Soluble chromogenic substrates for the assay of endo-1,4-β-xylanases and endo-1,4-β-glucanases
Abstract
New soluble chromogenic substrates were prepared for specific and rapid assays of endo-1,4-β-xylanases and endo-1,4-β-glucanases. A soluble beechwood 4-O-methyl-d-glucurono-d-xylan was dyed with Remazol brilliant blue R, and hydroxyethylcellulose was coupled to Ostazin brilliant red H-3B. The assays are based on photometric measurements of the enzyme-released dyed fragments soluble in the presence of organic solvents which precipitate the original substrates and their high-molecular-weight fractions. The assays are advantageous for rapid analyses of large amount of samples and also permit evaluation of the activities of both enzymes in the presence of exo-β-glycanases and β-glycosidases, at a high level of reducing compounds and viable cells, on the cell surface and on cell membranes and organelles.
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Serratia marcescens NJ01 and Erwinia carotovora, two pathogenic bacteria involved in vegetable spoilage, can persist in leafy vegetables and subsequently form biofilms. The aim of this study was to evaluate the effect of propyl gallate (PG) on the susceptibility of antibiotics against the biofilms of S. marcescens NJ01 and E. carotovora and uncover its anti-virulence mechanism. PG notably inhibited the production of AHLs and enhanced the susceptibility of antibiotics against the biofilm formation of S. marcescens NJ01 and E. carotovora and significantly improved the disrupted effect on the preformed biofilms of S. marcescens NJ01. PG exposure decreased the minimum inhibitory concentrations of these five antibiotics and reduced the production of virulence factors. Further investigation indicated that PG exposure resulted in the downregulation of genes involved in virulence, biofilm formation, and antioxidant enzymes of S. marcescens NJ01. The suppression of antioxidant enzymes caused the enhancement of oxidative stress, increased membrane permeability, and ultimately promoted the susceptibility of bacterial/biofilm cells to antibiotics. The tomato and lettuce infection assay indicated that PG could notably attenuate the pathogenicity of S. marcescens NJ01 and E. carotovora. These findings suggest that PG has the potential to function as an antibiotic accelerant to defend against spoilage pathogens.
Cellulose- and xylan-degrading yeasts: Enzymes, applications and biotechnological potential
2022, Biotechnology AdvancesMicrobes and their carbohydrate-active enzymes are central for depolymerization of complex lignocellulosic polysaccharides in the global carbon cycle. Their unique abilities to degrade and ferment carbohydrates are also utilized in many industrial processes such as baking, brewing and production of biofuels and drugs. Effective degradation and utilization of cellulose and hemicelluloses is important for the shift towards green bioeconomy, and requires microbes equipped with proper sets of carbohydrate-active enzymes (CAZymes). Knowledge of cellulolytic and xylanolytic CAZymes has mainly been generated from bacteria and filamentous fungi, while yeasts have been largely overlooked and may represent an untapped resource in natural CAZymes with industrial relevance. Cellulose and xylan-degrading yeasts with the ability to ferment saccharides are also promising candidates for consolidated bioprocesses (CBPs), as they can degrade lignocellulose and utilize its constituents to produce desired products at the same time. Cellulolytic yeasts able to utilize insoluble crystalline cellulose are rare while xylanolytic yeasts are rather widespread in nature. The lack of particular enzymes in yeasts can be remediated by introducing the missing enzymes into strains having outstanding product-forming attributes.
In this review, we provide a comprehensive overview of the cellulose- and xylan-degrading ascomycetous and basidiomycetous yeasts known to date. We describe how these yeasts can be identified through bioprospecting and bioinformatic approaches and summarize available growth and enzymatic assays for strain characterization. Known and predicted CAZymes are extensively analyzed, both in individual species and in a phylogenetic perspective. We also describe the strategies used for construction of recombinant cellulolytic and xylanolytic strains as well as current applications for polysaccharide-degrading yeasts. Finally, we discuss the great potential of these yeasts as industrial cell factories, identify open research questions and provide suggestions for future investigations.
A solid-state nanopore-based single-molecule approach for label-free characterization of plant polysaccharides
2021, Plant CommunicationsPolysaccharides are important biomacromolecules existing in all plants, most of which are integrated into a fibrillar structure called the cell wall. In the absence of an effective methodology for polysaccharide analysis that arises from compositional heterogeneity and structural flexibility, our knowledge of cell wall architecture and function is greatly constrained. Here, we develop a single-molecule approach for identifying plant polysaccharides with acetylated modification levels. We designed a solid-state nanopore sensor supported by a free-standing SiNx membrane in fluidic cells. This device was able to detect cell wall polysaccharide xylans at concentrations as low as 5 ng/μL and discriminate xylans with hyperacetylated and unacetylated modifications. We further demonstrated the capability of this method in distinguishing arabinoxylan and glucuronoxylan in monocot and dicot plants. Combining the data for categorizing polysaccharide mixtures, our study establishes a single-molecule platform for polysaccharide analysis, opening a new avenue for understanding cell wall structures, and expanding polysaccharide applications.
A novel GH30 xylobiohydrolase from Acremonium alcalophilum releasing xylobiose from the non-reducing end
2020, Enzyme and Microbial TechnologyXylanases of the GH30 family are grouped to subfamilies GH30-7 and GH30-8. The GH30-8 members are of bacterial origin and well characterized, while the GH30-7 members are from fungal sources and their properties are quite diverse. Here, a heterologous expression and characterization of the GH30-7 xylanase AaXyn30A from a cellulolytic fungus Acremonium alcalophilum is reported. From various polymeric and oligomeric substrates AaXyn30A generates xylobiose as the main product. It was proven that xylobiose is released from the non-reducing end of all tested substrates, thus the enzyme behaves as a typical non-reducing-end acting xylobiohydrolase. AaXyn30A is active on different types of xylan, exhibiting the highest activity on rhodymenan (linear β-1,3-β-1,4-xylan) from which also an isomeric xylotriose Xyl-β-1,3-Xyl-β-1,4-Xyl is formed. Production of xylobiose from glucuronoxylan is at later stage accompanied by a release of aldouronic acids differing from those liberated by the bacterial GH30-8 glucuronoxylanases.
Increase in the thermostability of GH11 xylanase XynJ from Bacillus sp. strain 41M-1 using site saturation mutagenesis
2019, Enzyme and Microbial TechnologyGH11 xylanase XynJ from Bacillus sp. strain 41M-1 has a β-jellyroll fold composed of eight β strands with a deep active-site cleft. We hypothesized that the thermostability of XynJ will increase if the flexibility of the β strands in the jellyroll structure is decreased without impairing activity. To verify this hypothesis, we introduced random mutations into Tyr13–Arg104 and Gly169–Tyr194, both of which are located in the β-jellyroll fold of XynJ, to construct a site saturation mutagenesis library. By screening 576 clones followed by site saturation mutation analysis of Thr82, T82A was selected as the most thermostable variant. In the hydrolysis of beechwood xylan at pH 7.8, the temperatures required to reduce initial activity by 50% in 15 min were 61 °C for the wild-type XynJ (WT) and 65 °C for T82A. The optimum hydrolysis temperatures were 60 °C for WT and 65 °C for T82A. There was little difference in the kcat and Km values and the pH dependence of activity between WT and T82A. Crystallographic analysis of WT and T82A revealed that thermostabilization by the T82A mutation might result from the removal of unfavorable van der Waals interactions. Thus, a highly thermostable XynJ variant was generated without impairing activity using this mutation strategy.
One-step process for producing prebiotic arabino-xylooligosaccharides from brewer's spent grain employing Trichoderma species
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