*3.3. Substrate Specificity*

The enzyme substrate specificity results showed that MFE-5E was a multifunctional enzyme that displayed 17.189, 13.471, 7.483 and 6.831 U/mg protein specific activity on CMC, wheat flour xylan, beechwood xylan and xyloglucan, respectively. These findings demonstrate that MFE-5E had a higher propensity to hydrolyse amorphous cellulose followed by various xylan substrates and xyloglucan (Table 2). In addition, MFE-5H exhibited the highest specific activity on xyloglucan (84.975 U/mg protein), followed by wheat flour xylan, beechwood xylan and CMC with 67.365, 52.015 and 28.584 U/mg protein, respectively. Both MFE-5E and MFE-5H were classified under GH family 5 (with accession numbers AMO13175 and AMO13178, respectively), which consists of some of the enzymes displaying diverse activities on various carbohydrate substrates (http://www.cazy.org/GH5.html). This explains the broad range of MFE-5E and MFE-5H activity on various carbohydrate substrates. The results in Table 2 also shows that the MFE-45 displayed highest specific activity on xyloglucan (12.86 U/mg protein). MFE-45 showed similar activity during hydrolysis of beechwood xylan and CMC, which were 4.55 and 4.75 U/mg protein, respectively. MFE-45 is reported to belong to GH family 45 and its accession number is AMO13193. According to the CAZy Database (http://www.cazy.org/GH45\_bacteria.html), enzymes in family 45 possess endoglucanase activity, xyloglucanase activity but not xylanase activity, which suggests that MFE-45 possesses a novel xylanase activity. The MFEs did not show any activity on Avicel, and the mannan substrate, locus bean gum, and several *p*-nitrophenyl-substrates (Table 2). However, MFEs presented in this study were true multifunctional enzymes that displayed specific activities on various substrates with their backbones linked by β-1,4-glycosic linkages, and possessed different sidechains.


**Table 2.** Substrate specificity of enzymes on polymeric and *p*-nitrophenyl substrates.

Values represent means ± SD and *n* = 3; N/A means there was no activity; # - means not tested on this substrate; pNP-A, G, -X, -C and M represents *p*-nitrophenyl-arabinofuranoside, -glucopyranoside, -xylopyranoside, -cellobioside and –mannopyranoside, respectively; Concentrations of polymeric and *p*-nitrophenyl substrates were 2% and 4 mM, respectively; U represents µmol/min.
