N. G. Karanth

Production of fungal rennet by Mucor miehei using solid state fermentation

SummaryInvestigations have been carried out on the production of fungal rennet using a thermophilic strain ofMucor miehei under solid state fermentation conditions. A high milk clotting enzyme activity (58000 Soxhlet units/g) was achieved when optimum conditions were used. Further, a high ratio of 6.6:1 between milk clotting and proteolytic activities for this enzyme was obtained. Cheese prepared using this enzyme was also found to be acceptable in organoleptic quality. Large scale production of the enzyme in trays using the optimum conditions gave milk-clotting enzyme activities comparable to those in flask experiments.

Preparation of stearoyl lactic acid ester catalyzed by lipases from Rhizomucor miehei and porcine pancreas optimization using response surface methodology

Abstract The esterification reaction between stearic acid and lactic acid using Rhizomucor miehei lipase and porcine pancreas lipase was optimized for maximum esterification using response surface methodology. The formation of the ester was found to depend on three parameters namely enzyme/substrate ratio, lactic acid (stearic acid) concentration and incubation period. The maximum esterification predicted by theoretical equations for both lipases matched well with the observed experimental values. In the case of R. miehei lipase, stearoyl lactic acid ester formation was found to increase with incubation period and lactic acid (stearic acid) concentrations with maximum esterification of 26.9% at an enzyme/substrate (E/S) ratio of 125u2009gu2009mol−1. In the case of porcine pancreas lipase, esterification showed a steady increase with increase in incubation period and lactic acid (stearic acid) concentration independent of the E/S ratios employed. In the case of PPL, a maximum esterification of 18.9% was observed at an E/S ratio of 25u2009gu2009mol−1 at a lactic acid (stearic acid) concentration of 0.09u2009M after an incubation period of 72u2009h.

Evidence that the glucoamylases and α‐amylase secreted by Aspergillus niger are proteolytically processed products of a precursor enzyme

A 125‐kDa starch hydrolysing enzyme of Aspergillus niger characterised by its ability to dextrinise and saccharify starch [Suresh et al. (1999) Appl. Microbiol. Biotechnol. 51, 673–675] was also found to possess activity towards raw starch. Segregation of these activities in the 71‐kDa glucoamylase and a 53‐kDa α‐amylase‐like enzyme supported by antibody cross‐reactivity studies and the isolation of mutants based on assay screens for the secretion of particular enzyme forms revealed the 125‐kDa starch hydrolysing enzyme as their precursor. N‐terminal sequence analysis further revealed that the 71‐kDa glucoamylase was the N‐terminal product of the precursor enzyme. Immunological cross reactivity of the 53‐kDa amylase with antibodies raised against the precursor enzyme but not with the 71‐ and 61‐kDa glucoamylase antibodies suggested that this enzyme activity is represented by the C‐terminal fragment of the precursor. The N‐terminal sequence of the 53‐kDa protein showed similarity to the reported Taka amylase of Aspergillus oryzae. Antibody cross‐reactivity to a 10‐kDa non‐enzymic peptide and a 61‐kDa glucoamylase described these proteins as products of the 71‐kDa glucoamylase. Identification of only the precursor starch hydrolysing enzyme in the protein extracts of fungal protoplasts suggested proteolytic processing in the cellular periplasmic space as the cause for the secretion of multiple forms of amylases by A. niger.

Characterisation of a starch-hydrolysing enzyme of Aspergillus niger

Abstract A UV-induced mutant strain of Aspergillus niger (CFTRI-1105-U9) overproduced a starch-hydrolysing enzyme with properties characteristically different from the known amylases of the fungus. The purified enzyme of 4.0 pI had an apparent molecular mass of 125u2009kDa and it dextrinised starch and then saccharified the dextrins. Patterns of the enzyme activity on starch, resulting in glucose at 60u2009°C and glucose, maltose and maltodextrins at 70u2009°C as primary products, suggested significant applications for the enzyme in starch-processing industries.

Study on the production of 6-pentyl-α-pyrone using two methods of fermentation

Abstractu2002The lactone 6-pentyl-α-pyrone has a characteristic coconut aroma and is produced by Trichoderma species. A study on the fermentative production of 6-pentyl-α-pyrone in both surface and submerged conditions by Trichoderma harzianum was carried out. Maximum concentrations of 455u2009mg/l and 167u2009mg/l after 96u2009h and 48u2009h of fermentation in surface and submerged conditions, respectively, were obtained without using any additional recovery operations. The resultant yields are higher than those previously reported in the literature, which may be attributable to strain characteristics in combination with the choice of fermentation conditions employed in the present study. Enough scope exists for further improvement in the yields by optimizing the cultural and nutritional parameters.

A lipoxygenase inhibitor from Aspergillus niger.

Abstract. A lipoxygenase-1 (LOX-1) inhibitor was isolated from the fermented broth of Aspergillus niger CFTRIxa01105. It was purified, using column and preparative thin layer chromatography. 1H NMR and GC-MS examination revealed the structure of the inhibitor to be 2-(2′-methyl, 4′-hydroxyphenyl), 2-(4′′hydroxyphenyl)-propane with a molecular weight ofxa0242 and the molecular formula C16H18O2. This bisphenol-derivative inhibitor shows 50% inhibition of soybean LOX-1 at 0.98xa0mM concentration. The activity of this inhibitor was compared with commercial bisphenol A and its structural analogues, butylhydroxyanisole and butylhydroxytoluene in an attempt to understand the role of functional groups affecting lipoxygenase activity.

High-cell-density fermentation of recombinant Saccharomyces cerevisiae using glycerol

To obtain a high cell density of recombinant Saccharomyces cerevisiae (INVSc 1 strain bearing a 2 μm plasmid, pYES2 containing a GAL1 promoter for expression of the β‐galactosidase gene), the yeast was grown with glycerol as the substrate by fed‐batch fermentation. The feeding strategy was based on an on‐line response of the medium pH to the consumption of glycerol. The approach was to feed excess carbon into the medium to create a benign environment for rapid biomass buildup. During cell growth in the presence of glycerol, the release of protons in the medium caused a decrease in pH and the consumption rate of ammonium phosphate served as an on‐line indicator for the metabolic rate of the organism. The extent of glycerol feeding in a fed‐batch mode with pH control at 5.0 ± 0.1 was ascertained from the automatic addition of ammonium phosphate to the medium. The glycerol feeding to ammonium phosphate addition ratio was found to be 2.5–3.0. On the basis of the experiments, a maximum dry cell biomass of 140 g per liter and a productivity of 5.5 g DCW/L/h were achieved. The high cell density of S. cerevisiae obtained with good plasmid stability suggested a simple and efficient fermentation protocol for recombinant protein production.

An enzyme-linked immunosorbent assay for the estimation of fungal biomass during solid-state fermentation

Abstract An enzyme-linked immunosorbent assay for sensitive, specific and quantitative estimation of fungal biomass during solid-state fermentation is described. Using this method, differential growth rates and colonization of the substrate can be studied. The assay has potential application for the efficient monitoring of solid-state fermentation involving specific fungus, for which available methods are not adequate.

Downstream processing of microbial rennet from solid state fermented moldy bran.

In recent years due to acute shortage of calf-rennet, microbial rennets seem to be an effective alternative and are commercially produced. Mucor miehei was cultivated under the solid state fermentation conditions, and the moldy bran was extracted using a semicontinuous multiple contact forced percolation method. The treated extract was then filtered through 5% R16 clay which enabled easy and efficient removal of impurities such as lipase and protease without involving costly chemical treatments. The ethyl alcohol precipitated enzyme was dried and made into powder form having activity of 1.5 x 10(5)Soxhlet units/gm.

Isomerization of non-insecticidal α-hexachlorocyclohexane (HCH) to insecticidal γ-HCH by Pseudomonas strain Ptm+

Pseudomonas strain Ptm+ grew on α-hexa-chlorocyclohexane (HCH, CAS no. 319846), using it as the sole source of carbon and energy. In a replacement-culture study, with the non-insecticidal α-HCH, γ-HCH (CAS no. 58899) was the first metabolite noticed at 6u2009h, and transient accumulation of insecticidal γ-HCH occurred for up to 18u2009h. Although delta- (CAS no. 319868) and beta-isomers (CAS no. 319857) were also detected, their concentrations were very low. By 18u2009h of incubation, about 23% of the α-HCH added was transformed into the gamma-isomer. Subsequently, the concentration of γ-HCH in the medium fell. Thin-layer chromatography, gas chromatography, gas chromatography-mass spectrometry and mosquito-larval bioassay analyses confirmed the formation of γ-HCH. This was associated with the formation of three more metabolites.