A thermostable bacterial lytic polysaccharide monooxygenase with high operational stability in a wide temperature range

Abstract

Background Lytic polysaccharide monooxygenases (LPMOs) are oxidative, copper-dependent enzymes that function as powerful tools in the turnover of various biomasses, including lignocellulosic plant biomass. While LPMOs are considered to be of great importance for biorefineries, little is known about industrial relevant properties such as the ability to operate at high temperatures. Here, we describe a thermostable, cellulose-active LPMO from a high-temperature compost metagenome (called mgLPMO10). Results MgLPMO10 was found to have the highest apparent melting temperature (83 °C) reported for an LPMO to date, and is catalytically active up to temperatures of at least 80 °C. Generally, mgLPMO10 showed good activity and operational stability over a wide temperature range. The LPMO boosted cellulose saccharification by recombinantly produced GH48 and GH6 cellobiohydrolases derived from the same metagenome, albeit to a minor extent. Cellulose saccharification studies with a commercial cellulase cocktail (Celluclast®) showed that the performance of this thermostable bacterial LPMO is comparable with that of a frequently utilized fungal LPMO from Thermoascus aurantiacus (TaLPMO9A). Conclusions The high activity and operational stability of mgLPMO10 are of both fundamental and applied interest. The ability of mgLPMO10 to perform oxidative cleavage of cellulose at 80 °C and the clear synergy with Celluclast® make this enzyme an interesting candidate in the development of thermostable enzyme cocktails for use in lignocellulosic biorefineries.

Structural and functional variation of chitin-binding domains of a lytic polysaccharide monooxygenase from Cellvibrio japonicus

“…The apparent melting temperatures of the isolated CBMs were 57.2 °C for CjCBM5 and 75.4 °C for CjCBM73, whereas the apparent melting temperature of the CjLPMO10A CD was 70.2 °C. In accordance with previous studies on the effect of copper binding on the stability of AA10 (30,31) and AA9 (32) LPMOs, the apo variant of CjLPMO10A CD showed reduced stability (Tm,app = 56.6 °C).…”

Lytic Polysaccharide Monooxygenase from Talaromyces amestolkiae with an Enigmatic Linker-like Region: The Role of This Enzyme on Cellulose Saccharification

“…Moreover, the thermostability of other LPMOs of the AA9 family, like those from Scytalidium thermophilum, Malbranchea cinnamomea, Myceliophthora thermophila [44,45], or Talaromyces cellulolyticus [46] are similar to that determined for TamAA9A. However, the most thermostable LPMOs are the bacterial LPMOAA10s found in a compost metagenome (mgLPMO10), with a melting temperature of 83 • C [47]. Despite this, due to the combination of thermostability and good pH tolerance of T. amestolkiae LPMO variants, we postulate they may be promising catalysts for lignocellulosic biomass valorization, since they are stable at the operational temperature used in industrial saccharification, where substrates are usually pretreated with alkali or diluted acid [48].…”

Defining the Frontiers of Synergism between Cellulolytic Enzymes for Improved Hydrolysis of Lignocellulosic Feedstocks

“…Even though Forsberg et al [79] did not measure the DS values for synergism during these studies, our calculations from their glucose/gluconic acid data suggest that a DS value of about 2.3 AU was achieved. In addition, Tuveng et al [26] also demonstrated that LPMO10 synergized with Cel48A and Cel6B, displaying a DS value of about 1.2 AU during Avicel hydrolysis. It is worth noting that, to date, most studies have focused on fungal cellulase-LPMO synergism-but few have focused on bacterial cellulase-LPMO synergism.…”

“…Both bacterial and fungal LPMOs have similar structures and mechanisms of action [23]. The catalytic domain of LPMOs can also be attached to CBMs by a flexible linker [24,26]. Furthermore, Isaksen et al [27] demonstrated that the fungal LPMO-AA9C, sourced from Neurospora crassa, was capable of cleaving polymeric cellulose and cello-oligosaccharides with a degree of polymerization of about four to six.…”

“…Furthermore, Isaksen et al [27] demonstrated that the fungal LPMO-AA9C, sourced from Neurospora crassa, was capable of cleaving polymeric cellulose and cello-oligosaccharides with a degree of polymerization of about four to six. The LPMOs cleaved the C1 of cellulose or cello-oligosaccharides to produce aldouronic acids; however, when they acted on the C4, they produced gem-diol [26,27]. The LPMOs from families AA9, AA10, and AA16 have been reported to synergize with cellulases to enhance the saccharification of cellulose [25,28,29].…”