Abstract
Cellulases from Bacillus and Geobacillus bacteria are potentially useful in the biofuel and animal feed industries. One of the unique characteristics of these enzymes is that they are usually quite thermostable. We previously identified a cellulase, GsCelA, from thermophilic Geobacillus sp. 70PC53, which is much more thermostable than its Bacillus homolog, BsCel5A. Thus, these two cellulases provide a pair of structures ideal for investigating the mechanism regarding how these cellulases can retain activity at high temperature. In the present study, we applied the SCHEMA non-contiguous recombination algorithm as a novel tool, which assigns protein sequences into blocks for domain swapping in a way that lessens structural disruption, to generate a set of chimeric proteins derived from the recombination of GsCelA and BsCel5A. Analyzing the activity and thermostability of this designed library set, which requires only a limited number of chimeras by SCHEMA calculations, revealed that one of the blocks may contribute to the higher thermostability of GsCelA. When tested against swollen Avicel, the highly thermostable chimeric cellulase C10 containing this block showed significantly higher activity (22%-43%) and higher thermostability compared to the parental enzymes. With further structural determinations and mutagenesis analyses, a 310 helix was identified as being responsible for the improved thermostability of this block. Furthermore, in the presence of ionic calcium and crown ether (CR), the chimeric C10 was found to retain 40% residual activity even after heat treatment at 90°C. Combining crystal structure determinations and structure-guided SCHEMA recombination, we have determined the mechanism responsible for the high thermostability of GsCelA, and generated a novel recombinant enzyme with significantly higher activity.
Highlights
Cellulases, including endoglucanases (EC3.2.1.4), cellobiohydrolases (EC3.2.1.91) and betaglucosidases (EC 3.2.1.21), convert cellulosic materials into renewable energy and commodity chemicals [1]
Thermophilic cellulases are desirable in such applications since their activity at higher temperatures could result in shorter hydrolysis times [2], decreased risk of contamination [3], facilitated recovery of volatile products such as ethanol [4], and lower costs for cooling after thermal pretreatment [5, 6]
Two bacterial GH5-family enzymes were chosen as parents for noncontiguous SCHEMA recombination: BsCel5A [11] from the thermophile Bacillus subtilis 168, and GsCelA from the highly thermophilic Geobacillus sp.53 [7] (Fig 1A)
Summary
Cellulases, including endoglucanases (EC3.2.1.4), cellobiohydrolases (EC3.2.1.91) and betaglucosidases (EC 3.2.1.21), convert cellulosic materials into renewable energy and commodity chemicals [1]. Thermophilic cellulases are desirable in such applications since their activity at higher temperatures could result in shorter hydrolysis times [2], decreased risk of contamination [3], facilitated recovery of volatile products such as ethanol [4], and lower costs for cooling after thermal pretreatment [5, 6]. Thermophilic bacteria belonging to the strains Bacillus, Geobacillus, Caldibacillus, Acidothermus, Caldocellum and Clostridium are known to produce thermostable cellulases [8, 9]. Bacillus and Geobacillus strains are industrial thermophilic bacterial strains widely used in the production of value-added vitamins, enzymes and proteins [8, 9]. The GsCelA enzyme considered in this study belongs to a particular group of Geobacillus. In contrast to its full-length sequence, the catalytic core of GsCelA has 60% homology with that of BsCel5A from Bacillus subtilis 168
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