Figure 1.
Error-prone PCR strategy used in the study.
PCR1: error prone-PCR performed on paman5a (HM357135) and paman26a (HM357136); PCR2: PCR without mutation performed on Ura3d1 (selection marker), pPOX2 (inducible promotor of acyl-coA oxidase 2) and prepro Lip2 (secretion signal sequence); PCR3: overlapping PCR to reconstruct the entire sequence between zeta platforms. Primers used are listed in Table 3.
Figure 2.
Screening strategy and mutant selection.
The number of variants screened at each step is indicated at the top (PaMan5A) and at the bottom (PaMan26A) of the diagram.
Table 1.
Mannanase activity of selected Y. lipolytica variants.
Table 2.
Kinetic constants of wild-type enzymes and selected variants toward galactomannan, mannohexaose (M6) and mannopentaose (M5).
Figure 3.
Structural view of PaMan5A (PDB 3ZIZ) exhibiting substituted amino-acids.
A. Surface view of the catalytic cleft of PaMan5A with mannotriose modelled in the −2 and −3 subsites and mannobiose modelled in the +1 and +2 subsites. The structures of GH5 from T. reesei and T. fusca in complex with mannobiose and mannotriose, respectively, were superimposed on the top of the structure of PaMan5A to map the substrate-binding subsites. The two catalytic glutamate residues, E177 and E283, are coloured in red. The substituted amino-acids are labelled and coloured in yellow. B. Structural based sequence alignment of the region around position 311 (according to PaMan5A numbering) from Podospora anserina (PaMan5A), Aplysia kurodai (AkMan, PDB 3VUP), Mytilus edulis (MeMan5A, PDB 2C0H), Cellvibrio mixtus (CmMan5A, PDB 1UUQ), Trichoderma reesei (TrMan5A, PDB 1QNR), Lycopersicon esculentum (LeMan4A, PDB 1RH9) and Thermomonospora fusca (TfMan5, PDB 2MAN). Secondary structure elements, α-helix α7 and β-strand β8, are indicated below the sequences as a cylinder and an arrow, respectively. Strictly conserved residues, G311 and W315 (according to PaMan5A numbering), are shown with a yellow and a grey background, respectively. C. Surface view of PaMan5A rotated of about 90° along the horizontal axis. The front clipping plane has been moved in order to visualize the location of G311 inside the molecule. The zoom shows a compact hydrophobic core in the vicinity of G311.
Figure 4.
Progress curves of the manno-oligosaccharides generated by the wild-type PaMan5A and the PaMan5A-K139R/Y223H variant upon hydrolysis of mannohexaose.
18.2 nM of the wild-type PaMan5A (A) and the PaMan5A-K139R/Y223H variant (B) were incubated with 1 mM of mannohexaose in acetate buffer pH 5.2 at 40°C. The amount of each manno-oligosaccharide, i.e., mannobiose (full circles), mannotriose (full squares), mannotetraose (crosses), and mannohexaose (full diamonds), is indicated during the course of the reaction.
Figure 5.
Structural view of PaMan26A (PDB 3ZM8) exhibiting substituted amino-acids.
The central panel shows a surface view of the entire PaMan26A structure, which is composed of a carbohydrate binding module (CBM) belonging to the CBM35 family in cyan, a linker in violet and a catalytic domain belonging to the GH26 family in green. The two catalytic glutamate residues, E300 and E390, are coloured in red. The two substituted amino-acids, P140 and D416, are labelled and coloured in yellow. The top view represents the surface view of the catalytic cleft of PaMan26A rotated about 90° along the horizontal axis with mannotriose modelled into the −2 to −4 subsites. The structure of GH26 from C. fimi in complex with mannotriose was superimposed on the top of the structure of PaMan26A to map the substrate-binding subsites. The bottom view displays the PaMan26A linker (from residue 131 to residue 141) in stick representation. The molecule has been rotated of about 90° along the horizontal axis and in the opposite direction compared to the top view. The proline residues of the linker are labelled.
Table 3.
List of primers used in the study.