here, we examined the effects of aprotinin, a potent inhibitor of trypsin proteinase and the kallikrein-kinin system, and the PAR-4 antagonist YPGKF-NH₂ (tcY-NH₂) on neutrophil recruitment in response to carrageenan and trypsin in the pleural cavity of mice. Methods: BALB/c mice were intrapleurally injected with trypsin or PAR-4-activating peptide AY-NH₂, pretreated with aprotinin or tcY-NH₂ (1 µg/cavity) prior to an intrapleural injection of trypsin or carrageenan, or pretreated with leukotriene B₄ antagonist U-75302 (3 µg/cavity) prior to a trypsin injection. The number of infiltrating neutrophils was evaluated after 4 h. Results: PAR-4-activating peptide AY-NH₂ and trypsin-induced neutrophil recruitment was inhibited by aprotinin, tcY-NH₂ or U-75302. Aprotinin and tcY-NH₂ also inhibited neutrophil recruitment induced by carrageenan. Conclusion: These data suggest a key role for PAR-4 in mediating neutrophil recruitment in a mouse model of pleurisy induced by the activity of trypsin or trypsin-like enzymes.

Allergen‐derived proteinases: Isolation, characterization and signaling via proteinase‐activated receptors (PARs)

ObjectiveTo identify proteolytic enzymes in insect and mould asthma allergens and assess their ability to signal via the PARs.MethodsAllergen‐derived trypsin‐like enzymes known to contribute to lung inflammation were visualized (western blot) with serine proteinase activity‐based probes (ABP). Enzymes were purified using ion‐exchange chromatography and analyzed for their ABP reactivity & substrate‐inhibitor kinetic profiles. Each enzyme was tested for its activation of PAR–dependent calcium signaling in PAR1 & PAR2‐expressing KNRK cells. ABP‐labeled enzymes were isolated by avidin affinity chromatography and their peptide sequences determined (mass spectrometry).ResultsBoth allergens contained ABP‐labeled proteinases with MWs of 20–26 kDa. Each enzyme had distinct kinetic substrate (Km) and inhibitor (Ki) profiles. The enzymes all caused PAR2 calcium signaling, and differentially activated PAR1. Sequencing revealed that the allergen proteinases were homologous with previously described trypsin‐like enzymes.ConclusionsDiverse allergens contain multiple biochemically distinct trypsin‐like proteinases that are able to target PARs 1 and 2. Thus, PARs and their activating allergen proteinases may be attractive therapeutic targets for the treatment of allergen‐induced asthma.Support: Canadian Institutes of Health Research, NIH USA and Lung Association of Alberta & NWT

PURIFICATION AND CHARACTERISTICS OF TRYPSIN FROM THE PANCREAS OF TIBETAN SHEEP

ABSTRACT Trypsin from the pancreas of Tibetan sheep was purified to 7.4-fold by ammonium sulphate and acetone precipitation, followed by Sephacryl S-200 (Whatman, Maidstone, England) and Sephadex G-75 (Whatman) gel filtration, with an 23.2-fold increase in specific activity and 13.6% yield. The final enzyme preparation was nearly homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the molecular weight of the enzyme was estimated to be approximately 27 kDa by SDS-PAGE. Trypsin-like enzymes had maximal activities at around pH 9.0 and 60C for the hydrolysis of Nα-ρ-Tosyl–L–arginine methyl ester hydrochloride (TAME). Trypsin was unstable above 60C and below pH 4.0, and was stabilized by calcium ions. Purified trypsin had a Michaelis–Menten constant (Km) and catalytic constant (Kcat) of 0.53 mM and 206 s−1, respectively, when TAME was used as the substrate. The specific trypsin inhibitors, soybean trypsin inhibited, Nα-ρ-tosyl-L-lysine chloromethyl ketone and phenyl methyl sulfonyl fluoride, strongly inhibited the activity of trypsin, while other protease inhibitors exhibited negligible inhibition. The result suggests that major proteinase in the pancreas of Tibetan sheep was trypsin-like serine proteinase. PRACTICAL APPLICATIONS Tibetan sheep is one of the unique livestock resources in western China. It was found that the trypsin extraction and purification from waste pancreas have the biological characteristics of the heat resistance, alkali resistance, high specific activity and high hydrolysis efficiency. It does not only serve as a better research tool enzyme, but is also widely applied in the food, pharmaceutical, textile and other industries. This paper provides the scientific research basis of the development of the waste pancreas for the Tibetan sheep, therefore reducing environmental pollution.

In vitro fertilization of the rock shrimp, Rhynchocinetes typus (Decapoda, Caridea): a review.

This review compiles all the research done on gametes and fertilization in the rock shrimp, R. typus, and describes the sequence of events from the first gamete interaction to zygote formation and the first cleavage of the embryo, with light, fluorescence confocal and electron microscopes. Early studies showed that sperm from the vas deferens have a tack-shape with a "needle-like process" or rigid spike (RS) that extends from a semi-spherical body that contains the arms with chromatin and spines. Upon contact with seawater and by action of Na +, the arms and spines extend, producing an inverted umbrella form of the spermatozoa. The first sperm-oocyte interaction occurs between protein receptors type lectins of the sperm RS and oocyte chorion sperm ligands. These ligands contain residues of α-Glu, Man (α 1-3) Man, α and β-GlcNAc and α-GalNA terminal residues. It was found that α-Man and GlcNAc residues are the ligands that are directly related to the adhesion process and further penetration of sperm. After this first interaction, the RS enters the oocyte envelope by the action of a trypsin-like enzyme, rhynchocinecine, present in the acicular process. Later, arms and spines penetrate the oocyte cytoplasm, where the chromatin of the arms begin to migrate to the central area of the sperm, condensing in a cup-shaped structure near the connecting piece, which forms the male pronucleus.