Soybean Trypsin Inhibitor Research Articles

Purification and Characterization of Trypsin From the Intestine of Genetically Improved Nile Tilapia (Oreochromis niloticus)

Trypsin, with molecular weight of 28 kDa from the intestine of genetically improved Nile tilapia (Oreochromis niloticus), was purified by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography. Purified trypsin had maximal activity at pH 8.0 and 60°C for hydrolysis of N α-p-tosyl-L-arginine methyl ester. The enzyme was stable at temperatures up to 50°C and pH range of 6.0–11.0. Its activity was strongly inhibited by metal ions such as Pb2+ and Fe3+ and protease inhibitors including soybean trypsin inhibitor and phenylmethylsulfonyl fluoride. Also, the ion Ca2+ slightly inhibited this activity. The Michaelis-Menten constant (K m) and catalytic constant (K cat) of purified trypsin were 0.036 mM and 152 s−1, respectively. Furthermore, trypsin contained low amounts of hydrophobic and aromatic amino acids as well as β-sheet (20.2%) and β-turn (25.0%).

Calcium and Calmodulin-dependent Serine/Threonine Protein Kinase Type II (CaMKII)-mediated Intramolecular Opening of Integrin Cytoplasmic Domain-associated Protein-1 (ICAP-1α) Negatively Regulates β1 Integrins

Focal adhesion turnover during cell migration is an integrated cyclic process requiring tight regulation of integrin function. Interaction of integrin with its ligand depends on its activation state, which is regulated by the direct recruitment of proteins onto the β integrin chain cytoplasmic domain. We previously reported that ICAP-1α, a specific cytoplasmic partner of β1A integrins, limits both talin and kindlin interaction with β1 integrin, thereby restraining focal adhesion assembly. Here we provide evidence that the calcium and calmodulin-dependent serine/threonine protein kinase type II (CaMKII) is an important regulator of ICAP-1α for controlling focal adhesion dynamics. CaMKII directly phosphorylates ICAP-1α and disrupts an intramolecular interaction between the N- and the C-terminal domains of ICAP-1α, unmasking the PTB domain, thereby permitting ICAP-1α binding onto the β1 integrin tail. ICAP-1α direct interaction with the β1 integrin tail and the modulation of β1 integrin affinity state are required for down-regulating focal adhesion assembly. Our results point to a molecular mechanism for the phosphorylation-dependent control of ICAP-1α function by CaMKII, allowing the dynamic control of β1 integrin activation and cell adhesion.

Development of an In Situ Detachment Protocol of Vero Cells Grown on Cytodex1 Microcarriers Under Animal Component-Free Conditions in Stirred Bioreactor

Subcultivation of Vero cells grown in a proprietary animal component-free medium named IPT-AFM, on microcarriers, was studied. TrypLE Select, a non-animal-derived protease, was used as an alternative to trypsin for cell passaging. We first studied the effect of increasing concentrations of TrypLE Select toward cell growth and then studied the inactivation of the protease using either soybean trypsin inhibitor (STI) or the soy hydrolysate Hypep 1510, in six-well plates. Data showed that cell growth was impaired by residual level of TrypLE Select; STI was identified as an efficient agent to neutralize this effect. To restore cell growth and inactivate TrypLE Select, STI should be added to the medium at least at 0.2 g L(-1). Cells were also grown in spinner flask on 2 g L(-1) Cytodex1 in IPT-AFM. In these conditions, the cell detachment yield was equal to 78 ± 8 %. Furthermore, cells exhibited a typical growth profile when using the dislodged cells to seed a new culture. A cell detachment yield of 70 ± 19 % was also achieved when the cells were grown in a 2-L stirred bioreactor in IPT-AFM, on 3 g L(-1) Cytodex1. This protocol can be of great interest to scale-up the process of Vero cells cultivation in IPT-AFM on Cytodex1 from one stirred bioreactor culture to another.

Pancreatic secretory trypsin inhibitor causes autocrine-mediated migration and invasion in bladder cancer and phosphorylates the EGF receptor, Akt2 and Akt3, and ERK1 and ERK2

Pancreatic secretory trypsin inhibitor (PSTI) is expressed in most bladder carcinomas, where its pathophysiological relevance is unclear. Using recombinant normal sequence PSTI/tumor-associated trypsin inhibitor (TATI), a variant associated with familial pancreatitis (N34S), an active site-inactivated variant (R18/V19), and immunoneutralization and RNA interference-mediated knockdown techniques, we investigated the actions of PSTI/TATI on cell migration (wounding monolayers), collagen invasion (gel invasion assays), and proliferation (Alamar blue) on 253J, RT4, and HT1376 human bladder carcinoma cell lines. All three forms of PSTI/TATI stimulated migration twofold, and normal sequence PSTI/TATI showed synergistic promigratory effects when added with EGF. Addition of structurally unrelated soybean trypsin inhibitor had no promigratory activity. Similar results were seen using collagen invasion assays, although the active site mutated variant had no proinvasive activity, probably due to reduced Akt2 activation. PSTI/TATI did not stimulate proliferation despite acting, at least partially, through the EGF receptor, as effects of PSTI/TATI were truncated by the addition of an EGF receptor blocking antibody or the tyrosine kinase inhibitor tyrphostin. Cell lines produced endogenous PSTI/TATI, and PSTI/TATI RNA interference knockdown or the addition of PSTI/TATI, EGF receptor, or tyrphostin blocking agents reduced migration and invasion below baseline. PSTI/TATI induced phosphorylation of the EGF receptor, ERK1 and ERK2, Akt2 and Akt3, JNK1, MKK3, and ribosomal protein S6 kinase 1. This profile was more limited than that induced by EGF and did not include Akt1, probably explaining the lack of proproliferative activity. Our findings of autocrine stimulation and synergistic responses between EGF and PSTI/TATI at concentrations found in urine and tissue suggest that PSTI/TATI has pathophysiological relevance.

A comparison study of the impact of boiling and high pressure steaming on the stability of soybean trypsin inhibitor

SummaryThe aim of the study is to compare the effect of boiling and high pressure steaming (HPS) on the degradation, inhibitory activity reduction and gastric digestibility of soybean trypsin inhibitor (STI). Thermal stability analysis showed that HPS treatment was effective in eliminating the inhibitory activity of STI than boiling. SDS‐PAGE and Western blot analysis indicated that boiling has less impact on the gastric digestibility of STI than HPS. More importantly, boiling‐pretreated STI revealed high inhibitory activity against trypsin even after digestion by pepsin in simulated gastric fluid (SGF), while HPS treatment was more effective. SDS‐PAGE analysis further verified that after boiling, STI still revealed strong binding ability to trypsin, while STI could be completely degraded by trypsin after HPS treatment for 30 min.

Secretion of miraculin through the function of a signal peptide conserved in the Kunitz-type soybean trypsin inhibitor family

Miraculin, a glycoprotein that modifies sour tastes into sweet ones, belongs to the Kunitz-type soybean trypsin inhibitor (STI) family. To clarify the functional relation of miraculin with Kunitz-type STIs, we investigated its subcellular localization and trypsin inhibitory activity. In transgenic Arabidopsis thaliana, miraculin, fused to yellow fluorescent protein, localized to and outside the plasma membrane depending on the putative secretion signal peptide. When transgenic seedlings were cultured in liquid medium, miraculin was present in the supernatant only after cellulase treatment. No trypsin inhibitory activity was detected in native or recombinant miraculin. In conclusion, miraculin is secreted outside the plasma membrane through the function of a signal peptide, conserved in Kunitz-type STIs, whereas its trypsin inhibitory activity may be lost during its evolution.

Trypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991: Its purification and characterization

Pterygoplichthys disjunctivus viscera trypsin was purified by fractionation with ammonium sulphate, gel filtration, affinity and ion exchange chromatography (DEAE-Sepharose). Trypsin molecular weight was approximately 27.5kDa according to SDS–PAGE, shown a single band in zymography. It exhibited maximal activity at pH 9.5 and 40°C, using N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulphonyl fluoride (PMSF) (100%), N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK) (85.4%), benzamidine (80.2%), and soybean trypsin inhibitor (75.6%) and partially inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (10.3%), ethylendiaminetetraacetic acid (EDTA) (8.7%) and pepstatin A (1.2%). Enzyme activity was slightly affected by metal ions (Fe2+>Hg2+>Mn2+>K+>Mg2+>Li+>Cu2+). Trypsin activity decreased continuously as NaCl concentration increased (0–30%). Km and kcat values were 0.13mM and 1.46s−1, respectively. Results suggest the enzyme have a potential application where room processing temperatures (25–35°C) or high salt (30%) concentration are needed, such as in fish sauce production.

Determining the role(s) of prime‐side residues in macromolecular inhibition of trypsin‐fold serine proteases

Trypsin‐fold serine proteases are among the most abundant of all proteases. By identifying residues that are important for enzyme‐inhibitor interaction in trypsin, we may advance the development of protease‐based therapies in general. Lysine‐60 (K60) is a highly conserved prime‐side residue that may play many roles in trypsin. In co‐crystals, K60 hydrogen bonds to tyrosine‐39 (Y39) and is positioned to restrict conformational mobility of phenylalanine‐41 (F41), possibly limiting the observed hydrogen bond interactions between those residues and macromolecular inhibitors. Substitution of K60 with other amino acid residues may disrupt these interactions and provide insight to their significance with respect to inhibitor binding. To test this hypothesis, we created trypsin variants K60G, K60A, K60V, K60I, and K60R and characterized them with respect to their activities and sensitivities to the macromolecular inhibitors soybean trypsin inhibitor (SBTI) and bovine pancreatic trypsin inhibitor (BPTI). Our initial results show that, compared to wild‐type trypsin, K60A and K60V are catalytically indistinguishable, more resistant to autolysis and more sensitive to inhibition by SBTI and BPTI. Further experiments with ecotin and other trypsin variants can provide additional insight on specific interactions. This work was supported by NSF CAREER Award MCB‐0643988–02 and NIH MARC T34‐GM008574.

Rice Os9BGlu31 Is a Transglucosidase with the Capacity to Equilibrate Phenylpropanoid, Flavonoid, and Phytohormone Glycoconjugates

Glycosylation is an important mechanism of controlling the reactivities and bioactivities of plant secondary metabolites and phytohormones. Rice (Oryza sativa) Os9BGlu31 is a glycoside hydrolase family GH1 transglycosidase that acts to transfer glucose between phenolic acids, phytohormones, and flavonoids. The highest activity was observed with the donors feruloyl-glucose, 4-coumaroyl-glucose, and sinapoyl-glucose, which are known to serve as donors in acyl and glucosyl transfer reactions in the vacuole, where Os9BGlu31 is localized. The free acids of these compounds also served as the best acceptors, suggesting that Os9BGlu31 may equilibrate the levels of phenolic acids and carboxylated phytohormones and their glucoconjugates. The Os9BGlu31 gene is most highly expressed in senescing flag leaf and developing seed and is induced in rice seedlings in response to drought stress and treatment with phytohormones, including abscisic acid, ethephon, methyljasmonate, 2,4-dichlorophenoxyacetic acid, and kinetin. Although site-directed mutagenesis of Os9BGlu31 indicated a function for the putative catalytic acid/base (Glu(169)), catalytic nucleophile residues (Glu(387)), and His(386), the wild type enzyme displays an unusual lack of inhibition by mechanism-based inhibitors of GH1 β-glucosidases that utilize a double displacement retaining mechanism.

Use of Protease Inhibitors in Composite Polyelectrolyte Microparticles in Order to Increase the Bioavailability of Perorally Administered Encapsulated Proteins

Protein protease inhibitors (aprotinin, soybean Bowman–Birk inhibitor, and Kunitz soybean trypsin inhibitor) possessing different specificity with respect to trypsin, chymotrypsin, and elastase were encapsulated together with a cargo protein in polyelectrolyte microparticles using layer-by-layer (LbL) deposition techniques. The most efficient inclusion of the inhibitors occurred at the formation stage of the insoluble protein complex with the polyanion. Simultaneous immobilization of the inhibitor and protein did not influence the physicochemical properties of the microparticles, specifically their pH-sensitive behavior under conditions modeling the passage through various parts of the human gastrointestinal tract after peroral administration. The most effective protection against the action of proteolytic enzymes of pancreatic juice and the small intestine was achieved for simultaneous release of cargo protein and inhibitor from the microparticles. Soybean Bowman–Birk inhibitor, which is most similar to insulin with respect to physicochemical properties, in addition to the soybean extract enriched with protease inhibitors were the most suitable agents for protection of human insulin or its rapidly acting analogs (lispro and aspart). These findings suggested that simultaneous microencapsulation of both protein and protein protease inhibitor was a promising way to increase the protein bioavailability upon peroral administration of polyelectrolyte microparticles.

Compensating Effects of Urea and Trimethylamine-N-Oxide on the Heteroassociation of α-Chymotrypsin and Soybean Trypsin Inhibitor

An assay for the determination of the equilibrium constant for heteroassociation of α-chymotrypsin and soybean trypsin inhibitor via fluorescence depolarization is described. Results obtained at neutral pH in saline buffer were consistent with prior determinations via sedimentation equilibrium and static light scattering. The dependence of the association equilibrium constant upon the concentrations of urea and trimethylamine-N-oxide (TMAO) added individually and in mixtures was determined at several temperatures. It was found that subdenaturing concentrations of urea decrease the extent of heteroassociation and that added TMAO increases the extent of heteroassociation. The effects of both cosolutes in mixtures upon the equilibrium heteroassociation of α-chymotrypsin and soybean trypsin inhibitor appear to be additive. A thermodynamic analysis of the combined results is presented.

Combined effects of three crystalline toxins from Bacillus thuringiensis with seven proteinase inhibitors on beet armyworm, Spodoptera exigua Hübner (Lepidoptera: Noctuidae)

Spodoptera exigua is a highly polyphagous pest and causes extensive damage to many field and truck crops. Since the larvae are not or only sublethally affected by Cry1Ac, the fused Cry1Ac/Cry1Ab, or the stacked Cry1Ac+CpTI transgenic cottons that are widely planted in China, S. exigua has become a major economic pest of cotton across a wide distribution since the commercialization of Bt cottons in 1997 in China. Proteinase inhibitors are potential candidates for enhancing Bt toxicity against, and for expanding control spectrum for insect pests. In the present paper, we first found that S. exigua larval midgut fluids could remarkably degrade activated Cry1Ca, and slightly hydrolyze Cry1Ac and Cry1Ab. Subsequently, we investigated interactions between the 3 Cry toxins and 7 proteinase inhibitors, i.e., phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, tannic acid, N-α-tosyl-l-phenylalanine chloromethyl ketone, N-α-tosyl-l-lysine chloromethyl ketone, elastatinal and ethylenediaminetetraacetic acid (EDTA), by monitoring larval growth and mortality rate. A 6-day dietary exposure of the newly molted 2nd instars to either the inhibitors (3 test concentrations) or the toxins (Cry1Ac, 31.30ng/cm2; Cry1Ab, 3.2ng/cm2; Cry1Ca, 0.6ng/cm2) alone only slightly affected larval growth. In contrast, exposure to the mixtures containing an inhibitor and a toxin, with the exception of those containing EDTA, synergistically reduced larval weight. In general, the synergisms were more obvious at higher inhibitor concentrations. Regarding larval mortality, the inhibitors except EDTA at the highest test concentration showed significant synergism to both Cry1Ab (0.2μg/cm2) and Cry1Ca (0.04μg/cm2). As the inhibitor concentration reduced, however, the synergistic effects decreased. These results indicated that trypsin-, chymotrypsin-, and elastase-like proteinases in S. exigua larval midgut were involved in proteolytical hydrolyzation of the 3 activated Cry toxins, and protection of Bt Cry toxins from proteinase degradation in the midgut by inhibitors may greatly enhance toxicity against S. exigua larvae.

The mutual influence of proteins from Varroa destructor extracts and from honeybee haemolymph on their proteolytic activity — in vitro study

The influence of extracts from Varroa destructor, a parasitic mite of the honeybee Apis mellifera, on the proteinase activity of worker bee haemolymph was analysed in vitro, along with the influence of bee haemolymph on the proteolytic activity of V. destructor extract. The study was conducted in three different environments: pH 7.5 (high activity of bee enzymes and very low activity of parasite enzymes), pH 5 (moderate activity of enzymes from both sources) and pH 3.5 (limited activity of bee proteinases and high activity of mite proteinases). Based on electrophoretic studies, the inhibition of the activity of bee haemolymph proteinases by V. destructor extracts was observed at each pH. The study at pH 7.5 with commercial inhibitors of the 4 main classes of proteinases (pepstatin A, ethylenediaminetetraacetic acid (EDTA), E-64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane), soybean trypsin inhibitor and Kunitz inhibitor) suggested that parasite extracts mainly inhibited serine proteinases and, to a lower degree, cysteine and aspartyl proteinases. At pH 3.5 and pH 5, a decrease of approximately 40% in parasite proteinase activity was also observed in the presence of bee haemolymph. The result points to the presence of aspartyl proteinase inhibitors in bee haemolymph, which may be an important defence element for bees during food intake by a mite. It was demonstrated that trypsin and trypsin inhibitors are active in the excretion/secretion products of V. destructor, the proteinases of which may assist the parasite in food suckling by preventing haemolymph coagulation, among other things.

Quantitative Characterization of the Interaction between Sucrose and Native Proteins via Static Light Scattering

Compatible osmolytes, such as sucrose, are small organic compounds that are synthesized in cells to protect proteins and other macromolecules from the effect of osmotic stress. Our present work is to quantitatively characterize the interactions between native globular proteins and sucrose, which has long been known to stabilize proteins against denaturation by heat or chaotropes, and has been shown to attenuate structural fluctuations in native proteins. The composition-dependent static light scattering of binary mixtures of each of four dilute globular proteins--bovine serum albumin (BSA), ovalbumin, ovomucoid, soybean trypsin inhibitor (STI), and sucrose were measured over a broad range of sucrose concentrations. A conventional analysis of the dependence of excess light scattering of a single macrosolute in a continuum solvent yields unphysical results. Then the analysis is based upon multicomponent scattering theory, treating sucrose as well as protein as a scattering species, and is shown to yield reliable information about the nature and magnitude of solute-solute interactions in protein-sucrose mixtures. The results could be satisfactorily accounted for by an effective hard particle model that indicates the nature of the underlying interactions between sucrose and each protein.

Protein-modified silver nanoplates for the complementary analytical method of localised surface plasmon resonance and matrix assisted laser desorption/ionisation mass spectrometry

A complementary analytical methodology combining localised surface plasmon resonance (LSPR) sensing and matrix assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) has been developed by using triangular silver nanoplates (Ag NPLs). Ag NPLs with near-IR LSPR absorbance were prepared via a two-step photo-mediated growth process from Ag nanoparticles. These could be utilised as a common platform for LSPR/MALDI-MS, as they were suitable for LSPR sensing via the analysis of surface plasmon absorption bands and as the assisting material for LDI-MS. For practical use, the detection of analytes by LSPR sensing can be achieved by using specific biomolecular recognition techniques that involve the surface modification of metal NPs with large molecules such as proteins. We investigated the effect on LSPR sensitivity (i.e. refractive index unit, RIU) of modifying the Ag surface with molecules of different sizes, and found that the RIU values were proportional to the amplitude of the cube root of the molecular weight, Mw1/3. We demonstrated the utility of this complementary LSPR and MALDI-MS analysis methodology by evaluating the binding of a soybean trypsin inhibitor to the Ag NPL substrate covalently modified with trypsin. This specific biomolecule recognition phenomenon was first detected using the LSPR technique, and then successfully identified by MALDI-MS.

Influence of γ-Radiation on the Structure and Function of Soybean Trypsin Inhibitor

Soybean trypsin inhibitor (STI) is a known antinutrient and food allergen present in soybean. γ-Radiation has the potential to inactivate the TI protein. However, a systematic study on the influence of different moisture levels during γ radiation on structure and function of the molecule has not been reported. Pure STI was irradiated up to 200 kGy, in dry state, with 50% moisture and in aqueous solution. The radiation damage in molecular structure was assessed using, SDS-PAGE, size exclusion chromatography, fluorescence measurement, and circular dichroism, while functional damage was assessed by the TI assay. In aqueous solution, both the structure and function of TI were almost destroyed at the 10 kGy dose. While with 50% moisture and in dry state, the loss in functional and structural attributes was discernible only at 30 and 100 kGy, respectively. The TI activity was found to be unaffected in dry and soaked seeds of soybean as well as other legumes up to irradiation doses of 100 and 50 kGy, respectively.

Structural basis for dual inhibitory role of tamarind Kunitz inhibitor (TKI) against factor Xa and trypsin

A Kunitz type dual inhibitor (TKI) of factor Xa (FXa) and trypsin was found in tamarind. It also shows prolongation of blood coagulation time. The deduced 185 amino acid sequence of TKI by cDNA cloning and sequence analysis revealed that it belongs to the Kunitz type soybean trypsin inhibitor (STI) family; however, it has a distorted Kunitz signature sequence due to insertion of Asn15 in the motif. TKI exhibited a competitive inhibitory activity against both FXa (K(i) =220nm) and porcine pancreatic trypsin (K(i) =3.2nm). The crystal structure of TKI shows a β-trefoil fold similar to Kunitz STI inhibitors; however, a distinct mobile reactive site, an inserted residue and loop β7β8 make it distinct from classical Kunitz inhibitors. The crystal structure of TKI-trypsin and a 3D model of TKI-FXa complex revealed that the distinct reactive site loop probably plays a role in dual inhibition. The reactive site of TKI interacts with an active site and two exosites (36 loop and autolysis loop) of FXa. Apart from Arg66 (P1), Arg64 (P3) is one of the most important residues responsible for the specificity of TKI towards FXa. Along with the reactive site loop (β4β5), loops β1 and β7β8 also interact with FXa and could further confer selectivity for FXa. We also present the role of inserted Asn15 in the stabilization of complexes. To the best of our knowledge, this is the first structure of FXa inhibitor belonging to the Kunitz type inhibitor family and its unique structural and sequence features make TKI a novel potent inhibitor. The complete nucleotide of TKI was deposited in the NCBI gene databank with accession no. HQ385502. The atomic coordinates and structure factor files for the structure of TKI and TKI:PPT complex have been deposited in the Protein Data Bank with accession numbers 4AN6 and 4AN7, respectively TKIandTKIbindbyx-ray crystallography(View interaction) TKIandPPTbindbyx-ray crystallography(View interaction).

Differential proteolysis of insulin-like growth factor binding protein-1 (IGFBP-1) in pregnancy

The insulin-like growth factors and their binding proteins are important for placental and foetal growth. In this study, we have investigated the presence of proteolytic activity directed against insulin-like growth factor binding protein-1 (IGFBP-1) in pregnancy. In addition, the effect of protease activity on IGFBP-1 immunoreactivity and IGF binding was characterised. 125I-IGFBP-1 was incubated with maternal and foetal serum, amniotic fluid and placental extracts. Breakdown of 125I-IGFBP-1 was determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. The size distribution of endogenous IGFBP-1 was determined by Western immunoblotting. Protease inhibitor studies characterised the proteolytic activity, and Western ligand blotting with 125I-IGF-I was used to determine IGF binding capacity of proteolysed IGFBP-1. Amniotic fluid samples collected after labour onset contained proteolytic activity that generated 12- and 19-kDa IGFBP-1 fragments that did not bind to 125I-IGF-I. This activity was not detected in amniotic fluid collected prior to labour onset or in other tissues. Activity was blocked by aprotinin, leupeptin, phenyl methyl sulphonyl fluoride, and Kunitz soybean trypsin inhibitor but not by ethylene diamine tetraacetic acid or pepstatin. Incubation of IGFBP-1 with trypsin generated fragments of a similar size to the amniotic fluid protease. In conclusion, we have demonstrated the presence in vivo of a trypsin-like proteolytic activity that alters the IGF-binding function of IGFBP-1 in pregnancy.

Molecular and functional properties of gelatin from the skin of unicorn leatherjacket as affected by extracting temperatures

Gelatins extracted from the skin of unicorn leatherjacket at different temperatures (45, 55, 65 and 75°C) in the presence and the absence of soybean trypsin inhibitor (SBTI; 100units/g pretreated skin) for 12h were characterised. In general, the addition of SBTI resulted in the lower yield, regardless of extraction temperature. Higher yield was obtained when higher extraction temperature was used (P<0.05). Gelatin from skin extracted at 75°C in the absence of SBTI showed the highest yield (10.66±0.41%) (based on dry weight). The highest α-amino group content was observed in gelatin extracted at 55°C without SBTI incorporated. The band intensity of β-chain and α-chains increased as the extraction temperature increased, particularly above 55°C. Gelatin extracted at 65°C with and without SBTI incorporation exhibited the highest gel strength (178.00±7.50g and 170.47±1.30g, respectively). FTIR spectra indicated that a greater loss of molecular order of triple helix with a higher degradation was found in gelatin extracted at 55°C in the absence SBTI. Gelatin extracted at 65°C, either with or without SBTI, had the highest EAI and ESI with high foam expansion and stability. Thus, the extraction of gelatin from the skin of unicorn leatherjacket at temperature sufficiently high could render the gelatin with less degradation.

Characterization of serine proteases of Lumbriculus variegatus and their role in regeneration

AbstractSerine proteases, ubiquitous enzymes known to function in digestion and immune protection in both vertebrates and invertebrates and implicated in regeneration in some species, were investigated in the California blackworm, Lumbriculus variegatus. Several serine proteases, rather than a single enzyme with broad specificity, were present in tissue extracts from the worms. Extracts were treated with a fluorescein‐labeled peptide chloromethyl ketone that specifically binds to trypsin/thrombin‐like proteases. Denaturing gel electrophoresis of labeled extracts showed several serine proteases with their molecular weight ranging 28,000–38,000 daltons. The trypsin/thrombin‐like activity was localized, using the fluorescein‐conjugated reagent, to the pharynx and digestive tract of L. variegatus. Movement of cells labeled by the reagent into regenerating tissues suggests that some differentiated endodermal tissues were used for reformation of digestive structures during regeneration in L. variegatus. The types of serine proteases in the extracts were further characterized by inhibitor studies. Presence of plasmin‐like activity was indicated by degradation of fibrin by tissue homogenates from the worms and the inhibitory effect of aprotinin on enzymes in these extracts. The ability of L. variegatus extracts to generate clots when incubated with rabbit plasma and partial inhibition of extract activity by phenylmethylsulfonyl fluoride and hirudin indicated presence of thrombin‐like activity. Consistent with the detection of trypsin, chymotrypsin, and plasmin‐like enzymes in the extracts was partial inhibition of L. variegatus serine protease activity by aminoethyl benzenesulfonyl fluoride and soybean trypsin inhibitor. Selective inhibition of chymotrypsin‐like activity by N‐tosyl‐l‐phenylalanine chloromethyl ketone and chymostatin as well as trypsin‐like activity by N‐tosyl‐l‐lysine chloromethyl ketone was observed. A potential role during regeneration for serine proteases is suggested by blockage of formation of head and tail structures by aminoethyl benzenesulfonyl fluoride, an inhibitor of these proteases.