Effect of polysaccharides on the stability and renaturation of soybean trypsin (Kunitz) inhibitor

Thermal denaturation and renaturation of soybean trypsin (Kunitz) inhibitor (STI) were studied by high-sensitivity differential scanning calorimetry in the presence of polysaccharides (dextran, ι- and κ-carrageenans, gum arabic, pectins and dextran sulfate). This study was carried out under conditions of both thermodynamic incompatibility and complex formation of STI and polysaccharides. The presence of polysaccharides did neither influence the denaturation temperature nor the denaturation enthalpy of STI under conditions of their incompatibility with the protein. No polysaccharide (except gum arabic) affected the ability of STI to renature and recover its inhibitory activity after thermal denaturation. At acidic pH values, the protein was shown to form electrostatic complexes with pectins and dextran sulfate. Substantial destabilisation of STI bound to dextran sulfate was observed. In the case of STI/pectin complexes, either a decrease or increase in the stability of STI was observed depending on the complex composition and esterification degree of pectin. The mechanism behind the changes in stability of STI bound to the polysaccharide matrix is discussed. Thermal denaturation of STI in complexes with dextran sulfate and pectin was completely irreversible. This observation indicates a possibility of suppressing antinutritional activities of trypsin inhibitors in soy products.

Schematic presentation of the denaturation of a protein (P) bound to a polymer matrix (M): (A) loose protein occupancy, (B) dense protein occupancy.     

聚碳酸乙烯撑酯与双端氨基聚乙二醇交联体系固定化酶载体的合成与表征

将聚碳酸乙烯撑酯(PVCA)与α,ω-双端氨基聚乙二醇(H₂N-PEG-NH₂)溶于DMF,于液蜡中进行交联反应制得亲水性固定化酶载体,将其与胰蛋白酶进行偶联反应制备了固定化胰蛋白酶.酶蛋白的比活力及其于载体上的结合量与反应条件有关,当w(PVCA)/w(H₂N-PEG-NH₂)为0.5时,二者均处于最高值.此固定化酶酶促反应的最适pH值和Km值均较之溶液酶有显著提高,但二者的最适酶促反应温度却相当一致.     

Membrane affinity chromatography used for the separation of trypsin inhibitor.

Polysulphone (PS) was chemically modified by acrylation-amination and by chloromethylation-amination, respectively. An ultrafiltration membrane of chemically modified polysulphone (CMPS) was prepared by the phase inversion method. Trypsin was then covalently bonded onto the CMPS membrane by diazotization. The activity of immobilized trypsin reaches up to 10200 U/g; 15 mg trypsin was immobilized on 1 g CMPS membrane. Separation of soybean trypsin inhibitor was carried out on the affinity membrane, yielding 6.5 mg pure trypsin inhibitor in one run. The enzyme membrane has good activity and stability.     

Characterization of p-aminobenzamidine-based sorbent and its use for high-performance affinity chromatography of trypsin-like proteases

An affinity sorbent, hydrophilic polymer-based carrier of different pore size (Toyopearl) with immobilized p-aminobenzamidine (ABA), has been prepared. Its basic properties and some applications for protein purification were studied. ABA, which is a synthetic inhibitor for trypsin-like proteases, was covalently immobilized to Toyopearl by reductive amination. The ligand density and binding capacity for porcine trypsin varied depending on the pore size of Toyopearl. The maximum binding capacity of the immobilized p-aminobenzamidine Toyopearl (ABA-Toyopearl) for trypsin was more than 40 mg/ml gel. ABA-Toyopearl thus obtained was very stable below pH 8 and was successfully used for high-performance affinity chromatography of trypsin-like proteases such as trypsin, thrombin, tissue-type plasminogen activator or urokinase in a single step at 25 degrees C.     

A novel reversible pH-triggered release immobilized enzyme system

A novel immobilized enzyme system supported by poly(acrylic acid/N,N’-methylene-bisacryl-amide) hydrogel microspheres was prepared. This system exhibited characteristics of reversible pH-triggered release. The morphology, size, and chemical structure were examined through optical microscopy, particle size analyzer, and Fourier transform infrared spectrometer. Immobilization and release features were further investigated under different conditions, including pH, time, and microsphere quantity. Results showed the microspheres were regularly spherical with 3.8 ~ 6.6 μm diameter. Loading efficiencies of bovine serum albumin immobilized by gel entrapment and adsorption methods were 93.9% and 56.2%, respectively. The pH-triggered protein release of the system occurred when medium pH was above 6.0, while it was hardly detected when medium pH was below 6.0. Release efficiencies of entrapped and adsorbed protein were 6.38% and 95.0%, respectively. Hence, adsorption method was used to immobilize trypsin. Loading efficiency of 77.2% was achieved at pH 4.0 in 1 h. Release efficiency of 91.6% was obtained under optimum pH catalysis condition set at 8.0 and trypsin was free in solutions with retention activity of 63.3%. And 51.5% of released trypsin could be reloaded in 10 min. The results indicate this kind of immobilized enzyme system offers a promising alternative for enzyme recovery in biotechnology.     

复合纤维素膜固定化胰蛋白酶反应器及其应用于蛋白质酶解

合成了甲基丙烯酸缩水甘油酯-纤维素复合膜,并以此膜为基质共价键合固定化胰蛋白酶,以N-苯甲酰-L-精氨酰乙酯(BAEE)为底物,应用高效液相色谱系统测定了酶固定化膜柱的催化反应特性。研究结果表明:温度、pH值、离子强度、有机溶剂及蛋白变性剂等都对固定化酶的活力有一定的影响。在最适条件下,固定化胰蛋白酶的活力为17800U/g干膜,蛋白载量为3.6mg/g(≈0.15μmol/g)干膜,活性回收率达到52%.固定化酶表现出较高的使用和储藏稳定性,在40℃下,水解BAEE底物24h活力无显着变化。固定化酶膜柱在4℃冷藏保存100d仍保存90%以上的水解活力。固定化酶反应器被应用于蛋白质酶解的肽谱实验。     

壳聚糖固定化胰蛋白酶的研究

以壳聚糖为载体，戊二醛为交联剂，采用两种方法制备了固定化胰蛋白酶。考察了固定化反应中pH值，戊二醛的浓度，以及给酶量对固定化胰蛋白酶活力的影响，并研究了这两种固定化胰蛋白酶的性质。实验结果表明，以戊二醛预交联的网状壳聚糖为载体制备的固定化胰蛋白酶具有更加优良的性能，在最佳固定化反应条件下，酶的活性加收率可达56％。此固定化胰蛋白酶的最适pH为7．0－8．5，最适温度为60℃，Km值为2．52mol/L，固定化胰蛋白酶表现出较好的热稳定性，pH贮存稳定性，以及在乙醇水溶液中的稳定性。     

Imidazole—a new ligand for metal affinity precipitation

Kunitz soybean trypsin inhibitor (STI) was specifically coprecipitated during precipitation of Cu(II)-loaded copolymers induced by increase in temperature and ionic strength. The copolymers used consisted of 1-vinylimidazole andN-vinylcaprolactam orN- isopropylacrylamide. The elution of STI was achieved by solubilization of the STI-Cu(II)-polymer complex in the presence of an excess of the competing ligand, imidazole, and a subsequent precipitation of the polymer with STI remaining free in solution in a purified form as judged by Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To the best of our knowledge this is the first reported successful metal affinity precipitation of protein in a heterobifunctional format.     

Immobilization of trypsin in polycation-polyanion complexes

Trypsin was immobilized in quasi-soluble polyanion-polycation complexes with retention of the enzyme activity. The activity of the immobilized enzyme strongly depends on pH of the prepared solution, composition, relative concentrations and molar masses of the polyelectrolytes. The highest activity of trypsin (about 93%) was found in the case of the complexes Na-poly(styrenesulfonate)-poly(diallyldimethylammonium chloride): trypsin = 10:1, prepared at pH 3.0, with high molar mass of the polyanion (about one million). This method can be proposed for immobilization of various serine proteases.     

Characterization of a monolithic immobilized trypsin microreactor with on-line coupling to ESI-MS

The preparation and characterization of a miniaturized trypsin reactor using on-line coupling with an ESI-TOF mass spectrometer are described. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-trypsin was covalently immobilized on poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith prepared in a 75 microm ID fused silica capillary resulting in a bioreactor with high local concentration of the proteolytic enzyme. Covalent immobilization of trypsin on this support was performed using the epoxide functional groups in either a one- or a multistep reaction. For on-line protein digestion-MS analysis the bioreactor was coupled with the mass spectrometer using a liquid junction microelectrospray interface. The performance of the reactor was tested using an on-line flow through the system with flow rates of 50-300 nL/min. The resulting protein consumption was in the atto- to low femtomole range. Proteolytic activity was characterized in a wide range of conditions with respect to the flow rate, pH, and temperature. Complete protein digestion was achieved in less than 30 s at 25 degrees C with the sequence coverage of 80% (cytochrome c), which is comparable to 3 h digestion in solution at 37 degrees C. Besides the good performance at laboratory temperature, the immobilized trypsin in the bioreactor also performed well at lower pH compared to the standard in-solution protocols.     

Application and properties of butyl acrylate/pentaerythrite triacrylate copolymers and cellulose-based Granocel as carriers for trypsin immobilization

The main point was the search for a proper carrier and the kind of carrier activation for trypsin (EC 3.4.21.4) immobilization. The acrylic and cellulose-based carriers were specially prepared in that they possessed the most often used anchor groups: -OH, -NH(2), DEAE and/or -COOH. The immobilization procedures were selected to apply mainly to protein amine groups and appropriate anchor groups on the carrier. As activity tests low (N-benzoyl-dl-arginine-p-nitroanilide, BAPNA) and high (casein) molecular weight substrates were used. It was found, as a rule, that trypsin bound to -COOH groups with the help of carbodiimide was less active and that the amount of bound protein and measured activity (BAPNA) are considerably higher when protein is immobilized via divinyl sulfone. Both rules were observed irrespective of the nature of the polymer matrix. Both types of carriers were found suitable for trypsin immobilization and they were far better than the corresponding Eupergit C-bound enzyme preparations. Taking into account storage stability and activity for both substrates, the divinylsulfone linkage formed between unmodified Granocel and trypsin was the most effective method for the enzyme immobilization. For this preparation, BAPNA and casein conversion, thermal stability at 60 degrees C and estimated kinetic parameters were compared with those obtained for the native enzyme. It was shown that mass transport limitations could be effectively eliminated by suitable conditions and immobilized trypsin was considerably more stable. The values k(cat)/K(m) indicated that the immobilized enzyme was even better as amidase activity was regarded and its potential for protein hydrolysis was only less than twice.     

利用聚合脂质体的沉淀可逆性固定STI及其PL-STI对胰蛋白酶的亲和作用

通过双功能试剂戊二醛、二甘醇二酸酐和1,4-丁二醇双缩水甘油醚等将大豆胰蛋白酶抑制剂STI固定到由二十五-10,12-二块-1-醇磷脂乙醇胺形成的聚合脂质体上.同时探讨了其作为亲和沉淀吸附剂对对胰蛋白酶的亲和作用.在PL沉淀过程中,胰蛋白酶有20%～60%从PL-STI上解离下来.PL-STI可作为酶的亲和沉淀吸附剂.     

Development of microwave-assisted protein digestion based on trypsin-immobilized magnetic microspheres for highly efficient proteolysis followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis

In this study, very easily prepared trypsin-immobilized magnetic microspheres were applied in microwave-assisted protein digestion and firstly applied for proteome analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Magnetic microspheres with small size were synthesized and modified by 3-glycidoxypropyltrimethoxysilane (GLYMO). Trypsin was immobilized onto magnetic microspheres through only a one-step reaction of its amine group with GLYMO. When these easily prepared trypsin-immobilized magnetic microspheres were applied in microwave-assisted protein digestion, the magnetic microspheres not only functionalized as substrate for trypsin immobilization, but also as an excellent microwave absorber and thus improved the efficiency of microwave-assisted digestion greatly. Cytochrome c was used as a model protein to verify its digestion efficiency. Without any additives such as organic solvents or urea, peptide fragments produced in 15 s could be confidently identified by MALDI-TOF-MS and better digestion efficiency was obtained comparing to conventional in-solution digestion (12 h). Besides, with an external magnet, trypsin could be used repeatedly and at the same time no contaminants were introduced into the sample solution. It was verified that the enzyme maintained high activity after seven runs. Furthermore, reversed-phase liquid chromatography (RPLC) fractions of rat liver extract were also successfully processed using this novel method. These results indicated that this fast and efficient digestion method, which combined the advantages of immobilized trypsin and microwave-assisted protein digestion, will greatly hasten the application of top-down proteomic techniques for large-scale analysis in biological and clinical research.     

Immobilization of Horseradish Peroxidase on Nonwoven Polyester Fabric Coated with Chitosan

The immobilization of horseradish peroxidase (HRP) on composite membrane has been investigated. This membrane was prepared by coating nonwoven polyester fabric with chitosan glutamate in the presence of glutraldehyde as a crosslinking agent. The physico-chemical properties of soluble and immobilized HRP were evaluated. The soluble HRP lost 90% of its activity after 4 weeks of storage at 4°C, whereas the immobilized enzyme retained 85% of its original activity at the same time. A reusability study of immobilized HRP showed that the enzyme retained 54% of its activity after 10 cycles of reuse. Soluble and immobilized HRP showed the same pH optima at pH 5.5. The immobilized enzyme had significant stability at different pH values, where it had maximum stability at pH 3.0 and 6.0. The kinetic properties indicated that the immobilized enzyme had more affinity toward substrates than soluble enzyme. The soluble and immobilized enzymes had temperature optima at 30 and 40°C and were stable up to 40 and 50°C, respectively. The stability of HRP against metal ion inactivation was improved after immobilization. Immobilized HRP exhibited high resistance to proteolysis by trypsin. The immobilized HRP was more resistant to inactivation induced by urea, Triton X-100, and organic solvents compared to its soluble counterpart. The immobilized HRP showed very high yield of immobilization and markedly high stabilization against several forms of denaturants that offer potential for several applications.     

工艺条件对酸性大豆蛋白固体饮料品质的影响

对影响酸性大豆蛋白固体饮料品质的因素进行了研究,并优化了酸性大豆蛋白固体饮料制备的工艺条件,研究发现最佳制备工艺条件为大豆蛋白与大豆多糖添加质量比为1∶0.22、干燥前调节混合液p H值为7.0、先混和大豆多糖和麦芽糊精再与大豆蛋白混合、喷雾干燥并取收集桶中样品,在此条件下制备的新型大豆蛋白固体饮料溶解性较好,且调制成的酸性大豆蛋白液体饮料的沉淀率较低,仅为0.95%。     

新型亲水聚合物修饰硅胶颗粒固定化酶的制备及在蛋白质组鉴定中的应用

采用原子转移自由基聚合法制备了亲水聚合物修饰的硅胶颗粒作为一种新型固定化酶载体,在实现胰蛋白酶高密度固定的同时,显著降低了载体材料非特异性吸附导致的样品损失。因此,该固定化酶材料兼具高酶解效率和高回收率的特性。以标准蛋白质牛血清白蛋白(BSA)为样本,使用该固定化酶1 min即可完成酶解,鉴定到肽段对BSA的氨基酸序列覆盖率可达90%以上。该固定化酶材料成功应用于酵母菌全蛋白质复杂样本的酶解,从3 min酶解产物中鉴定到666个蛋白质,超过同样条件下溶液酶解12 h的鉴定结果。     

A Trypsin Immobilized Sol-Gel for Protein Indentification in MALDI-MS Applications

The proteolytic enzyme trypsin was chemically immobilized to an amine-functionalized sol-gel using adipoyl chloride under nonaqueous conditions and a nitrogen atmosphere. In the synthesis of the sol-gel, tetraethyl orthosilicate (TEOS), and 3-(2-aminoethylamino) propyldimethoxymethylsilane (AEAPMS) (50:50, v/v) were used, which provided convenient physical and chemical conditions to maintain catalytic activity of immobilized trypsin molecules for the digestion of proteins in proteomics applications. Bovine serum albumin was used as a model protein to perform enzymatic digestion using the trypsin immobilized sol-gel. The resulting peptides were analyzed by matrix-assisted laser desorption/ionization-mass spectrometry to evaluate the digestion performance and specificity of the sol-gel material. The trypsin immobilized sol-gel showed superior enzymatic activity in protein digestion and it was determined that the sol-gel material could be repeatedly used at least 25 times without significant activity loss in long-term use. Additionally, autocatalysis was prevented by immobilization of trypsin. The peptide digest having the highest purity was obtained for protein identification studies.     

Single-step Purification and Immobilization of MBP–phytase Fusion on Starch Agar Beads: Application in Dephytination of Soy Milk

Periplasmic phytase, appA from E. coli has been noticed as a superior feed and food additive owing to its high specific activity, acidic pH optimum and resistance to gastric proteases. E. coli phytase was expressed as a fusion protein with maltose-binding protein, affinity-purified to homogeneity and, subsequently, immobilized in one step using a cost-effective matrix prepared from starch agar bead. Immobilized enzyme revealed an activity optimum at pH 6, while that of free enzyme was observed at pH 4. Both the immobilized and free enzyme showed a temperature optimum at 60?°C. Cleavage of 87?kDa fusion protein using factor Xa released 45?kDa appA. Hydrolysis of soy milk using immobilized enzyme led to 10% increase in release of inorganic phosphate at 50?°C relative to free fusion protein. This study suggests the usability of MBP as an immobilizing linker to other food enzymes for economical use in industry.     

Development of an open-tubular trypsin reactor for on-line digestion of proteins

A study was initiated to construct a micro-reactor for protein digestion based on trypsin-coated fused-silica capillaries. Initially, surface plasmon resonance was used both for optimization of the surface chemistry applied in the preparation and for monitoring the amount of enzyme that was immobilized. The highest amount of trypsin was immobilized on dextran-coated SPR surfaces which allowed the covalent coupling of 11 ng mm⁻² trypsin. Fused-silica capillaries were modified in a similar manner and the resulting open-tubular trypsin-reactors having a pH optimum of pH 8.5, display a high activity when operated at 37 °C and are stable for at least two weeks when used continuously. Trypsin auto-digestion fragments, sample carry-over, and loss of signal due to adsorption of the protein were not observed. On-line digestion without prior protein denaturation, followed by micro-LC separation and photodiode array detection, was tested with horse-heart cytochrome C and horse skeletal-muscle myoglobin. The complete digestion of 20 pmol μL⁻¹ horse cytochrome C was observed when the average residence time of the protein sample in a 140 cm ×50 μm capillary immobilized enzyme reactor (IMER) was 165 s. Mass spectrometric identification of the injected protein on the basis of the tryptic peptides proved possible. Protein digestion was favorable with respect to reaction time and fragments formed when compared with other on-line and off-line procedures. These results and the easy preparation of this micro-reactor provide possibilities for miniaturized enzyme-reactors for on-line peptide mapping and inhibitor screening.     

SPR Study of the Blocking Activity of Soybean Trypsin Inhibitor by Macrocyclic Complexes of Nickel Immobilized on a Gold Surface

A method is proposed for the modification of metallic gold surfaces by self-organization of a nickel(II) macrocyclic complex containing a thiol substituent. Effective blocking of biologically active soybean trypsin inhibitor by the immobilized complex was observed. The prospects of using the given surface to create sensitive elements for sensors capable of detecting both small molecules (thiocyanate ions) and some protein substrates is demonstrated.