Date of Award

Winter 11-20-2020

Semester of Degree

December

Document Type

Restricted Access Dissertation

Degree Name

Ph.D. in Paper and Bioprocess Engineering

Department

Paper & Bioprocess Engineering

Major Professor

Shijie Liu

Steering Committee Member

Chang Geun Yoo

Steering Committee Member

Bandaru Ramarao

Steering Committee Member

Zhenhong Yuan

Abstract

Escherichia coli was genetically modified for the production of D-psicose. A modified plasmid containing an extra copy of gene expression elements, including Ribosomal Binding Site (RBS), T7 promoter, multiple restriction enzyme cloning site, was constructed for the inserting the encoding sequences of D-glucose isomerase (GI) from E. coli MG1655 and D-psicose 3-epimerase (DPE) from Agrobacterium tumefaciens CGMCC 1.1488. The recombinant plasmid was introduced into E. coli BL21(DE3) for the co-expression of GI and DPE. The resulting stain showed capacity for the bioconversion of D-glucose to D-psicose. The characterization and optimization of the D-psicose production were carried out.

The optimal conditions were found to be weak alkaline (pH 7.0-7.5), mesothermal (40- 50°C), with the co-existence of Mg2+ and Co2+ . The transformation equilibrium with an 16% conversion rate was reached at 24h under the optimal conditions, with the highest rate during the first 12h. The increase of initial substrate concentration required higher enzyme input to reach the maximum transformation rate. The enzymatic hydrolysate of sugarcane bagasse and microalgae have been used as the substrate for D-psicose production, which gave out rare sugar yields of 5.7% and 6.5%, respectively.

A kinetic model based on fast equilibrium step assumption was built. The regression was carried out and the rate constants were determined with the experimental data of sugar concentrations accumulating with reaction time. The enzyme loading experimental data showed good agreement with the predicted data calculated by the model based on the derived constants.

Three deep eutectic solvents (DES), including ChCl/glycerol, ChCl/urea, and ChCl/ethylene glycol, have been proved to be capable of improving the conversion efficiency of D-psicose by the whole cell catalysts. The increase of nucleic acid and protein content in the reaction solution indicated the extracellular penetration of intracellular substance. TEM results verified the cell membrane permeability by the DESs, which eliminated the substrate transportation and accelerated the reaction rate.

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