Mina Mukherjee

Purification and characterization of a growth-regulating laccase from Pleurotus florida

Pleurotus florida produces two laccase enzymes (L1 and L2) within which L2 is associated with the vegetative growth of the fungus. In the present investigation the L2 has been purified to homogeneity and characterized. The molecular mass of the enzyme has been determined to be 73 kDa and 70 kDa by gel filtration chromatography and SDS‐PAGE, respectively. The purified enzyme shows a pI value of 4.2. The optimum reaction temperature is 50 °C. Spectroscopic analysis reveals that L2 has two copper atoms, a type I copper and a type II copper. The Km and some other kinetic parameters of L2 has been determined.

Laccase production in solid-state and submerged fermentation by Pleurotus ostreatus

A solid‐state fermentation (SSF) system for production of an industrially important enzyme laccase by Pleurotus ostreatus was developed by using potato dextrose yeast extract medium and polyurethane foam as a supporting material. The maximum laccase production in the SSF system was as high as 3×105 U/L. Addition of inducers, such as copper and ferulic acid, further enhanced the laccase production in SSF. Moreover, the time required for the maximum laccase production was reduced to 6 days compared to 10 days reported earlier. The improvement achieved by the SSF system was investigated by comparing it to a submerged fermentation system (SmF), both experimentally and by using a standard theoretical model along with a parameter sensitivity analysis. Laccase production in SSF was found to be twice of that in SmF. One of the main reasons for higher laccase production in SSF compared to SmF was possibly due to the presence of higher proteolytic activity in SmF. Strong proteolytic activity in SmF presumably caused subsequent laccase degradation, which lowered the ultimate laccase production in SmF compared to SSF.

Purification and characterization of laccase-1 from Pleurotus florida.

Pleurotus florida (ITCC 3308) produces two laccase enzymes (L1 and L2) in potato-dextrose media containing 0.5% yeast extract. Concentrated culture filtrate was separated on DEAE-Sephadex (A-50) column into two enzyme peaks, subsequently named L1 and L2. The L1 enzyme has been purified to homogeneity by ion-exchange and gel-permeation chromatography. L1 is a monomeric glycoprotein with a molar mass of 77 and 82 kDa as determined by SDS-PAGE and gelfiltration chromatography, respectively. The pI value of L1 has been determined to be 4.1. The optimum reaction temperature of the enzyme is 50°C. TheKm and some other kinetic parameters of L1 have been determined. Cyanide and azide completely inhibit the enzyme activity. The enzyme was fully active in 1: 1 (V/V) buffer-chloroform for at least 2 h. Spectroscopic analysis revealed that the enzyme has four copper atoms, a type 1 copper, a type 2 copper and a type 3 binuclear copper.

Accumulation of a Natural Substrate of Laccase in Gills of Pleurotus florida During Sporulation

During sporulation, laccase activity of Pleurotus florida decreased to a minimum level in spite of increase in the number of isozymes. An endogenous laccase substrate was detected especially in the gill structure of the sporophore, which competitively inhibited oxidation of guaiacol by the enzyme during in vitro assay. Appearance of the laccase substrate in the gill structure may be linked with the sporulation phenomenon.

Regulation of endo-1,4-β-glucanase secretion fromTermitomyces clypeatus by carbon catabolic product(s)

Secretion of CMCase byTermitomyces clypeatus was only observed in the presence of a gluconeogenic amino acid, a citrate-cycle acid, maleate, subinhibitory concentrations of glucosamine, or fluoride in the medium. The enzyme was not secreted in the presence of caffeine or IBMX or theophylline, and these phosphodiesterase inhibitors lowered the secretion of CMCase by glutamate. The presence of both glucosamine and glutamate in a cellulose medium were, however, antagonistic to CMCase secretion. In a growth medium, xylose and glucose were equivalent carbon source for the fungus while succinate was a poor source and strongly repressed growth at higher concentrations. Growth ofT. clypeatus was highly favored in media containing xylose/glucose with succinate/glutamate. During growth ofT. clypeatus in a glucose medium, the intracellular glucose level was stabilized by the presence of succinate, glutamate or glucosamine in the medium. All these observation suggested that a negative cellular regulation, mediated by carbon catabolic product(s), existed inT. clypeatus which regulated the secretion of CMCase. A transient but significant increase of intracellular cAMP and cGMP levels was observed at the onset of mycelial growth in glucose and glucose/maleate media, respectively.

Role of potato extract in extracellular laccase production of Pleurotus florida

Pleurotus florida produces extracellular laccase when grown on potato‐dextrose medium but not in the other minimal media containing only dextrose. When potato extract has been fractionated in G‐25 column two types of fractions were obtained with respect to iodine colouration. Fractions of the first peak (P1) gives blue colouration with iodine indicating the presence of starch whereas the fractions of the other region (P2) decolourise the iodine colour indicating presence of one or more reducing compound(s). The P2 fractions give red colour after reacting with diazonized sulphanilic acid suggesting phenolic nature of the compound(s). Addition of fractions of either P1 or P2 in the minimal media can not support the laccase production in P. florida but combination of the two fractions is sufficient to induce laccase production. Similarly minimal media containing starch from the other two sources can stimulate the laccase production in P. florida only in presence of P2 fraction. On the basis of these observation we propose that phenolic compound(s) present in potato extract is capable of inducing laccase synthesis in P. florida.

Study of laccase production by Pleurotus ostreatus in a 5 l bioreactor and application of the enzyme to determine the antioxidant concentration of human plasma

Aims:  To achieve high laccase production from Pleurotus ostreatus in a bench top bioreactor and to utilize the enzyme for determination of the total antioxidant concentration (TAC) of human plasma.

Preparation and regeneration of mycelial protoplasts ofPleurotus florida andP. ostreatus

A method for isolation of higher frequency of regenerated protoplast fromPleurotus florida andP. ostreatus is reported.

Carboxymethyl xylan-a specific substrate directly differentiating backbone-hydrolysing and side chain-reacting β-d-(1→4)-xylanases of the mushroom Termitomyces clypeatus

Abstract The mushroom Termitomyces clypeatus produces two endoxylanases (D) and (X) when grown in media containing dextrin and xylan as carbon sources, respectively. Endoxylanase (D) showed wide variation in its activity on different lots of xylan preparations, and its activity was found to be dependent upon the composition of xylans. The xylose-liberating endoxylanase (X) did not discriminate between different xylans. The activity of xylanase (D) was found to decrease as the proportion of xylose in different xylan preparations increased. The dialyzable oligosaccharides from the digestion of xylan by enzyme (D) contained constituent sugars of xylan, whereas xylose was the main constituent sugar of the undialyzable fraction. Enzyme (D) also could not liberate any reducing group from carboxymethyl xylan (CMX), a suitable substrate for viscometric and colorimetric assays of endolytic activity of xylanases. CMX was found to be modified preferentially at the substituent sugars of xylan rather than at backbone residues. Thus CMX proved to be a specific substrate for colorimetric assay of true endoxylanase activity.

Natural resistance of the mycelial culture of the mushroom,Panaeolus papillonaceus, towards growth inhibition by polyene antibiotics

The mycelial culture of the mushroomPanaeolus papillonaceus showed high tolerance (150 μg/ml) of polyene antibiotics (nystatin, amphotercinin B) present in the growth medium and protoplast of the fungus regenerated normally in the presence of the antibiotics. Both antibiotics inhibited growth of other mushroom strains at concentrations from 10 μg/ml to 20 μg/ml. Because polyene antibiotics interact with free membrane sterol of the sensitive fungi, the sterol present in the mycelia ofP. papillonaceus was studied. Extraction of sterol from the mushroomP. papillonaceus required primary treatment of the dried mycelia with alkali, and only ergosterol was identified as present as the extracted sterol. No sterol or sterol conjugate (fatty acid ester) could be extracted directly from the mycelia by petroleum ether, chloroform, or methanol without prior alkali treatment. Homogenization of the mycelia and subsequent treatment of the homogenate with detergent or chaotropic ions did not release any sterol conjugate in the aqueous phase. The unique nature of the sterol component present in the mycelia ofP. papillonaceus was indicated.