Efficient saccharification of lignocellulosic biomass requires concerted development of a pretreatment method, an enzyme cocktail and an enzymatic process, all of which are adapted to the feedstock. Recent years have shown great progress in most aspects of the overall process. In particular, increased insights into the contributions of a wide variety of cellulolytic and hemicellulolytic enzymes have improved the enzymatic processing step and brought down costs. Here, we review major pretreatment technologies and different enzyme process setups and present an in-depth discussion of the various enzyme types that are currently in use. We pay ample attention to the role of the recently discovered lytic polysaccharide monooxygenases (LPMOs), which have led to renewed interest in the role of redox enzyme systems in lignocellulose processing. Better understanding of the interplay between the various enzyme types, as they may occur in a commercial enzyme cocktail, is likely key to further process improvements.

Lignin is a natural aromatic polymer that is not easily degraded, hindering the efficient use of lignocellulose-rich biomass resources, such as straw. Biodegradation is a method of decomposing lignin that has recently received increasing attention.

Lignocellulosic biomass is an abundant and renewable resource that potentially contains large amounts of energy. It is an interesting alternative for fossil fuels, allowing the production of biofuels and other organic compounds. In this paper, a review devoted to the processing of lignocellulosic materials as substrates for fermentation processes is presented. The review focuses on physical, chemical, physicochemical, enzymatic, and microbiologic methods of biomass pretreatment. In addition to the evaluation of the mentioned methods, the aim of the paper is to understand the possibilities of the biomass pretreatment and their influence on the efficiency of biofuels and organic compounds production. The effects of different pretreatment methods on the lignocellulosic biomass structure are described along with a discussion of the benefits and drawbacks of each method, including the potential generation of inhibitory compounds for enzymatic hydrolysis, the effect on cellulose digestibility, the generation of compounds that are toxic for the environment, and energy and economic demand. The results of the investigations imply that only the stepwise pretreatment procedure may ensure effective fermentation of the lignocellulosic biomass. Pretreatment step is still a challenge for obtaining cost-effective and competitive technology for large-scale conversion of lignocellulosic biomass into fermentable sugars with low inhibitory concentration.

With a set of perfect extracellular lignocellulolytic enzymes, white-rot fungus has been recognized as playing an important role in the degradation of lignocellulose materials, which leads to the possibility of creating a composite enzymatic system with high hydrolysis efficiency in vitro. Echinodontium taxodii is a promising white-rot fungus for biologic pretreatment. In this study, we extracted the extracellular products of E. taxodii under solid-state fermentation conditions, mixed the extracellular products with cellulase to build a composite enzymatic system, and systematically evaluated the effect of this system on the hydrolysis of acid-pretreated and raw maize stovers. We found that the extracellular products from E. taxodii could significantly improve the hydrolysis efficiency of cellulase, with a synergistic action between the extracellular products and cellulase. Corn stovers treated with extracellular products were suitable for the enzymatic hydrolysis of cellulase. Furthermore, we found that pure proteins from the extracellular products were not sufficient to generate synergistic action. This finding suggests that non-protein substances may also be involved in the synergistic action between the extracellular products and cellulase.

Lytic polysaccharide monooxygenases (LPMOs) are essential for enzymatic conversion of lignocellulose-rich biomass in the context of biofuels and platform chemicals production. Considerable insight into the mode of action of LPMOs has been obtained, but research on the cellulose specificity of these enzymes is still limited. Hence, we studied the product profiles of four fungal Auxiliary Activity family 9 (AA9) LPMOs during their oxidative cleavage of three types of cellulose: bacterial cellulose (BC), Avicel PH-101 (AVI), and regenerated amorphous cellulose (RAC). We observed that attachment of a carbohydrate-binding module 1 (CBM1) did not change the substrate specificity of LPMO9B from C1 (LPMO9B) but stimulated the degradation of all three types of cellulose. A detailed quantification of oxidized ends in both soluble and insoluble fractions, as well as characterization of oxidized cello-oligosaccharide patterns, suggested that LPMO9B generates mainly oxidized cellobiose from BC, while producing oxidized cello-oligosaccharides from AVI and RAC ranged more randomly from DP2-8. Comparable product profiles, resulting from BC, AVI, and RAC oxidation, were found for three other AA9 LPMOs. These distinct cleavage profiles highlight cellulose specificity rather than an LPMO-dependent mechanism and may further reflect that the product profiles of AA9 LPMOs are modulated by different cellulose types.© 2021 The Authors. Published by American Chemical Society.

Efficient enzymatic conversion of crystalline polysaccharides is crucial for an economically and environmentally sustainable bioeconomy but remains unfavorably inefficient. We describe an enzyme that acts on the surface of crystalline chitin, where it introduces chain breaks and generates oxidized chain ends, thus promoting further degradation by chitinases. This enzymatic activity was discovered and further characterized by using mass spectrometry and chromatographic separation methods to detect oxidized products generated in the absence or presence of H(2)(18)O or (18)O(2). There are strong indications that similar enzymes exist that work on cellulose. Our findings not only demonstrate the existence of a hitherto unknown enzyme activity but also provide new avenues toward more efficient enzymatic conversion of biomass.

Our understanding of fungal cellulose degradation has shifted dramatically in the past few years with the characterization of a new class of secreted enzymes, the lytic polysaccharide monooxygenases (LPMO). After a period of intense research covering structural, biochemical, theoretical and evolutionary aspects, we have a picture of them as wedge-like copper-dependent metalloenzymes that on reduction generate a radical copper-oxyl species, which cleaves mainly crystalline cellulose. The main biological function lies in the synergism of fungal LPMOs with canonical hydrolytic cellulases in achieving efficient cellulose degradation. Their important role in cellulose degradation is highlighted by the wide distribution and often numerous occurrences in the genomes of almost all plant cell-wall degrading fungi. In this review, we provide an overview of the latest achievements in LPMO research and consider the open questions and challenges that undoubtedly will continue to stimulate interest in this new and exciting group of enzymes. © The Author 2014. Published by Oxford University Press.

Cellulose-degrading auxiliary activity family 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) are known to be widely distributed among filamentous fungi and participate in the degradation of lignocellulose via the oxidative cleavage of celluloses, cello-oligosaccharides, or hemicelluloses. AA9 LPMOs have been reported to have extensive interactions with not only cellulases but also oxidases. The addition of AA9 LPMOs can greatly reduce the amount of cellulase needed for saccharification and increase the yield of glucose. The discovery of AA9 LPMOs has greatly changed our understanding of how fungi degrade cellulose. In this review, apart from summarizing the recent discoveries related to their catalytic reaction, functional diversity, and practical applications, the stability, expression system, and protein engineering of AA9 LPMOs are reviewed for the first time. This review may provide a reference value to further broaden the substrate range of AA9 LPMOs, expand the scope of their practical applications, and realize their customization for industrial utilization.Key Points• The stability and expression system of AA9 LPMOs are reviewed for the first time.• The protein engineering of AA9 LPMOs is systematically summarized for the first time.• The latest research results on the catalytic mechanism of AA9 LPMOs are summarized.• The application of AA9 LPMOs and their relationship with other enzymes are reviewed.

The thermophilic fungus Malbranchea cinnamomea contains a host of enzymes that enable its ability as an efficient degrader of plant biomass and that could be mined for industrial applications. This thermophilic fungus has been studied and found to encode eight lytic polysaccharide monooxygenases (LPMOs) from auxiliary activity family 9 (AA9), which collectively possess different substrate specificities for a range of plant cell-wall-related polysaccharides and oligosaccharides. To gain greater insight into the molecular determinants defining the different specificities, structural studies were pursued and the structure of McAA9F was determined. The enzyme contains the immunoglobulin-like fold typical of previously solved AA9 LPMO structures, but contains prominent differences in the loop regions found on the surface of the substrate-binding site. Most significantly, McAA9F has a broad substrate specificity, with activity on both crystalline and soluble polysaccharides. Moreover, it contains a small loop in a region where a large loop has been proposed to govern specificity towards oligosaccharides. The presence of the small loop leads to a considerably flatter and more open surface that is likely to enable the broad specificity of the enzyme. The enzyme contains a succinimide residue substitution, arising from intramolecular cyclization of Asp10, at a position where several homologous members contain an equivalent residue but cyclization has not previously been observed. This first structure of an AA9 LPMO from M. cinnamomea aids both the understanding of this family of enzymes and the exploration of the repertoire of industrially relevant lignocellulolytic enzymes from this fungus.

褐藻寡糖有着丰富的生物学功能,酶法制备功能性褐藻寡糖具有重要实践应用价值。为发掘高活性及稳定性的褐藻寡糖制备酶,对浅海热液嗜热菌Yeosuana marina sp. JLT21中的海藻酸裂解酶YMA-1的基因在大肠杆菌中进行表达、纯化及酶活鉴定。结果发现YMA-1由306个氨基酸残基构成,是多糖裂解酶家族7（PL7）新成员;重组YMA-1酶的最适催化条件是55℃,pH 9.0,比活力1.3×10⁴U/mg,Cu²⁺ 可有效促进酶活;在37℃,pH 9.0 条件下,该酶对海藻酸钠、聚甘露糖醛酸和聚古罗糖醛酸的比活力分别达到（5 201.21±86.46）U/mg、（6 399.73±253.12）U/mg和（3 751.68±116.25）U/mg,酶解海藻酸钠终产物多为不饱和三糖和四糖,表现出内切双功能型海藻酸裂解酶活性。YMA-1酶作为PL7家族中较宽底物谱、高活性及稳定性的内切海藻酸裂解酶,在高效绿色生产功能性褐藻寡糖上有着潜在应用价值。

SAGA复合体是一种多功能蛋白复合物,负责细胞内10%以上的基因转录。Spt7作为SAGA复合体的核心蛋白,维持SAGA复合体的稳定。除酵母之外的真菌中并未有Spt7相关的研究报道。本研究对不同黑曲霉、丝衣霉等丝状真菌来源的Spt7的氨基酸序列进行多序列比对及蛋白结构的分析,发现Spt7在不同物种一致性较低,但均存在保守型的Bromo结构域。以黑曲霉1062为出发菌株,通过农杆菌转化法将过表达spt7的质粒转入1062菌株中,获得黑曲霉OE spt7转化子。通过对OE spt7转化子及对照组在CM培养基生长形态进行观察分析发现,过表达Spt7有益于菌体的生长及分生孢子的生成,在72 h时OE spt7转化子和对照组的菌落直径分别达到2.9 cm和2.8 cm,且转化子的孢子数量达到（5.8-6.3）×10⁷/cm²,较对照组（2.3×10⁷/cm²）提高1.5-1.7倍,且转化子菌丝分支较对照组多。另外,与对照组相比,OE spt7转化子在15 mmol/L H₂O₂条件下孢子萌发更快,菌落直径是对照组4倍,且在15% NaCl高渗以及39℃高温的条件下较对照组生长更加鲜活。通过qRT-PCR分析参与菌体抗氧化胁迫的过氧化物酶及耐高温的热休克蛋白基因的转录水平,发现除了CatR转录水平下调3.56倍,其他的过氧化物酶SOD、CpeB、GPX分别上调了3.8、1.89、3.56倍,除Hsp40及Hsp70较对照组无显著差异,Hsp90的转录水平上调19倍。

Background: Lytic polysaccharide monooxygenases (LPMO) release a spectrum of cleavage products from their polymeric substrates cellulose, hemicellulose, or chitin. The correct identification and quantitation of these released products is the basis of MS/HPLC-based detection methods for LPMO activity. The duration, effort, and intricate analysis allow only specialized laboratories to measure LPMO activity in day-to-day work. A spectrophotometric assay will simplify the screening for LPMO in culture supernatants, help monitor recombinant LPMO expression and purification, and support enzyme characterization.Results: Based on a newly discovered peroxidase activity of LPMO, we propose a fast, robust, and sensitive spectrophotometric activity assay using 2,6-dimethoxyphenol (2,6-DMP) and H2O2. The fast enzymatic assay (300 s) consists of 1 mM 2,6-DMP as chromogenic substrate, 100 mu M H2O2 as cosubstrate, and an adequate activity of LPMO in a suitable buffer. The high molar absorption coefficient of the formed product coerulignone (epsilon(469) = 53,200 M-1 cm(-1)) makes the assay sensitive and allows reliable activity measurements of LPMO in concentrations of approx. 0.5-50 mg L-1.Conclusions: The activity assay based on 2,6-DMP detects a novel peroxidase activity of LPMO. This activity can be accurately measured and used for enzyme screening, production, and purification, and can also be applied to study binding constants or thermal stability. However, the assay has to be used with care in crude extracts, because other enzymes such as laccase or peroxidase will interfere with the assay. We also want to stress that the peroxidase activity is a homogeneous reaction with soluble substrates and should not be correlated to heterogeneous LPMO activity on polymeric substrates.

Lytic Polysaccharide Monooxygenases (LPMOs) are auxiliary accessory\nenzymes that act synergistically with cellulases and which are increasingly being used in secondgeneration\nbioethanol production from biomasses. Several LPMOs have been identified in various\nfilamentous fungi, including Aspergillus fumigatus. However, many LPMOs have not been characterized\nyet.

Cellulose is the most abundant polysaccharide in lignocellulosic biomass, where it is interlinked with lignin and hemicellulose. Bioethanol can be produced from biomass. Since breaking down biomass is difficult, cellulose-active enzymes secreted by filamentous fungi play an important role in degrading recalcitrant lignocellulosic biomass. We characterized a cellobiohydrolase (AfCel6A) and lytic polysaccharide monooxygenase LPMO (AfAA9_B) from Aspergillus fumigatus after they were expressed in Pichia pastoris and purified. The biochemical parameters suggested that the enzymes were stable; the optimal temperature was ~60 °C. Further characterization revealed high turnover numbers (kcat of 147.9 s−1 and 0.64 s−1, respectively). Surprisingly, when combined, AfCel6A and AfAA9_B did not act synergistically. AfCel6A and AfAA9_B association inhibited AfCel6A activity, an outcome that needs to be further investigated. However, AfCel6A or AfAA9_B addition boosted the enzymatic saccharification activity of a cellulase cocktail and the activity of cellulase Af-EGL7. Enzymatic cocktail supplementation with AfCel6A or AfAA9_B boosted the yield of fermentable sugars from complex substrates, especially sugarcane exploded bagasse, by up to 95%. The synergism between the cellulase cocktail and AfAA9_B was enzyme- and substrate-specific, which suggests a specific enzymatic cocktail for each biomass by up to 95%. The synergism between the cellulase cocktail and AfAA9_B was enzyme- and substrate-specific, which suggests a specific enzymatic cocktail for each biomass.

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).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).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.