Showing papers by "Ashok Pandey published in 2005"

Microbial cellulases ─ Production, applications and challenges

Abstract: Microbial cellulases find applications in various industries and constitute a major group of the industrial enzymes. Recently, there is resurgence in utilization of biomass for fuel production employing cellulases and hence forth in obtaining better yields and novel activities. Improving the economics of such processes will involve cost reduction in cellulase production which may be achieved by better bioprocesses and genetic improvement of cellulase producers to yield more of the enzyme. The review discusses the current knowledge on cellulase production by microorganisms and the genetic controls exercised on it. It discusses the industrial applications of cellulases and the challenges in cellulase research especially in the direction of improving the process economics of enzyme production.

Biopigments from Monascus: strains selection, citrinin production and color stability

Abstract: Fungi form the genus Monascus are a promising source for natural color additives. However, before effectively applying Monascus to foods, it is important to select strains which produce large amounts of biopigments but little or no citrinin, a mycotoxin usually also produced by these fungi. Also, color stability of these pigments should be properly investigated. In order to compare Monascus strains for biopigment production in solid substrate fermentation (SSF), 4 strains (NRRL 1991, NRRL 2897, CCT 3802 and LPB 31) were cultivated over PDA in Petri dishes, and compared for radial growth velocity. Also, these strains were cultivated over cooked rice, and compared in relation to their capacity to produce biopigments and citrinin. The results showed that the strain LPB 31 is the best strain for biopigment production in SSF, giving both higher pigment concentration and lower citrinin concentration on the extracts, showing that it is a promising strain for production of this bioproduct. Biopigmentassays for heat and pH stability, show that these biopigments are unstable at low pH and high temperatures, but may be successfully used at near-neutrality pH's and in non-thermal processed foods.

Purification and Characterization of Tannin Acyl Hydrolase from Aspergillus niger ATCC 16620

Abstract: Summary Tannin acyl hydrolase produced extracellularly by the fungal strain Aspergillus niger ATTC 16620 in solid state fermentation was purified from the cell free culture broth by ammonium sulphate fractionation followed by DEAE–Sephadex A-50 chromatography. SDS-PAGE analysis indicated that the enzyme protein molecular mass was 168 kDa. Enzyme activity was stable up to the temperature of 40 °C and the enzyme activity was optimal at pH=6. Tannase activity was maximal at 0.01 M concentration of the substrate. The addition of metal ions like Zn 2+ ,M n 2+ ,C u 2+ ,C a 2+ ,M g 2+ and Fe 2+ inhibited the enzyme activity. Only K + ions enhanced tannase activity, and an activity of 4.31 U/mL was reported here. Enzyme activity was maximal after 15–20 min of incubation time, with an activity of 3.9 U/mL. Km was found to be 1.03 mM and Vmax=4.25 mmol/min. Since the enzyme is active over a wide range of pH and temperature it could find potential use in the food-processing industry.

Production and Partial Purification of a Neutral Metalloprotease by Fungal Mixed Substrate Fermentation

Abstract: Summary Five strains of fungi belonging to Aspergillus sp. were evaluated by casein agar plate assay and a wheat bran-based solid-state fermentation for selecting a neutral protease-producing culture. Based on the results, A. oryzae NRRL 2217 was selected for further studies. Sixteen different agro-industrial residues were evaluated for their potential to serve as a substrate for neutral protease production by this fungal strain. Results showed that a combination of coconut oil cake and wheat bran in the mass ratio of 1:3 was the best substrate for enzyme production. Various process parameters influencing protease production including fermentation time, initial moisture content, and fermentation temperature were optimised. The medium was supplemented with different nutrients in the form of organic and inorganic nitrogen and carbon sources. Supplementation of chitin increased the enzyme production significantly. Ammonium nitrate as inorganic nitrogen supplement slightly enhanced enzyme production. No organic nitrogen supplement was effective enhancer of enzyme production. Fermentation was performed under optimised conditions (initial moisture content V/m = 50 %, temperature 30 °C, 48 h). Partial purification of the enzyme resulted in a 3-fold increase in the specific activity of the enzyme. The partially purified enzyme was characterised by various features that govern the enzyme activity such as assay temperature, assay pH and substrate concentration. The effect of various metal ions and known protease inhibitors on the enzyme activity was also studied. The enzyme was found to be stable in pH range 7.0–7.5, and at temperature of 50 °C for 35 min. By the activating effect of divalent cations (Mg 2+ ,C a 2+ ,F e 2+ ) and inhibiting effect of certain chelating agents (EGTA, EDTA), the enzyme was found to be a metalloprotease.

L(+)-lactic acid production using Lactobacillus casei in solid-state fermentation.

Abstract: Lactobacillus casei was grown at 37 degrees C on sugarcane bagasse (5 g) soaked with cassava starch hydrolysate (final moistening volume 34 ml) containing 3 g reducing sugar in a solid-state condition. The maximum yield of L-lactic acid after various process optimisations was 2.9 g/5 g initial substrate corresponding to 97% conversion of sugar to lactic acid with initial substrate moisture of 72%.

l-leucine aminopeptidase production by filamentous Aspergillus fungi.

Abstract: K.M. NAMPOOTHIRI, V. NAGY, K. KOVACS, G. SZAKACS AND A. PANDEY. 2005. Aims: To screen various filamentous fungi belonging to Aspergillus spp. producing leucine and methionine aminopeptidases. Methods and Results: Twenty-eight Aspergillus strains representing 14 species within the genus were screened for L-leucine aminopeptidase (LAP) production in two media in shake flask fermentation. Two Aspergillus sojae (NRRL 1988 and NRRL 6271) and one Aspergillus oryzae (NRRL 6270) strains were selected as the best producers for further studies. The peak LAP activities were 2AE61, 2AE59 and 1AE30 IU ml )1 for the three fungi on days 2, 5 and 4 respectively. In addition to LAP, L-methionine aminopeptidase (MAP) activity was also detected. Apart from submerged fermentation, the highest LAP yields by solid-state fermentation were 11AE39, 17AE40 and 13AE02 IU g )1 dry matter for the above fungi. The temperature and pH optimum of the enzyme was found to be in the range of 65‐75� Ca t pH 8AE0‐9AE0 for all three fungi. Metal ions, Co 2+ and Fe 2+ in 2 mmol l )1 concentration apparently enhanced the relative enzyme activity and heat stability. Conclusions: Two A. sojae (NRRL 1988 and NRRL 6271) and one A. oryzae (NRRL 6270) strains were found to be the best producers of LAP and MAP. The preliminary characterization studies revealed that the enzyme is considerably thermostable and belongs to the class metalloenzymes. Significance and Impact of the Study: A good number of aspergilli were screened and the ability of the fungal aminopeptidase to release a particular N-terminal amino acid along with its high thermal stability, makes them interesting for controlling the degree of hydrolysis and flavour development for a wide range of substrate.

Partial purification and characterization of α-amylase produced by Aspergillus oryzae using spent-brewing grains

Abstract: Solid-state fermentation (SSF) was carried out to produce α-amylase from Aspergillus oryzae (IFO 30103) using spent brewing grains (SBG) as substrate. A maximum of 11296 U/gds amylase activity was obtained after 48 h of fermentation. The extracted enzyme was subjected to partial purification by ammonium sulphate fractionation. Maximum specific activity was obtained with 40-70% fraction. SDS-PAGE of the corresponding sample revealed an approximate 66 kDa band, which was confirmed by activity staining as α-amylase. It was optimally active at pH 5 and 50°C by using 1% starch as substrate concentration. The partially purified α-amylase loses activity rapidly above 50C but it can be retained in the presence of Ca. Presence of Mn and Fe enhanced the enzyme activity and is almost doubled in presence of Mn. However, in the presence of Hg and Cu the activity is reduced. Hg reduced the enzyme activity by half.

Microbial synthesis of chitinase in solid cultures and its potential as a biocontrol agent against phytopathogenic fungus Colletotrichum gloeosporioides

Abstract: Antifungal activity of chitinase can be effectively utilized in biologic pest control strategies. Because solid-state cultivation has been termed a cost-effective means for fungal growth and metabolite production, chitinase production by Trichoderma harzianum was studied using wheat bran-based solid medium containing 1% colloidal chitin. Chitinase synthesis was found to be growth associated because maximum enzyme (5.4 U/g of dry substrate) and biomass production occurred at 72h. Substrate moisture had a critical impact on chitinase production; five grams of medium having an initial moisture content of 68.4% when incubated for 72 h increased the enzyme yield to 9.3 U/g of dry substrate. Optimization of colloidal chitin concentration showed that improvements in chitinase yield and maximum activity were attained with a 2% (w/w) concentration. Supplementation of additional nitrogen sources also influenced enzyme production, and the best yield was obtained with yeast extract. The effect of crude chitinase on hyphal morphology of the phytopathogenic fungus Collelotrichum gloeosporioides was swelling as well as lysis of hyphal wall, depending on the age of the mycelium. Studies of pH and thermal stability showed that crude culture filtrate was active over pH 4.0–6.0 and retained about 48.2% activity after 40 min of incubation at 40°C.

Comparative study of amidase production by free and immobilized Escherichia coli cells.

Abstract: Escherichia coli NCIM 2569 was evaluated for its potential for amidase production under submerged fermentation. Among the various amide compounds screened, maximum substrate specificity and enzyme yield (8.1 U/mL) were obtained by using 1% acetamide. Fermentation was carried out at 30°C in shake-flask culture under optimized process conditions. A maximum of 0.52 U/mL of intracellular amidase activity was also obtained from cells incubated for 24 h. Studies were also performed to elucidate the optimal conditions (gel concentration, initial biomass, curing period of beads, and calcium ion concentration in the production medium) for immobilization of whole cells. By using E. coli cells entrapped in alginate, a maximum of 6.2 U/mL of enzyme activity was obtained after 12 h of incubation under optimized conditions. Using the immobilized cells, three repeated batches were carried out successfully, and 85% of the initial enzyme activity was retained in the second and third batches. The study indicated that the immobilized E. coli cells offered certain advantages such as less time for maximum enzyme production, more stability in the enzyme production rate, and repeated use of the biocatalyst.