Dataset Information

Production of four Neurospora crassa lytic polysaccharide monooxygenases in Pichia pastoris monitored by a fluorimetric assay.

ABSTRACT:

Unlabelled

Background

Recent studies demonstrate that enzymes from the glycosyl hydrolase family 61 (GH61) show lytic polysaccharide monooxygenase (PMO) activity. Together with cellobiose dehydrogenase (CDH) an enzymatic system capable of oxidative cellulose cleavage is formed, which increases the efficiency of cellulases and put PMOs at focus of biofuel research. Large amounts of purified PMOs, which are difficult to obtain from the native fungal producers, are needed to study their reaction kinetics, structure and industrial application. In addition, a fast and robust enzymatic assay is necessary to monitor enzyme production and purification.

Results

Four pmo genes from Neurospora crassa were expressed in P. pastoris under control of the AOX1 promoter. High yields were obtained for the glycosylated gene products PMO-01867, PMO-02916 and PMO-08760 (>300 mg L-1), whereas the yield of non-glycosylated PMO-03328 was moderate (~45 mg L-1). The production and purification of all four enzymes was specifically followed by a newly developed, fast assay based on a side reaction of PMO: the production of H2O2 in the presence of reductants. While ascorbate is a suitable reductant for homogeneous PMO preparations, fermentation samples require the specific electron donor CDH.

Conclusions

P. pastoris is a high performing expression host for N. crassa PMOs. The pmo genes under control of the native signal sequence are correctly processed and active. The novel CDH-based enzyme assay allows fast determination of PMO activity in fermentation samples and is robust against interfering matrix components.

SUBMITTER: Kittl R

PROVIDER: S-EPMC3500269 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Publications

Production of four Neurospora crassa lytic polysaccharide monooxygenases in Pichia pastoris monitored by a fluorimetric assay.

Kittl Roman R Kracher Daniel D Burgstaller Daniel D Haltrich Dietmar D Ludwig Roland R

Biotechnology for biofuels 20121026 1

<h4>Unlabelled</h4><h4>Background</h4>Recent studies demonstrate that enzymes from the glycosyl hydrolase family 61 (GH61) show lytic polysaccharide monooxygenase (PMO) activity. Together with cellobiose dehydrogenase (CDH) an enzymatic system capable of oxidative cellulose cleavage is formed, which increases the efficiency of cellulases and put PMOs at focus of biofuel research. Large amounts of purified PMOs, which are difficult to obtain from the native fungal producers, are needed to study t ...[more]

PMID: 23102010

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Project description:Family AA9 lytic polysaccharide monooxygenases (LPMOs) are abundant in fungi, where they catalyze oxidative depolymerization of recalcitrant plant biomass. These AA9 LPMOs cleave cellulose and some also act on hemicelluloses, primarily other (substituted) β-(1→4)-glucans. Oxidative cleavage of xylan has been shown for only a few AA9 LPMOs, and it remains unclear whether this activity is a minor side reaction or primary function. Here, we show that Neurospora crassa LPMO9F (NcLPMO9F) and the phylogenetically related, hitherto uncharacterized NcLPMO9L from N. crassa are active on both cellulose and cellulose-associated glucuronoxylan but not on glucuronoxylan alone. A newly developed method for simultaneous quantification of xylan-derived and cellulose-derived oxidized products showed that NcLPMO9F preferentially cleaves xylan when acting on a cellulose-beechwood glucuronoxylan mixture, yielding about three times more xylan-derived than cellulose-derived oxidized products. Interestingly, under similar conditions, NcLPMO9L and the previously characterized McLPMO9H, from Malbranchea cinnamomea, showed different xylan-to-cellulose preferences, giving oxidized product ratios of about 0.5:1 and 1:1, respectively, indicative of functional variation among xylan-active LPMOs. Phylogenetic and structural analysis of xylan-active AA9 LPMOs led to the identification of characteristic structural features, including unique features that do not occur in phylogenetically remote AA9 LPMOs, such as four AA9 LPMOs whose lack of activity toward glucuronoxylan was demonstrated in the present study. Taken together, the results provide a path toward discovery of additional xylan-active LPMOs and show that the huge family of AA9 LPMOs has members that preferentially act on xylan. These findings shed new light on the biological role and industrial potential of these fascinating enzymes. IMPORTANCE Plant cell wall polysaccharides are highly resilient to depolymerization by hydrolytic enzymes, partly due to cellulose chains being tightly packed in microfibrils that are covered by hemicelluloses. Lytic polysaccharide monooxygenases (LPMOs) seem well suited to attack these resilient copolymeric structures, but the occurrence and importance of hemicellulolytic activity among LPMOs remain unclear. Here, we show that certain AA9 LPMOs preferentially cleave xylan when acting on a cellulose-glucuronoxylan mixture, and that this ability is the result of protein evolution that has resulted in a clade of AA9 LPMOs with specific structural features. Our findings strengthen the notion that the vast arsenal of AA9 LPMOs in certain fungal species provides functional versatility and that AA9 LPMOs may have evolved to promote oxidative depolymerization of a wide variety of recalcitrant, copolymeric plant polysaccharide structures. These findings have implications for understanding the biological roles and industrial potential of LPMOs.

Project description:The lytic polysaccharide monooxygenases (LPMOs) are copper metalloenzymes that can enhance polysaccharide depolymerization through an oxidative mechanism and hence boost generation of biofuel from e.g. cellulose. By employing density functional theory in a combination of quantum mechanics and molecular mechanics (QM/MM), we report a complete description of the molecular mechanism of LPMOs. The QM/MM scheme allows us to describe all reaction steps with a detailed protein environment and we show that this is necessary. Several active species capable of abstracting a hydrogen from the substrate have been proposed previously and starting from recent crystallographic work on a substrate-LPMO complex, we investigate previously suggested paths as well as new ones. We describe the generation of the reactive intermediates, the abstraction of a hydrogen atom from the polysaccharide substrate, as well as the final recombination step in which OH is transferred back to the substrate. We show that a superoxo [CuO2]+ complex can be protonated by a nearby histidine residue (suggested by recent mutagenesis studies and crystallographic work) and, provided an electron source is available, leads to formation of an oxyl-complex after cleavage of the O-O bond and dissociation of water. The oxyl complex either reacts with the substrate or is further protonated to a hydroxyl complex. Both the oxyl and hydroxyl complexes are also readily generated from a reaction with H2O2, which was recently suggested to be the true co-substrate, rather than O2. The C-H abstraction by the oxyl and hydroxy complexes is overall favorable with activation barriers of 69 and 94 kJ mol-1, compared to the much higher barrier (156 kJ mol-1) obtained for the copper-superoxo species. We obtain good structural agreement for intermediates for which structural data are available and the estimated reaction energies agree with experimental rate constants. Thus, our suggested mechanism is the most complete to date and concur with available experimental evidence.

Dataset Information

Production of four Neurospora crassa lytic polysaccharide monooxygenases in Pichia pastoris monitored by a fluorimetric assay.

Unlabelled

Background

Results

Conclusions

Publications

Production of four Neurospora crassa lytic polysaccharide monooxygenases in Pichia pastoris monitored by a fluorimetric assay.

Similar Datasets

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