Enzyme‐mediated hydrogel encapsulation of single cells for high‐throughput screening and directed evolution of oxidoreductases

Abstract

Directed evolution of oxidoreductases to improve their catalytic properties is being ardently pursued in the industrial, biotechnological, and biopharma sectors.Hampering this pursuit are current enzyme screening methods that are limited in terms of throughput, cost, time, and complexity. We present a directed evolution strategy that allows for large-scale one-pot screening of glucose oxidase (GOx) enzyme libraries in well-mixed homogeneous solution. We used GOx variants displayed on the outer cell wall of yeasts to initiate a cascade reaction with horseradish peroxidase (HRP), resulting in peroxidase-mediated phenol crosscoupling and encapsulation of individual cells in well-defined fluorescent alginate hydrogel shells within~10 min in mixed cell suspensions. Following application of denaturing stress to whole-cell GOx libraries, only cells displaying GOx variants with enhanced stability or catalytic activity were able to carry out the hydrogel encapsulation reaction. Fluorescence-activated cell sorting was then used to isolate the enhanced variants. We characterized three of the newly evolved Aspergillus niger GOx enzyme sequences and found up to~5-fold higher specific activity, enhanced thermal stability, and differentiable glycosylation patterns. By coupling intracellular gene expression with the rapid formation of an extracellular hydrogel capsule, our system improves high-throughput screening for directed evolution of H 2 O 2producing enzymes many folds. K E Y W O R D Scell encapsulation, directed evolution, glucose oxidase, hydrogels, yeast display

Chemical Structure–Antioxidant Activity Relationship of Water–Based Enzymatic Polymerized Rutin and Its Wound Healing Potential

“…In addition, water soluble PR had a positive influence on the viability of the human skin derived fibroblasts and HaCaT cells, proving its potential for further studies related to its topological wound healing application, i.e., in the form of bioactive wound dressings. The knowledge on the chemical structure of PR and on its chemical structure–antioxidant activity relationship, obtained in this study, could be in the future transferred also in the new approach of enzymatic phenol polymerization, which has been developed recently and use the genetic manipulation to achieve the displaying of initiating the enzyme, synthesized by the cells themselves, on the cell surface, where the enzyme serves for radical polymerization of added polysaccharides with phenol moieties and encapsulation of the cell themselves [45,46]. The synthesis of PR according to recently described approach could be potentially useful for in vivo cellular therapies and it could represent a new approach for protection of cells against oxidative stress in wound healing therapies.…”

Antioxidant Activity of Synthetic Polymers of Phenolic Compounds

“…To keep the concentration of oxidant low, several methods for in-situ generation of H 2 O 2 have been reported in the literature [23][24][25]. Uyama et al and others reported a bio-enzymatic system for the polymerization of phenols using peroxidase as catalyst and glucose oxidase as in situ H 2 O 2 generation catalyst [26][27][28][29].…”

Advances in single mammalian cells encapsulation technologies

“…Wilhelm T. S. Huck等人采用类似的策略借助单细胞微 胶囊封装系统实现了单细胞水平上的细胞因子分 泌 [148] 和mRNA定量分析 [149] . 此外, 单细胞微胶囊封装 系统为单细胞水平上的全基因组 [150] 、脂质含量 [151] 以 及蛋白酶分泌 [152] 等分析提供了平台. [159,160] .…”