## 9 Properties of Processive Endocellulases in Hydrolyzing Pre-Treated Cellulosic Substrates  
While Avicel is widely used as a pure crystalline cellulose substrate with estimated CrI at 0.5-0.6, it is a microcrystalline cellulose with relatively much small molecular weights (DP of 150-500 glucose units) in comparison with other crystalline cellulose substrates such as Whatman No. 1 filter paper (DP of 750-2800 glucose units) and Solka-Floc(\({}^{\text{\textregistered}}\)) (DP of 750-1500 glucose units) [4]. Thus, GH5-p4818 Cel5_2A activity reported by [PERSON] et al. [45] was also determined at 0.127 \(\upmu\)mol D-glucose/mg protein.min by using Solka-Floc(\({}^{\text{\textregistered}}\)) and this activity value was about 70% lower than the value measured with Avicel (Table 1). Differences in porosity and molecular weights, as reflected by their different DP values, were likely responsible for this discrepancy in the GH5-p4818 Cel5_2A activities measured between these two crystalline cellulosic substrates. Due to the fact that biological pretreatment of biomass materials is not well established, various physiochemical pre-treatments of biomass materials have been established and optimized to release semi-pure pretreated cellulosic substrates for potential industrial scale of saccharification and subsequent fermentation of ethanol [10,24]. Physicochemical pre-treatments of biomass feedstock are expensive and represent about 21% of the total expense of producing lignocellulosic ethanol [24]. It should be pointed out that Solka-Floc(\({}^{\text{\textregistered}}\)) is a semi-pure crystalline cellulose product commercially available from the International Fibre Corporation. Solka-Floc(\({}^{\text{\textregistered}}\)) has properties comparable to pretreated cellulosic substrates including CrI values (CrI for both 0.4-0.7) and DP (DP of 750-1500 vs. 400-1000 glucose units) [4]. Thus, the GH5-p4818 Cel5_2A activity determined by using Solka-Floc(\({}^{\text{\textregistered}}\)) reported by [PERSON] et al. [45] should represent specific activity of this novel enzyme in hydrolyzing pretreated crystalline cellulosic substrates. Amorphous disordered insoluble celluloses and dissolved soluble celluloses are different entities, which is different from crystalline celluloses in terms of physicochemical properties and rates of hydrolysis by cellulases for industrial applications [124]. While needing to be further optimized for potential industrial scale of applications, both amorphous insoluble cellulose and soluble cellulose substrates have been developed and widely used in cellulase activity tests [4,143]. Acid swollen cellulose, primarily in the form of phosphoric acid swollen cellulose (PASC), is a typical insoluble amorphous disordered cellulose with CrI at 0.00-0.04 and DP of 100-1000 glucose units [19,124]. Regenerated amorphous celluloses (RAC) are soluble amorphous cellulose substrates and have been generated by using concentrated phosphoric acid and cellulose solvents [143], which representsfurther treated readily degradable next generation cellulosic substrates for commercial ethanol production [76; 143]. The tCel5A1 processive endoglucanase reported at 24.8 umol D-glucose/mg protein.min by using RAC at 70 \({}^{\circ}\)C but not at the physiological pH by [PERSON] and [PERSON] [127]. The GH5-p4818 Cel5_2A activity reported by [PERSON] et al. [45] was determined at 1.39 umol D-glucose/mg protein.min by using RAC at 50 \({}^{\circ}\)C and this activity was about 5.4-fold higher than the value measured with Avicel in this study (Table 1). This result of GH5-p4818 Cel5_2A activity on RAC was consistent with previous studies in showing that endocellulases with activities on crystalline celluloses would have much amplified hydrolytic activities in hydrolyzing amorphous cellulose substrates frequently demonstrated with PASC or RAC [118; 120; 131; 133]. Hence, these tCel5A1 and the GH5-p4818 Cel5_2A endoglucanase activities determined by RAC, as reported by [PERSON] and [PERSON] [127] and [PERSON] et al.