Abstract
In the process of yielding biofuels from cellulose degradation, traditional enzymatic hydrolysis, such as β-glucosidase catalyzing cellobiose, can barely resolve the contradiction between cellulose degradation and bioenergy conservation. However, it has been shown that cellobiose phosphorylase provides energetic advantages for cellobiose degradation through a phosphorolytic pathway, which has attracted wide attention. Here, the cellobiose phosphorylase gene from Caldicellulosiruptor bescii (CbCBP) was cloned, expressed, and purified. Analysis of the enzymatic properties and kinetic mechanisms indicated that CbCBP catalyzed reversible phosphorolysis and had good thermal stability and broad substrate selectivity. In addition, the phosphorolytic reaction of cellobiose by CbCBP proceeded via an ordered Bi Bi mechanism, while the synthetic reaction proceeded via a ping pong Bi Bi mechanism. The present study lays the foundation for optimizing the degradation of cellulose and the synthesis of functional oligosaccharides.
Highlights
In the process of yielding biofuels from cellulose degradation, traditional enzymatic hydrolysis, such as β-glucosidase catalyzing cellobiose, can barely resolve the contradiction between cellulose degradation and bioenergy conservation
The results showed that CbCBP had good thermal stability, high glucose tolerance, and broad substrate selectivity
The analysis of the enzymatic properties and kinetic mechanisms revealed that the half-life of the CbCBP was 6 h and 4 h at 65 °C and 75 °C, indicating that good thermal stability
Summary
In the process of yielding biofuels from cellulose degradation, traditional enzymatic hydrolysis, such as β-glucosidase catalyzing cellobiose, can barely resolve the contradiction between cellulose degradation and bioenergy conservation. The exoglucanase degrades the reducing or non-reducing ends of the cellulose chain, producing mainly c ellobiose[14]. Β-glucosidase acts only on the non-reducing ends, hydrolyzing cellobiose and cello-oligosaccharides to g lucose[15]. Cellobiose is an important intermediate in the cellulose degradation process. Β-glucosidase cleaves cellobiose into two glucose molecules in the presence of water, followed by the metabolism of glucose molecules through the glycolytic pathway to produce e thanol[17]. This method consumes two ATP molecules for the hydrolysis of one cellobiose molecule. Given the importance of bioenergy storage, it is imperative to explore a highperformance pathway that consumes less ATP than the cellulose hydrolysis pathway with two ATP molecules
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