Andler R, Steinbüchel A
Institute of Molecular Microbiology and Biotechnology, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany.
Institute of Molecular Microbiology and Biotechnology, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany; Environmental Sciences Department, King Abdulaziz University, Jeddah, Saudi Arabia.
J Biotechnol. 2017 Jan 10;241:184-192. doi: 10.1016/j.jbiotec.2016.12.008. Epub 2016 Dec 7.
Aiming at finding feasible alternatives for rubber waste disposal, the partial enzymatic degradation of poly(cis-1,4-isoprene)-containing materials represents a potential solution. The use of rubber-degrading enzymes and the biotransformation of rubber into new materials is limited by the high costs associated with the production and purification of the enzyme and the complexity of the process. This study presents a simple and low-cost procedure to obtain purified latex clearing protein (Lcp), an enzyme capable of cleaving the double bonds of poly(cis-1,4-isoprene) in presence of oxygen to produce different size of oligomers with terminal aldehyde and ketone groups, respectively. The gene coding for Lcp1 from Gordonia polyisoprenivorans strain VH2 was overexpressed in Escherichia coli C41 (DE3), and by using an auto-induction medium high protein yields were obtained. The cultivation process was described and compared with an IPTG-inducible medium previously used. Purification of the enzyme was performed using salting out precipitation with ammonium sulfate. Different salt concentrations and pH were tested in order to find the optimal for purification, obtaining a concentration of 60mg Lcp per l. The enzymatic activity of the purified enzyme was measured by an oxygen consumption assay in the presence of polyisoprene latex. Volumetric activities of 0.16Uml were obtained at optimal conditions of temperature and pH. The results showed an active and partial purified fraction of Lcp1, able to cleave the backbone of poly(cis-1,4-isoprene) and to produce degradation products that were identified with staining methodologies (Schiff reagent for aldehyde groups and 2,4-DNPH for carbonyl groups) and characterized using nuclear magnetic resonance (NMR). Thirteen different storage conditions were tested for the purified enzyme analyzing the enzymatic activity after 1 and 3 months. Lcp1, as an ammonium sulfate precipitate, was stable, easy to handle and sufficiently active for further analysis. The described methodology offers the possibility to upscale the process and to produce large amounts of this protein.
为了寻找橡胶废料处理的可行替代方法，含聚（顺式-1,4-异戊二烯）材料的部分酶促降解是一种潜在的解决方案。橡胶降解酶的使用以及橡胶向新材料的生物转化受到与酶的生产和纯化相关的高成本以及该过程复杂性的限制。本研究提出了一种简单且低成本的方法来获得纯化的乳胶澄清蛋白（Lcp），该酶能够在氧气存在下裂解聚（顺式-1,4-异戊二烯）的双键，分别产生具有末端醛基和酮基的不同大小的低聚物。来自戈登氏多聚异戊二烯ivorans菌株VH2的Lcp1编码基因在大肠杆菌C41（DE3）中过表达，并通过使用自诱导培养基获得了高蛋白产量。描述了培养过程并与先前使用的IPTG诱导培养基进行了比较。使用硫酸铵盐析沉淀法进行酶的纯化。测试了不同的盐浓度和pH值以找到纯化的最佳条件，每升获得60mg Lcp的浓度。通过在聚异戊二烯乳胶存在下的耗氧测定法测量纯化酶的酶活性。在最佳温度和pH条件下获得了0.16U/ml的体积活性。结果显示了Lcp1的活性和部分纯化级分，能够裂解聚（顺式-1,4-异戊二烯）的主链并产生降解产物，这些产物通过染色方法（用于醛基的席夫试剂和用于羰基的2,4-二硝基苯肼）进行鉴定，并使用核磁共振（NMR）进行表征。测试了纯化酶的十三种不同储存条件，分析1个月和3个月后的酶活性。作为硫酸铵沉淀的Lcp1是稳定的，易于处理并且具有足够的活性用于进一步分析。所描述的方法提供了扩大该过程规模并大量生产这种蛋白质的可能性。
