A C4-oxidizing Lytic Polysaccharide Monooxygenase Cleaving Both Cellulose and Cello-oligosaccharides

Lignocellulosic biomass is a renewable resource that significantly can substitute fossil resources for the production of fuels, chemicals, and materials. Efficient saccharification of this biomass to fermentable sugars will be a key technology in future biorefineries. Traditionally, saccharification was thought to be accomplished by mixtures of hydrolytic enzymes. However, recently it has been shown that lytic polysaccharide monooxygenases (LPMOs) contribute to this process by catalyzing oxidative cleavage of insoluble polysaccharides utilizing a mechanism involving molecular oxygen and an electron donor. These enzymes thus represent novel tools for the saccharification of plant biomass. Most characterized LPMOs, including all reported bacterial LPMOs, form aldonic acids, i.e., products oxidized in the C1 position of the terminal sugar. Oxidation at other positions has been observed, and there has been some debate concerning the nature of this position (C4 or C6). In this study, we have characterized an LPMO from Neurospora crassa (NcLPMO9C; also known as NCU02916 and NcGH61–3). Remarkably, and in contrast to all previously characterized LPMOs, which are active only on polysaccharides, NcLPMO9C is able to cleave soluble cello-oligosaccharides as short as a tetramer, a property that allowed detailed product analysis. Using mass spectrometry and NMR, we show that the cello-oligosaccharide products released by this enzyme contain a C4 gemdiol/keto group at the nonreducing end.     

AA10家族裂解多糖单加氧酶的模块化组成及底物选择性的研究进展

AA10家族裂解多糖单加氧酶(lytic polysaccharide monooxygenases, LPMOs)主要分布于细菌中,因其具有催化纤维素和几丁质等结晶多糖氧化降解的特性,在工业生物质转化过程中具有极强的应用潜力,从而受到广泛关注。然而,AA10家族不同LPMOs作用的底物种类及氧化位点和氧化产物也不尽相同,LPMOs的结构与组成对其底物选择性的影响机制有待进一步探究。因此,本文综述了AA10家族LPMOs的模块化结构组成及其催化机制,梳理了AA10家族LPMOs的底物谱,系统总结了AA10家族LPMOs的结构、关键作用残基及多模块组合对底物选择性影响的最新进展,并展望了LPMOs在生物质转化和生物燃料工业中广阔的应用前景。     

裂解性多糖单加氧酶及其应用研究进展北大核心

裂解多糖单加氧酶(lytic polysaccharide monooxygenases,LPMOs)是近几年新发现的氧化酶,该酶在生物质酶解方面发挥着重要的作用,因此,被描述为生物质解构助推器。LPMOs与底物的结合具有特异性,催化机理尚未完全阐明。虽然关于LPMOs的研究很多,但真正投入到工业生物质转化中的却很少,这对它们的表达、调控和应用都提出了挑战。本文首先系统综述了LPMOs的发现与分类、催化机制、构效关系,其次探讨了LPMOs的活性测定方法及重组表达技术,最后协同综述了LPMOs在不同领域的应用并对未来的研究方向进行了展望。本综述有助于加深对LPMOs的系统认识,推动LPMOs及其酶工程的研究,以期为LPMOs的研究和应用提供参考。     

Overproduction and characterization of a lytic polysaccharide monooxygenase in Bacillus subtilis using an assay based on ascorbate consumption

Lytic polysaccharide monooxygenases (LPMOs) are copper ion-containing enzymes that degrade crystalline polysaccharides, such as cellulose or chitin, through an oxidative mechanism. To the best of our knowledge, there are no assay methods for the direct characterization of LPMOs that degrade substrates without coupled enzymes. As such, in this study, a coupled enzyme-free assay method for LPMOs was developed, which is based on measuring the consumption of ascorbic acid used as an external electron donor for LPMOs. To establish this new assay method, a chitin-active LPMO from Bacillus atrophaeus (BatLPMO10) was cloned as a model enzyme. An expression system using B. subtilis as the host cell yielded a simple purification process without complicated periplasmic fractionation, as well as improved productivity by 3.7-fold higher than that of Escherichia coli BL21(DE3). At the optimum pH determined using a newly developed assay, BatLPMO10 showed the highest activity in terms of promoting chitin degradation by a chitinase. In addition, the assay method indicated that BatLPMO10 was inhibited by sodium ions, and BatLPMO10 and a chitinase mutually enhanced each other’s activities upon degrading chitin as the substrate. In conclusion, this hydrolase-free ascorbate assay allows quantitative analysis of BatLPMO10 without a coupled enzyme.     

Production of recombinant lytic polysaccharide monooxygenases and evaluation effect of its addition into Aspergillus fumigatus var. niveus cocktail for sugarcane bagasse saccharification

Lignocellulosic biomass is a promising alternative for producing biofuels, despite its recalcitrant nature. There are microorganisms in nature capable of efficiently degrade biomass, such as the filamentous fungi. Among them, Aspergillus fumigatus var. niveus (AFUMN) has a wide variety of carbohydrate-active enzymes (CAZymes), especially hydrolases, but a low number of oxidative enzymes in its genome. To confirm the enzymatic profile of this fungus, this study analyzed the secretome of AFUMN cultured in sugarcane bagasse as the sole carbon source. As expected, the secretome showed a predominance of hydrolytic enzymes compared to oxidative activity. However, it is known that hydrolytic enzymes act in synergy with oxidative proteins to efficiently degrade cellulose polymer, such as the Lytic Polysaccharide Monooxygenases (LPMOs). Thus, three LPMOs from the fungus Thermothelomyces thermophilus (TtLPMO9D, TtLPMO9H, and TtLPMO9O) were selected, heterologous expressed in Aspergillus nidulans, purified, and used to supplement the AFUMN secretome to evaluate their effect on the saccharification of sugarcane bagasse. The saccharification assay was carried out using different concentrations of AFUMN secretome supplemented with recombinant T. thermophilus LPMOs, as well as ascorbic acid as reducing agent for oxidative enzymes. Through a statistic design created by Design-Expert software, we were able to analyze a possible cooperative effect between these components. The results indicated that, in general, the addition of TtLPMO9D and ascorbic acid did not favor the conversion process in this study, while TtLPMO9O had a highly significant cooperative effect in bagasse saccharification compared to the control using only AFUMN secretome.     

Activation of bacterial lytic polysaccharide monooxygenases with cellobiose dehydrogenase

Lytic polysaccharide monooxygenases (LPMOs) represent a recent addition to the carbohydrate‐active enzymes and are classified as auxiliary activity (AA) families 9, 10, 11, and 13. LPMOs are crucial for effective degradation of recalcitrant polysaccharides like cellulose or chitin. These enzymes are copper‐dependent and utilize a redox mechanism to cleave glycosidic bonds that is dependent on molecular oxygen and an external electron donor. The electrons can be provided by various sources, such as chemical compounds (e.g., ascorbate) or by enzymes (e.g., cellobiose dehydrogenases, CDHs, from fungi). Here, we demonstrate that a fungal CDH from Myriococcum thermophilum (MtCDH), can act as an electron donor for bacterial family AA10 LPMOs. We show that employing an enzyme as electron donor is advantageous since this enables a kinetically controlled supply of electrons to the LPMO. The rate of chitin oxidation by CBP21 was equal to that of cosubstrate (lactose) oxidation by MtCDH, verifying the usage of two electrons in the LPMO catalytic mechanism. Furthermore, since lactose oxidation correlates directly with the rate of LPMO catalysis, a method for indirect determination of LPMO activity is implicated. Finally, the one electron reduction of the CBP21 active site copper by MtCDH was determined to be substantially faster than chitin oxidation by the LPMO. Overall, MtCDH seems to be a universal electron donor for both bacterial and fungal LPMOs, indicating that their electron transfer mechanisms are similar.     

Characterization of a lytic polysaccharide monooxygenase from Aspergillus fumigatus shows functional variation among family AA11 fungal LPMOs

The discovery of oxidative cleavage of recalcitrant polysaccharides by lytic polysaccharide monooxygenases (LPMOs) has affected the study and industrial application of enzymatic biomass processing. Despite being widespread in fungi, LPMOs belonging to the auxiliary activity (AA) family AA11 have been understudied. While these LPMOs are considered chitin active, some family members have little or no activity toward chitin, and the only available crystal structure of an AA11 LPMO lacks features found in bacterial chitin-active AA10 LPMOs. Here, we report structural and functional characteristics of a single-domain AA11 LPMO from Aspergillus fumigatus, AfAA11A. The crystal structure shows a substrate-binding surface with features resembling those of known chitin-active LPMOs. Indeed, despite the absence of a carbohydrate-binding module, AfAA11A has considerable affinity for α-chitin and, more so, β-chitin. AfAA11A is active toward both these chitin allomorphs and enhances chitin degradation by an endoacting chitinase, in particular for α-chitin. The catalytic activity of AfAA11A on chitin increases when supplying reactions with hydrogen peroxide, showing that, like LPMOs from other families, AfAA11A has peroxygenase activity. These results show that, in stark contrast to the previously characterized AfAA11B from the same organism, AfAA11A likely plays a role in fungal chitin turnover. Thus, members of the hitherto rather enigmatic family of AA11 LPMOs show considerable structural and functional differences and may have multiple roles in fungal physiology.     

A rapid quantitative activity assay shows that the Vibrio cholerae colonization factor GbpA is an active lytic polysaccharide monooxygenase

The discovery of the copper-dependent lytic polysaccharide monooxygenases (LPMOs) has revealed new territory for chemical and biochemical analysis. These unique mononuclear copper enzymes are abundant, suggesting functional diversity beyond their established roles in the depolymerization of biomass polysaccharides. At the same time basic biochemical methods for characterizing LPMOs, such as activity assays are not well developed. Here we describe a method for quantification of C1-oxidized chitooligosaccharides (aldonic acids), and hence LPMO activity. The method was used to quantify the activity of a four-domain LPMO from Vibriocholerae, GbpA, which is a virulence factor with no obvious role in biomass processing.     

Lytic polysaccharide monooxygenases promote oxidative cleavage of lignin and lignin–carbohydrate complexes during fungal degradation of lignocellulose

Overcoming lignocellulosic biomass recalcitrance, especially the cleavage of cross-linkages in lignin–carbohydrate complexes (LCCs) and lignin, is essential for both the carbon cycle and industrial biorefinery. Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that play a key role in fungal polysaccharide oxidative degradation. Nevertheless, comprehensive analysis showed that LPMOs from a white-rot fungus, Pleurotus ostreatus, correlated well with the Fenton reaction and were involved in the degradation of recalcitrant nonpolysaccharide fractions in this research. Thus, LPMOs participated in the extracellular Fenton reaction by enhancing iron reduction in quinone redox cycling. A Fenton reaction system consisting of LPMOs, hydroquinone, and ferric iron can efficiently produce hydroxy radicals and then cleave LCCs or lignin linkages. This finding indicates that LPMOs are underestimated auxiliary enzymes in eliminating biomass recalcitrance.     

Novel enzymes for the degradation of cellulose

ABSTRACT: The bulk terrestrial biomass resource in a future bio-economy will be lignocellulosic biomass, which is recalcitrant and challenging to process. Enzymatic conversion of polysaccharides in the lignocellulosic biomass will be a key technology in future biorefineries and this technology is currently the subject of intensive research. We describe recent developments in enzyme technology for conversion of cellulose, the most abundant, homogeneous and recalcitrant polysaccharide in lignocellulosic biomass. In particular, we focus on a recently discovered new type of enzymes currently classified as CBM33 and GH61 that catalyze oxidative cleavage of polysaccharides. These enzymes promote the efficiency of classical hydrolytic enzymes (cellulases) by acting on the surfaces of the insoluble substrate, where they introduce chain breaks in the polysaccharide chains, without the need of first "extracting" these chains from their crystalline matrix.     

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

Among the extensive repertoire of carbohydrate-active enzymes, lytic polysaccharide monooxygenases (LPMOs) have a key role in recalcitrant biomass degradation. LPMOs are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds in polysaccharides such as cellulose and chitin. Several LPMOs contain carbohydrate-binding modules (CBMs) that are known to promote LPMO efficiency. However, structural and functional properties of some CBMs remain unknown, and it is not clear why some LPMOs, like CjLPMO10A from the soil bacterium Cellvibrio japonicus, have multiple CBMs (CjCBM5 and CjCBM73). Here, we studied substrate binding by these two CBMs to shine light on their functional variation and determined the solution structures of both by NMR, which constitutes the first structure of a member of the CBM73 family. Chitin-binding experiments and molecular dynamics simulations showed that, while both CBMs bind crystalline chitin with K_d values in the micromolar range, CjCBM73 has higher affinity for chitin than CjCBM5. Furthermore, NMR titration experiments showed that CjCBM5 binds soluble chitohexaose, whereas no binding of CjCBM73 to this chitooligosaccharide was detected. These functional differences correlate with distinctly different arrangements of three conserved aromatic amino acids involved in substrate binding. In CjCBM5, these residues show a linear arrangement that seems compatible with the experimentally observed affinity for single chitin chains. On the other hand, the arrangement of these residues in CjCBM73 suggests a wider binding surface that may interact with several chitin chains. Taken together, these results provide insight into natural variation among related chitin-binding CBMs and the possible functional implications of such variation.     

Quantification of the catalytic performance of C1-cellulose-specific lytic polysaccharide monooxygenases

Applied Microbiology and Biotechnology - Lytic polysaccharide monooxygenases (LPMOs) have recently been shown to significantly enhance the degradation of recalcitrant polysaccharides and are of...     

H2O2 feeding enables LPMO-assisted cellulose saccharification during simultaneous fermentative production of lactic acid

Simultaneous saccharification and fermentation (SSF) is a well-known strategy for valorization of lignocellulosic biomass. Because the fermentation process typically is anaerobic, oxidative enzymes found in modern commercial cellulase cocktails, such as lytic polysaccharide monooxygenases (LPMOs), may be inhibited, limiting the overall efficiency of the enzymatic saccharification. Recent discoveries, however, have shown that LPMOs are active under anoxic conditions if they are provided with H₂O₂ at low concentrations. In this study, we build on this concept and investigate the potential of using externally added H₂O₂ to sustain oxidative cellulose depolymerization by LPMOs during an SSF process for lactic acid production. The results of bioreactor experiments with 100 g/L cellulose clearly show that continuous addition of small amounts of H₂O₂ (at a rate of 80 µM/h) during SSF enables LPMO activity and improves lactic acid production. While further process optimization is needed, the present proof-of-concept results show that modern LPMO-containing cellulase cocktails such as Cellic CTec2 can be used in SSF setups, without sacrificing the LPMO activity in these cocktails.     

Re-interpreting the role of endo-��-mannanases as mannan endotransglycosylase/hydrolases in the plant cell wall

Background

Mannans are hemicellulosic polysaccharides in the plant primary cell wall with two major physiological roles: as storage polysaccharides that provide energy for the growing seedling; and as structural components of the hemicellulose–cellulose network with a similar function to xyloglucans. Endo-β-mannanases are hydrolytic enzymes that cleave the mannan backbone. They are active during seed germination and during processes of growth or senescence. The recent discovery that endo-β-mannanase LeMAN4a from ripe tomato fruit also has mannan transglycosylase activity requires the role of endo-β-mannanases to be reinterpreted.

Aims

In this review, the role of endo-β-mannanases as mannan endotransglycosylase/hydrolases (MTHs) in remodelling the plant cell wall is considered by analogy to the role of xyloglucan endotransglucosylase/hydrolases (XTHs). The current understanding of the reaction mechanism of these enzymes, their three-dimensional protein structure, their substrates and their genes are reported.

Future outlook

There are likely to be more endohydrolases within the plant cell wall that can carry out hydrolysis and transglycosylation reactions. The challenge will be to demonstrate that the transglycosylation activities shown in vitro also exist in vivo and to validate a role for transglycosylation reactions during the growth and development of the plant cell wall.Key words: Cell wall, endo-β-mannanase, endohydrolase, mannan, endotransglycosylase     

A hierarchical classification of polysaccharide lyases for glycogenomics

Carbohydrate-active enzymes face huge substrate diversity in a highly selective manner using only a limited number of available folds. They are therefore subjected to multiple divergent and convergent evolutionary events. This and their frequent modularity render their functional annotation in genomes difficult in a number of cases. In the present paper, a classification of polysaccharide lyases (the enzymes that cleave polysaccharides using an elimination instead of a hydrolytic mechanism) is shown thoroughly for the first time. Based on the analysis of a large panel of experimentally characterized polysaccharide lyases, we examined the correlation of various enzyme properties with the three levels of the classification: fold, family and subfamily. The resulting hierarchical classification, which should help annotate relevant genes in genomic efforts, is available and constantly updated at the Carbohydrate-Active Enzymes Database (http://www.cazy.org).     

Effect of uncouplers and inhibitors of oxidative phosphorylation on the reduced and oxidized forms of mitochondiral ATPase.

A series of uncouplers and inhibitors of oxidative phosphorylation have been studied with regard to their effect on the hydrolytic activity of the reduced and oxidized forms of isolated or membrane-bound mitochondrial ATPase. Uncouplers (2,4-dinitrophenol, dicoumarol), which are also activators of the hydrolytic activity of ATPase, were more potent activators on the oxidized form of the enzyme. Inhibitors of oxidative phosphorylation (oligomycin, azide and amytal) had a more potent inhibitory effect on the hydrolytic activity of ATPase in its reduced form. Purified F1-ATPase, oligomycin insensitive in the oxidized form of the enzyme, became sensitive to oligomycin in the reduced form. An interpretation of the results suggests the presence of a mechanism that unifies the action of these different compounds on the synthesis and hydrolysis of ATP catalyzed by mitochondrial ATPase.     

Structural and Functional Characterization of a Lytic Polysaccharide Monooxygenase with Broad Substrate Specificity

The recently discovered lytic polysaccharide monooxygenases (LPMOs) carry out oxidative cleavage of polysaccharides and are of major importance for efficient processing of biomass. NcLPMO9C from Neurospora crassa acts both on cellulose and on non-cellulose β-glucans, including cellodextrins and xyloglucan. The crystal structure of the catalytic domain of NcLPMO9C revealed an extended, highly polar substrate-binding surface well suited to interact with a variety of sugar substrates. The ability of NcLPMO9C to act on soluble substrates was exploited to study enzyme-substrate interactions. EPR studies demonstrated that the Cu²⁺ center environment is altered upon substrate binding, whereas isothermal titration calorimetry studies revealed binding affinities in the low micromolar range for polymeric substrates that are due in part to the presence of a carbohydrate-binding module (CBM1). Importantly, the novel structure of NcLPMO9C enabled a comparative study, revealing that the oxidative regioselectivity of LPMO9s (C1, C4, or both) correlates with distinct structural features of the copper coordination sphere. In strictly C1-oxidizing LPMO9s, access to the solvent-facing axial coordination position is restricted by a conserved tyrosine residue, whereas access to this same position seems unrestricted in C4-oxidizing LPMO9s. LPMO9s known to produce a mixture of C1- and C4-oxidized products show an intermediate situation.     

Electrophoretic approaches to the analysis of complex polysaccharides

Complex polysaccharides, glycosaminoglycans (GAGs), are a class of ubiquitous macromolecules exhibiting a wide range of biological functions. They are widely distributed as sidechains of proteoglycans (PGs) in the extracellular matrix and at cellular level. The recent emergence of enhanced analytical tools for their study has triggered a virtual explosion in the field of glycomics. Analytical electrophoretic separation techniques, including agarose-gel, capillary electrophoresis (HPCE) and fluorophore-assisted carbohydrate electrophoresis (FACE), of GAGs and GAG-derived oligosaccharides have been employed for the structural analysis and quantification of hyaluronic acid (HA), chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), heparan sulfate (HS), heparin (Hep) and acidic bacterial polysaccharides. Furthermore, recent developments in the electrophoretic separation and detection of unsaturated disaccharides and oligosaccharides derived from GAGs by enzymatic or chemical degradation have made it possible to examine alterations of GAGs with respect to their amounts and fine structural features in various pathological conditions, thus becoming applicable for diagnosis. In this paper, the electromigration procedures developed to analyze and characterize complex polysaccharides are reviewed. Moreover, a critical evaluation of the biological relevance of the results obtained by these electrophoresis approaches is presented.     

保肝降酶的天然药物研究进展

天然药物治疗肝损伤的功效及其低毒性已得到广泛的认可。由于大多肝损伤均有氧化应激的参与,天然药物的保肝作用通常与其抗氧化特性或激发体内抗氧化防御系统的能力有关。然而,越来越多的证据表明,除了抗氧化,天然药物还具有许多其他保肝机制。文章综述了近年来保肝降酶的天然药物的研究进展,将国内外已报道的天然药物分黄酮类、生物碱类、皂苷类、多糖类、木脂素类、萜类及其他类并分别列举其治疗机制及效果,为天然药物保肝降酶相关性的研究提供文献依据和研究思路,并对未来保肝降酶的新药研发方向进行展望。     

保肝降酶的天然药物研究进展

天然药物治疗肝损伤的功效及其低毒性已得到广泛的认可。由于大多肝损伤均有氧化应激的参与,天然药物的保肝作用通常与其抗氧化特性或激发体内抗氧化防御系统的能力有关。然而,越来越多的证据表明,除了抗氧化,天然药物还具有许多其他保肝机制。文章综述了近年来保肝降酶的天然药物的研究进展,将国内外已报道的天然药物分黄酮类、生物碱类、皂苷类、多糖类、木脂素类、萜类及其他类并分别列举其治疗机制及效果,为天然药物保肝降酶相关性的研究提供文献依据和研究思路,并对未来保肝降酶的新药研发方向进行展望。