Marianne Hayn

Production of Trichoderma cellulase in laboratory and pilot scale

Abstract Trichoderma reesei mutants QM 9414, MCG 77, MCG 80, RUT C 30, CL-847, VTT-D, and SVG are the preferred microorganism for the production of a complete cellulase which can hydrolyze crystalline cellulose. All these strains secrete cellulolytic proteins (exocellulases, endocellulases, and cellobiase) when they are grown on a mineral medium supplemented with pure cellulose (Solka Floc, cotton, Avicel) or pretreated lignocellulose (wood, straw). Some of the mutants produce cellulase with lactose as the sole carbon source. Submersed shake culture fermentations were mainly used for strain selection and preliminary optimization of culture conditions. Laboratory fermentations were performed in stirred tank reactors with volumes up to 3000 liters. On average, 250 mg of cellulase protein is formed per gram of metabolized carbon source, the enzyme has a specific activity of about 0·5 to 1·0 filter paper units per mg protein. The volumetric productivity rate is in the range of 50–150 FPU liter h −1 . The enzyme concentration strongly depends on the substrate concentration and increases from 2–3 FPU ml −1 at 2% to 7–18 FPU ml −1 at 8%. High enzyme concentrations and volumetric rates are obtained by fed batch fermentations. If compared under identical conditions, all T. reesei mutants developed after 1978 show approximately the same capacity to produce cellulase. The very great differences reported in the literature result from modifications of the fermentation parameters and difficulties in objectively measuring the enzyme concentration FPU ml −1 . On an industrial scale, Trichoderma cellulases are produced by solid state as well as by submersed fermentation. A list of commercially available cellulases and their potential application is given. If compared on a protein base (mg cellulase protein g −1 cellulose), the different cellulases show very different capacities for hydrolyzing cellulose.

Purification and characterization of two extracellular β-glucosidases from Trichoderma reesei

A major beta-glucosidase I and a minor beta-glucosidase II were purified from culture filtrates of the fungus Trichoderma reesei grown on wheat straw. The enzymes were purified using CM-Sepharose CL-6B cation-exchange and DEAE Bio-Gel A anion-exchange chromatography steps, followed by Sephadex G-75 gel filtration. The isolated enzymes were homogeneous in SDS-polyacrylamide gel electrophoresis and isoelectric focusing. beta-Glucosidase I (71 kDa) was isoelectric at pH 8.7 and contained 0.12% carbohydrate; beta-glucosidase II (114 kDa) was isoelectric at pH 4.8 and contained 9.0% carbohydrate. Both enzymes catalyzed the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside (pNPG). The Km and kcat/Km values for cellobiose were 2.10 mM, 2.45.10(4) s-1 M-1 (beta-glucosidase I) and 11.1 mM, 1.68.10(3) s-1 M-1 (beta-glucosidase II). With pNPG as substrate the Km and kcat/Km values were 182 microM, 7.93.10(5) s-1 M-1 (beta-glucosidase I) and 135 microM, 1.02.10(6) s-1 M-1 (beta-glucosidase II). The temperature optimum was 65-70 degrees C for beta-glucosidase I and 60 degrees C for beta-glucosidase II, the pH optimum was 4.6 and 4.0, respectively. Several inhibitors were tested for their action on both enzymes. beta-Glucosidase I and II were competitively inhibited by desoxynojirimycin, gluconolactone and glucose.

Molecular Cloning of the Full-length cDNA of (S)-Hydroxynitrile Lyase from Hevea brasiliensis FUNCTIONAL EXPRESSION IN ESCHERICHIA COLI AND SACCHAROMYCES CEREVISIAE AND IDENTIFICATION OF AN ACTIVE SITE RESIDUE

The full-length cDNA of (S)-hydroxynitrile lyase (Hnl) from leaves of Hevea brasiliensis (tropical rubber tree) was cloned by an immunoscreening and sequenced. Hnl from H. brasiliensis is involved in the biodegradation of cyanogenic glycosides and also catalyzes the stereospecific synthesis of aliphatic, aromatic, and heterocyclic cyanohydrins, which are important as precursors for pharmaceutical compounds. The open reading frame identified in a 1.1-kilobase cDNA fragment codes for a protein of 257 amino acids with a predicted molecular mass of 29.2 kDa. The derived protein sequence is closely related to the (S)-hydroxynitrile lyase from Manihot esculenta (Cassava) and also shows significant homology to two proteins of Oryza sativa with as yet unknown enzymatic function. The H. brasiliensis protein was expressed in Escherichia coli and Saccharomyces cerevisiae and isolated in an active form from the respective soluble fractions. Replacement of cysteine 81 by serine drastically reduced activity of the heterologous enzyme, suggesting a role for this amino acid residue in the catalytic action of Hnl.

Risk factors for microangiopathy-related cerebral damage in the Austrian stroke prevention study

Microangiopathy-related cerebral damage (MARCD) represents a common incidental MRI observation in the elderly. The risk factors of such findings are widely unknown. We therefore performed MRI in 349 randomly selected volunteers (ages 50 to 70 years) without neuropsychiatric disease, and evaluated the association of MARCD with conventional and recently suggested cerebrovascular risk factors such as apolipoprotein E genotypes, plasma concentrations of essential antioxidants and anticardiolipin antibody titres. MARCD was defined as evidence of early confluent and confluent deep white matter hyperintensities and lacunes. It was present in 71 (20.3%) subjects. Individuals with MARCD were older than those without such findings (62.7 years vs 59.6 years; P=0.0001). They had a higher rate of arterial hypertension (45.1% vs 28.1%; P=0.006) and cardiac disease (50.7% vs 37.1%; P=0.04), higher systolic blood pressure readings at exam (144.4 mmHg vs 136.7 mmHg; P=0.004), and higher serum fibrinogen concentrations (327.1 mg/dl vs 292.5 mg/dl; P=0.001). Their levels of total cholesterol (217.6 mg/dl vs 231.2; P=0.009), apolipoprotein A-I (167.3 mg/dl vs 177.4 mg/dl, P=0.02), lycopene (0.17 micromol/l vs 0.24 micromol/l; P=0.003), retinol (1.91 micromol/l vs 2.10 micromol/l; P=0.02) and alpha-tocopherol (27.55 micromol/l vs 31.14 micromol/l; P=0.001) were significantly lower. Forward stepwise regression analysis created a model of independent predictors of MARCD with age entering first (odds ratio 2.01/10 years), fibrinogen second (odds ratio 2.45/100 mg/dl), alpha-tocopherol third (odds ratio 0.55/10 micromol/l), and arterial hypertension fourth (odds ratio 1.96). The association of MARCD with various treatable clinical conditions may have preventive implications.

Cloning and characterization of the gene for the thermostable xylanase XynA from Thermomyces lanuginosus

A thermostable xylanase from the filamentous fungus Thermomyces lanuginosus (DSM 5826) was purified. This enzyme has an apparent molecular weight of 24-26 kDa as determined by SDS polyacrylamide gel electrophoresis. cDNA and genomic DNA fragments coding for this enzyme were cloned and sequenced. The cDNA contains an open reading frame encoding a polypeptide of 225 amino acids and was functionally expressed in E. coli as a LacZ fusion protein. Comparison of the cDNA sequence with the genomic DNA sequence showed that the xylanase was encoded by two exons interrupted by an intron of 106 bp. Comparison of the deduced amino acid sequence to other published xylanases revealed high homology to xylanases of the family G glycanases.

A rapid assay for catechol oxidase and laccase using 2-nitro-5-thiobenzoic acid

Abstract The reaction between 4-methyl-1,2-benzoquinone or p -benzoquinone with 2-nitro-5-thiobenzoic acid anion (TNB) consumes 1 mol of quinone per mol of thiol yielding almost colorless Michael-type adducts. Based on this reaction a convenient and sensitive assay for polyphenol oxidases was developed. The method depends on following spectrophotometrically (λ = 412 nm) the decrease in the absorbance of the yellow-colored TNB due to a coupled reaction with the quinones generated by enzymatic oxidation of 4-methylcatechol (catechol oxidase) or 1,4-dihydroxybenzene (laccase). Catechol oxidase activity was measured in enzyme preparations from potato, muchroom, apple, quince, and spinach; laccase was assayed in preparations from banana, spruce needles, and Rhus atomaria . For both enzymes the absorbance at 412 nm decreased linearly with time over the major part of the reaction. The relation between rate and enzyme concentration was linear for all catechol oxidases. With laccases, however, a linear dependence was observed only for low concentrations. A comparison of the TNB assay with other presently used assays (O 2 electrode, spectrophotometric, and chronometric methods) showed that the different tests give results for enzyme activity which are in good agreement with each other. Based on oxygen consumption measured by the O 2 electrode method and quinone production measured with the TNB test, it was calculated that at the very initial stage of the reaction the various enzymes consume approximately 0.5 ml of O 2 per mol of quinone formed.

Determination of fatty acids in the main lipoprotein classes by capillary gas chromatography : BF3/methanol transesterification of lyophilized samples instead of folch extraction gives higher yields

The amount of individual fatty acids contained in the main human lipoproteins VLDL, LDL, lipoprotein (a), HDL2, and HDL3 were determined by two different methods. In Method I, the lipids were first extracted by the classical Folch procedure and then transesterified with BF3/methanol and separated by capillary GC. In Method II the lipoprotein solution was freeze dried prior to transesterification with BF3/methanol. In all lipoproteins except VLDL significantly more fatty acids were found with Method II as compared to Method I. For total fatty acids the increase was up to 17.5%, for polyunsaturated fatty acids up to 24.5%. The total fatty acid content determined by Method II resembled closely the content independently derived from the enzymatically determined lipid composition. The results indicate that in case of lipoproteins quantification of fatty acids should be made with freeze-dried samples rather than with Folch extracts.

Relationships among Oxidation of Low-Density Lipoprotein, Antioxidant Protection, and Atherosclerosis

Publisher Summary Cardiovascular diseases (CVD) are the main cause of death in most developed countries. CVD is a global term for a wide range of diseases. According to the World Health Organization (WHO) classification system ICD-9 and ICD-10, they are divided into 69 subgroups. The underlying primary cause of most cardiovascular diseases is believed to be atherosclerosis, a progressive multifactorial disease of the artery wall. The lipid hypothesis suggests that lipid deposits are formed in the subendothelial space because of increased plasma low-density lipoprotein (LDL), endothelial dysfunction and permeability, and an imbalance of cholesterol influx and efflux in the early stages of atherosclerosis. The development of early lipid deposits, fatty streaks, and advanced plaques is a slow process involving a complex interplay of many factors. The most important risk factors that influence atherosclerosis are dyslipidemia, hypertension, diabetes, and smoking. A decrease of oxidative stress and antioxidant protection of LDL might therefore be a strategy with great promise for the prevention of atherosclerosis-associated cardiovascular disease. Evidence shows that an improvement of the antioxidant status by simply eating antioxidant-rich food/vegetables and fruits more frequently could significantly reduce the risk of cancer. This chapter describes the LDL oxidation hypothesis, the possible influence of oxidized LDL (oxLDL) and antioxidants on atherogenesis, and the assessment and relevance of in vitro-measured LDL oxidation indices. There is also a summary of epidemiological studies on relationships of antioxidant consumption and blood antioxidant concentration with cardiovascular disease. The oxidative stress posed on LDL in assays employing 1 μM Cu2+ or more is relatively strong and causes complete oxidation within several hours. Epidemiological studies on the relationship between dietary antioxidant intake and plasma concentrations of antioxidants and cardiovascular disease also support that low levels of antioxidants, particularly vitamin E, are associated with an increased incidence of atherosclerosis.

Purification and properties of an acidic α-mannanase from Sclerotium rolfsii

The plant pathogen basidiomycete Sclerotium rolfsii secretes five different β-d-mannanases into the growth medium when cultivated in the presence of Amorphophallus konjak glucomannan as an inducer. Out of these enzymes an acidic endo-1,4-β-mannanase was purified to electrophoretic homogeneity by means of ultrafiltration, anion exchange chromatography and gel filtration. This enzyme has a molecular mass of 61.2 kDa and the pI value is in the acidic region at 3.5. The pH and temperature optima are 2.9 and 74 °C, respectively. The mannanase is stable at low pH values and retains 50% of its activity after 4 h incubation at pH 3.0 and 50 °C. In addition to β-1,4-linked mannans the mannanase of S. rolfsii also hydrolyzes glucomannans, galactomannans and manno-oligosaccharides not smaller than mannotetraose, but the enzyme does not show any mannosidase, xylanase, or endoglucanase activities. During the hydrolysis of mannotetraose in the presence of mannose, the monomer is transferred to other oligosaccharides, which has been proved by using 14C-labeled mannose.

Mannan-degrading enzymes from Sclerotium rolfsii: Characterisation and synergism of two endo β-mannanases and a β-mannosidase

Abstract For the degradation of α-mannans and β-mannans Sclerotium rolfsii produces multi-enzyme systems. When cultivated in the presence of glucomannan this fungus secrets at least two β-mannosidases (EC 3.2.1.25) and five β-mannanases (EC 3.2.1.78). The degradation of different mannans by two purified β-mannanases (61.2 kDa, pI 3.5; 41.9 kDa, pI 3.2) and a mannosidase (57.5 kDa, pI 4.5) is discussed in this paper. All three enzymes were most stable at pH 4.5 and when they were incubated for 1 h at this pH and 65°C they retained about 50% of their activity. Mannans, gluco- and galactomannans were completely hydrolysed by the 58 kDa β-mannosidase, which only liberated monomers from the mannans. The activity of this enzyme was enhanced by addition of either one of the β-mannanases. The two β-mannanases randomly cleaved fragments larger than mannobiose from the mannans. The K M values of the β-mannanases showed that galactose substituents of galactomannans seem to be required for the enzyme substrate binding, whilst acetyl groups inhibited both enzymes.