Abstract
Microbial mannanases are biotechnologically important enzymes since they target the hydrolysis of hemicellulosic polysaccharides of softwood biomass into simple molecules like manno-oligosaccharides and mannose. In this study, we have implemented a strategy of molecular engineering in the yeast Yarrowia lipolytica to improve the specific activity of two fungal endo-mannanases, PaMan5A and PaMan26A, which belong to the glycoside hydrolase (GH) families GH5 and GH26, respectively. Following random mutagenesis and two steps of high-throughput enzymatic screening, we identified several PaMan5A and PaMan26A mutants that displayed improved kinetic constants for the hydrolysis of galactomannan. Examination of the three-dimensional structures of PaMan5A and PaMan26A revealed which of the mutated residues are potentially important for enzyme function. Among them, the PaMan5A-G311S single mutant, which displayed an impressive 8.2-fold increase in kcat/KM due to a significant decrease of KM, is located within the core of the enzyme. The PaMan5A-K139R/Y223H double mutant revealed modification of hydrolysis products probably in relation to an amino-acid substitution located nearby one of the positive subsites. The PaMan26A-P140L/D416G double mutant yielded a 30% increase in kcat/KM compared to the parental enzyme. It displayed a mutation in the linker region (P140L) that may confer more flexibility to the linker and another mutation (D416G) located at the entrance of the catalytic cleft that may promote the entrance of the substrate into the active site. Taken together, these results show that the directed evolution strategy implemented in this study was very pertinent since a straightforward round of random mutagenesis yielded significantly improved variants, in terms of catalytic efiiciency (kcat/KM).
Highlights
Hemicellulose is a combined designation of a diverse set of abundant non-crystalline carbohydrate polymers, among which mannans are the major component in softwoods [1] and are present in certain plant seeds [2]
The yeast Y. lipolytica was chosen as host to perform molecular engineering of PaMan5A and PaMan26A since a very high reproducibility in protein expression level was demonstrated [25]
The two genes encoding PaMan5A and PaMan26A were inserted into the Y. lipolytica expression vector in frame with the yeast preprolip2 secretion peptide under the control of the oleic acidinducible promoter POX2 (Figure 1)
Summary
Hemicellulose is a combined designation of a diverse set of abundant non-crystalline carbohydrate polymers, among which mannans are the major component in softwoods [1] and are present in certain plant seeds [2]. Mannans comprise molecules constituted either by a backbone of b-1,4-linked D-mannose residues, known as mannan, or by a heterogeneous combination of b-1,4-D-mannose and b-1,4-D-glucose units, termed glucomannan. Both can be decorated with a-1,6-linked galactose side chains, and these polysaccharides are referred to as galactomannan and galactoglucomannan, respectively. Mannans are hydrolyzed by the coordinated action of several types of glycoside hydrolases (GH) among which endo-b-1,4 mannanases (EC 3.2.1.78) are the key enzymes that depolymerize the mannan backbone. They are encountered in families GH5, GH26 and GH113 in the CAZy database [4,5]. Enzymatic hydrolysis of lignocellulosic biomass is one of the major bottlenecks due to the recalcitrance of the plant cell wall and the high cost of enzymes, mainly due to the fact that large amounts are required to breakdown lignocellulose to fermentable sugars [12,13,14]
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