Trypsiligase-Catalyzed Labeling of Proteins on Living Cells

Fluorescent fusion proteins are powerful tools for studying biological processes in living cells, but universal application is limited due to the voluminous size of those tags, which might have an impact on the folding, localization or even the biological function of the target protein. The designed biocatalyst trypsiligase enables site-directed linkage of small-sized fluorescence dyes on the N terminus of integral target proteins located in the outer membrane of living cells through a stable native peptide bond. The function of the approach was tested by using the examples of covalent derivatization of the transmembrane proteins CD147 as well as the EGF receptor, both presented on human HeLa cells. Specific trypsiligase recognition of the site of linkage was mediated by the dipeptide sequence Arg-His added to the proteins’ native N termini, pointing outside the cell membrane. The labeling procedure takes only about 5 minutes, as demonstrated for couplings of the fluorescence dye tetramethyl rhodamine and the affinity label biotin as well.     

Trypsin Inhibitor Activity In Vitro Digestibility and Sensory Quality of Meat-Like Yuba Products as Affected by Processing

ABSTRACT: :
Soybeans ( Glycine max ) were soaked and ground to obtain soymilk. The soymilk was cooked in an open tank and held at 85 to 90 deg;C. Yuba films were picked up in 20 min intervals and dried for 20 min. Yuba films were soaked in chicken-flavor solutions (25% and 35%), and baking soda (BS) solutions (0%, 1%, 2%, and 3% BS), and cooked at 100 °C for 30 min, 60 min, and 90 min. TIA decreased (p < 0.05) with the increase of heating time and BS concentration. In vitro protein digestibility (IVPD) decreased with heating time and BS concentration (p < 0.05). Sensory characteristics were affected by flavor concentration. By using 0% BS, 25% of the chicken flavor concentration, and a short heating time method, meat-like products with low TIA, high IVPD, and good sensory characteristics were obtained.     

Trypsin inhibitor activity of soybean as affected by genotype and fertilisation

The presence of protease inhibitors in soybean prohibits the utilisation of the raw beans for food and feed. However, little information is available about environmental influences and the effects of nitrogen and sulphur supply on the antinutritional constituents of soybean. As these factors may influence protease inhibitors, soybean genotypes segregated according to the presence or absence of the Kunitz trypsin inhibitor have been evaluated for trypsin inhibitor activity (TIA) in field trials. TIA was affected significantly by environment (geographical location), fertilisation treatment and genotype. Environmental means of TIA were between 69.5 and 104.8 mg g^?1. Nitrogen application, which caused an increase in seed protein content, resulted in a reduction in TIA by about 15% as compared with the control. Remarkably, simultaneous application of nitrogen and sulphur in the form of ammonium sulphate had a similar reductive effect on TIA to that of nitrogen application alone, although soybean protease inhibitors are rich in sulphur amino acids. Significant genetic variation in TIA was found both within the genotype class with the Kunitz inhibitor present as well as within the class lacking this inhibitor. The results suggest that TIA of soybean may be modified considerably by genetic improvement and appropriate agronomic management. Copyright © 2003 Society of Chemical Industry     

Protein modelling using a chimera reference protein derived from exons

Bovine pancreatic /S-trypsin (PDB ID-code: 1TPO) which is registeredin the Brookhaven Protein Data Bank (PDB) consists of four exons.The results of homology searches for each exon in the PDB showedthat homologous proteins were tonin (PDB ID-code: 1TON), ratmast cell protease (PDB ID-code: 3RP2_A), kaffikrein A (PDBID-code: 2PKA_B) and kallikrein A (2PKA_B) respectively. Thus,for the three-dimensional structure prediction of 1TPO, a chimeraprotein was constructed from the three proteins mentioned aboveand the 3-D structure prediction was performed using this chimerareference protein. The modelled structure of 1TPO was energeticallyoptimized by molecular mechanics and molecular dynamics simulationand was compared with its X-ray crystal structure registeredin the PDB. The root mean square deviations (r.m.s.d.) of mainchain atoms and the neighbouring active site (5 sphere fromHis57, AsplO2 and Serl95) between the modelled structure andthe X-ray structure were 1.66 and 0.94 respectively. Porcinepancreatic elastase (PDB ID-code: 3EST) which is registeredin the PDB was used as the reference protein and the modelledstructure from 3EST was also compared with the X-ray data. Ther.m.s.d. of main chain atoms and that of the active site were2.14 and 1.18 respectively. These results dearly support thepropriety of this method using the chimera reference protein.     

新型功能高分子材料及其应用

功能高分子是一类具有特殊用途的高分子材料 ,印迹高分子、敏感性水凝胶和固定化酶是三种较有特色的功能高分子材料。该文将对上述三种功能高分子材料以及它们在生化分离、生物催化、物质分析与检测以及药物控制释放中的应用做一介绍 ,同时也对它们的不足和发展前景进行了评述     

Some compositional properties of seeds and oils of eightAmaranthus species

Grain of 21Amaranthus accessions (eight species) was analyzed for crude fat, fatty acid profiles (FAP), and vitamin E (tocopherols and tocotrienols). Contents of (1→3), (1→4) β-glucan were determined in 12 accessions (four species), and trypsin inhibitor activity (TIA) in 20 accessions (six species). FAP and vitamin E profiles were compared to those of barley, buckwheat, corn, lupin, oat, and wheat oils. Crude fat content ranged from 5.2 to 7.7%, and of the oils examined, amaranth oil was most similar in FAP to corn and buckwheat oils. Amaranth was higher than all but wheat and lupin in tocopherol content but was virtually devoid of tocotrienols, which have been shown to have hypocholesterolemic activity. Amaranth grain did not contain (1→3), (1→4) β-glucans and was low in trypsin inhibitor activity (≤4.3 trypsin units inhibited/mg). Any hypocholesterolemic effects of dietary amaranth are apparently due to substances other than (1→3), (1→4) β-glucans or tocotrienols.     

Acidolysis of babassu fat catalyzed by immobilized lipase

The lipase-catalyzed interesterification of oils and fats gives products that are unobtainable by chemical interesterification methods. Acidolysis of babassu fat and palmitic acid, catalyzed by immobilized lipase (Lipozyme; Novo Industri, Bagsveard, Denmark), was studied. The reactions were performed at 65°C with 5% w/w enzyme for 4 h. The molar proportions of babassu fat/palmitic acid were 1∶0.1, 1∶0.3 and 1∶0.5. At the end of the reaction period, the catalyst particles were removed by filtration, and the residual oil was extracted with organic solvent (diethyl ether). The recovered particles were then reused. The palmitic acid content of babassu fat before and after acidolysis changed from 10 to 22% at a molar proportion of 1∶0.5. The equilibrium was attained in about 4 h. The original water and enzymatic activities of Lipozyme were maintained after acidolysis. Water sorption isotherms of the immobilized enzyme were determined at 25, 35 and 45°C. From the temperature dependence of the isotherms, isosteric heats of sorption were evaluated by means of the Clausius Clapeyron equation. Monolayer moisture content was calculated by means of the B.E.T. and Guggenhein-Anderson-De Boer analyses. Paper presented at the International Meeting on Fats and Oils Technology, Universidade Estadual de Campinas, Brazil, 1991.     

Synthesis of 2‐monoglycerides by alcoholysis of palm oil and tuna oil using immobilized lipases

Commercial immobilized lipases were used for the synthesis of 2‐monoglycerides (2‐MG) by alcoholysis of palm and tuna oils with ethanol in organic solvents. Several parameters were studied, i.e., the type of immobilized lipases, water activity, type of solvents and temperatures. The optimum conditions for alcoholysis of tuna oil were at a water activity of 0.43 and a temperature of 60 °C in methyl‐tert‐butyl ether for ～12 h. Although immobilized lipase preparations from Pseudomonas sp. and Candida antarctica fraction B are not 1, 3‐regiospecific enzymes, they were considered to be more suitable for the production of 2‐MG by the alcoholysis of tuna oil than the 1, 3‐regiospecific lipases (Lipozyme RM IM from Rhizomucor miehei and lipase D from Rhizopus delemar). With Pseudomonas sp. lipase a yield of up to 81% 2‐MG containing 80% PUFA (poly‐unsaturated fatty acids) from tuna oil was achieved. The optimum conditions for alcoholysis of palm oil were similar as these of tuna oil alcoholysis. However, lipase D immobilized on Accurel EP100 was used as catalyst at 40 °C with shorter reaction times (<12 h). This lead to a yield of ～60% 2‐MG containing 55.0‐55.7% oleic acid and 18.7‐21.0% linoleic acid.     

固定化紫草细胞生产紫草宁衍生物

用海藻胶包埋紫草细胞,在紫草色素生产培养基M_9中,常温、黑暗下培养不同时间,收集培养液并提取色素,进行紫外—可见全波长分光光度扫描和TLC分析。结果表明:固定化紫草细胞可连续分泌紫草色素,其主要成分与天然成分基本相同,产量已达到一定水平。此外对海藻胶包埋条件、固定化珠粒的稳定性以及1L固定化植物细胞反应器生产紫草色素工艺进行了讨论。