Directed Evolution of a Thermophilic β-glucosidase for Cellulosic Bioethanol Production

Abstract

Characteristics that would make enzymes more desirable for industrial applications can be improved using directed evolution. We developed a directed evolution technique called random drift mutagenesis (RNDM). Mutant populations are screened and all functional mutants are collected and put forward into the next round of mutagenesis and screening. The goal of this technique is to evolve enzymes by rapidly accumulating mutations and exploring a greater sequence space by providing minimal selection pressure and high-throughput screening. The target enzyme was a β-glucosidase isolated from the thermophilic bacterium, Caldicellulosiruptor saccharolyticus that cleaves cellobiose resulting from endoglucanase hydrolysis of cellulose. Our screening method was fluorescence-activated cell sorting (FACS), an attractive method for assaying mutant enzyme libraries because individual cells can be screened, sorted into distinct populations and collected very rapidly. However, FACS screening poses several challenges, in particular, maintaining the link between genotype and phenotype because most enzyme substrates do not remain associated with the cells. We employed a technique where whole cells were encapsulated in cell-like structures along with the enzyme substrate. We used RNDM, in combination with whole cell encapsulation, to create and screen mutant β-glucosidase libraries. A mutant was isolated that, compared to the wild type, had higher specific and catalytic efficiencies (k _cat/K _M) with p-nitrophenol-glucopyranoside and -galactopyranoside, an increased catalytic turnover rate (k _cat) with cellobiose, an improvement in catalytic efficiency with lactose and reduced inhibition (K _i) with galactose and lactose. This mutant had three amino acid substitutions and one was located near the active site.