B. C. Bhattacharyya

Microbial production of gallic acid by modified solid state fermentation

Bioconversion of tannin to gallic acid from powder of teri pod (Caesalpinia digyna) cover was achieved by the locally isolated fungus, Rhizopus oryzae, in a bioreactor with a perforated float for carrying solid substrate and induced inoculum. Modified Czapek-Dox medium, put beneath the perforated float, with 2% tannic acid at pH 4.5, temperature 32°C, 93% relative humidity, incubated for 3 days with 3-day-old inoculum was optimum for the synthesis of tannase vis-à-vis gallic acid production. Conversion of tannin to gallic acid was 90.9%. Diethyl ether was used as the solvent for extraction of gallic acid from the fermented biomass.

Optimization of tannase biosynthesis by a newly isolated Rhizopus oryzae

A strain isolated locally and identified as Rhizopus oryzae (RO, IIT KGP) was found to synthesise an extracellular enzyme, tanin acyl hydrolase, showing its degradability of tannic acid to gallic acid. For maximizing the enzyme secretion in the fermented broth, the influencing parameters were optimized in shake flask culture. Experiments showed that modified Czapek dox medium with 2% tannic acid, 1% glucose, 0.05% sodium nitrate incubated for 4 days with 2 days old inoculum was the optimum for the synthesis of tannase by Rhizopus oryzae (RO, IIT KGP). Maximum enzyme activity was found to be 6.12 U/ml.

Protease extraction in solid state fermentation of wheat bran by a local strain of Rhizopus oryzae and growth studies by the soft gel technique

Studies on the extraction of protease in the solid state fermentation of wheat bran by Rhizopus oryzae, were conducted. Using distilled water (DW) as extractant, the optimum volume was 5 ml:g bran. An extraction or soaking time o f2ha t 4°C was sufficient to extract out nearly 90% (52.5 U:g bran) of enzyme while complete extraction was attained in 10 h (58.7 U:g bran). With the optimum extractant volume of 5 ml DW:g bran, most of the enzyme was recovered in one extraction (60 U:g bran) while repeated extractions yielded very little enzyme (5 and 2.8 U:g bran in the second and the third extractions, respectively). An attempt has been made to explain these results with the leaching or dissolving equation. Among other studies, while extractant pH did not affect recovery, extraction with neutral potassium phosphate buffer resulted in increased yields compared to DW and was also affected by the molarity of the buffer, with the maximum increase (1.42 times) being effected by 0.6 M buffer. Another major study was the investigation of protease production in relation to growth as seen in ‘soft gel culture’. Growth in soft gel culture was almost linear although colony growth on malt extract agar was radial. Production did not appear to be associated with growth.

Effect of additives on the behavioural properties of tannin acyl hydrolase

Abstract The effect of metal ions on tannase activity was studied. One mM Mg +2 or Hg + activated tannase activity. Ba +2 , Ca +2 , Zn +2 , Hg +2 and Ag + inhibited tannase activity at 1.0 mM concentration and Fe +3 and Co +2 completely inhibited tannase activity. Ag + , Ba +2 , Zn +2 and Hg +2 competitively inhibited tannase activity. Among the anions studied 1 mM Br − or S 2 O 3 −2 enhanced tannase activity. Tween 40 and Tween 80 enhanced tannase activity whereas Tween 60 inhibited tannase activity. Sodium lauryl sulfate and Triton X-100 inhibited tannase activity. Urea stimulated tannase activity at a concentration of 1.5 M. Among the chelators chosen for the present study, 1 mM EDTA or 1,10- o -phenanthrolein inhibited tannase activity Dimethyl sulphoxide and β-mercaptoethanol inhibited tannase activity at 1 mM concentration whereas soybean extract inhibited tannase activity at concentrations varying from 0.05 to 1.0% (w/v). Among the nitrogen sources selected ammonium ferrous sulfate, ammonium sulfate, ammonium nitrate and ammonium chloride enhanced tannase activity at 0.1% (w/v) concentration.

Optimization of n variable biological experiments by evolutionary operation-factorial design technique

Since the traditional approach to optimization of biological systems based on the single variable search technique is incapable of detecting the true optimum when a number of chemicals are used together as inducers, the Evolutionary Operation (EVOP)-factorial design technique was applied successfully to optimize the concentrations of vitamin (biotin), metal ion (CaCl(2)) and plant hormone (NAA) for maximizing enzyme (protease) production by Rhizopus oryzae (RO IIT RB-13, NRRL-21498) in solid state fermentation. Addition of inducers to an optimal level (biotin-2.5 ppm, CaCl(2)-20 ppm and NAA-12.5 ppm) resulted in a approximately 1.9-fold increase in protease production.

Production of tannase by solid-state fermentation

An attempt has been made to optimize the production of enzyme tannase by solid state fermentation (SSF) using the organism Rhizopus oryzae. The best favourable conditions for enzyme production include initial pH 5 with 4 days of incubation period at 40°C and 72% humidity, and 10 g wheat bran soaked in 2.5% tannic acid.

Use of low-cost gelling agents and support matrices for industrial scale plant tissue culture

The efficacies of sago (from Metroxylon sagu Rottb.) and isubgol (from Plantago ovata Forsk.) as gelling agents and those of filter paper, nylon cloth, polystyrene foam and glass wool cloth as support matrices have been tested for the propagation of plantlets of chrysanthemum (Dendranthema grandiflora Tzvelev). The performances of these low-cost gelling agents and matrices were found satisfactory and could compare well with that of agar. Glass wool cloth was, however, found to be the best matrix. Comparative cost estimations of the matrices and gelling agents have been presented and their merits and/or demerits have been discussed. For a given quantity of a medium, sago and isubgol cost about 1/18th and 1/10th respectively, compared to agar (e.g. Sigma, purified agar, No. A. 7921). The corresponding costs of the matrices are also less than that of agar. The results showed the potential of the substitutes for economic commercial application, replacing the costliest, though not indispensable, gelling agent agar.

Rapid mass propagation of Chrysanthemum morifolium by callus derived from stem and leaf explants

A procedure for rapid multiplication of Chrysanthemum morifolium RAMAT cv. Birbal Sahni using leaf callus and stem (nodal/internodal) callus as well as node and apical shoots has been developed. Murashige and Skoogs medium (1962) supplemented with 2mg/1 2,4-D yielded good green calli from both leaf and stem segments within 2 weeks. About 1 cm × 1 cm callus regenerated 2–3 shoots after 3 weeks on MS solid medium supplemented with 0.1 mg/l IAA and 0.2 mg/l BAP. Each of the regenerated shoots when transferred to the same shooting medium without agar yielded about 150 new shoots, which in turn regenerated roots after another week in MS half strength or modified Whites media (Rangaswamy, 1961). It has been estimated that about 1014 plantlets could be produced in a year from one expiant following the proposed protocol.

Extracellular alkaline protease of newly isolatedRhizopus oryzae

SummaryA fungal isolate identified asRhizopus oryzae, produces an extracellular alkaline serine protease. Maximum protease formation was after six days in shake flask culture at two different conditions of pH and temperature optimum (pH 5 at 30°C and pH 10 at 37°C). AgNO3 and Tween 80 increased protease synthesis. The enzyme is stable between pH 3 and pH 11 and has a temperature optimum of 60°C.

Purification of alkaline protease of Rhizopus oryzae by foam fractionation

Studies on the cost effective purification method is very much essential for the industrially important enzyme like protease. As foam fractionation is an important technique in downstream processing of the biologicals, purification of the crude proteolytic enzyme produced by Rhizopus oryzae has been attempted using converging-diverging foam fractionator. The effects of different parameters studied on the purification efficiency by foam fractionation were pH of the broth, air flow rate, initial liquid height. The results obtained were found quite encouraging at pH 10 with 1 LPM of air flow rate at low initial liquid height.