琼脂糖凝胶提取纯化抑肽酶的研究

采用琼脂糖凝胶为载体,共价偶联牛胰蛋白酶,制成抑肽酶亲和吸附剂,将其用于亲和层析牛肺提取液,分离纯化高比活抑肽酶。通过实验研究,计算抑肽酶抑制比活单位(BAEE单位/mg胰蛋白酶)为79000,酶活性回收率76.63%。该工艺具有操作简便、收率高、产品质量好、污染低等优点,适于工业化,有一定的工业应用前景。     

交联壳聚糖微球纯化抑肽酶研究

采用反相悬浮法制备交联壳聚糖微球,共价偶联胰蛋白酶,制成抑肽酶亲和吸附剂,将其直接亲和层析牛肺粗提液,制备高比活抑肽酶。结果表明,制备的亲和吸附剂单位活力1270KIU·g-1,蛋白质偶联率52.4%,酶活性回收率50.8%。制备的抑肽酶比活力达5650KIU·mg-1,并且该吸附剂具有非特异性吸附小、能反复使用、机械强度高、活化简单、价格低廉等优点。     

快速膜乳化法制备温敏微球及其固定胰蛋白酶

采用快速膜乳化法并结合低温聚合法制备了尺寸均一、重复性较好的聚(异丙基丙烯酰胺-丙烯酸) [P(NIPAM-co-AAc)]微球. 结果表明,所制微球平均粒径为5.2 mm,多分散性指数为0.0323. 对微球温敏响应性质的研究表明,加入亲水性单体会降低微球的低临界共溶解温度(LCST),且加入量越多LCST降低程度越大,交联剂加入量增大,微球的LCST升高. 加入亲水性共聚单体,微球的响应时间增加. P(NIPAM-co-AAc)微球用于胰蛋白酶固定化,在1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐浓度0.9 mg/mL、胰蛋白酶浓度1.8 mg/mL及磷酸盐缓冲液浓度70 mmol/L时,可得到固载量为276 mg/g、活性回收率达75.07%的最优值,固定化胰蛋白酶的最适pH值为8、最适温度为37℃.     

固定化七钼酸铵催化剂在葡萄糖差向异构中的应用条件优化研究

本文对自制固定化七钼酸铵催化剂在不同的反应条件下进行葡萄糖差向异构研究试验,得到最佳异构化反应条件:底物浓度为45～50%,反应温度为126～130℃,pH 2.5～3.0,催化剂添加量为1.25‰.反应时间为40min,催化剂粒径为150～200目数。固定化七钼酸铵催化剂与游离态的七钼酸铵催化剂催化活性差异不明显,但同条件下重复10次葡萄糖差向异构试验,结果固定化七钼酸铰催化剂的活性仍保持94%以上,说明固定化七钼酸铵催化剂活性保持比较稳定,可重复应用于工业化生产,降低成本。     

亲和胶质气体泡沫的制备及其对胰蛋白酶的吸附分离特性

Triton X-114, an non-ionic surfactant, was modified with the affinity ligand of trypsin, paminobenzamidine (PAB) and the affinity surfactant (PAB-TX) was synthesized. Then, the affinity surfactant was used to prepare affinity-based colloidal gas aphrons (CGA). The stability of the affinity CGA was investigated at different temperatures and compared with that of the CGA prepared from Triton X-114. Compared with the CGA from Triton X-114, the affinity CGA showed high selective adsorption property for trypsin. In the separation of a protein mixture, recovery yield higher than 74% were achieved for trypsin and the separation factor reached over 1.5. The results showed that the affinity CGA possessed promising selectivity for separating trypsin from a protein mixture.     

酶催化剂的固定化研究及其在催化反应中的应用

酶催化剂具有高效性,多样性,底物专一性,区域选择性、化学选择性、对映选择性以及反应条件温和的特点。而酶的固定化后除了保持原有的特点外,易与反应物和产物分离,可回收重复使用,降低生产成本。本文对酶催化剂的固定化方法以及在有机催化反应中的应用作了部分简述。并对固定化方法进行了比较和评价。     

胰蛋白酶酶解酪蛋白工艺条件的优化

本文选取酪蛋白为实验原料,胰蛋白酶为水解酶,以水解度和多肽含量为双评价指标,通过单因素和正交试验设计研究了胰蛋白酶酶解酪蛋白的条件。单因素实验研究了反应时间,底物质量,酶的添加量,p H值和温度对水解的影响,确定了主要影响因素及水平范围,通过L9(34)正交试验获得最佳的酪蛋白酶解条件。研究结果表明:胰蛋白酶酶解酪蛋白的最佳条件为:温度55℃,p H值8.0,时间120 min,底物质量0.5 g,酶添加量0.06 g。在此条件下,酪蛋白的水解度可达23.65%,多肽含量为54.79%。     

外加剂对提高固定化细胞制备L-苯丙氨酸能力的作用

在固定化细胞制备L -苯丙氨酸的研究体系中 ,以卡拉胶为固定化载体 ,所得的固定化细胞在使用过程中的催化稳定性较差 ,难以满足大规模生产的需要。故在卡拉胶包埋固定E .coli细胞过程中 ,对交联剂的处理方式和吸附剂的添加进行了研究。研究发现 ,复合交联剂对固定化细胞的处理能使固定化细胞的转化能力明显提高 :多乙烯多胺 (质量分数 0 .2 5 % )和已二胺 (质量分数 0 .2 5 % )对固定化细胞的组合处理 ,固定化细胞的转化能力提高了 3 1.6%。在细胞包埋过程中添加质量分数 4%的吸附剂E ,固定化细胞的转化能力提高了近 5 2 %。     

生物催化剂的固定化及其在精细化工中的应用

一前言生物技术越来越多地应用于化学工业,据预测,将来生物技术可能取代20％的化学工业工艺。目前在精细化学品和药物制造,以及食品工业中都已较成熟地利用了生物催化剂。但是,在日益发展的工业应用中,这些生物催化剂也日渐暴露出它的缺陷。对于酶来说,它对热、有机溶剂和酸、碱溶液均不稳定,且会受许多酶抑制剂和蛋白分解酶的影响,加上酶溶解在水溶液中,因此,反应后回收     

细胞固定化技术及其在废水处理中的应用研究

对细胞固定化技术进行了较为全面的介绍,指出理想的细胞固定化载体应该具备对微生物无毒、性质稳定、传质性能良好、强度高、寿命长、价格低廉等条件。详细阐述了细胞固定化技术在处理氨氮废水、含酚废水、重金属废水中的研究与应用现状,认为寻找高效、抗毒性强的生物及性质优良的固定化载体,开发高效的固定化反应器,进行生物再生是实现细胞固定化技术在废水处理中广泛应用的主要研究方向。特别指出的是这项技术在重金属废水处理中有着广阔的应用前景。     

B型肉毒神经毒素的纯化及胰蛋白酶对其作用分析

目的纯化B型肉毒神经毒素,并比较未激活和用胰蛋白酶激活的B型肉毒神经毒素的分子特性,分析在酸性和碱性条件下B型肉毒毒素复合物的完整性。方法取未激活、胰蛋白酶激活和热处理激活的B型肉毒毒素复合物,采用阴离子交换柱层析纯化神经毒素(分别为A、B和C组)。在酸性条件下溶解激活的B型肉毒毒素复合物(D组)。对各组进行纯度(特异毒性)测定和SDS-PAGE分析。结果柱层析后,神经毒素纯度均比复合物的纯度高,激活的神经毒素比未激活的高2～3倍;在非还原和还原条件下,A组的SDS-PAGE显示神经毒素、重链和轻链3条带;B组显示重链和轻链2条带;C组在非还原条件下显示重链和轻链2条带,在还原条件下只显示轻链1条带。在酸性条件下,B型肉毒毒素复合物由神经毒素与非毒性成分构成,在碱性条件下,神经毒素与非毒性成分分离。结论已成功纯化了B型肉毒神经毒素,经胰蛋白酶处理后,单链裂解为双链,比活性提高。     

Trypsin Inhibitor Assay: Expressing,Calculating, and Standardizing Inhibitor Activity in Absolute Amounts of Trypsin Inhibited or Trypsin Inhibitors

For expressing trypsin inhibitor activity (TIA), trypsin units inhibited (TUI), trypsin inhibited, and trypsin inhibitors have been used. Although the last two units are preferred, their calculations in current practices require refinement. With the proposed AOCS method Ba 12a-2020, four experiments were conducted, using four trypsin preparations having specific activity of 11,625, 12,602, 13,728, and 14,926 Nα-benzoyl-L-arginine ethyl ester (BAEE) units/mg protein, respectively. Experiment 1 determined the relationship between absorbance at 410 nm (A410) and trypsin concentration. Experiment 2 involved assaying raw and heated soybeans, expressing TIA as TUI/mg sample and μg trypsin inhibited/mg sample, and determining conversion factors between the two units. Experiment 3 resembled Experiment 2 except for using purified soybean Kunitz inhibitor (KTI) and Bowman-Birk inhibitor (BBI). Conversion factors determined correlated highly with trypsin-specific activity (R² = 0.9789). After standardizing against a reference trypsin having 15,000 BAEE units/mg protein, a standardized conversion factor of 0.03 A410 (1.5 TUI) = 1 μg trypsin inhibited was determined. It remained consistent regardless of trypsin specific activity, with or without inhibitors, and type of inhibitor samples. By using purified inhibitors (Experiment 3), conversion values between TUI and μg trypsin inhibitor and between μg trypsin inhibited and μg trypsin inhibitor could also be calculated, enabling expression of TIA in amounts of pure KTI, BBI or their equivalents. Furthermore, when the AOCS method was modified with half substrate concentration, half trypsin concentration or half both (Experiment 4), TIA values in TUI could change with modifications but values in mg trypsin inhibited (standardized) or trypsin inhibitor remained consistent.     

Isolation of Trypsin from Bovine Pancreas Using Immobilized Benzamidine and Peptide CTPR Ligands in Expanded Beds

Abstract

Peptide CTPR and p‐amino benzamidine (PAB) immobilized on Streamline? were utilized as the chromatographic matrices for trypsin purification from bovine pancreas. By using a clarified pancreas extract, maximum capacity for CTPR‐Streamline was 47.4 mg/mL and for PAB‐Streamline 78.9 mg/mL while Kd values were 0.39 and 0.38 respectively. Dynamic capacity was 23.0 and 46.0 mg/mL for CTPR‐ and PAB‐Streamline respectively. When the purification process was applied to unclarified pancreas extract in the expanded‐bed adsorption mode, 80% trypsin recovery with a purification factor of 18.7 was achieved. Cationic and anionic trypsin obtained from the affinity column were separated by ion‐exchange chromatography.     

A Comparison of the ISO and AACC Methods for Determining the Activity of Trypsin Inhibitors in Soybean Meal

The international standard method for the determination of trypsin inhibitor activity (TIA) in soya products, ISO 14902, was compared with the American Association of Cereal Chemists’ standard AACC 22‐40.01 as modified by Hamerstrand in 1981 (AACC‐based method), using soybean meals as matrices. TIA, expressed as milligram of inhibited trypsin per gram of sample, was determined by both methods in each of 30 samples of soybean meal. TIA values according to ISO 14902 were significantly lower (P < 0.001) than those afforded by the AACC‐based method. This difference, which means that AACC‐based method and ISO 14902 TIA values are not directly comparable, is attributable to between methods differences, in decreasing order of influence: particle size (P < 0.01), trypsin inhibitor extraction method (P < 0.05), and trypsin substrate (P < 0.01). N‐benzoyl‐l ‐arginine‐4‐nitroanilide hydrochloride, the ISO 14902 trypsin substrate, affords TIA values 6.4 % higher than the racemic mixture used by the AACC method, but it seems unlikely that in most contexts this advantage would outweigh the disadvantage of its greater cost.     

抑肽酶的提取及活性研究

以牛肺为原料,经酸沉淀,胰蛋白酶亲和层析,分离提纯抑肽酶。并建立了二种抑肽酶活性测定方法(UKIU法及PKAIU法)。提纯后抑肽酶纯度提高88.3倍。得率为96.8％。活性抑制试验表明,该抑肽酶可抑制PKA对前激肽释放酶(PK)的作用,以及尿激酶对纤溶酶原的作用,对纤溶酶原的活化及纤溶酶活性也有较强抑制作用。     

Optimization of L-Asparaginase Immobilization onto Calcium Alginate Beads

In this study, anti-leukemic enzyme L-asparaginase (E.C.3.5.1.1) from Escherichia coli ATCC 11303 was modified by the microencapsulation technique onto calcium alginate beads. Using response surface methodology (RSM), a three-level full factorial design, the values of concentration of sodium alginate, concentration of calcium chloride, and enzyme loading were investigated to obtain the highest residual L-asparaginase (L-ASNase) activity % (immobilized enzyme activity/free enzyme activity). The effects of the studied factors on immobilization were evaluated The predicted values by the model were close to the experimental values, indicating suitability of the model. The results presented that an increase in sodium alginate concentration increased the percent of residual activity of L-ASNase at any given calcium chloride concentration and the moderate amount of enzyme loading increased the percent residual activity. The optimal immobilization conditions were as follows: sodium alginate 1.98% (w/v), calcium chloride concentration 3.70% (w/v), and enzyme load 46.91% (v/v). The highest residual L-ASNase activity % obtained was 34.49%.     

变压吸附技术在气体分离提纯中的应用

介绍了变压吸附技术的基本原理、发展概况及基本工作过程,并阐述了该技术在氢气的分离与提纯、二氧化碳的分离与提纯、一氧化碳的分离、空气分离制氧、空气分离制氮、碳的脱除等工业过程中的应用,对变压吸附技术今后的发展提出了展望。     

氧化葡萄糖酸杆菌的固定化及在鼓泡式反应器中制备乙醇酸

考察了包埋法固定氧化葡萄糖酸杆菌的过程,并且研究了利用该固定化细胞在鼓泡塔生物反应器中转化乙二醇制备乙醇酸。固定化的最优条件为:聚乙烯醇的最佳质量分数为8%,海藻酸钠为0.8%,最适湿细胞包埋量为每毫升凝胶0.27 g。固定化细胞对酸碱的抗性和热稳定性均较游离细胞有所提高。利用固定化细胞在鼓泡式反应器中转化乙二醇制备乙醇酸,在乙二醇质量浓度70 g/L的条件下,首批转化率达到92.6%,并且可以实现连续转化6个批次,平均转化率在85%以上。     

Trypsin Inhibitor and Urease Activity of Soybean Meal Products from Different Countries and Impact of Trypsin Inhibitor on Ileal Amino Acid Digestibility in Pig

Trypsin inhibitor (TI) and urease activity (UA) are the most relevant quality aspects of soybean meal (SBM) for monogastric animals. This study’s objective was to determine the levels of TI and UA in SBM collected from feed mills in different countries, whether TI and UA are correlated since they are both heat-labile, and impact of TI on pig ileal amino acid digestibility. TI was 1.20–8.37 mg g⁻¹ with 36.4% samples <3 mg g⁻¹, UA was 0–0.311 ΔpH unit with 64.2% samples <0.05 ΔpH unit in 847 solvent-extracted SBM samples. TI and UA results varied by countries and world areas. By country, mean TI was highest in Germany and lowest in Philippines, mean UA was highest in India and lowest in Philippines. By world areas, mean TI was highest in Latin America and lowest in North America, UA was highest in Asia and lowest in Latin America. TI and UA results vary in different soybean products, TI ranked as raw soybeans > expeller SBM > full-fat-extruded (FFE) SBM ≥ heat-inactivated full-fat (HIFF) SBM > solvent-extracted SBM; UA ranked as raw soybeans > HIFF SBM > FFE SBM ≥ solvent-extracted SBM ≥ expeller SBM. TI was not correlated with UA in 847 solvent-extracted SBM, suggesting that UA may not be used as a surrogate indicator for TI. A diet contained 38% SBM with 8.78 mg g⁻¹ TI significantly reduced apparent ileal digestibility of amino acids and crude protein by 13.3–26.0% and 23.3% points, respectively, in cannulated pigs compared to SBM with 2.51 mg g⁻¹ TI.     

超氧化物歧化酶的固定及其活性检测

目的探讨超氧化物歧化酶(SOD)的固定及固定化SOD活性的检测方法。方法以淀粉为载体,对SOD进行固定,采用改进的邻苯三酚测活法(以Vc为终止剂),并结合过滤或离心的方法去除沉淀,测定固定化SOD的活性。比较过滤法与离心法,及过滤法与经典邻苯三酚法测定酶活性的差异,并检测过滤法的线性、精密性和准确性。结果过滤法检测SOD活性的精密性较离心法高,与经典邻苯三酚法检测结果差异无统计学意义,在SOD浓度为6~60μg/ml范围内,SOD活性值与浓度线性关系良好(r=0.999 5),精密性及准确性良好。固定化SOD的比活性为1 730.07 U/g,酶活回收率为53.3%。结论以淀粉为载体固定化SOD效果好,经改进的邻苯三酚法可用于固定化SOD的活性测定。