nan Ikram-ul-Haq

Production of alpha amylase by Bacillus licheniformis using an economical medium.

The present study is concerned with the selection of new medium for the production of alpha amylase by Bacillus licheniformis. Different agricultural by-products such as wheat bran, sunflower meal, cotton seed meal, soybean meal, rice husk or rice bran were tested for the production of alpha amylase. Among different agricultural by-products evaluated, wheat bran was found to be the best basal and standardized medium for optimal production of alpha amylase. The production was increased 2-folds when soluble starch was replaced with pearl millet starch at 1% level and nutrient broth concentrations was reduced from 1% level to 0.5%. The newly selected fermentation medium containing (% w/v) wheat bran 1.25, nutrient broth 0.5, pearl millet starch 1.0, lactose 0.5, NaCl 0.5, CaCl2 0.2 in 100 ml of phosphate buffer. The kinetic values of Y(p/x), Y(p/s), and Q(p) indicated that the production of enzyme was greater in newly selected medium than the conventional more expensive medium.

Mutation of Aspergillus niger for hyperproduction of citric acid from black strap molasses

Spore suspensions of Aspergillus niger GCB 75, which produced 31.1 g/l citric acid from 15% sugars in molasses, were subjected to u.v.-induced mutagenesis. Among three variants, GCM 45 was found to be the best citric acid producer and was further improved by chemical mutagenesis using NTG. Out of 3 deoxy-D-glucose-resistant variants, GCM 7 was selected as the best mutant which produced 86.1 ± 1.5 g/l citric acid after 168 h of fermentation of potassium ferricyanide + H2SO4-pretreated black strap molasses (containing 150 g sugars/l) in Vogels medium. On the basis of comparison of kinetic parameters, namely the volumetric substrate uptake rate (Qs), and specific substrate uptake rate (qs), the volumetric productivity, theoretical yield and specific product formation rate, it was observed that the mutants were faster growing organisms and had the ability to overproduce citric acid.

High performance microbiological transformation of L-tyrosine to L-dopa by Yarrowia lipolytica NRRL-143

BackgroundThe 3,4-dihydroxy phenyl L-alanine (L-dopa) is a drug of choice for Parkinsons disease, controlling changes in energy metabolism enzymes of the myocardium following neurogenic injury. Aspergillus oryzae is commonly used for L-dopa production; however, potential improvements in ease of handling, growth rate and environmental impact have led to an interest in exploiting alternative yeasts. The two important elements required for L-dopa production are intracellular tyrosinases (thus pre-grown yeast cells are required for the transformation of L-tyrosine to L-dopa) and L-ascorbate, which acts as a reducing agent.ResultsPre-grown cells of Yarrowia lipolytica NRRL-143 were used for the microbiological transformation of L-tyrosine to L-dopa. Different diatomite concentrations (0.5–3.0 mg/ml) were added to the acidic (pH 3.5) reaction mixture. Maximum L-dopa biosynthesis (2.96 mg/ml L-dopa from 2.68 mg/ml L-tyrosine) was obtained when 2.0 mg/ml diatomite was added 15 min after the start of the reaction. After optimizing reaction time (30 min), and yeast cell concentration (2.5 mg/ml), an overall 12.5 fold higher L-dopa production rate was observed when compared to the control. Significant enhancements in Yp/s, Qs and qs over the control were observed.ConclusionDiatomite (2.0 mg/ml) addition 15 min after reaction commencement improved microbiological transformation of L-tyrosine to L-dopa (3.48 mg/ml; p ≤ 0.05) by Y. lipolytica NRRL-143. A 35% higher substrate conversion rate was achieved when compared to the control.

Biosynthesis of l-DOPA by Aspergillus oryzae

The present investigation deals with the biosynthesis of L-DOPA by parental (GCB-6) and mutant (UV-7) strains of Aspergillus oryzae. There was a marked difference between the mycelial morphology and pellet type of parental and UV-irradiated mutant culture. The mutant strain of A. oryzae UV-6 exhibited pellet-like mycelial morphology and improved tyrosinase activity. Mould mycelium was used for biochemical conversion of L-tyrosine to L-DOPA because tyrosinase is an intracellular enzyme. The mutant was found to yield 3.72 fold higher production of L-DOPA than the parental strain. The mutant strain is stable and D-glc-resistant. The comparison of kinetic parameters was also done which showed the greater ability of the mutant to yield L-DOPA (i.e., Yp/x 40.00+/-0.01 d mg/mg with parent and 182.86+/-0.02a mg/mg in case of mutant). When cultures grown for various incubation periods, were monitored for Qp, Qs and q(p), there was significant enhancement (p < 0.0025-0.005) in these variables by the mutant strain of A. oryzae UV-7 over GCB-6 on all the rates. L-DOPA (3,4-dihydroxy phenyl L-alanine) is a drug of choice in the treatment of Parkinsons disease and myocardium following neurogenic injury.

Double mutant of Aspergillus oryzae for improved production of l‐dopa (3,4‐dihydroxyphenyl‐l‐alanine) from l‐tyrosine

Aspergillus oryzae mutant strain UV‐7 was further improved for the production of l‐dopa from l‐tyrosine using chemical mutation. Different putative mutant strains of the organism were tested for the production of l‐dopa in triplicate shake‐flask cultures. Among these putative mutants, the strain designated SI‐12 gave a maximal production of l‐dopa (444±14 mg of l‐dopa/g of cells). The regulation of l‐dopa from different carbon source solutions [initial substrate concentration (S0)=30 g/l] by the mutant culture was investigated. At an initial pH (pH0) of 5.0 and a temperature (T) of 30 °C, 100% of sugars were utilized for product and cell mass formation, corresponding to final l‐dopa product yield of 189±8 mg/g of substrate utilized and maximum volumetric and specific productivities of 145±5 mg/h per litre and 155±8 mg/h per g of cells respectively. There was up to 3‐fold enhancement in product formation rate. This enhancement is, to our knowledge, the highest reported in the literature. To explain the kinetic mechanism of l‐dopa formation and its thermal inactivation, the thermodynamic parameters were determined with the application of the Arrhenius model. Activation enthalpy and entropy for product formation, in the case of the mutant derivative, were 40 kJ/mol and 0.076 kJ·mol−1·K−1 for its production and 116 kJ/mol and 0.590 kJ·mol−1·K−1 for thermal inactivation respectively. The respective values for product formation and product de‐activation were lower than the respective values for the parental culture. Therefore the mutant strain was thermodynamically more resistant to thermal denaturation during the product‐formation process.

Production of an extracellular lipase from Candida lipolytica and parameter significance analysis by Plackett‐Burman design

The enzyme lipase (triacylglycerol hydrolase, EC 3.1.1.3) hydrolyses triacylglycerols to fatty acids, mono‐ and di‐acylglycerols along with a glycerol moiety and are important biocatalysts in industrial applications. In this study, the production of a novel extracellular lipase by Candida lipolytica NRRL‐Y‐1095 using shaking culture was performed. Different chemically enriched fermentation media were evaluated for enzyme activity and one was found to be optimal. Temperature (30°C), initial pH (6) and rate of incubation (48 h) were found complimentary for better enzyme secretion from the yeast cells. The size of inoculum (4 mL, 16 h old) was also optimized. Urea and olive oil at a level of 2 and 1.5 (% w/v) were found to be the best sole nitrogen and carbon sources, respectively. Over 50‐fold enhancement in lipase production (4.01 U) was achieved by the culture when the process parameters including incubation period, sucrose level, initial pH, inoculum size and addition of urea were further identified using the 2‐factorial Plackett–Burman experimental design and response surface methodology. In particular, the addition of 5 g/L sugar under the optimized conditions depicted that the results were highly significant (p≤0.05). Further studies will aim at scale‐up studies in a rotary or drum bioreactor as a pre‐requisite for the commercial exploitation of the batch process.

Purification and characterisation of α-amylase produced by mutant strain of Aspergillus oryzae EMS-18

α-Amylase produced by a mutant strain of Aspergillus oryzae EMS-18 has been purified to homogeneity as judged by sodium dodecyle sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified by using 70% ammonium sulphate precipitation followed by anion exchange chromatography on DEAE-Sephadex column and gel filtration on Sephadex G-100. An enzyme purification factor of 9.5-fold was achieved with a final specific activity of 1987.7 U/mg protein and overall yield of 23.8%. The molecular weight of purified α-amylase was estimated to be 48 kDa by SDS-PAGE. The purified enzyme revealed an optimum assay temperature and pH 40°C and 5.0, respectively. Except Ca++ all other metal ions such as Mg, Mn, Na, Zn, Ni, Fe, Cu, Co and Ba were found to be inhibitory to enzyme activity.

L‐Ascorbate, a strong inducer of L‐dopa (3,4‐dihydroxy‐L‐phenylalanine) production from pre‐grown mycelia of Aspergillus oryzae NRRL‐1560

The inductive effect of L‐ascorbate on the microbiological production of L‐dopa (3,4‐dihydroxy‐L‐phenylalanine) from Aspergillus oryzae NRRL‐1560 was investigated. All biochemical reactions were performed aerobically using mould mycelia as a source of enzyme tyrosinase and acetate buffer (pH 3.0) as an extractant. L‐Tyrosine as a substrate was added at a level of 2.5 mg/ml. Maximal L‐dopa production (1.876 mg/ml) was achieved when L‐ascorbate (5.0 mg/ml) was added 6 min after the initiation of the biochemical reaction at 50 °C, consuming 2.144 mg/ml L‐tyrosine. The performance of fuzzy‐logic control of the reaction was found to be highly promising for improvement of the substrate conversion rate (∼80%). After optimizing the reaction conditions, particularly the addition of L‐ascorbate, an increase in L‐dopa yield of 22.96% was achieved compared with the control (without ascorbate addition) when the process variables, namely buffer pH, L‐tyrosine concentration and reaction temperature, were further identified using a two‐factorial Plackett–Burman design.

Kinetic characterization of extracellular α-amylase from a derepressed mutant ofBacillus licheniformis

Three strains ofBacillus licheniformis were isolated and screened for α-amylase production by solid-state fermentation. Of these, IS-2 gave relatively higher enzyme production (32±2.3 U/[g·min]) and was selected for improvement after treatment withN-methylN-nitroN-nitroso guanidine (NG) or nitrous acid (NA) to enhance its hydrolytic potential. Among the mutant variants, NA-14 gave higher enzyme production (98±1.6 U/[g·min]), and hence, was selected for kinetic and thermal characterization. M1 as a moistening agent (pH 7.0, optimized) supported 2.65-fold improved amylolytic activity by the derepressed mutant 72 h after inoculation. The values of product yield coefficient (Yp/x=1833.3 U/g) and specific rate constant (qp=25.46 U/[g·h]) with starch were severalfold improved over those from other carbon sources and the other cultures. The purified enzyme from NA-14 was most active at 40°C; however, the activity remained almost constant up to 44°C. The NA-induced random mutagenesis substantially improved the enthalpy (ΔHD=94.5±11 kJ/mol) and entropy of activation (ΔS=−284±22 J/[mol·K]) for α-amylase activity and substrate binding for starch hydrolysis. The results of this study (117.8±5.5 U/[g·min]) revealed a concomitant improvement in the endogenous metabolism of the mutant culture for α-amylase production.

The kinetic basis of the role of Ca++ ions for higher yield of citric acid in a repeated-batch cultivation system

The present investigation deals with role of Ca++ ions in increasing the yield of citric acid in a repeated-batch cultivation system (working volume 9-1) and its kinetic basis. Five different hyper-producing strains of Aspergillus niger were evaluated for citric acid production using clarified cane-molasses as basal substrate. Among the cultures, NGGCB101 (developed by u.v./chemical mutation in our labs) gave maximum production of citric acid i.e., 87.98 g/1, 6 days after mycelial inoculation. The addition of CaCl2 to the culture medium promoted the formation of small rounded fluffy pellets (1.55 mm, diameter), which were desirable for citric acid productivity. CaCl2 at a level of 2.0 μM, added during inoculation time, was optimized for commercial exploitation of molasses. During repeated-batch culturing, a yield of citric acid monohydrate of 128.68 g/1 was obtained when the sampling vs. substrate feeding was maintained at 4-1 (44.50% working volume). The incubation period was reduced from 6 to only 2 days. The values of kinetic parameters such as substrate consumption and product formation rates revealed the hyperproducibility of citric acid by the selected Aspergillus niger NGGCB101 (LSD = 0.456a, HS). Case studies are highly economical because of higher yield of product, lower energy consumption and the use of raw substrate without any additional supplementation.