Polysaccharide lyases: recent developments as biotechnological tools

Polysaccharide lyases, which are polysaccharide cleavage enzymes, act mainly on anionic polysaccharides. Produced by prokaryote and eukaryote organisms, these enzymes degrade (1,4) glycosidic bond by a beta elimination mechanism and have unsaturated oligosaccharides as major products. New polysaccharides are cleaved only by their specific polysaccharide lyases. From anionic polysaccharides controlled degradations, various biotechnological applications were investigated. This review catalogues the degradation of bacterial, plant and animal polysaccharides (neutral and anionic) by this family of carbohydrate acting enzymes.     

Aspergillus Enzymes Involved in Degradation of Plant Cell Wall Polysaccharides

Degradation of plant cell wall polysaccharides is of major importance in the food and feed, beverage, textile, and paper and pulp industries, as well as in several other industrial production processes. Enzymatic degradation of these polymers has received attention for many years and is becoming a more and more attractive alternative to chemical and mechanical processes. Over the past 15 years, much progress has been made in elucidating the structural characteristics of these polysaccharides and in characterizing the enzymes involved in their degradation and the genes of biotechnologically relevant microorganisms encoding these enzymes. The members of the fungal genus Aspergillus are commonly used for the production of polysaccharide-degrading enzymes. This genus produces a wide spectrum of cell wall-degrading enzymes, allowing not only complete degradation of the polysaccharides but also tailored modifications by using specific enzymes purified from these fungi. This review summarizes our current knowledge of the cell wall polysaccharide-degrading enzymes from aspergilli and the genes by which they are encoded.     

Aspergillus enzymes involved in degradation of plant cell wall polysaccharides.

Degradation of plant cell wall polysaccharides is of major importance in the food and feed, beverage, textile, and paper and pulp industries, as well as in several other industrial production processes. Enzymatic degradation of these polymers has received attention for many years and is becoming a more and more attractive alternative to chemical and mechanical processes. Over the past 15 years, much progress has been made in elucidating the structural characteristics of these polysaccharides and in characterizing the enzymes involved in their degradation and the genes of biotechnologically relevant microorganisms encoding these enzymes. The members of the fungal genus Aspergillus are commonly used for the production of polysaccharide-degrading enzymes. This genus produces a wide spectrum of cell wall-degrading enzymes, allowing not only complete degradation of the polysaccharides but also tailored modifications by using specific enzymes purified from these fungi. This review summarizes our current knowledge of the cell wall polysaccharide-degrading enzymes from aspergilli and the genes by which they are encoded.     

Fungal enzyme sets for plant polysaccharide degradation

Enzymatic degradation of plant polysaccharides has many industrial applications, such as within the paper, food, and feed industry and for sustainable production of fuels and chemicals. Cellulose, hemicelluloses, and pectins are the main components of plant cell wall polysaccharides. These polysaccharides are often tightly packed, contain many different sugar residues, and are branched with a diversity of structures. To enable efficient degradation of these polysaccharides, fungi produce an extensive set of carbohydrate-active enzymes. The variety of the enzyme set differs between fungi and often corresponds to the requirements of its habitat. Carbohydrate-active enzymes can be organized in different families based on the amino acid sequence of the structurally related catalytic modules. Fungal enzymes involved in plant polysaccharide degradation are assigned to at least 35 glycoside hydrolase families, three carbohydrate esterase families and six polysaccharide lyase families. This mini-review will discuss the enzymes needed for complete degradation of plant polysaccharides and will give an overview of the latest developments concerning fungal carbohydrate-active enzymes and their corresponding families.     

Functional Diversity of Carbohydrate-Active Enzymes Enabling a Bacterium to Ferment Plant Biomass

Microbial metabolism of plant polysaccharides is an important part of environmental carbon cycling, human nutrition, and industrial processes based on cellulosic bioconversion. Here we demonstrate a broadly applicable method to analyze how microbes catabolize plant polysaccharides that integrates carbohydrate-active enzyme (CAZyme) assays, RNA sequencing (RNA-seq), and anaerobic growth screening. We apply this method to study how the bacterium Clostridium phytofermentans ferments plant biomass components including glucans, mannans, xylans, galactans, pectins, and arabinans. These polysaccharides are fermented with variable efficiencies, and diauxies prioritize metabolism of preferred substrates. Strand-specific RNA-seq reveals how this bacterium responds to polysaccharides by up-regulating specific groups of CAZymes, transporters, and enzymes to metabolize the constituent sugars. Fifty-six up-regulated CAZymes were purified, and their activities show most polysaccharides are degraded by multiple enzymes, often from the same family, but with divergent rates, specificities, and cellular localizations. CAZymes were then tested in combination to identify synergies between enzymes acting on the same substrate with different catalytic mechanisms. We discuss how these results advance our understanding of how microbes degrade and metabolize plant biomass.     

Plant glycoside hydrolases involved in cell wall polysaccharide degradation.

The cell wall plays a key role in controlling the size and shape of the plant cell during plant development and in the interactions of the plant with its environment. The cell wall structure is complex and contains various components such as polysaccharides, lignin and proteins whose composition and concentration change during plant development and growth. Many studies have revealed changes in cell walls which occur during cell division, expansion, and differentiation and in response to environmental stresses; i.e. pathogens or mechanical stress. Although many proteins and enzymes are necessary for the control of cell wall organization, little information is available concerning them. An important advance was made recently concerning cell wall organization as plant enzymes that belong to the superfamily of glycoside hydrolases and transglycosidases were identified and characterized; these enzymes are involved in the degradation of cell wall polysaccharides. Glycoside hydrolases have been characterized using molecular, genetic and biochemical approaches. Many genes encoding these enzymes have been identified and functional analysis of some of them has been performed. This review summarizes our current knowledge about plant glycoside hydrolases that participate in the degradation and reorganisation of cell wall polysaccharides in plants focussing particularly on those from Arabidopsis thaliana.     

Golgi enzymes that synthesize plant cell wall polysaccharides: finding and evaluating candidates in the genomic era

Although the synthesis of cell wall polysaccharides is a critical process during plant cell growth and differentiation, many of the wall biosynthetic genes have not yet been identified. This review focuses on the synthesis of non-cellulosic matrix polysaccharides formed in the Golgi apparatus. Our consideration is limited to two types of plant cell wall biosynthetic enzymes: glycan synthases and glycosyltransferases. Classical means of identifying these enzymes and the genes that encode them rely on biochemical purification of enzyme activity to obtain amino acid sequence data that is then used to identify the corresponding gene. This type of approach is difficult, especially when acceptor substrates for activity assays are unavailable, as is the case for many enzymes. However, bioinformatics and functional genomics provide powerful alternative means of identifying and evaluating candidate genes. Database searches using various strategies and expression profiling can identify candidate genes. The involvement of these genes in wall biosynthesis can be evaluated using genetic, reverse genetic, biochemical, and heterologous expression methods. Recent advances using these methods are considered in this review.     

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.     

Regulation of <Emphasis Type="Italic">Aspergillus</Emphasis> genes encoding plant cell wall polysaccharide-degrading enzymes; relevance for industrial production

The genus Aspergillus is widely used for the production of plant cell wall polysaccharide-degrading enzymes. The range of enzymes purified from these fungi covers nearly every function required for the complete degradation of cellulose, xyloglucan, xylan, galacto(gluco)mannan and pectin. This paper describes the Aspergillus enzymes involved in the degradation of these polysaccharides and discusses the regulatory systems involved in the expression of the genes encoding these proteins. The latter is of major importance in the large-scale production of these enzymes for industrial applications.     

Plant protein inhibitors of cell wall degrading enzymes

Plant cell walls, which consist mainly of polysaccharides (i.e. cellulose, hemicelluloses and pectins), play an important role in defending plants against pathogens. Most phytopathogenic microorganisms secrete an array of cell wall degrading enzymes (CWDEs) capable of depolymerizing the polysaccharides in the plant host wall. In response, plants have evolved a diverse battery of defence responses including protein inhibitors of these enzymes. These include inhibitors of pectin degrading enzymes such as polygalacturonases, pectinmethyl esterases and pectin lyases, and hemicellulose degrading enzymes such as endoxylanases and xyloglucan endoglucanases. The discovery of these plant inhibitors and the recent resolution of their three-dimensional structures, free or in complex with their target enzymes, provide new lines of evidence regarding their function and evolution in plant-pathogen interactions.     

Carbohydrate-binding domains: multiplicity of biological roles

Insoluble polysaccharides can be degraded by a set of hydrolytic enzymes formed by catalytic modules appended to one or more non-catalytic carbohydrate-binding modules (CBM). The most recognized function of these auxiliary domains is to bind polysaccharides, bringing the biocatalyst into close and prolonged vicinity with its substrate, allowing carbohydrate hydrolysis. Examples of insoluble polysaccharides recognized by these enzymes include cellulose, chitin, β-glucans, starch, glycogen, inulin, pullulan, and xylan. Based on their amino acid similarity, CBMs are grouped into 55 families that show notable variation in substrate specificity; as a result, their biological functions are miscellaneous. Carbohydrate or polysaccharide recognition by CBMs is an important event for processes related to metabolism, pathogen defense, polysaccharide biosynthesis, virulence, plant development, etc. Understanding of the CBMs properties and mechanisms in ligand binding is of vital significance for the development of new carbohydrate-recognition technologies and provide the basis for fine manipulation of the carbohydrate–CBM interactions.     

Protease-catalyzed synthetic reactions and immobilization-activation of the enzymes in hydrophilic organic solvents.

The catalytic feature of serine proteases for synthetic reactions in hydrophilic organic solvents and effects of immobilization by complexation with polysaccharides are described. Free alpha-chymotrypsin and subtilisin Carlsberg catalyze esterification, transesterification, and peptide synthesis in hydro-organic cosolvents with less than 10% water. Subtilisin BPN' is catalytically less active. The medium effects on the reaction kinetics and product yield were investigated in terms of the nature of solvent and water content in the reaction systems. The substrate- and stereo-specificities of the enzymes suggest that the enzymes maintain their native conformations in these low-water organic solvents. The catalytic activities of the proteases markedly increase by immobilization or complexation with polysaccharides, such as chitin or chitosan. The results of the rate measurements suggest that the primary role of the support materials is the activation of the enzymes and the increase in substrate concentration at reaction sites.     

Histochemical studies of alpha and beta-glucosidases in the germinating pollen grains ofPortulaca grandiflora

The paper deals with the distribution of alpha and beta-glucosidases in the germinating pollen grains ofPortulaca grandiflora. Both these enzymes are localized in the pollen wall and the cytoplasmic granules. The latter are distributed throughout the pollen cytoplasm, pollen tube and stigma hair. In non-germinating pollen grains, enzymes are concentrated in the pollen wall. Stigma hair sheaths are completely free from enzymes. The functional significance of these enzymes in the hydrolysis of phenolic glycosides and polysaccharides is discussed.     

生物质多糖的高效降解与降解酶（系）的精确定制

自然界中多糖类生物质资源十分丰富,然而其复杂的抗降解屏障限制了生物转化的进程.近年来,随着生物质多糖结构的快速解析以及大量多糖降解酶的鉴定研究,针对不同底物结构或产物需求,仿制高效微生物多糖代谢途径,精确定制多糖降解酶系,促进生物质高效转化已成为可能.本文分析中性多糖(纤维素和木聚糖)、碱性多糖(几丁质和壳聚糖)以及酸性多糖(褐藻胶)的精细结构组成与基团性质,总结3类多糖主要降解酶的活性架构特征及其底物精确结合模式.文章还阐述蛋白质工程设计与定制策略,针对酶分子不同功能区的分析,可为酶分子的功能快速设计与改造提供靶点,以获得适宜于工业应用的高效酶分子,此外,根据微生物胞外降解酶系的降解次序与协同关系,可基于应用需求精确定制复杂多糖降解酶系,实现生物质的高效与高值降解转化.     

Modulatory effect of Ganoderma lucidum polysaccharides on serum antioxidant enzymes activities in ovarian cancer rats

In the present study, we isolated polysaccharides from Ganoderma lucidum and investigated its effect on serum antioxidant enzymes activity in ovarian cancer rats to explore the mechanism underlying the pharmacological anti-cancer activity of the polysaccharides. Rats were grouped into the control, model and polysaccharides-treated groups. After experiment ended, serum antioxidant enzymes activity in rats were measured. Results showed that polysaccharides from G. lucidum significantly reduced MDA production and increased serum antioxidant enzymes activity. These results suggest that the antioxidant activity of polysaccharides from G. lucidum might be benefical towards ovarian cancer therapy.     

Complete genome sequence of the complex carbohydrate-degrading marine bacterium, Saccharophagus degradans strain 2-40 T

The marine bacterium Saccharophagus degradans strain 2-40 (Sde 2-40) is emerging as a vanguard of a recently discovered group of marine and estuarine bacteria that recycles complex polysaccharides. We report its complete genome sequence, analysis of which identifies an unusually large number of enzymes that degrade >10 complex polysaccharides. Not only is this an extraordinary range of catabolic capability, many of the enzymes exhibit unusual architecture including novel combinations of catalytic and substrate-binding modules. We hypothesize that many of these features are adaptations that facilitate depolymerization of complex polysaccharides in the marine environment. This is the first sequenced genome of a marine bacterium that can degrade plant cell walls, an important component of the carbon cycle that is not well-characterized in the marine environment.     

Pectic substances,pectic enzymes and haze formation in fruit juices

The pectic substances, located primarily in the middle lamella between cells in higher plant tissues, are complex polysaccharides. They include the negatively charged rhamnogalacturonans, and the neutral arabinogalactans I and II and l-arabinans. These polysaccharides add viscosity to juices but may also form hazes and precipitates and retard maximum recovery of juices from the fruit. The rhamnogalacturonans are degraded by the enzymes pectin methylesterase and polygalacturonase normally present in plant tissues and by these enzymes and pectate lyase in microbially derived commercial pectic enzymes added during processing. The presence of a?abinofuranosidase, which degrades l-arabinans, in commercial pectic enzyme preparations, can cause haze formation in juices such as apple and pear.     

Characterization and localization of the cell wall autolysis substrate in Pisum sativum epicotyls

The products released in cell wall autolysis from 4-day-old epicotyls of Pisum sativum elute in gel filtration chromatography (Bio Gel P.2) as two components, mono and polysaccharides, in a practically constant ratio over the time of incubation. The polysaccharides are mainly composed of arabinose and galactose, with smaller amounts of xylose and glucose, whereas the monosaccharide are almost exclusively composed of galactose. The same results were obtained when inactive cell walls were hydrolyzed by the enzymes extracted from the cell wall with LiCl. The hydrolysis of the different cell wall fractions by these enzymes shows that the autolytic substrates are preferentially located on the pectic fractions.     

Genomic Potential for Polysaccharide Deconstruction in Bacteria

Glycoside hydrolases are important enzymes that support bacterial growth by enabling the degradation of polysaccharides (e.g., starch, cellulose, xylan, and chitin) in the environment. Presently, little is known about the overall phylogenetic distribution of the genomic potential to degrade these polysaccharides in bacteria. However, knowing the phylogenetic breadth of these traits may help us predict the overall polysaccharide processing in environmental microbial communities. In order to address this, we identified and analyzed the distribution of 392,166 enzyme genes derived from 53 glycoside hydrolase families in 8,133 sequenced bacterial genomes. Enzymes for oligosaccharides and starch/glycogen were observed in most taxonomic groups, whereas glycoside hydrolases for structural polymers (i.e., cellulose, xylan, and chitin) were observed in clusters of relatives at taxonomic levels ranging from species to genus as determined by consenTRAIT. The potential for starch and glycogen processing, as well as oligosaccharide processing, was observed in 85% of the strains, whereas 65% possessed enzymes to degrade some structural polysaccharides (i.e., cellulose, xylan, or chitin). Potential degraders targeting one, two, and three structural polysaccharides accounted for 22.6, 32.9, and 9.3% of genomes analyzed, respectively. Finally, potential degraders targeting multiple structural polysaccharides displayed increased potential for oligosaccharide deconstruction. This study provides a framework for linking the potential for polymer deconstruction with phylogeny in complex microbial assemblages.     

Plant cell walls: Protecting the barrier from degradation by microbial enzymes

Plant cell walls are predominantly composed of polysaccharides, which are connected in a strong, yet resilient network. They determine the size and shape of plant cells and form the interface between the cell and its often hostile environment. To penetrate the cell wall and thus infect plants, most phytopathogens secrete numerous cell wall degrading enzymes. Conversely, as a first line of defense, plant cell walls contain an array of inhibitors of these enzymes. Scientific knowledge on these inhibitors significantly progressed in the past years and this review is meant to give a comprehensive overview of plant inhibitors against microbial cell wall degrading enzymes and their role in plant protection.