Giant Amazonian fish pirarucu (Arapaima gigas): Its viscera as a source of thermostable trypsin

A trypsin was purified from pyloric caeca of pirarucu (Arapaima gigas). The effect of metal ions and protease inhibitors on its activity and its physicochemical and kinetic properties, as well its N-terminal sequence, were determined. A single band (28.0 kDa) was observed by SDS–PAGE. Optimum pH and temperature were 9.0 and 65 °C, respectively. The enzyme was stable after incubation for 30 min in a wide pH range (6.0–11.5) and at 55 °C. The kinetic parameters K_m, k_cat and k_cat/K_m were 0.47 ± 0.042 mM, 1.33 s⁻¹ and 2.82 s⁻¹ mM⁻¹, respectively, using BApNA as substrate. This activity was shown to be very sensitive to some metal ions, such as Fe²⁺, Hg²⁺, Zn²⁺, Al³⁺, Pb²⁺, and was highly inhibited by trypsin inhibitors. The trypsin N-terminal sequence IVGGYECPRNSVPYQ was found. The features of this alkaline peptidase suggest that it may have potential for industrial applications (e.g. food and detergent industries).     

Biochemical properties of two isoforms of trypsin purified from the Intestine of skipjack tuna (Katsuwonus pelamis)

Two trypsins (A and B) from the intestine of skipjack tuna (Katsuwonus pelamis) were purified by Sephacryl S-200, Sephadex G-50 and DEAE-cellulose with a 177- and 257-fold increase in specific activity and 23% and 21% recovery for trypsin A and B, respectively. Purified trypsins revealed a single band on native-PAGE. The molecular weights of both trypsins were 24 kDa as estimated by size exclusion chromatography and SDS–PAGE. Trypsin A and B exhibited the maximal activity at 55 °C and 60 °C, respectively, and had the same optimal pH at 9.0. Both trypsins were stable up to 50 °C and in the pH range from 6.0 to 11.0. Both trypsin A and B were stabilised by calcium ion. Activity of both trypsins continuously decreased with increasing NaCl concentration (0–30%) and were inhibited by the specific trypsin inhibitors – soybean trypsin inhibitor and N-p-tosyl-l-lysine chloromethyl ketone. Apparent K_m and K_cat of trypsin A and B were 0.22–0.31 mM and 69.5–82.5 S⁻¹, respectively. The N-terminal amino acid sequences of the first 20 amino acids of trypsin A and B were IVGGYECQAHSQPPQVSLNA and IVGGYECQAHSQPPQVSLNS, respectively.     

Purification and characterization of trypsin from the pyloric caeca of brownstripe red snapper (Lutjanus vitta)

Trypsin was purified from the pyloric caeca of brownstripe red snapper (Lutjanus vitta) by ammonium sulphate (40–60% saturation) precipitation, soybean trypsin inhibitor (SBTI)-Sepharose 4B column and DEAE-Sephacel column chromatography. Purified trypsin showed a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) and native-PAGE. A yield of 4.9% with the purification-fold of 20 was obtained. Trypsin had an apparent molecular weight of 23 kDa. SBTI and N-ρ-tosyl-l-lysine-chloromethylketone (TLCK) showed a strong inhibitory effect on the purified trypsin, while other protease inhibitors exhibited negligible inhibition. Trypsin had maximal activity at pH 8.5 and 60 °C for the hydrolysis of α-N-benzoyl-dl-arginine-ρ-nitroanilide (BAPNA). It was stable within the temperature range of 25–55 °C and pH range of 7.0–10.0. Purified trypsin had a Michaelis–Menten constant (K_m) and catalytic constant (k_cat) of 0.507 mM and 4.71 s⁻¹, respectively, when BAPNA was used as the substrate. For the hydrolysis of α-N-ρ-tosyl-l-arginine methyl ester (TAME), K_m and k_cat were 0.328 mM and 112 s⁻¹, respectively.     

Effects of limited enzymatic hydrolysis with trypsin on the functional properties of hemp (Cannabis sativa L.) protein isolate

Effects of limited enzymatic hydrolysis induced by trypsin on the physicochemical and functional properties of hemp (Cannabis sativa L.) protein isolate (HPI) were investigated. The enzymatic hydrolysis was confirmed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC). SEC and differential scanning calorimetry (DSC) analyses confirmed the presence of aggregates in the corresponding hydrolysates (with the degree of hydrolysis of 2.3–6.7%). Functional properties, including protein solubility (PS), thermal properties, emulsifying and foaming properties, and water holding and fat adsorption capacities (WHC and FAC) were evaluated. The PS was remarkably improved by the limited enzymatic hydrolysis at all tested pH values. However, the enzymatic hydrolysis led to the marked decreases in emulsifying activity index, foaming capacity and foam stability, WHC and FAC. These decreases were to a great extent related to the presence of aggregates in the hydrolysates.     

胰蛋白酶在合成高分子阴离子交换树脂上的固定化作用

研究了胰蛋白酶在合成高分子聚苯乙烯阴离子交换树脂上的固定化技术,固定化酶的动力学性质以及稳定性特征。阴离子交换树指GM201经预处理除去杂质后与偶联剂成二醛反应,再与胰蛋白酶反应,可把酶固定化在载体上。动力学分析表明:固定化胰蛋白酶的K_m(米氏常数)值高于溶液酶,最适温度及最适pH值均有较大变化。稳定性试验表明:固定化酶的热稳定性低于溶液酶,在微酸性,中性及碱性介质中的稳定性高于溶液酶。用固定化胰蛋白酶水解酪蛋白及提取大豆胰蛋白酶抑制剂的试验结果表明,固定化酶具有良好的操作稳定性。     

胰蛋白酶水解酪蛋白进程研究

研究了碱性务件下胰蛋白酶对酪蛋白的水解，用甲醛固定法对蛋白质的水解度进行测定，并用水解度（DH）表征其反应历程。分析了底物浓度、酶浓度、时间、pH和温度对DH、水解速度的影响。     

Gallic acid oxidizes Met residues in peptides released from bovine β-lactoglobulin by in vitro digestion

Phenolic compounds (PCs) are frequently present in foods. However, little is known about the effect of PCs on enzymatic digestion process of food proteins and their products. In this study, the effect of gallic acid (GA) on in vitro digestion of β-lactoglobulin (β-LG) was investigated as a model system for analysis of the interaction between PCs and food proteins. GA showed no effect on the initial rate of β-LG digestion. However, after 1.5 h of digestion, the observed degree of hydrolysis of β-LG was lower in the presence than in the absence of GA. The peptides released from β-LG were characterized by LC/IT-TOF-MS and thirty peptides were identified. In particular, four new peaks were obtained following in vitro digestion of β-LG in the presence of GA. Met(7), Met(24) and Met(145) in the peptides corresponding to these peaks were oxidized to methionine sulfoxide residues.     

Effective Diffusivity and Kinetics of Urease Inactivation and Color Change During Processing of Soybeans with Superheated-Steam Fluidized Bed

Abstract

Influences of temperature and moisture content on diffusional transport property and kinetics of both urease inactivation and color change during soybean treatment with superheated steam in the temperatures of 120–150°C were investigated. The experimental results have shown that the effective diffusivity and apparent rates of such reactions are related to temperature in a way that the rates of water transport, urease inactivation, and brown pigment formation are higher with higher temperature. In addition, these rates are also enhanced by increasing moisture content, except for the browning reaction that is inhibited by this factor due to the dilution effect. The reduction in urease activity is reasonably described by modified first-order reaction and the color change of soybeans is characterized by three Hunter parameters in which the changes of L-, a- and b-values are followed according to zero-order, modified Monod and first-order reactions, respectively. The kinetics of inactivation and color change, along with the experimental data of protein solubility and lysine content, have been suggested that the soybean should be treated with superheated steam at the temperature ranging from 120 to 135°C. The superheated-steam fluidized-bed technique can simultaneously be applied for both drying and inactivating such components in soybeans by a single operation when the initial moisture content falls within a suitable range, approximately lower than 20%d.b. At elevated initial moisture contents, two-stage drying technique is recommended in order to avoid excessive cooked soybeans.     

Changes of trypsin in activity and secondary structure induced by complex with trypsin inhibitors and tea polyphenol

To reveal the relationships between the activity of trypsin and its structural change, changes of trypsin in biological activity induced by complex with Bowman-Birk trypsin inhibitor (BBTI), Kunitz soybean trypsin inhibitor (KSTI, type I-S) and tea polyphenol (TP) were detected and their relationship with the secondary structure changes were studied by far-UV circular dichroism (CD) spectra measurement. BBTI and KSTI were also irradiated by ultrasonic to compare the effects on trypsin. The rank was found as KSTI > BBTI > TP according to their inhibitory activities against trypsin. Yet BBTI exhibited much stronger resistance against ultrasonic irradiation than KSTI. BBTI, KSTI and TP were found inactivate trypsin by modifying the secondary structures and far-UV spectrum of trypsin. Complex of trypsin with ultrasonic-treated BBTI and native BBTI and KSTI exhibited the similar modified effects in secondary structures, decrease of α-helix and β-turn content, increase of β-sheet content and unchanged random coil content basically. But complex of trypsin with ultrasonic-treated KSTI exhibited less modified effects because of inactivation by ultrasonic irradiation. The changes of trypsin in secondary structure induced by complex with TP showed different from those induced by complex with BBTI and KSTI, increase of α-helix content, decrease of random coil content and unchanged β-sheet and β-turn content basically.     

Trypsins from the pyloric ceca of jacopever (Sebastes schlegelii) and elkhorn sculpin (Alcichthys alcicornis): Isolation and characterization

Trypsins from the pyloric ceca of jacopever (Sebastes schlegelii), TR-J, and elkhorn sculpin (Alcichthys alcicornis), TR-E, were purified by gel filtration on Sephacryl S-200 and Sephadex G-50. The molecular weights of TR-J and TR-E were estimated to be 24,000 Da by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. TR-J and TR-E revealed optimum temperatures of 60 and 50 °C, respectively, and showed the same optimum pH (pH 8.0) for hydrolysis of N-p-tosyl-l-arginine methyl ester. TR-J and TR-E were unstable at above 50 and 40 °C, respectively, and were more stable at alkaline pH than at acidic pH. Thermal stabilities of TR-J and TR-E were highly calcium dependent. These purified trypsin enzymes were inhibited by serine protease inhibitors, such as TLCK and soybean trypsin inhibitor. The N-terminal amino acid sequences of TR-J and TR-E were also investigated. The N-terminal amino acid sequences of TR-J, IVGGYECKPYSQPHQVSLNS, and TR-E, IVGGYECTPHSQAHQVSLNS, were found, and these sequences showed highly homology to other fish trypsins.