Temperature optima were 102, 96, 98, 85, 94, and 95☌ for TCel1, TCel2, Tcel3, TCel4, TCel5, and TCel6, respectively. Table 2 shows the optimum temperatures (also shown in Table 1) for catalytic ac- tivity for all six cellulases (TCel1 to -6) and two beta-glucosidases (TCel11 and TCel12) along with percentages of maximum activity at 60, 45, and 20☌. Both TCel11 and TCel12 were true ␤ -glucosidases even though they have distinct physical properties: molecular masses of 81.3 and 51.5 kDa, pIs of 5.38, 5.84, and net charges at pH 7 of Ϫ 16.90 and Ϫ 9.10, respectively (Table 1). Finally, we also assayed beta-glucosidases (TCel11 and TCel12) for the ability to hydrolyze p -nitrophenol- ␤ - D -glucoside (pNPG), a colorimetric substrate commonly used to determine specific activity for beta-glucosidases, and found that both were active, at 694 and 609 U/mg protein, respectively (Table 1). Thus, TCel enzymes function on insoluble cellulosic substrates with specific activities equivalent to those of fungal cellobiohydrolases. On the other hand, endoglucanase showed high specific activity toward CMC (221.94 mU/mg protein). As expected, cellobiohydrolase showed low activity on CMC (78.4 mU/mg protein) and comparable activities on insoluble substrates (27.08, 27.29, and 0.01 mU/mg protein with PASC, CL, and FP, respectively). Table 1 compares the specific enzyme activities of our TCel enzymes on various cellulosic substrates with those of bench- mark commercial enzymes, namely, Megazyme’s cellobiohydrolase (E.C.

TCel4 overlapped both groups and showed reduced activities across all substrates tested (Table 1). On the other hand, TCel1 and TCel3 efficiently hydrolyzed PASC (44.03 and 33.68 mU/mg protein, respectively), cotton linters (9.44 and 5.83 mU/mg protein, respectively), and filter paper (7.29 and 5.97 mU/mg protein, respectively) and were less active on CMC than the other enzymes (TCel2, TCel5, and TCel6). TCel2, TCel5, and TCel6 efficiently hydrolyzed soluble carboxymethylcellulose (162.64, 91.11, and 116.18 mU/mg protein, respectively) but were much less active on insoluble substrates such as PASC (5.62, 23.68, and 14.3 mU/mg protein, respectively), cotton linters (1.53, 4.72, and 3.54 mU/mg protein, respectively), and filter paper (2.01, 5.55, and 3.54 mU/mg protein, respectively). Table 1 describes cellulase specific activities for our enzyme collection on carboxymethylcellulose (CMC) (a soluble substi- tuted cellulose substrate) or more crystalline (insoluble) forms of cellulose, such as cotton linters (CL), filter paper (FP), and phosphoric acid-swollen Avicel (PASC). The two ␤ -glucosidases TCel11 and TCel12 belong to CAZy families GH3 and GH1, respectively. Two clear groups of cellulases appear in Table 1, i.e., CAZy family GH12, with molecular masses of 31 to 38 kDa and pI 4.8 to 5.7, and CAZy family GH5, with molecular masses of 52 to 60 kDa and pI 6.5 to 7. Table 1 describes the physical and biochemical properties derived from the predicted amino acid sequence of all eight proteins as well as the temperature and pH optima found experimentally. Recovered proteins were further analyzed for biochemical activity as well as other physical and biochemical properties (see Materials and Methods in the supplemental material). The proteins were purified by His 6 tag nickel-chelate affinity chromatography of boiled crude cell extracts. Cloned TCel genes were expressed and proteins produced in E. Genes noted as TCel1 through TCel6 correspond to putative cellulases, and TCel11 and TCel12 correspond to putative ␤ -glucosidases. Based on the relatedness of cellulases displayed in the tree, we chose six putative cellulases and two clearly identified ␤ -glucosidases (not shown in the tree) for further biochemical studies. Figure 1 displays a neighbor-joining phylogenetic tree (38) of all BLASTp-identified archaeal and bacterial putative cellulases. The search resulted in 32 prokaryotic species with at least three hits in one of the four functional categories of carbohydrate-active enzymes defined by CAZy (data not shown) however, only nine genomes showed significant carbohydrate-hydrolyzing enzyme content. Bacterial carbohydrate-active enzyme protein models (glycosyl hydrolases, lyases, esterases, and carbohydrate-binding do- main-containing enzymes) were derived from CAZy (7) and used in BLASTp surveys of sequenced archaeal and thermophilic bacterial genomes (GenBank release 161.0).

Tcel cleson