Date of Award

Spring 2026

Document Type

Thesis

Publisher

Santa Clara : Santa Clara University, 2026

Degree Name

Master of Science (MS)

Department

Bioengineering

First Advisor

Michelle E. McCully

Abstract

The enzyme Endo-β-1,4-Glucanase from Bacillus subtilis, commonly known as BsCel5A, catalyzes the hydrolysis of β-1,4-glycosidic bonds in cellulose aiding in biomass deconstruction and its conversion into fermentable sugars. Efficient, thermostable cellulases are critical for reducing the cost and energy demands of lignocellulosic bioprocessing. Unfortunately, many naturally occurring enzymes are limited by activity rate and stability in bioprecessing conditions, especially high temperatures. In this study, we compared the catalytic and thermal properties of wild-type BsCel5A (BsCel5A‑WT) and a truncated catalytic core variant (BsCel5A‑CC) to evaluate their efficiency and thermostability. The catalytic and thermal properties were investigated through the hydrolysis of a small, soluble, artificial cellulose analog p‑nitrophenyl‑β‑D‑cellobioside (pNPC) as a model reaction. We found that the catalytic core alone retained endoglucanase activity and exhibited increased catalytic efficiency relative to the wild-type enzyme, while both constructs show similar patterns of activity loss upon prolonged heat treatment. These findings suggest that non‑catalytic regions are not essential for BsCel5A activity on soluble substrates and that truncation can enhance its catalytic performance. This work establishes a convenient pNPC based assay for comparing BsCel5A variants and provides a foundation for future efforts to improve enzyme thermostability.

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