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Kinetic modeling of phosphorylase-catalyzed iterative β-1,4-glycosylation for degree of polymerization-controlled synthesis of soluble cello-oligosaccharides
Biotechnology for Biofuels ( IF 6.1 ) Pub Date : 2021-06-10 , DOI: 10.1186/s13068-021-01982-2
Mario Klimacek , Chao Zhong , Bernd Nidetzky

Cellodextrin phosphorylase (CdP; EC 2.4.1.49) catalyzes the iterative β-1,4-glycosylation of cellobiose using α-d-glucose 1-phosphate as the donor substrate. Cello-oligosaccharides (COS) with a degree of polymerization (DP) of up to 6 are soluble while those of larger DP self-assemble into solid cellulose material. The soluble COS have attracted considerable attention for their use as dietary fibers that offer a selective prebiotic function. An efficient synthesis of soluble COS requires good control over the DP of the products formed. A mathematical model of the iterative enzymatic glycosylation would be important to facilitate target-oriented process development. A detailed time-course analysis of the formation of COS products from cellobiose (25 mM, 50 mM) and α-d-glucose 1-phosphate (10–100 mM) was performed using the CdP from Clostridium cellulosi. A mechanism-based, Michaelis–Menten type mathematical model was developed to describe the kinetics of the iterative enzymatic glycosylation of cellobiose. The mechanistic model was combined with an empirical description of the DP-dependent self-assembly of the COS into insoluble cellulose. The hybrid model thus obtained was used for kinetic parameter determination from time-course fits performed with constraints derived from initial rate data. The fitted hybrid model provided excellent description of the experimental dynamics of the COS in the DP range 3–6 and also accounted for the insoluble product formation. The hybrid model was suitable to disentangle the complex relationship between the process conditions used (i.e., substrate concentration, donor/acceptor ratio, reaction time) and the reaction output obtained (i.e., yield and composition of soluble COS). Model application to a window-of-operation analysis for the synthesis of soluble COS was demonstrated on the example of a COS mixture enriched in DP 4. The hybrid model of CdP-catalyzed iterative glycosylation is an important engineering tool to study and optimize the biocatalytic synthesis of soluble COS. The kinetic modeling approach used here can be of a general interest to be applied to other iteratively catalyzed enzymatic reactions of synthetic importance.

中文翻译:

磷酸化酶催化的迭代β-1,4-糖基化的动力学建模,用于可溶性纤维寡糖的聚合度控制合成

纤维糊精磷酸化酶 (CdP;EC 2.4.1.49) 使用 α-d-葡萄糖 1-磷酸作为供体底物,催化纤维二糖的迭代 β-1,4-糖基化。聚合度 (DP) 高达 6 的纤维寡糖 (COS) 是可溶的,而聚合度 (DP) 较大的纤维寡糖 (COS) 自组装成固体纤维素材料。可溶性 COS 因其用作提供选择性益生元功能的膳食纤维而备受关注。可溶性 COS 的有效合成需要对形成的产物的 DP 进行良好控制。迭代酶促糖基化的数学模型对于促进以目标为导向的工艺开发很重要。从纤维二糖(25 mM,50 mM) 和 α-d-葡萄糖 1-磷酸 (10–100 mM) 使用来自纤维素梭菌的 CdP 进行。开发了基于机制的 Michaelis-Menten 型数学模型来描述纤维二糖的迭代酶促糖基化的动力学。机械模型与 COS 依赖 DP 的自组装成不溶性纤维素的经验描述相结合。如此获得的混合模型用于根据从初始速率数据导出的约束进行的时程拟合的动力学参数确定。拟合的混合模型很好地描述了 DP 范围 3-6 中 COS 的实验动力学,并解释了不溶性产物的形成。混合模型适用于解开所用工艺条件(即底物浓度、供体/受体比例、反应时间)和获得的反应输出(即可溶性 COS 的产率和组成)。以富含 DP 4 的 COS 混合物为例,展示了用于合成可溶性 COS 的操作窗口分析的模型应用。 CdP 催化的迭代糖基化混合模型是研究和优化生物催化的重要工程工具可溶性 COS 的合成。这里使用的动力学建模方法可以广泛应用于其他具有合成重要性的迭代催化酶促反应。
更新日期:2021-06-11
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