Generally speaking, rational engineering refers to planned biochemical changes to a protein through the use of protein sequence and structure information, which in theory corresponds to a physiological or functional change in the proteins behavior. The engineered changes are usually predicted using computational biology and protein sequence data. However, there is limited struc­tural information available for enzymes, for example, in structure—function relationship—so predictions on behavioral changes after rational engineeringstill remain in a trial-like state (Maki et al., 2009). Nonetheless, with increasing knowledge of biomass substrates and a rigorous test of our knowledge about enzyme interac­tions with plant-based biomass, rational engineering can be a valuable tool in the economical production of biofuels and value-added by-products.

Briefly, rational design of proteins can be summed up in three simple steps: (1) a suitable enzyme is chosen based on desired characteristics, (2) using computational biology or a high resolution crystallographic structure, the amino acid sites to be changed are identified, and

(3) mutants produced from rationally engineered pro­teins are characterized (Percival Zhang et al., 2006).

Moreover, rational modifications to enzymes often include amino acids substitutions using site-directed mutagenesis, which can be used to increase the stability of enzymes (i. e. thermostability), substrate specificity, cofactor specificity, and the elucidation of enzymatic mechanisms (Bornscheuer and Pohl, 2001). In the field of biomass conversion to biofuels and bioproducts, the use of rational design has pioneering examples as out­lined here.

For the most part, there are numerous reviews that summarize studies that revealed the mechanism of cellulase and other biomass-converting genes through the use of site-directed mutagenesis (Schulein, 2000; Wilson, 2004; Wither, 2001). On the contrary, very few researchers have reported increasing cellulase and other biomass-converting activities or enhancing properties through site-directed mutagenesis. However, Baker et al. were able to improve the activity of endoglucanase

Cel5A of Acidothermus celluloyticus toward microcrystal­line cellulose by 20% (Baker et al., 2005). This was accomplished utilizing a high-resolution crystallo­graphic structure (Sakon et al., 1996) to determine a se­ries of mutations designed to alter the active cleft through a change in chemistry of the product-leaving side. As a result, structural information allowed end — product inhibition to be alleviated by a substitution of a nonaromatic residue at site 245; a Y245G mutant increased the KI of cellobiose by 15-fold.

In a similar study, site-directed mutagenesis was used to improve the catalytic activity of endo/exocellulase Cel9A in Thermobifida fusca by 40% with soluble and amorphous cellulose, such as carboxymethyl cellulose (CMC) and swollen cellulose. Through the use of com­puter modeling, the conserved phenylalanine residue F476 (one of three residues) was found at the end of the carbohydrate binding module and appeared to play an important role in the initial binding of the cellulase to substrate. Also, computer modeling was used to predict that a new hydrogen bond could be created as a result of mutating the conserved phenylalanine residue F476 to a tyrosine. Therefore, the observed increase in catalytic ac­tivity of mutant F476Y is thought to be attributed to better binding properties, which are key for placing the soluble and amorphous cellulose chains in the carbohydrate binding domain (Escovar-Kousen et al., 2004).

Rational engineering of enzymes can also be used to improve characteristics such as thermostability and alkalinity in addition to specific activity. The roles of highly conserved residues (Asp 60, Tyr 35 and Glu 141), near the catalytic site, were investigated in the pH — dependent activity of xylanase XYL1p from Scytalidium acidophilum using site-directed mutagenesis. In doing so, three single mutants, D60N, Y35W and E141A, were created and the activities of three combined xyla — nase mutants DN/YW, DN/EA and YW/EA were eval­uated at different pHs and temperatures. An increased pH optimum of 0.5—1.5 pH units and lower specific activities were observed in all the mutants except one. Mutant E141A exhibited a 50% increase in specific activ­ity at pH 4.0 and had an overall higher catalytic effi­ciency than wild-type enzyme (Al Balaa et al., 2009). This work presents some important knowledge in acid­ophilic adaptation and, at the same time, is a prime example of how rational engineering can lead to the development of enzymes more suitable for the biocon­version industry environment, with competitive cata­lytic efficiency maintained.

Finally, the possibility of using rational engineering to improve the pH optimum and catalytic efficiency of lac — case enzymes, involved in the oxidation of lignin, has been increasing as several researchers explore important residues conserved in laccases from fungi (Rogers et al.,

2009) . In one compelling example, researchers replaced an Asp residue in position 206 with an Asn residue in a laccase from T. versicolor, using site-directed mutagen­esis. Upon expression of mutants in the yeast Yarrowia lipolytica, it was noted that catalytic activity was signifi­cantly affected as the pH optimum was raised by 1.4 pH units (Madzak et al., 2006), highlighting the interac­tion between the reducing substrate and the binding pocket of laccase. This study, like those discussed previ­ously, pave the way for future development of efficient biomass-converting enzymes.