Available Technology

A Single Multi-Functional Enzyme for Efficient Biomass Conversion

Lignocellulosic biomass is an abundant source of fermentable sugars, and biofuels derived from these renewable sources represent one of the best alternatives to petroleum-based fuels. Efficient conversion of lignocellulosic biomass, however, remains a challenge due to its inherent recalcitrance. Given the current state of technology of simultaneous saccharification and fermentation (SFF) and the commercial enzyme cocktails available, various chemical and thermal pretreatment steps are required to achieve meaningful conversation of biomass. In nature, most cellulolytic organisms are of two types: those with non-complexed cellulases, xylanases, and hemicellulases produced by aerobic fungi and most bacteria; and those where cellulases, xylanases, and hemicellulases are complexed on a protein scaffold. This latter case is known only for a few anaerobic bacteria and fungi. In both cases, the enzymes secreted have a wide range of complexity but are mostly equipped with a single catalytic domain. An alternate enzymatic system, midway between the two previous paradigms, is one where the most abundant enzymes secreted are not only multi-modular but possess more than one catalytic domain. This strategy could present several advantages; it allows the synergistic effects between several catalytic domains usually found in cellulosomal systems but also lessen problems that cellulosomes may encounter due to their size. NREL scientists have made a discovery based on a microorganism that was found in 1990 on the Kamchatka Peninsula in Russia. It has now been found that this microorganism can digest cellulose almost twice as fast as the current leading component cellulose enzyme on the market. The name of this bacterium is Caldicellulosiruptor bescii, and it secretes the cellulose CelA. It has a complex arrangement of two catalytic domains separated by linked peptides and cellulose binding modules.
NREL researchers put CelA to the test and found that it produced more sugars than the most abundant cellulase in the leading commercial mixtures, Cel7A, when acting on Avicel, which is an industry standard to test cellulose degradation. They found that CelA not only can digest cellulose in the more common surface removal, but that it also creates cavities in the material, which leads to greater synergy with more conventional cellulases, resulting in higher sugar release. If an enzyme can produce sugars more efficiently, it means lower cost for the enzyme cocktail, which is a major cost driver in the process of converting biomass into fuel. NREL Scientists have also shown that CelA can efficientialy hydrolize crystalline cellulose to glucose and cellobiose, this enzyme can also efficient hydrolyze the xylan in natural plant cell walls to xylose. This could potentially lead to lower costs as fewer enzymes or simpler cocktails could be used. This single enzyme converts both the glucan and xylan components of the biomass materials. Typically multiple enzymes are required to achieve significant levels of glucan and xylan conversion. CelA is a single enzyme can convert both glucan and xylan. The invention reduces the number of individual components in an enzyme preparation from three enzymes to one single enzyme simplifying enzyme preparations and reducing costs. The reduction in the number of critical enzymes in a commercial enzyme formulation reduces likelihood for loss of activity under storage/use conditions and may substantially reduce the cost to produce the product. An article relating to the findings of the invention can be found in the journal Science: “Revealing Nature’s Cellulase Diversity: Digestion Mechanism of Caldicellulosiruptor bescii CelA”
CelA exhibits high specific activity at elevated temperatures: -When acting alone on switchgrass, corn stover and xylan from switchgrass, CelA is substantially more active than current formulations of T. reesei Cel7A cellulase formulations at 85C, 75C and 60C. -Compatibility: CelA activity is applicable with several biomass pretreatment schemes (alkaline peroxide, dilute-acid and ammonia fiber expansion) but actually performed better on untreated substrate. -Formulation: The addition of β-D-glucosidase substantially improves the performance of CelA: -Glucan conversion increased 75% -CelA can achieve 60% conversion of xylan from native switchgrass
Internal Laboratory Ref #: 
NREL ROI 12-28
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