METHODS: In this study, the gene encoding a cellobiohydrolase B (cbhB) from A. niger ATCC 10574 was cloned and expressed in the methylotrophic yeast Pichia pastoris X-33. The recombinant CBHB was purified and characterised to study its biochemical and kinetic characteristics. To evaluate the potential of CBHB in assisting biomass conversion, CBHB was supplemented into a commercial cellulase preparation (Cellic(®) CTec2) and was used to hydrolyse oil palm empty fruit bunch (OPEFB), one of the most abundant lignocellulosic waste from the palm oil industry. To attain maximum saccharification, enzyme loadings were optimised by response surface methodology and the optimum point was validated experimentally. Hydrolysed OPEFB samples were analysed using attenuated total reflectance FTIR spectroscopy (ATR-FTIR) to screen for any compositional changes upon enzymatic treatment.
RESULTS: Recombinant CBHB was over-expressed as a hyperglycosylated protein attached to N-glycans. CBHB was enzymatically active towards soluble substrates such as 4-methylumbelliferyl-β-D-cellobioside (MUC), p-nitrophenyl-cellobioside (pNPC) and p-nitrophenyl-cellobiotrioside (pNPG3) but was not active towards crystalline substrates like Avicel(®) and Sigmacell cellulose. Characterisation of purified CBHB using MUC as the model substrate revealed that optimum catalysis occurred at 50 °C and pH 4 but the enzyme was stable between pH 3 to 10 and 30 to 80 °C. Although CBHB on its own was unable to digest crystalline substrates, supplementation of CBHB (0.37%) with Cellic(®) CTec2 (30%) increased saccharification of OPEFB by 27%. Compositional analyses of the treated OPEFB samples revealed that CBHB supplementation reduced peak intensities of both crystalline cellulose Iα and Iβ in the treated OPEFB samples.
DISCUSSION: Since CBHB alone was inactive against crystalline cellulose, these data suggested that it might work synergistically with other components of Cellic(®) CTec2. CBHB supplements were desirable as they further increased hydrolysis of OPEFB when the performance of Cellic(®) CTec2 was theoretically capped at an enzyme loading of 34% in this study. Hence, A. niger CBHB was identified as a potential supplementary enzyme for the enzymatic hydrolysis of OPEFB.
RESULTS: All HPs of B. lehensis G1 were grouped according to their predicted functions based on the presence of functional domains in their sequences. From the metal-binding group of HPs of the cluster, an HP termed Bleg1_2507 was discovered to contain a thioredoxin (Trx) domain and highly-conserved metal-binding ligands represented by Cys69, Cys73 and His159, similar to all prokaryotic and eukaryotic Sco proteins. The built 3D structure of Bleg1_2507 showed that it shared the βαβαββ core structure of Trx-like proteins as well as three flanking β-sheets, a 310 -helix at the N-terminus and a hairpin structure unique to Sco proteins. Docking simulations provided an interesting view of Bleg1_2507 in association with its putative cytochrome c oxidase subunit II (COXII) redox partner, Bleg1_2337, where the latter can be seen to hold its partner in an embrace, facilitated by hydrophobic and ionic interactions between the proteins. Although Bleg1_2507 shares relatively low sequence identity (47%) to BsSco, interestingly, the predicted metal-binding residues of Bleg1_2507 i.e. Cys-69, Cys-73 and His-159 were located at flexible active loops similar to other Sco proteins across biological taxa. This highlights structural conservation of Sco despite their various functions in prokaryotes and eukaryotes.
CONCLUSIONS: We propose that HP Bleg1_2507 is a Sco protein which is able to interact with COXII, its redox partner and therefore, may possess metallochaperone and redox functions similar to other documented bacterial Sco proteins. It is hoped that this scientific effort will help to spur the search for other physiologically relevant proteins among the so-called "orphan" proteins of any given organism.
RESULTS: Phylogenetic analysis revealed at least four distinct DENV3/III lineages. Two of the lineages (DENV3/III-B and DENV3/III-C) are current actively circulating whereas the DENV3/III-A and DENV3/III-D were no longer recovered since the 1980s. Selection pressure analysis revealed strong evidence of positive selection on a number of amino acid sites in PrM, E, NS1, NS2a, NS2b, NS3, NS4a, and NS5. The Malaysian DENV3/III isolates recovered in the 1980s (MY.59538/1987) clustered into DENV3/III-B, which was the lineage with cosmopolitan distribution consisting of strains actively circulating in the Americas, Africa, and Asia. The Malaysian isolates recovered after the 2000s clustered within DENV3/III-C. This DENV3/III-C lineage displayed a more restricted geographical distribution and consisted of isolates recovered from Asia, denoted as the Asian lineage. Amino acid variation sites in NS5 (NS5-553I/M, NS5-629 T, and NS5-820E) differentiated the DENV3/III-C from other DENV3 viruses. The codon 629 of NS5 was identified as a positively selected site. While the NS5-698R was identified as unique to the genome of DENV3/III-C3. Phylogeographic results suggested that the recent Malaysian DENV3/III-C was likely to have been introduced from Singapore in 2008 and became endemic. From Malaysia, the virus subsequently spread into Taiwan and Thailand in the early part of the 2010s and later reintroduced into Singapore in 2013.
CONCLUSIONS: Distinct clustering of the Malaysian old and new DENV3/III isolates suggests that the currently circulating DENV3/III in Malaysia did not descend directly from the strains recovered during the 1980s. Phylogenetic analyses and common genetic traits in the genome of the strains and those from the neighboring countries suggest that the Malaysian DENV3/III is likely to have been introduced from the neighboring regions. Malaysia, however, serves as one of the sources of the recent regional spread of DENV3/III-C3 within the Asia region.