Characteristics and N-terminal amino acid sequence of a manganese peroxidase purified from Lentinula edodes cultures grown on a commercial wood substrate

Summary Extracellular culture filtrates from ligninolytic cultures of the lignin-degrading basidiomycete Lentinula (syn. Lentinus) edodes (Berk.) Pegler contained one major peroxidase when grown on a commercial oak-wood substrate. The peroxidase was purified by polyethylenimine clarification, anion-exchange chromatography, and hydrophobic-interaction HPLC. The enzyme (MnP1) was a heme-iron protein with an apparent molecular weight of 44 600 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and an isoelectric point of pH 3.2. The native enzyme had an absorption maximum at 407 nm, which shifted to 420 nm upon H₂O₂ addition. The pyridine-hemochrome-absorption spectrum indicated that one heme group was present per enzyme as protoporphyrin IX. N-terminal amino acid sequencing showed that MnP1 had higher sequence homology with manganese peroxidases than with lignin peroxidases reported from Phanerochaete chrysosporium. L. edodes MnP1 was capable of oxidizing lignin and lignin-model compounds in the presence of manganese and H₂O₂.On leave from the Department of Biochemistry, University of Otago, P. O. Box 56, Dunedin, New Zealand.Research carried out while a visiting scientist at the USDA Forest Products Laboratory from the National Chemistry Laboratory, Pune, India 41 1008 Offprint requests to: I. T. Forrester     

Occurrence of a procellulase in the culture filtrates of Penicillium janthinellum

The endo-1,4-β-d-glucanase [cellulase, 1,4-(1,3:1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] activity of two-day old culture filtrates of Penicillium janthinellum has been enhanced four-fold by incubating with a 10-day old culture filtrate of Penicillium funiculosum grown on the same medium. An inactive protein isolated by fractionation of two-day old culture filtrate of P. janthinellum using preparative isoelectric focusing, showed 30- to 50-fold enhancement of endo-1,4-β-d-glucanase activity. This fraction has been designated the ‘procellulase’ in the present paper. The purity of the procellulase was confirmed by analytical isoelectric focusing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. It had a molecular weight of 68 000 and an isoelectric point of pH 3.7.     

Effect of nitrogen, salt, and iron content in the growth medium and light intensity on lipid production by microalgae isolated from freshwater sources in Thailand

Four green microalgae (TRG, KB, SK, and PSU) identified as Botryococcus spp. by morphological criteria were isolated from lakes and freshwater ponds in southern Thailand. In nitrogen-rich medium the strains achieved a lipid content of 25.8%, 17.8%, 15.8% and 5.7%, respectively. A combination of nitrogen deficiency, moderately high light intensity (82.5 μE m(-2) s(-1)) and high level of iron (0.74 mM) improved lipid accumulation in TRG, KB, SK, and PSU strains up to 35.9%, 30.2%, 28.4% and 14.7%, respectively. The lipid contents and plant oil-like fatty acid composition of the microalgae suggested their potential as biodiesel feedstock.     

Production of xylanolytic enzymes in association with the cellulolytic activities of Penicillium funiculosum

Penicillium funiculosum produced 16 and 0.4 units ml^?1 of d-xylanase (1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) and β-d-xylosidase (1,4-β-d-xylan xylohydrolase, EC 3.2.1.37), respectively, in shake flasks. Both enzymes were 100% stable when heated at 50°C for 30 min and on prolonged heating d-xylanase and β-d-xylosidase showed 46 and 20% loss, respectively. Maximum hydrolysis (75%) of d-xylan was obtained when the end products were removed. The addition of β-d-xylosidase markedly influenced the degree of hydrolysis of d-xylan. End-product analysis of the d-xylan hydrolysate showed the presence of d-xylose, d-xylobiose, d-xylotriose, d-xylotetraose, d-xylopentose and l-arabinose. The fractionation of culture filtrate of Penicillium funiculosum grown on cellulose powder or in a combination of cellulose powder and wheat bran indicated the presence of two d-xylanases. The role of cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and d-xylanase on the overall hydrolysis of pure cellulose and lignocellulosic substrates is discussed.     

Immobilization of Penicillium funiculosum cellulase on a soluble polymer

The three cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] components of Penicillium funiculosum have been immobilized on a soluble, high molecular weight polymer, poly(vinyl alcohol), using carbodiimide. The immobilized enzyme retained over 90% of cellulase [1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4], and exo-β-d-glucanase [1,4-β-d-glucan cellobiohydrolase, EC 3.2.1.91] and β-d-glucosidase [β-d-glucoside glucohydrolase, EC 3.2.1.21] activities. The bound enzyme catalysed the hydrolysis of alkali-treated bagasse with a greater efficiency than the free cellulase. The potential for reuse of the immobilized system was studied using membrane filters and the system was found to be active for three cycles.     

Production of ethanol from wood and agricultural residues by Neurospora crassa

The feasibility of utilizing the rapidly growing tropical woods for ethanol production by Neurospora crassa has been studied. Hydrolysis of cold alkali pretreated wood gave a saccharification of 68% based on the available carbohydrate. The direct fermentation of pretreated wood (20 g l^?1) by Neurospora crassa gave quantitative conversion of available hemicellulose/cellulose to ethanol in 5 days. Increasing the substrate concentration to 50 g l^?1lowered the conversion to 40–60% yielding 12 g l^?1of ethanol. Fermentation of wood (50 g l^?1) pretreated with hot 1 m NaOH followed by neutralization with HCl gave only 6 g l^?1of ethanol.     

Properties and applications of Penicillium funiculosum cellulase immobilized on a soluble polymer

Summary The cellobiase and xylanase activities of Penicillium funiculosum were immobilized on a soluble polymer poly(vinyl alcohol) (PVA). The kinetic parameters and the adsorption characteristics of the bound and free enzymes were compared. The Km value of the immobilized preparation was the same as the free enzyme. The hydrolysis of different cellulosic substrates by the bound enzyme is investigated.     

Characterization of a major xylanase purified from Lentinula edodes cultures grown on a commercial solid lignocellulosic substrate

Summary The white-rot basidiomycete Lentinula (syn. Lentinus) edodes (Berk.) Pegler is the dominant edible mushroom cultivated on wood. The major xylanase detected in cultures grown on a commercial oak wood medium was extracted, purified, and characterized. The enzyme was a non-debranching endo--d-xylanase (1,4--d-xylan xylanohydrolase; E.C.3.2.1.8) highly specific for xylans, with a molecular weight of 41 000 (on sodium dodecyl sulfate gels) and an isoelectric point of 3.6. With aspen glucuronoxylan as substrate, the enzyme showed optimal activity at pH 4.5–5.0 and 60° C, with a K _m of 0.66 mg/ml and specific activity of 310 units/mg protein at 40° C. It was capable of hydrolyzing (forming reducing sugars from) 40%–50% of the hydrolyzable linkages in either glucuronoxylan or arabinoxylan. The enzyme produced xylose and major identifiable products in the xylobiose or xylotriose (and presumably larger) size range including xylobiose and xylooligosaccharides, but neither glucuronic acid nor arabinose. Products were also produced from arabinoxylan that appeared to be arabinoxylobiose and arabinoxylotriose.Research carried out while a visiting scientist from the National Chemistry Laboratory, Pune, India 411008The Forest Products Laboratory is maintained in cooperation with the University of Wisconsin. This article was written and prepared by US Government employees on official time, and it is therefore in the public domain and not subject to copyright Offprint requests to: G. F. Leatham     

Glucohydrolase from Penicillium funiculosum

Summary A 1,4--d-glucan glucohydrolase (EC 3.2.1.74) was isolated from culture filtrates of Penicillum funiculosum and purified by isoelectric focussing. The purified enzyme was homogeneous as indicated by electrophoresis on sodium dodecyl sulphate-polyacrylamide gels. The enzyme had a molecular weight of 20 000 and the pI was 4.45. The hydrolysis of Avicel by the purified enzyme and culture broth using equal amounts of Walseth units were comparable. The glucohydrolase did not act in synergism with endoglucanase or cellobiohydrolase from the same culture. The enzyme had little ability to attack carboxymethyl cellulose. It showed activity towards Avicel, Walseth cellulose and cellooligosaccharides (G₃-G₅), producing glucose as the end product, indicating that the enzyme is a -1–4 glucan glucohydrolase. The enzyme exhibited transglucosidase activity, producing higher oligosaccharides from cellobiose.NCL Communication no. 3899