Isolation and characterization of extra- and intra-cellular metal proteinases produced in the spawn-running process ofHypsizygus marmoreus

Isolation and characterization of extra-(PE-1) and intra-cellular (PE-2) metal proteinases produced during the spawn-running process ofHypsizygus marmoreus were carried out. These enzymes were the most active toward Hammarsten casein at pH 7.0 (PE-1) and pH 6.5–7.5 (PE-2). The molecular weight and pl value of PE-1 were 29,500, 8.8 and those of PE-2 were 21,500, 8.4. Km values against the synthetic peptide substrate Z-Gly-l-Leu-NH₂ were 0.9×10⁻³M (PE-1) and 1.2×10⁻³M (PE-2). PE-1 was strongly inhibited by phosphoramidon, whereas PE-2 was weakly inhibited. These enzymes are considered to play an important role in providing nitrogenous substrates during fruit-body formation.     

High-level production of D-mannitol with membrane cell-recycle bioreactor

Ten heterofermentative lactic acid bacteria were compared in their ability to produce D-mannitol from D-fructose in a resting state. The best strain, Leuconostoc mesenteroides ATCC-9135, was examined in high cell density membrane cell-recycle cultures. High volumetric mannitol productivity (26.2 g l⁻¹ h⁻¹) and mannitol yield (97 mol%) were achieved. Using the same initial biomass, a stable high-level production of mannitol was maintained for 14 successive bioconversion batches. Applying response surface methodology, the temperature and pH were studied with respect to specific mannitol productivity and yield. Moreover, increasing the initial fructose concentration from 100 to 120 and 140 g l⁻¹ resulted in decreased productivities due to both substrate and end-product inhibition of the key enzyme, mannitol dehydrogenase (MDH). Nitrogen gas flushing of the bioconversion media was unnecessary, since it did not change the essential process parameters. Journal of Industrial Microbiology & Biotechnology (2002) 29, 44–49 doi:10.1038/sj.jim.7000262 Received 12 November 2001/ Accepted in revised form 30 March 2002     

Metabolic function of glycogen phosphorylase and trehalose phosphorylase in fruit-body formation ofFlammulina velutipes

Glycogen phosphorylase in the vegetative mycelium ofFlammulina velutipes converts glycogen to α-glucose 1-phosphate (G1P) in the colony during fruit-body development. Glycogen may contribute to the synthesis of trehalose as the starting material in the vegetative mycelium during the fruiting process of the colony, and the trehalose produced is translocated into the fruit-bodies as the main carbohydrate substrate for their development. Trehalose phosphorylase activity in the vegetative mycelium was at a relatively high level until fruit-body initiation, suggesting the turnover of this disaccharide during the vegetative stage of the colony development. Trehalose phosphorylase activity in the stipes showed a peak level at the early phase of fruit-body development, suggesting the continuing phosphorolysis of trehalose by this enzyme. The stipes also showed a high specific activity of phosphoglucomutase at a sufficient level to facilitate the conversion of G1P to α-glucose 6-phosphate (G6P). In the pilei a large amount of G1P remained until the growth of the fruit-bodies ceased. Trehalase activities in the stipes and pilei were at a very low level, and this enzyme may not contribute to the catabolism of trehalose in the fruit-body development.     

Cultivation characteristics ofIsaria japonica

Cultivation characteristics of fruit-body (synnema) formation ofIsaria japonica were examined using liquid and solid media in order to produce fruit-bodies on a large scale. Mycelia grew well at 18–28°C on PDA medium with an initial pH of 7.0. The formation of fruit-bodies ofI. japonica was induced by lowering temperature to below 20°C in PD liquid medium. In sawdust-rice bran basal medium mixed with pupal powder prepared from silkworms (Bombyx mori), the fresh weight of fruit-bodies increased with increasing content of pupal powder. The highest yields of fruit-bodies were obtained in carbon-rich barley grain medium supplemented with pupal powder. The fruit-bodies grown under CO₂ concentrations of 1,000 μl/L had coral-like, many-branched synnemata with numerous conidiospores, whereas those formed under high concentrations (9,000 μl/L) of CO₂ had unbranched and longer synnemata. High concentrations of CO₂ remarkably inhibited conidiospore formation on synnemata. Continuous high-intensity illumination at 2.93 W·m⁻² inhibited the elongation of synnemata, and low-intensity illumination at 0.088 W·m⁻² slightly inhibited the branching of synnemata. Fruit-bodies were produced on the pupa metamorphosed from living larvae ofAgrotis fucosa placed on the surface of a culture ofI. japonica incubated in sawdust-rice bran medium.     

Kinetic determination of tryptophan by using the B-Z oscillating chemical system

A simple and rapid method was devised for determination of tryptophan, based on the Belousov-Zhabotinskii (B-Z) oscillating chemical system. Changes in oscillating period and amplitude were linearly proportional to the negative logarithm of l-tryptophan concentration over the range of 6.44 × 10⁻⁷–2.55 × 10⁻⁴ M, with the regression coefficients of near unity and a lower detection limit of 6.5 × 10⁻⁸ M. d-tryptophan was also examined although it is rarely found in most biological fluids, and perhaps not at all in natural proteins. The change of period against to negative logarithm of d-tryptophan concentration over the range of 4.9 × 10⁻⁵–8.24 × 10⁻⁴ M is linear. Because the optimum conditions for determination of l- and d-tryptophan are not the same, a little amount of d-tryptophan does not affect the determination of l-tryptophan. Various influences were studied and a possible mechanism of perturbation to the B-Z oscillator by tryptophan was also discussed. Spectrophotometry and fluorescence spectrophotofluorimetry were used for comparision and confirmation of the results.     

Leaf bisection for the enzymatic isolation of mesophyll protoplasts fromSaintpaulia ionantha

We describe a rapid and efficient procedure for isolating leaf mesophyll protoplasts fromSaintpaulia ionantha (African violet). Because of the unusual surface properties of the leaf, efficient digestion required bisection of the lamina into abaxial and adaxial halves to expose the mesophyll. Enzymatic digestion occurred during a 3 h incubation with 2% (m/v) driselase in 0.50 M mannitol, yielding 2–3 × 10⁵ protoplasts g⁻¹ (f.m.), with 70% viability. Protoplasts ranged in size from 50 to 100μm, and were derived from mostly non-photosynthetic mesophyll parenchyma.     

In Vitro Culture of Rudimentary Embryos of Ilex paraguariensis: Responses to Exogenous Cytokinins

The effects of benzyladenine (BAP), kinetin (KIN), zeatin (ZEA), isopentenyladenine (2iP), and thidiazuron (TDZ) were studied on in vitro growth of rudimentary embryos of Ilex paraguariensis St. Hil. Heart stage zygotic embryos were removed from seeds of immature, light green fruits and cultured aseptically on quarter-strength Murashige and Skoog medium containing 3% sucrose, 0.65% agar, and supplemented with or without three concentrations of BAP, KIN, ZEA, 2iP, or TDZ. Cultures were incubated in darkness at 27 ± 2°C. Media containing 4.4 × 10⁻⁶ m BAP, 4.6 × 10⁻⁶ m KIN, or 4.9 × 10⁻⁶ m 2iP were totally ineffective in inducing embryo growth after culture for 28 days. However, lower concentrations of these compounds (4.4 × 10⁻⁸ m BAP, 4.6 × 10⁻⁸ m KIN, 4.5 × 10⁻⁸ m ZEA, or 4.9 × 10⁻⁸ m 2iP) promoted embryo growth. TDZ at 9.9 × 10⁻⁹ m, 9.9 × 10⁻⁸ m, or 9.9 × 10⁻⁷ m induced embryo growth at similar rates. The maximum percentage of embryos converted to seedlings was achieved when the medium was supplemented with 4.5 × 10⁻⁷ m ZEA. Received August 1, 1997; accepted February 19, 1998     

Biotechnological production of mannitol and its applications

Mannitol, a naturally occurring polyol (sugar alcohol), is widely used in the food, pharmaceutical, medical, and chemical industries. The production of mannitol by fermentation has become attractive because of the problems associated with its production chemically. A number of homo- and heterofermentative lactic acid bacteria (LAB), yeasts, and filamentous fungi are known to produce mannitol. In particular, several heterofermentative LAB are excellent producers of mannitol from fructose. These bacteria convert fructose to mannitol with 100% yields from a mixture of glucose and fructose (1:2). Glucose is converted to lactic acid and acetic acid, and fructose is converted to mannitol. The enzyme responsible for conversion of fructose to mannitol is NADPH- or NADH-dependent mannitol dehydrogenase (MDH). Fructose can also be converted to mannitol by using MDH in the presence of the cofactor NADPH or NADH. A two enzyme system can be used for cofactor regeneration with simultaneous conversion of two substrates into two products. Mannitol at 180 g l⁻¹ can be crystallized out from the fermentation broth by cooling crystallization. This paper reviews progress to date in the production of mannitol by fermentation and using enzyme technology, downstream processing, and applications of mannitol.     

Isolation of a new flavonol glycoside and its effects on the blue color of seed coats ofOphiopogon jaburan

From the blue seed coats ofOphiopogon jaburan, a new flavonol glycoside was isolated as needles and determined to be kaempferol 3-O-β-d-galactoside-4′-O-β-d-glucoside (OK-2) by UV and NMR spectral analyses. OK-2 and kaempfrol 3, 4′-di-O-β-d-glucoside (OK-1), which was detected previously, in the blue seed coat were present in a molar ratio of about 13:7. OK-2 was newly found as a factor causing the blueing effects on ophionin which is a main anthocyanin in the blue seed coats. The mixture of 4.8×10⁻³ M OK-2 and 2.5×10⁻³ M ophionin in Mcllvaine's buffer solution (pH 5.6) showed stable blue color, and the absorption spectrum of the mixture showed two absorption peaks and a shoulder in visible reasion, coinciding with that of the fresh blue seed coat. The effect of ophionin and OK-2 co-pigmentation on the blue color of seed coat ofO. jaburan was discussed.     

Comparative analysis of four <Emphasis Type="Italic">β</Emphasis>-galactosidases from <Emphasis Type="Italic">Bifidobacterium bifidum</Emphasis> NCIMB41171: purification and biochemical characterisation

Four different β-galactosidases (previously named BbgI, BbgII, BbgIII and BbgIV) from Bifidobacterium bifidum NCIMB41171 were overexpressed in Escherichia coli, purified to homogeneity and their biochemical properties and substrate preferences comparatively analysed. BbgI was forming a hexameric protein complex of 875 kDa, whereas BbgII, BbgIII and BbgIV were dimers with native molecular masses of 178, 351 and 248 kDa, respectively. BbgII was the only enzyme that preferred acidic conditions for optimal activity (pH 5.4–5.8), whereas the other three exhibited optima in more neutral pH ranges (pH 6.4–6.8). Na⁺ and/or K⁺ ions were prerequisite for BbgI and BbgIV activity in Bis–Tris-buffered solutions, whereas Mg⁺⁺ was strongly activating them in phosphate-buffered solutions. BbgII and BbgIII were slightly influenced from the presence or absence of cations, with Mg⁺⁺, Mn⁺⁺ and Ca⁺⁺ ions exerting the most positive effect. Determination of the specificity constants (k _cat/K _m) clearly indicated that BbgI (6.11 × 10⁴ s⁻¹ M⁻¹), BbgIII (2.36 × 10⁴ s⁻¹ M⁻¹) and especially BbgIV (4.01 × 10⁵ s⁻¹ M⁻¹) are highly specialised in the hydrolysis of lactose, whereas BbgII is more specific for β-d-(1→6) galactobiose (5.59 × 10⁴ s⁻¹ M⁻¹) than lactose (1.48 × 10³ s⁻¹ M⁻¹). Activity measurements towards other substrates (e.g. β-d-(1→6) galactobiose, β-d-(1→4) galactobiose, β-d-(1→4) galactosyllactose, N-acetyllactosamine, etc.) indicated that the β-galactosidases were complementary to each other by hydrolysing different substrates and thus contributing in a different way to the bacterial physiology.     

Uptake of amino acids by actidione-treated yeast cells

Uptake of glyeine,l-cysteine,l-leucine,l-methionine,l-aspartic acid andl-lysine was investigated in resting cells ofSaccharomyces cerevisiae treated with 0.3mm actidione for blocking protein synthesis. The amino acids were taken up against substantial concentration gradients (up to nearly 1,000∶1 for μm l-cysteine and glycine). They were present in the free form inside the cells. Their unidirectional transmembrane fluxes were under a negative feedback control by the intracellular concentration of the amino acid involved. The amino acids tested apparently employed more than one transport agéncies for their membrane passage, the half-saturation constants being 6.2–7.7×10⁻⁴ m for glycine, 2.5×10⁻⁴ m forl-cysteine, 6×10⁻⁵ and 4×10⁻⁴ m forl-lysine, 3×10⁻⁵ and 6×10⁻⁴ m forl-methionine, 7–18×10⁻⁵ and 1.6×10⁻³ m forl-aspartic acid and 6×10⁻⁵ and 2×10⁻³ m forl-leucine. The specificities of the transport systems are overlapping but there emerges a wide-affinity transport system for glycine, alanine, leucine, methionine, serine, cysteine, phenylalanine, aspartic acid, asparagine, glutamic acid and tryptophan (and possibly for other amino acids), and more specific systems for each of the following: glycine, lysine, methionine, histidine, arginine, and aspartic and glutamic acids. Proline had the peculiar effect of stimulating the transport of all the amino acids tested. The amino acids apparently interacted in the uptake not only by competition for the binding site but also by allotopic inhibition (e.g.l-cysteine) and possibly stimulation (l-proline). The initial rate of uptake of amino acids and their steady-state level of distribution were characterized by identical activation energies: 7.5 kcal/mole forl-lysine, 6.9 kcal/mole forl-aspartic acid, and 13.2 kcal/mole for glycine.     

Expression of glf Z.m. increases D-mannitol formation in whole cell biotransformation with resting cells of Corynebacterium glutamicum

A recombinant oxidation/reduction cycle for the conversion of D-fructose to D-mannitol was established in resting cells of Corynebacterium glutamicum. Whole cells were used as biocatalysts, supplied with 250 mM sodium formate and 500 mM D-fructose at pH 6.5. The mannitol dehydrogenase gene (mdh) from Leuconostoc pseudomesenteroides was overexpressed in strain C. glutamicum ATCC 13032. To ensure sufficient cofactor [nicotinamide adenine dinucleotide (reduced form, NADH)] supply, the fdh gene encoding formate dehydrogenase from Mycobacterium vaccae N10 was coexpressed. The recombinant C. glutamicum cells produced D-mannitol at a constant production rate of 0.22 g (g cdw)⁻¹ h⁻¹. Expression of the glucose/fructose facilitator gene glf from Zymomonas mobilis in C. glutamicum led to a 5.5-fold increased productivity of 1.25 g (g cdw)⁻¹ h⁻¹, yielding 87 g l⁻¹ D-mannitol from 93.7 g l⁻¹ D-fructose. Determination of intracellular NAD(H) concentration during biotransformation showed a constant NAD(H) pool size and a NADH/NAD⁺ ratio of approximately 1. In repetitive fed-batch biotransformation, 285 g l⁻¹ D-mannitol over a time period of 96 h with an average productivity of 1.0 g (g cdw)⁻¹ h⁻¹ was formed. These results show that C. glutamicum is a favorable biocatalyst for long-term biotransformation with resting cells. Dedicated to Prof. Hermann Sahm on the occasion of his 65th birthday.     

Study of the reactivity of quinohemoprotein alcohol dehydrogenase with heterocycle-pentacyanoferrate(III) complexes and the electron transfer path calculations

The reactivity of alcohol dehydrogenase IIG (ADH IIG) from Pseudomonas putida HK5 with new heterocycle-pentacyanoferrate(III) complexes and hexacyanoferrate(III) was determined at pH 7.2. The pentacyanoferrate(III) complexes contained imidazole, pyrazole, pyridine, their derivatives and 2-aminobenzothiazole as the sixth ligand. The largest reactivity of the complexes with ADH IIG was estimated for the complex containing pyridine. An apparent bimolecular constant (k _ox ) for this complex was 8.7 × 10⁵ M⁻¹s⁻¹. The lowest value of k _ox was estimated for the complex with benzotriazole (k _ox = 3.1 × 10⁴ M⁻¹s⁻¹). The investigation of the hexacyanoferrate(III) enzymatic reduction rate at different ionic strength gave a single negative charge of reduced ADH IIG. Docking calculations revealed two binding sites of the complexes in ADH-IIG structure. The first one is located at the entrance to the PQQ pocket, and the second is at the site of cytochrome domain. The calculations of electron transfer (ET) path indicated that the most effective ET takes place from heme to the complex docked at the entrance to the PQQ pocket. This shortest path is constructed of amino acids Ser607 and Cys606.     

Effect of an exo-polysaccharide from the culture broth of Hericium erinaceus on enhancement of growth and differentiation of rat adrenal nerve cells

It was found that an exo-biopolymer (M.W. 1,000,000, molar ratio of 1.5:1.7:1.2:0.6:0.9, glucose:galactose:xylose:mannose:fructose, purity 99%) purified from the liquid culture broth of Hericium erinaceus mycelium enhanced the growth of rat adrenal nerve cells. The polymer also improved the extension of the neurites of PC12 cell. Its efficacy was found to be higher than those from known nerve growth factors such as Nerve Growth Factor (NGF) and Brain-Derived Nerve Factor (BDNF). The effect of two standards has not been observed above 0.1 (mg l⁻¹) of supplementation; however, the polymer did show the effect of cell growth and neurite extension at up to 1.0 (mg l⁻¹) of addition. While the polymer improved both cell growth and neurite extension, NGF and BDNF did only outgrowth of the neurites. Maximum cell density and length of the neurites were observed as 1.5×10⁵ (viable cells ml⁻¹) and 230 μm, respectively in adding 0.8 (mg l⁻¹) of the biopolymer for 8 days cultivation. The control growth was observed only as 1.2×10⁵ (viable cell ml⁻¹) of maximum cell density and 140 μm of maximum length, respectively. It was also confirmed that the polymer reacted with the nerve cells within 30 min after adding the sample, compared to 80 min in adding two other growth factors. Number of neurite-bearing cells remained relatively steady in adding the polymer even when the cell growth started to be decreased. It was interesting that the polymer effectively delayed apoptosis of PC12 cells by dramatically reducing the ratio of apoptotic cells to 20% from 50% of the control. This revised version was published online in September 2006 with corrections to the Cover Date.     

Characterization of malate dehydrogenase from the hyperthermophilic archaeon <Emphasis Type="Italic">Pyrobaculum islandicum</Emphasis>

Native and recombinant malate dehydrogenase (MDH) was characterized from the hyperthermophilic, facultatively autotrophic archaeon Pyrobaculum islandicum. The enzyme is a homotetramer with a subunit mass of 33 kDa. The activity kinetics of the native and recombinant proteins are the same. The apparent K _m values of the recombinant protein for oxaloacetate (OAA) and NADH (at 80°C and pH 8.0) were 15 and 86 μM, respectively, with specific activity as high as 470 U mg⁻¹. Activity decreased more than 90% when NADPH was used. The catalytic efficiency of OAA reduction by P. islandicum MDH using NADH was significantly higher than that reported for any other archaeal MDH. Unlike other archaeal MDHs, specific activity of the P. islandicum MDH back-reaction also decreased more than 90% when malate and NAD⁺ were used as substrates and was not detected with NADP⁺. A phylogenetic tree of 31 archaeal MDHs shows that they fall into 5 distinct groups separated largely along taxonomic lines suggesting minimal lateral mdh transfer between Archaea.     

Kinetic study of mannitol production using cashew apple juice as substrate

The use of agriculture excess as substrate in industrial fermentations became an interesting alternative to reduce production costs and to reduce negative environmental impact caused by the disposal of these products. In this work, a kinetic study of mannitol production using cashew apple juice as substrate was studied. The carbohydrates of cashew apple juice are glucose and fructose. Sucrose addition favored the yield of mannitol (85%) at the expense of lower productivity. The best results were obtained applying only cashew apple juice as substrate, containing 50 g L⁻¹ of total reducing sugar (28 g L⁻¹ of fructose), yielding 18 g L⁻¹ of mannitol with 67% of fructose conversion into mannitol and productivity of 1.8 g L⁻¹ h⁻¹.     

Bioelectrical activity in the heart of the lugworm Arenicola marina

Standard microelectrode technique was used to study electrical activity of the isolated heart of the polychaete annelid, Arenicola marina. Typical pacemaker activity with slow diastolic depolarization was observed in all recordings. The average maximum diastolic potential (−58.4 ± 3.2 mV), the average amplitude of the action potential (28.7 ± 4.7 mV) and the average total duration of the action potential (2,434 ± 430 ms) were determined. There has been no gradient of automaticity observed in our studies, which suggests that all regions of the Arenicola heart could possess pacemaker functions. Acetylcholine (ACh) produced a concentration dependent (5 × 10⁻⁸–5 × 10⁻⁵ M) increase of the beating rate via increase in the rate of the diastolic depolarization. ACh (5 × 10⁻⁵ M) increased beating rate by 2.5-fold compared to the control rate. A stronger action of ACh resulted in depolarization, block of action potential generation and contracture of the heart. The non-hydrolysable ACh analog carbacholine (10⁻⁸–10⁻⁶ M) produced similar effects. All effects of ACh and carbacholine were abolished by 5 × 10⁻⁶ M atropine. d-Tubocurarine (5 × 10⁻⁵ M) did not significantly alter effects of ACh or carbacholine. Epinephrine (10⁻⁸–10⁻⁶ M) caused the slowing of pacemaker activity and marked decrease of action potential duration. 10⁻⁶ M epinephrine produced complete cardiac arrest. The effects of epinephrine were not significantly altered by the β-blocker propranolol (5 × 10⁻⁶ M). The β-agonist isoproterenol (10⁻⁷–10⁻⁵ M) and the α-agonist xylometazoline (10⁻⁶–10⁻⁵ M) did not produce significant effects. Thus, cholinergic effects in the Arenicola heart are likely to be mediated via muscarinic receptors, while the nature of adrenergic effects needs further investigation.     

Malic Dehydrogenase Heterogeneity in Malaria (Plasmodium lophurae and P. berghei)*

SYNOPSIS. Molecular heterogeneity of malic dehydrogenase (MDH) in malaria was shown by zone electrophoresis in potato starch, starch gel, and by enzymatic activity with analogs of the coenzyme diphosphopyridine nucletide. A single anodal peak of MDH characterized the normal duck red blood cell whereas P. lophurae free of the host cell had a cathodal form of the enzyme. Infected duck erythrocytes had a combination of these electrophoretic forms. The isolated enzymes had different pH optima with oxaloacetate as substrate: pH 7.4 for the duck red cell and 6.4 for the plasmodial enzyme. The Km of each enzyme for oxaloacetate varied with the pH. The Km at pH 7.4 was 4.1 and 4.4 × 10⁵ M for parasite and host, respectively, whereas at 6.4 it was 2.0 × 10⁵ M for P. lophurae and 6.3 × 10⁵M for the duck erythrocyte. At pH 7.4 both enzymes were inhibited by oxaloacetate concentrations greater than 10^?4 M. P. berghei MDH also had a different electrophoretic character from that of the mouse red blood cell. Quantitatively, MDH activity increased with parasitization, and erythrocyte-free P. lophurae contained approximately twice the activity found in the uninfected duck erythrocyte. The quantity of MDH activity of the infected cell was ca. 50% less than the sum of the activities of the parasite and the uninfected cell. It is suggested that these properties of the parasite MDH may give it a physiologic advantage over the red cell under the conditions which prevail intraerythrocytically.     

Effects of Lens Major Intrinsic Protein on Glycerol Permeability and Metabolism

Lens Major Intrinsic Protein (MIP) is a member of a family of membrane transport proteins including the Aquaporins and bacterial glycerol transporters. When expressed in Xenopus oocytes, MIP increased both glycerol permeability and the activity of glycerol kinase. Glycerol permeability (p _Gly ) was 2.3 ± 0.23 × 10⁻⁶ cm sec⁻¹ with MIP vs. 0.92 ± 0.086 × 10⁻⁶ cm sec⁻¹ in control oocytes. The p _Gly of MIP was independent of concentration from 5 × 10⁻⁵ to 5 × 10⁻² m, had a low temperature dependence, and was inhibited approximately 90%, 80% and 50% by 1.0 mm Hg⁺⁺, 0.2 mm DIDS (diisothiocyanodisulfonic stilbene), and 0.1 mm Cu⁺⁺, respectively. MIP-enhanced glycerol phosphorylation, resulting in increased incorporation of glycerol into lipids. This could arise from an increase in the total activity of glycerol kinase, or from an increase in its affinity for glycerol. Based on methods we present to distinguish these mechanisms, MIP increased the maximum rate of phosphorylation by glycerol kinase (0.12 ± 0.03 vs. 0.06 ± 0.01 pmol min⁻¹ cell⁻¹) without changing the binding of glycerol to the kinase (K _M ∼ 10 μm). Received: 23 May 1997/Revised: 4 August 1997     

Changes in low molecular weight carbohydrates composition and chitin throughout the cultivation stages ofPleurotus ostreatus

Changes in contents of soluble low molecular weight carbohydrates and chitin in a sawdust-rice bran medium during mycelial growth ofPleurotus ostreatus in bottle cultivation were examined in relation to fruit-body yield of nine stocks. Glucose, mannitol, inositol, sucrose, and trehalose were detected in cultures after mycelial spreading. No significant correlation was observed between contents of soluble low molecular weight carbohydrate during mycelial growth and the fruit-body yield. Negative correlation was found between trehalose content in post-harvest cultures and the fruit-body yield. Chitin content in cultures decreased in the fruiting stage. Positive correlation was detected between chitin content of fruit-bodies and the decrement of chitin in post-harvest culture caused by fruit-body growth.