unassigned: Fibronectin 3 (FN3) and Ig-like modules (Ig) are usually collocated beside modular cellulase catalytic domains. However, very few researchers have investigated the role of these modules. In a previous study, we have sequenced and analyzed bacterial metagenomic DNA in Vietnamese goats' rumen and found that cellulase-producing bacteria and cellulase families were dominant [1]. In this study, the properties of modular cellulases and the role of a FN3 in unique endoglucanase FN3-GH5 were determined.
unassigned: Based on Pfam analysis, the cellulases sequences containing FN3, Ig modules were extracted from 297 complete ORFs. The alkaline, thermostability, tertiary structure of deduced enzymes were predic... More
unassigned: Fibronectin 3 (FN3) and Ig-like modules (Ig) are usually collocated beside modular cellulase catalytic domains. However, very few researchers have investigated the role of these modules. In a previous study, we have sequenced and analyzed bacterial metagenomic DNA in Vietnamese goats' rumen and found that cellulase-producing bacteria and cellulase families were dominant [1]. In this study, the properties of modular cellulases and the role of a FN3 in unique endoglucanase FN3-GH5 were determined.
unassigned: Based on Pfam analysis, the cellulases sequences containing FN3, Ig modules were extracted from 297 complete ORFs. The alkaline, thermostability, tertiary structure of deduced enzymes were predicted by AcalPred, TBI softwares, Phyre2 and Swiss models. Then, whole and truncated forms of XFN3GH5 were expressed in E. coli and purified by His-tag affinity column for assessment of FN3 abilities to enhance enzyme activity, solubility and conformation.
unassigned: From 297 complete ORFs coding for cellulases, 148 sequences containing FN3, Ig were identified. Mostly FN3 appeared in 90.9% beta-glucosidase GH3 and situated downstream of catalytic domains. The Ig was found upstream of 100% endoglucanase GH9. Rarely FN3 was seen to be situated downstream of X domain and upstream of catalytic domain endoglucanase GH5. Whole enzyme (XFN3GH5) and truncate forms FN3, XFN3, FN3GH5, GH5 were cloned in pET22b(+) and pET22SUMO to be expressed in single and fusion forms with SUMO partner (S). The FN3, SFN3 increased GH5 solubility in FN3GH5, SFN3GH5. The SFN3 partly served for GH5 conformation in SFN3GH5, increased modules interaction and enzyme-soluble substrate affinity to enhance SXFN3GH5, SFN3GH5 activities in mixtures. Both SFN3 and SXFN3 did not anchor enzyme on filter paper but exfoliate and separate cellulose chains on filter paper for enzyme hydrolysis.
unassigned: Based on these findings, the presence of FN3 module in certain cellulase was confirmed and it assisted for enzyme conformation and activity in both soluble and insoluble substrate.
