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

One of natures most important biological processes is the degradation of lignocellulosic materials to carbon dioxide, water and humic substances. This implies possibilities to use biotechnology in the pulp and paper industry and consequently, the use of microorganisms and their enzymes to replace or supplement chemical methods is gaining interest. This chapter describes the structure of wood and the main wood components, cellulose, hemicelluloses and lignins. The enzyme and enzyme mechanisms used by fungi and bacteria to modify and degrade these components are described in detail. Techniques for how to assay for these enzyme activities are also described. The possibilities for biotechnology in the pulp and paper industry and other fiber utilizing industries based on these enzymes are discussed.

Microorganisms and enzymes involved in the degradation of plant fiber cell walls

Microbial mannanases have become biotechnologically important since they target the hydrolysis of complex polysaccharides of plant tissues into simple molecules like manno-oligosaccharides and mannoses The role of mannanases in the paper and pulp industry is well established and recently they have found application in the food and feed technology, coffee extraction, oil drilling and detergent industry Mannanses are enzymes produced mainly from microorganisms but mannanases produced from plants and animals have also been reported Bacterial mannanases are mostly extracellular and can act in a wide range of pH and temperature, though acidic and neutral mannanases are more common This review will focus on complex mannan structure and the microbial enzyme complex involved in its complete breakdown, mannanase sources, production conditions and their applications in the commercial sector The reference to plant and animal mannanases has been made to complete the overview However, the major emphasis of the review is on the microbial mannanases

Microbial mannanases: an overview of production and applications.

Purification and characterization of two β-mannanases from Trichoderma reesei

Glucomannan, a promising polysaccharide for biopharmaceutical purposes.

An alpha-D-galactosidase (EC 3.2.1.22) and a beta-D-mannanase (EC 3.2.1.78), which were secreted into the growth medium when Aspergillus tamarii was cultivated in the presence of galactomannan, were purified by a procedure including chromatography on hydroxyapatite and DEAE-cellulose columns. Each of these enzymes showed a single protein band, corresponding to their respective activities, on polyacrylamide-gel electrophoresis. Both enzymes were shown to be glycoproteins containing N-acetylglucosamine, mannose and galactose, with molar proportions of 1:6:1.5 for alpha-D-galactosidase and 1:13:8 for beta-D-mannanase. Mr values as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate and by the electrophoretic method of Hedrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164] were 56000 and 53000 respectively. The alpha-D-galactosidase differed markedly from the mycelial forms I and II studied in the preceding paper [Civas, Eberhard, Le Dizet & Petek (1984) Biochem. J. 219, 849-855] with regard to both its kinetic and structural properties.

Glycosidases induced in Aspergillus tamarii. Secreted alpha-D-galactosidase and beta-D-mannanase.

Two alpha-D-galactosidases (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) produced by Aspergillus tamarii were purified from the mycelial extract by a procedure including chromatography on hydroxyapatite, DEAE-cellulose and ECTEOLA-cellulose. Each of these enzymes showed a single protein band corresponding to the alpha-D-galactosidase activity when examined by polyacrylamide-gel electrophoresis. They catalysed the hydrolysis of o-nitrophenyl alpha-D-galactoside, melibiose, raffinose and stachyose, but did not attack the galactomannans. Their Mr values were respectively 265000 +/- 5000 and 254000 +/- 5000 by the method of Hedrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164]. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate in each case showed a single protein band, with Mr 88000 and 77500 respectively. The purified enzymes contained carbohydrate, consisting of N-acetylglucosamine, mannose, glucose and galactose in the estimated molar proportions of 1:9:5:8 in alpha-galactosidase I.

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Glycosidases induced in Aspergillus tamarii. Mycelial alpha-D-galactosidases.

Alpha interferon (IFN-alpha) and IFN-beta are able to interfere with viral infection. They exert a vast array of biologic functions, including growth arrest, cell differentiation, and immune system regulation. This regulation extends from innate immunity to cellular and humoral adaptive immune responses. A strict control of expression is needed to prevent detrimental effects of unregulated IFN. Multiple IFN-A subtypes are coordinately induced in human and mouse cells infected by virus and exhibit differences in expression of their individual mRNAs. We demonstrated that the weakly expressed IFN-A11 gene is negatively regulated after viral infection, due to a distal negative regulatory element, binding homeoprotein pituitary homeobox 1 (Pitx1). Here we show that the POU protein Oct-1 binds in vitro and in vivo to the IFN-A11 promoter and represses IFN-A expression upon interferon regulatory factor overexpression. Furthermore, we show that Oct-1-deficient MEFs exhibit increased in vivo IFN-A gene expression and increased antiviral activity. Finally, the IFN-A expression pattern is modified in Oct-1-deficient MEFs. The broad representation of effective and potent octamer-like sequences within IFN-A promoters suggests an important role for Oct-1 in IFN-A regulation.

The POU transcription factor Oct-1 represses virus-induced interferon A gene expression.

Mouse interferon-beta (Mu-INF-beta) induced in C-243 cells with Newcastle disease virus was purified in four steps including ammonium sulfate fractionation. DEAE-cellulose, monoclonal Mu-IFN-beta antibody affinity and Mono-S cation-exchange chromatographies. Specific activity of the purified Mu-IFN-beta ranged over 1.1-1.4 X 10(9) NIH units/mg protein. This preparation was submitted to pronase digestion and gel on Fractogel TSK HW-40. The permethylated and acetylated glycopeptide fraction was analyzed by chemical-ionization (ammonia) mass spectrometry. The major glycopeptide is composed of Gal, Man, GlcNAc and NeuAc with a molar ratio of 2.0:3.6:3.4:0.5. The GLC pattern of methyl derivatives obtained by methanolysis and acetylation of fully methylated glycopeptide identified 2,3,4,6-tetra-O-methylgalactose; 3,4,6-tri-O-methyl-mannose; 2,3,4- and 2,4,6-tri-O-methylgalactose; 2,4,di-O-methyl mannose and 3,6-di-O-methylglucosamine. These results when compared with data on N-glycans suggest the following structure for the carbohydrate moiety of Mu-INF-beta: (formula; see text).

Fahrettin Petek

Papers

Glycosidases induced in Aspergillus tamarii. Secreted alpha-D-galactosidase and beta-D-mannanase.

Glycosidases induced in Aspergillus tamarii. Mycelial alpha-D-galactosidases.

The POU transcription factor Oct-1 represses virus-induced interferon A gene expression.

Purification and carbohydrate structure of natural murine interferon-beta.

Purification et propriétés de deux (1 → 4)-β-d-glucosidases d'Aspergillus roseus