[P003] Poster

12:10 PM - 1:00 PM JST(3:10 AM - 4:00 AM UTC)

[P02-195] Systematic Reaction Engineering of a DnXR–GDH Cofactor Regeneration System for Improved Enzymatic Xylitol Production

○Akilandaeswari J¹, Sathyanarayana N Gummadi² (1. Ph.D Scholar, Indian Institute of Technology Madras (India), 2. Professor, Indian Institute of Technology Madras (India))

Keywords:

Cofactor regeneration system (CFR),Reaction engineering,Xylitol production,Xylose reductase,Glucose dehydrogenase

Enzymatic xylitol production using xylose reductase (DnXR) from Debaryomyces nepalensis NCYC 3413, coupled with glucose dehydrogenase (GDH) for in situ NADPH regeneration, offers a sustainable alternative to conventional energy-intensive processes. In our previous work, the DnXR–GDH cofactor regeneration (CFR) system achieved a xylitol titre of 13.62 ± 0.02 g/L and a productivity of 5.35 ± 0.05 g/L·h, representing one of the highest performances reported for two-enzyme NADPH-regenerating systems.Despite this, progressive pH decline due to gluconolactone accumulation was identified as a critical limitation, leading to premature saturation of xylose conversion. The present study systematically addresses this bottleneck through reaction engineering strategies. Increasing sodium phosphate buffer concentration beyond the standard 70 mM resulted in a 42% improvement in xylitol titre (19.35 ± 0.16 g/L), demonstrating that enhanced buffering capacity effectively mitigates pH drop and sustains enzyme activity. Further reaction saturation beyond 100 min further indicated additional constraints associated with cofactor stability and product/by-product inhibition. Ongoing investigations focus on the stability of NADPH/NADP⁺ under varying temperature and ionic strength, as well as the inhibitory effects of xylitol and gluconolactone on DnXR and GDH through kinetic (K_i ) analysis. These insights establish a mechanistic framework for the development of a pH-stat controlled, fed-batch CFR system with immobilised enzymes, enabling continuous and industrially viable xylitol production from lignocellulosic feedstocks.