The conversion of recalcitrant plant-derived cellulosic biomass into biofuels is dependent on highly efficient cellulase systems that produce near-quantitative levels of soluble saccharides. Similar ...to other fungal and bacterial cellulase systems, the multienzyme cellulosome system of the anaerobic, cellulolytic bacterium Clostridium thermocellum is strongly inhibited by the major end product cellobiose. Cellobiose-induced inhibition can be relieved via its cleavage to noninhibitory glucose by the addition of exogenous noncellulosomal enzyme β-glucosidase; however, because the cellulosome is adsorbed to the insoluble substrate only a fraction of β-glucosidase would be available to the cellulosome. Towards this end, we designed a chimeric cohesin-fused β-glucosidase (BglA-CohII) that binds directly to the cellulosome through an unoccupied dockerin module of its major scaffoldin subunit. The β-glucosidase activity is thus focused at the immediate site of cellobiose production by the cellulosomal enzymes. BglA-CohII was shown to retain cellobiase activity and was readily incorporated into the native cellulosome complex. Surprisingly, it was found that the native C. thermocellum cellulosome exists as a homooligomer and the high-affinity interaction of BglA-CohII with the scaffoldin moiety appears to dissociate the oligomeric state of the cellulosome. Complexation of the cellulosome and BglA-CohII resulted in higher overall degradation of microcrystalline cellulose and pretreated switchgrass compared to the native cellulosome alone or in combination with wild-type BglA in solution. These results demonstrate the effect of enzyme targeting and its potential for enhanced degradation of cellulosic biomass.
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
cellulosome is 70 °C,
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 K
and K
, 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 (T
) 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
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.
The thermostability of endoglucanase (E.C. 3.2.1.4) Cel8A, a major component of the cellulosome complex from Clostridium thermocellum, was significantly enhanced using a directed evolution strategy. ...To ensure that thermostability would not compromise enzyme activity, a two‐step screening strategy was employed that involved consecutive activity and thermostability assays. We have combined three of the mutations from the thermostability screen to obtain a Cel8A variant with a significant increase in thermal resistance without substantial alteration of kinetic parameters. One of the three mutations (S329G) provided the highest contribution to enzyme stability. This single mutation served to increase the Tm by 7.0 °C and the half‐life of activity by eight fold at 85 °C. Site‐saturation mutagenesis at position 329 revealed that only the glycine residue could confer thermostability. The structural changes responsible for the properties of the mutant enzymes are discussed.
Thermostable mutant enzymes: The X‐ray crystal structure of the Clostridium thermocellum Cel8A cellulase depicts the amino acid substitutions which enable the protein to withstand elevated temperatures while maintaining wild‐type levels of activity. The mutant enzyme may be used in combination with other selected glycoside hydrolases to efficiently degrade lignocellulosic materials used in the biofuel industry.
: This paper reviews the mechanism and assessment of regulated radiative heat dissipation, involving the circulatory system and the skin. It describes the quantitative assessment of skin temperature ...modulation. The main regulating process, which can be quantitatively monitored by fast and sensitive dynamic infrared imaging, involves autonomic nervous control of cutaneous and subcutaneous perfusion. This control is significantly affected by a variety of local or systemic pathologic conditions, including cancer and certain neuropathies. A potential clinical application that objectively assesses local attenuation of temperature modulation in the presence of breast cancer is described in some detail. Systemic aberrations in skin temperature modulation can be clinically useful also in neurology. It can be used also in psychology and psychiatry to evaluate transient effects of mental stress on the autonomic nervous system.
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