Andler R, Altenhoff A-L, Mäsing F, Steinbüchel A
Inst. für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Münster, D-48149, Germany.
Facultad de Ciencias Agrarias y Forestales, Universidad Católica del Maule, Talca, Chile.
Biotechnol Prog. 2018 Jul;34(4):890-899. doi: 10.1002/btpr.2631. Epub 2018 Apr 17.
Cleavage of the backbone of poly(cis-1,4-isoprene) (IR) in solid rubber material was accomplished by the addition of partially purified latex clearing protein (Lcp1 ) using a 200-mL enzyme reactor. Two strategies for the addition of Lcp1 were studied revealing that the daily addition of 50 µg mL of Lcp1 for 5 days was clearly a more efficient regime in comparison to a one-time addition of 250 µg of Lcp1 at the beginning. Soluble oligo(cis-1,4-isoprene) molecules occurred as degradation products and were identified by ESI-MS and GPC. Oxygenase activity of Lcp1 with solid IR particles as substrate was shown for the first time by measuring the oxygen consumption in the reaction medium. A strong decrease of the dissolved oxygen concentration was detected at the end of the assay, which indicates an increase in the number of cleavage reactions. The oligo(cis-1,4-isoprene) molecules comprised 1 to 11 isoprene units and exhibited an average molecular weight (M ) of 885 g mol . Isolation of the oligo(cis-1,4-isoprene) molecules was achieved by using silica gel column chromatography. The relative quantification of the isolated products was performed by HPLC-MS after derivatization with 2,4-dinitrophenilhydrazyne yielding a concentration of total degradation products of 1.62 g L . Analysis of the polymer surface in samples incubated for 3 days with Lcp1 via ATR-FTIR indicated the presence of carbonyl groups, which occurred upon the cleavage reaction. This study presents a cell-free bioprocess as an alternative rubber treatment that can be applied for the partial degradation of the polymer. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:890-899, 2018.
通过在200毫升酶反应器中添加部分纯化的乳胶清除蛋白（Lcp1），实现了固体橡胶材料中聚（顺式-1,4-异戊二烯）（IR）主链的裂解。研究了两种添加Lcp1的策略，结果表明，与一开始一次性添加250微克Lcp1相比，每天添加50微克/毫升Lcp1，持续5天显然是一种更有效的方式。可溶性低聚（顺式-1,4-异戊二烯）分子作为降解产物出现，并通过电喷雾电离质谱（ESI-MS）和凝胶渗透色谱（GPC）进行鉴定。通过测量反应介质中的氧气消耗，首次证明了以固体IR颗粒为底物时Lcp1的加氧酶活性。在测定结束时检测到溶解氧浓度大幅下降，这表明裂解反应的数量增加。低聚（顺式-1,4-异戊二烯）分子由1至11个异戊二烯单元组成，平均分子量（M）为885克/摩尔。通过硅胶柱色谱法实现了低聚（顺式-1,4-异戊二烯）分子的分离。在用2,4-二硝基苯肼衍生化后，通过高效液相色谱-质谱（HPLC-MS）对分离产物进行相对定量，得到总降解产物的浓度为1.62克/升。通过衰减全反射傅里叶变换红外光谱（ATR-FTIR）对用Lcp1孵育3天的样品中的聚合物表面进行分析，表明存在羰基，这是在裂解反应中产生的。本研究提出了一种无细胞生物工艺，作为一种可用于聚合物部分降解的替代橡胶处理方法。©2018美国化学工程师学会生物技术进展，34:890-899，2018年。
