酶分子化学修饰研究进展

酶是高效生物催化剂,在工业和临床医药上应用广泛。但由于酶是蛋白质,稳定性差,且在生物体内有较强的免疫原性,因而严重制约了其应用。对酶分子进行化学修饰是提高其稳定性和降低免疫原性的有效途径。简要介绍几种改进酶催化特性的方法、酶分子修饰效果的分析与评价、酶通过化学修饰获得的新性质及其原理、酶化学修饰的研究动态等。     

多聚唾液酸对L-天冬酰胺酶的修饰及修饰酶特性研究

来源于大肠杆菌 (Ｅ .ｃｏｌｉ)的Ｌ 天冬酰胺酶是治疗淋巴性白血病和恶性淋巴肿瘤的有效酶制剂 ,已应用于临床。该酶与其他蛋白质类药物一样 ,在临床应用中存在两个常见问题 :一是酶制剂在体内易被降解 ,导致半衰期短 ;二是免疫原性。为了解决上述问题 ,人们用亲水性的大分子如血清蛋白、右旋糖苷和单甲氧基聚乙二醇 (ｍＰＥＧ)对该酶进行修饰。其中ｍＰＥＧ[1] 修饰后的Ｌ 天冬酰胺酶的抗原抗体结合能力完全消失 ,免疫原性下降 ,且体内半衰期延长 ;但酶活力只有天然酶的 8%～ 14% ,且ｍＰＥＧ在人体组织中无法降解 ,目前尚难评估长期使用…     

化学修饰解除天冬酰胺酶抗原性研究

本文报道了用活化的单甲氧基聚乙二醇PEG_2在底物保护的条件下修饰天冬酰胺酶。结果,修饰酶在抗原抗体结合能力完全消失的同时,酶活力保持30％以上,且修饰酶的抗胰蛋白酶水解能力明显增强,体外半衰期延长17倍,免疫原性显著下降。     

麦芽糖的改性及其化学修饰胰蛋白酶的研究

目的:通过高碘酸钠氧化法以麦芽糖修饰胰蛋白酶,检验此方法是否能够改善胰蛋白酶的稳定性.方法:采用高碘酸钠改性麦芽糖,使其中具有能够与酶分子中氨基结合的醛基,从而在一定条件下化学修饰胰蛋白酶.结果:改性麦芽糖的最佳条件为,高碘酸钠浓度0.05g/mL,高碘酸钠与麦芽糖质量比1.1∶1,初始pH 1.0,反应时间3h,反应温度30℃;修饰胰蛋白酶的最佳条件为,pH6.0,改性麦芽糖与胰蛋白酶质量比1∶1,修饰温度4℃,修饰时间24h;在最佳条件下修饰酶后,pH在8.9～10.0时,原酶活力与修饰酶活力分别下降了其最高活力的15.8%和9.8%;将酶置于50℃ 2h后,原酶与修饰酶分别保留了其置于4℃ 2h后活力的88.7%和94.7%.结论:该方法能够提高胰蛋白酶的耐碱稳定性和热稳定性.     

蛋白质药物的聚乙二醇修饰

聚乙二醇被广泛应用于蛋白质药物的化学修饰,修饰后的蛋白质药物的性质发生多方面变化,如:增加了此类药物的溶解度和稳定性,耐酶水解的能力增强,减弱或消除免疫原性,增加药物在体内的半衰期等。近年来,聚乙二醇修饰剂的种类和修饰方法发展较快,蛋白质分子上的氨基、巯基、羧基均成为化学修饰的研究对象。本文综述了聚乙二醇修饰的原理与方法,修饰方法的比较与优化,修饰后产品的鉴定与检测。     

化学修饰法在酶分子改造中的应用

酶的体外不稳定性限制了其工业应用,而化学修饰法通过化学手段在分子水平上对酶进行改造,可有效地改善酶的性质,为酶的应用提供更广泛的前景.综述酶分子的化学修饰方法及其影响因素,并介绍修饰产物的常用检测评价方法.     

优质L-天冬酰胺酶的开发与应用及重组表达研究进展

L-天冬酰胺酶用于治疗急性淋巴细胞白血病与淋巴瘤,但高昂的价格限制了临床上的广泛应用,重组表达解决了成本的难题。L-天冬酰胺酶所具有的低谷氨酰胺酶活性是其副作用的根源,同时细菌来源的L-天冬酰胺酶能导致危及生命的过敏反应,寻找副作用更低的优质L-天冬酰胺酶及其修饰方法,成为研究的重点。对L-天冬酰胺酶的重组表达,及具有更优性质的L-天冬酰胺酶开发进展进行了综述,并阐明其在临床治疗及食品工业领域的巨大应用潜力。     

聚乙二醇对菠萝蛋白酶的化学修饰

方法:用琥珀酸酐法活化的聚乙二醇对菠萝蛋白酶进行化学修饰,得到菠萝蛋白酶的修饰酶,对比研究三种菠萝蛋白酶:修饰酶、混合酶、天然酶的热稳定性及酸碱稳定性,考察金属离子对三种菠萝蛋白酶的影响。结果:当在55℃水浴保温100min后天然酶活力只保留20%,混合酶活力保留37%,修饰酶活力保留58%;在pH3.0-4.5及pH6.0-7.0的条件下,修饰酶活力高于天然酶活力。当Ca2 的浓度达到0.05mg/mL时,修饰酶的活力高达257.66%;当Mg2 的浓度达到0.035mg/mL时,修饰酶的活力高达147.25%。一价离子Na 对三种菠萝蛋白酶无明显影响。结论:修饰的菠萝蛋白酶对温度和pH值的稳定性均比天然酶有很大程度的提高。混合酶的活力介于天然酶和修饰酶之间说明聚乙二醇对菠萝蛋白酶有一定的保护作用。二价离子Ca2 、Mg2 对三种菠萝蛋白酶活力均有不同程度的激活作用。     

功能化离子液体化学修饰猪胰脂肪酶的催化性能

选取氯化1-羧甲基-3-甲基咪唑、氯化1-羧甲基-3-乙基咪唑、氯化1-羧甲基-3-丁基咪唑3种离子液体对猪胰脂肪酶(PPL)进行化学修饰,得到3种修饰的脂肪酶分别命名为PPL-M、PPL-E、PPL-B。以三乙酸甘油酯水解为模型反应,考察离子液体修饰前后PPL的活力、热稳定性、耐有机溶剂性等酶学性质,并通过紫外光谱研究修饰对PPL空间结构的影响。结果表明:修饰后PPL的活力明显提高,对温度和pH的敏感度降低。修饰酶的热稳定性明显提高,在高浓度的甲醇及N,N-二甲基甲酰胺(DMF)中仍能保持游离酶活力的100%。修饰后酶的特征吸收峰发生红移,吸收强度增强,修饰后酶的微环境发生了改变。     

牛血清白蛋白对超氧化物歧化酶的化学修饰

目的：通过化学修饰提高超氧化歧化酶（SOD）的稳定性,考察金属离子在不同浓度下对SOD活性的影响。方法：用戊二醛作为交联剂,用牛血清白蛋白（BSA）将牛红细胞超氧化物歧化酶进行化学修饰,得到SOD的修饰酶。对比研究三种SOD：修饰酶,混合酶及天然酶的理化性质。结果：修饰酶等电点降低,对温度、pH的稳定性较天然酶有很大提高,对胰蛋白酶和胃蛋白酶也表现出很强的耐水解性。二价离子Mg^2 、Mn^2 对天种SOD活力均有不同程度的抵制作用,Ca^2 、Zn^2 、Cu^2 对修饰酶活力有激活作用,一价离子K^ 对三种OSD活力均无明显影响.结论:修饰酶较天然酶的稳定性有很大的提高,加入Ca^2 、Zn^2 、Cu^2 可提高修饰酶的活力。     

Glycosylation of Escherichia coli L-asparaginase.

Reductive coupling with sodium cyanoborhydride has been used with lactose and N-acetylneuraminyl lactose to prepare glycosylated Escherichia coli L-asparaginase. A substantial degree of modification can be achieved without significant loss of enzyme activity. The lactosylated enzyme shows increased thermal stability and resistance to proteolytic cleavage and is cleared more rapidly from the plasma of mice, compared to native asparaginase. The effect on clearance varies directly with the degree of lactosylation. Asparaginase modified with N-acetylneuraminyl lactose, in contrast, with approximately 13.6 mol of N-acetylneuraminyl lactose/mol of enzyme, is cleared more slowly, with a t 1/2 that is approximately twice that of the native enzyme.     

Purification and Some Biological Properties of Asparaginase from Azotobacter vinelandii

Asparaginase was found in the soluble fraction of cells of Azotobacter vinelandii, and its activity remained the same during growth of the organism in a nitrogen-free medium. The specific activity and the yield of A. vinelandii increased twofold in the presence of ammonium sulfate. Within limits, the temperature (30 to 37°C) and pH (6.5 to 8.0) of the medium showed little effect on the levels of enzyme activity. The enzyme was purified to near homogeneity by standard methods of enzyme purification, including affinity chromatography, and had optimum activity at pH 8.6 and 48°C. The approximate molecular weight was 84,000. The apparent K_m value for the substrate was 1.1 × 10^-4 M. Metal ions or sulfhydryl reagents were not required for enzyme activity. Cu²⁺, Zn²⁺, and Hg²⁺ showed concentration-dependent inhibition, whereas amino and keto acids had no effect on the enzyme activity. Asparaginase was stable when incubated with rat serum and ascites fluid. The enzyme had no effect on the membrane of sheep erythrocytes and did not inhibit the incorporation of radioactive precursors into deoxyribonucleic acid, ribonucleic acid, and protein in Yoshida ascites sarcoma cells. Asparaginase activity was not detected in the tumor cells.     

Asparaginase production by human clinical isolates of Vibrio succinogenes.

Three human isolates of Vibrio succinogenes produced asparaginase. Apparent Km's were 87,220, and 320 microM. The rate of glutamine hydrolysis was between 2.8 and 3.5% of the rate of asparagine hydrolysis. Asparaginase production was not induced by ammonium ions, and enzyme yields were lower than those obtained with the rumen strain.     

Asparaginase production by human clinical isolates of Vibrio succinogenes

Three human isolates of Vibrio succinogenes produced asparaginase. Apparent Km's were 87,220, and 320 microM. The rate of glutamine hydrolysis was between 2.8 and 3.5% of the rate of asparagine hydrolysis. Asparaginase production was not induced by ammonium ions, and enzyme yields were lower than those obtained with the rumen strain.     

Guine pig liver L-asparaginase. Separation, purification, and intracellular localisation of two distinct enzymatic activities.

Two distinct L-asparaginase (EC 3.5.1.1) activities were detected in guinea pig liver: Asparaginase 1 and Asparaginase 2. Asparaginase 1 has been purified 272 fold from the crude homogenate; its molecular weight was evaluated by gel filtration to be about 150 000. The purified preparation was shown to be homogeneous by cellulose acetate strip and polyacrylamide disc-gel electrophoresis. Asparaginase 2 has been purified 63.5 fold from the crude homogenate. Its molecular weight was evaluated by gel filtration to be about 21 500. Cellulose acetate strip electrophoresis demonstrated two bands, one of which corresponded to Asparaginase 1 and the other to Asparaginase 2. Cellular fractionation in the ultracentrifuge, showed Asparaginase 1 to be present only in the cytosol fraction. Asparaginase 2 which was unstable at 105 000 X g seemed mostly localized in the mitochondria and secondarily in the cytoplasmic fraction.     

Effect of medium composition on the growth and asparaginase production of Vibrio succinogenes.

Asparaginase synthesis by Vibrio succinogenes is induced by ammonium ions. Synthesis occurs throughout exponential phase, and in early stationary phase asparaginase accounts for about 5% of the total soluble protein. The organism grows best when fumarate is provided as the terminal electron acceptor of the formate-oxidizing cytochrome system. Yeast extract or enzyme-hydrolyzed proteins are effective nutrient sources. In an ammonium formate-sodium fumarate medium, where maximum growth and asparaginase synthesis occurs, the total enzyme yield (international units per liter of culture) is about one-tenth that obtainable with a good asparaginase-producing strain of Escherichia coli. The energetic inefficiency of V. succinogenes appears to cause a low yield of cells and therefore low total enzyme yield. However, the levels of asparaginase accumulated within cells raise questions about the organism's protein synthesizing system.     

Albumin/asparaginase capsules prepared by ultrasound to retain ammonia

Asparaginase reduces the levels of asparagine in blood, which is an essential amino acid for the proliferation of lymphoblastic malign cells. Asparaginase converts asparagine into aspartic acid and ammonia. The accumulation of ammonia in the bloodstream leads to hyperammonemia, described as one of the most significant side effects of asparaginase therapy. Therefore, there is a need for asparaginase formulations with the potential to reduce hyperammonemia. We incorporated 2 % of therapeutic enzyme in albumin-based capsules. The presence of asparaginase in the interface of bovine serum albumin (BSA) capsules showed the ability to hydrolyze the asparagine and retain the forming ammonia at the surface of the capsules. The incorporation of Poloxamer 407 in the capsule formulation further increased the ratio aspartic acid/ammonia from 1.92 to 2.46 (and 1.10 from the free enzyme), decreasing the levels of free ammonia. This capacity to retain ammonia can be due to electrostatic interactions and retention of ammonia at the surface of the capsules. The developed BSA/asparaginase capsules did not cause significant cytotoxic effect on mouse leukemic macrophage cell line RAW 264.7. The new BSA/asparaginase capsules could potentially be used in the treatment of acute lymphoblastic leukemia preventing hyperammonemia associated with acute lymphoblastic leukemia (ALL) treatment with asparaginase.     

Distribution and Properties of a Potassium-dependent Asparaginase Isolated from Developing Seeds of Pisum sativum and Other Plants

Asparaginase (EC 3.5.1.1) was isolated from the developing seed of Pisum sativum. The enzyme is dependent upon the presence of K⁺ for activity, although Na⁺ and Rb⁺ may substitute to a lesser extent. Maximum activity was obtained at K⁺ concentrations above 20 millimolar. Potassium ions protected the enzyme against heat denaturation. The enzyme has a molecular weight of 68,300.     

L-asparaginase from developing seeds of Lupinus arboreus.

Asparaginase (EC 3.5.1.1) activity reached a maximum 40 days post anthesis in developing seeds of Lupinus arboreus and this correlated with the appearance of other ammonia assimilatory enzymes. Asparaginase, purified from these developing seeds, was resolved into three isoforms, designated asparaginases A, B and C. A major protein species in asparaginase A preparations co-focussed with enzyme activity on an isoelectric focussing gel. When analysed by SDS-PAGE, asparaginase isoforms A and B each yielded several polypeptides with M(r)s in the 14,000 to 19,000 ranged. These peptides are fragmentation products of an M(r) 36,000 asparaginase subunit. Polyclonal antibodies raised against asparaginase isoforms A and B precipitated asparaginase activity from a partially purified L. arboreus seed extract. Immunoaffinity chromatography recovered polypeptides with M(r)s between 14,000 and 19,000. Partial protein sequences were obtained for these asparaginase polypeptides.     

Cloning, expression, and purification of Helicobacter pylori L-asparaginase

Asparaginase from Helicobacter pylori (HpA) has been cloned and expressed in E. coli cells. The recombinant strain stably expressed catalytically active HpA. Optimization of culturing and expression conditions resulted in the expression level of the recombinant enzyme amounting up to 6% of total protein of the producer strain. A method developed for HpA purification included a single chromatographic stage and provided more than 60%-yield of the active enzyme. Specific asparaginase activity was 92 U/mg of protein, whereas the rate of glutamine hydrolysis was just 8.3 × 10^?3 U/mg, respectively. Data obtained indicate that due to low glutaminase specificity HpA may be employed as a non-toxic enzyme preparation for treatment of leukemia.