Abstract

We previously reported an in vitro enzymatic pathway for conversion of nonfood cellulose to starch (PNAS,110 (18): 7182–7187, 2013), in which the two sequential enzymes cellobiose phosphorylase (CBP) from Clostridium thermocellum and potato alpha-glucan phosphorylase (PGP) from Solanum tuberosum were the two key enzymes responsible for the whole conversion rate. In this work CBP and PGP were fused to form a large enzyme and it turned out that the fusion protein could exhibit a good bifunctionality when PGP moiety was put at the N-terminus and CBP moiety at the C-terminus (designated as PGP-CBP). Although the coupled reaction rate of PGP-CBP was decreased by 23.0% compared with the free enzymes, substrate channeling between the two active sites in PGP-CBP was formed, demonstrated by the introduction of the competing enzyme of PGP to the reaction system. The potential of PGP-CBP fusion enzyme being applied to the conversion of cellulose to amylose was discussed.

摘要

我们之前报道了一种将非食用纤维素转化为淀粉的体外酶促途径（ PNAS，110（18）：7182–7187，2013），其中来自热纤梭菌的两种连续酶纤维二糖磷酸化酶（CBP）和马铃薯α-葡聚糖磷酸化酶(PGP) 来自马铃薯是负责整个转化率的两种关键酶。在这项工作中，CBP 和 PGP 融合形成一个大酶，结果表明，当 PGP 部分位于 N 端，CBP 部分位于 C 端时，融合蛋白可以表现出良好的双功能性（称为 PGP-CBP ）。尽管与游离酶相比，PGP-CBP 的偶联反应速率降低了 23.0%，但在 PGP-CBP 中的两个活性位点之间形成了底物通道，这可以通过将 PGP 竞争酶引入反应系统来证明。讨论了将 PGP-CBP 融合酶应用于纤维素转化为直链淀粉的潜力。