Abstract
β-Glucosidases are key enzymes in the process of cellulose utilization. It is the last enzyme in the cellulose hydrolysis chain, which converts cellobiose to glucose. Since cellobiose is known to have a feedback inhibitory effect on a variety of cellulases, β-glucosidase can prevent this inhibition by hydrolyzing cellobiose to non-inhibitory glucose. While the optimal temperature of the Clostridium thermocellum cellulosome is 70 °C, C. thermocellum β-glucosidase A is almost inactive at such high temperatures. Thus, in the current study, a random mutagenesis directed evolutionary approach was conducted to produce a thermostable mutant with Kcat and Km, similar to those of the wild-type enzyme. The resultant mutant contained two mutations, A17S and K268N, but only the former was found to affect thermostability, whereby the inflection temperature (Ti) was increased by 6.4 °C. A17 is located near the central cavity of the native enzyme. Interestingly, multiple alignments revealed that position 17 is relatively conserved, whereby alanine is replaced only by serine. Upon the addition of the thermostable mutant to the C. thermocellum secretome for subsequent hydrolysis of microcrystalline cellulose at 70 °C, a higher soluble glucose yield (243%) was obtained compared to the activity of the secretome supplemented with the wild-type enzyme.
Highlights
Cellulose, the major polymer in the plant cell wall, is the most abundant organic resource on Earth, and cellulosic is a primary feedstock for the production of ethanol-based biofuels
In order to generate thermostable mutants of C. thermocellum BglA, in vitro directed evolution was applied on the full-length open reading frame (ORF) of the Clo1313_2020 gene
In this report, directed evolution was conducted on the β-glucosidase A gene of the thermophilic bacterium, C. thermocellum, to produce a potent thermostable mutant (Mut 1), which contained two mutations: Alanine at position 17 was substituted with serine, and lysine at position 258 was substituted with asparagine
Summary
The major polymer in the plant cell wall, is the most abundant organic resource on Earth, and cellulosic is a primary feedstock for the production of ethanol-based biofuels. In order to utilize cellulose as a resource for biofuel production, the chain must first be enzymatically hydrolyzed into its primary monomeric glucose units. The glucose is used as a carbon source for alcoholic fermentation to produce bioethanol. Alternative fermentation processes can be used for the production of various other biochemicals such as butanol, acetone, lactic acid, succinic acid and more. Efficient enzymatic hydrolysis of the cellulose is crucial for increasing the cost-effectiveness of the bioethanol and biochemical production process [3,4]
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