Quratulain Syed

One-factor-at-a-time (OFAT) optimization of xylanase production from Trichoderma viride-IR05 in solid-state fermentation

Abstract The present study dealt with the production of enzyme xylanase by solid substrate fermentation using Trichoderma viride-IR05. Different substrates such as wheat bran, rice polish, rice husk, soybean meal, sunflower meal, sugarcane bagasse or corn cobs were evaluated for enzyme production. Of all the substrates evaluated, sugarcane bagasse was found to be best for enzyme synthesis. The substrate, sugarcane bagasse pretreated biologically, 2% H2SO4, 2.5% KOH or 3%H2O2. However 2.5% KOH gave maximum yield of enzyme as evidenced by the SEM analysis of the pretreated substrate. The cultural conditions were optimized for the production of xylanase in 250 ml Erlenmeyer flask such as incubation period (seven days), substrate concentration (10 g), liquid to solid ratio (11:10), initial pH of diluent (4.5), incubation temperature (30 °C) with inoculum size of 10%. Further supplementation of xylose, NaNO3 or tryptone and tween-80 as additional carbon source, nitrogen and surfactant improved (72.4 ± 1.42 U/g) the titer of xylanase by T. viride-IR05, respectively.

Effect of alkaline pretreatment on delignification of wheat straw.

This study was conducted to analyse structural changes through scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) after alkaline pretreatment of wheat straw for optimum steaming period. During the study, 2 mm size of substrate was soaked in 2.5% NaOH for 1 h at room temperature and then autoclaved at 121°C for various steaming time (30, 60, 90 and 120 min). Results revealed that residence time of 90 min at 121°C has strong effect on substrate, achieving a maximum cellulose content of 83%, delignification of 81% and hemicellulose content of 10.5%. Further SEM and FTIR spectroscopy confirmed structural modification caused by alkaline pretreatment in substrate. Maximum saccharification yield of 52.93% was achieved with 0.5% enzyme concentration using 2.5% substrate concentration for 8 h of incubation at 50°C. This result indicates that the above-mentioned pretreatment conditions create accessible areas for enzymatic hydrolysis.

Optimization of process parameters for xylanase production by Bacillus sp. in submerged fermentation

Abstract In this study an attempt was made to optimize the cultural and nutritional conditions for xylanase production by Bacillus species in submerge fermentation process. Whole fermentation process was carried out in 250 ml Erlenmeyer flask with agitation speed of 140 rpm. Bacillus subtilis exhibit maximum xylanase production at initial medium pH of 8, substrate concentration of 2% with inoculum size of 2% at 35 °C for 48 h of fermentation period. Further supplementation of sucrose, (NH4)2SO4 and peptone as a carbon and nitrogen sources favored enzyme production. The other strain Bacillus megaterium showed its peak xylanase production at initial medium pH of 8, inoculum size of 1.5% with substrate concentration of 1.5% at incubation temperature of 40 °C for 72 h of fermentation period. The best carbon and nitrogen sources are xylose, KNO3 and malt extract. Both strains can also utilize molasses at 0.5% concentration for xylanase production can grow in medium containing 0.2% NaCl (B. subtilis BS04) and 0.8% NaCl (B. megaterium BM07) respectively. The optimum temperature of xylanase was 50 °C and pH was 5 and 5.5 by B. subtilis BS04 and B. megaterium BM07 respectively.

Kinetics Study of Extracellular Detergent Stable Alkaline Protease from Rhizopus oryzae

ABSTRACT In this study, extracellular alkaline protease was produced from Rhizopus oryzae in submerged fermentation using dairy waste (whey) as a substrate. Fermentation kinetics was studied and various parameters were optimized. The strain produced maximum protease at initial medium pH of 6.0 medium depth of 26 mm, inoculum size of 2% at incubation temperature of 35 o C for 168 h of fermentation. Alkaline protease was purified to homogeneity by ammonium sulphate fractionation followed by sephadex G-100 chromatography. The molecular mass of alkaline protease was 69 kDa determined by 10% SDS-PAGE. The optimum pH and temperature of alkaline protease was 9.0 and 40 o C, respectively. Metal profile of the enzyme showed that the enzyme was non-metallic in nature. The K m , K cat , V max and K cat /K m values of purified protease were 7.0 mg/mL, 3.8 x10 2 S -1 , 54.30 µmol/min and 54.28 s -1 mg -1 .mL respectively, using casein as substrate. The purified alkaline protease had stability with commercial detergents.

On-site cellulase production by Trichoderma reesei 3EMS35 mutant and same vessel saccharification and fermentation of acid treated wheat straw for ethanol production.

Bioethanol production from lignocellulosic raw materials involves process steps like pre-treatment, enzymatic hydrolysis, fermentation and distillation. In this study, wheat straw was explored as feedstock for on-site cellulase production by T. reesei 3EMS35 mutant, and as a substrate for second generation bioethanol production from baker yeast. Scanning electron microscopy (SEM) and X-ray diffractography (XRD) of untreated wheat straw (UWS) and acid treated wheat straw (TWS) were done to understand the structural organization and changes in the cellulase accessibility and reactivity. The effect of delignification and structural modification for on-site cellulase enzyme production was comparably studied. The efficiency of crude cellulase enzyme for digestion of UWS and TWS and then production of ethanol from TWS was studied using same-vessel saccharification and fermentation (SVSF) technique, both in shaking flasks as well as in fermenters. Two different methods of operation were tested, i.e. the UWSEnz method, where UWS was used for on-site enzyme production, and TWSEnz method where TWS was applied as substrate for cellullase production. Results obtained showed structural modifications in cellulose of TWS due to delignification, removal of wax and change of crystallinity. UWS was better substrate than TWS for cellulase production due to the fact that lignin did not hinder the enzyme production by fungus but acted as a booster. On-site cellulase enzyme produced by T. reesei 3EMS35 mutant hydrolyzed most of cellulose (91 %) in TWS within first 24 hrs. Shake flasks experiments showed that ethanol titers and yields with UWSEnz were 2.9 times higher compared to those obtained with TWSEnz method respectively. Comparatively, titer of ethanol in shake flask experiments was 10 % higher than this obtained in 3 L fermenter with UWSEnz. Outcomes from this investigation clearly demonstrated the potential of on-site cellulase enzyme production and SVSF for ethanol production from wheat straw.

Isolation and purification of complex II from Proteus mirabilis strain ATCC 29245.

A respiratory complex was isolated from plasma membrane of pathogenic Proteus mirabilis strain ATCC 29245. It was identified as complex II consisting of succinate:quinone oxidoreductase (EC 1.3.5.1) containing single heme b. The complex II was purified by ion-exchange chromatography and gel filtration. The molecular weight of purified complex was 116.5 kDa and it was composed of three subunits with molecular weights of 19 kDa, 29 kDa and 68.5 kDa. The complex II contained 9.5 nmoles of cytochrome b per mg protein. Heme staining indicated that the 19 kDa subunit was cytochrome b. Its reduced form showed absorptions peaks at 557.0, 524.8 and 424.4 nm. The α-band was shifted from 557.0 nm to 556.8 nm in pyridine ferrohemochrome spectrum. The succinate: quinone oxidoreductase activity was found to be high in this microorganism.

Process optimisation for the biosynthesis of cellulase by Bacillus PC-BC6 and its mutant derivative Bacillus N3 using submerged fermentation

This study deals with optimisation of cultural conditions for enhanced production of cellulase by Bacillus PC-BC6 and its mutant derivative Bacillus N3. Influence of different variables including incubation time, temperature, inoculum size, pH, nitrogen sources and metal ions has been studied. The optimum conditions for cellulase production were incubation period of 72 h, inoculum size 4% incubation temperature 37°C, pH 7, 0.25% ammonium sulphate, 0.2% peptone as inorganic and organic nitrogen source in case of Bacillus PC-BC6. In case of mutant Bacillus N3, optimal conditions were incubation period of 48 h, incubation temperature 37°C, inoculum size 3%, pH 7, 0.2% ammonium chloride and 0.15% yeast extract. Presence of MnSO4 and CaCl2 enhances the enzyme production by Bacillus PC-BC6 and mutant Bacillus N3, respectively. This study was innovative and successful in producing cellulase economically by using cheap indigenous substrate Saccharum spontaneum.

Isolation, Characterization and Selection of Avermectin-Producing Streptomyces avermitilis Strains From Soil Samples.

Background: Streptomyces avermitilis, belonging to Actinomycetes, is specialized for production of avermectin, used as an anthelmintic and insecticidal agent. It is mostly found in soil and its isolation is very crucial for medically important avermectin production. Objectives: In the present study, 10 bacterial isolates lacking antimicrobial activities were isolated from the soil samples collected from different areas of Lahore, Pakistan. Materials and Methods: Three distinctive localities of Lahore were opted for soil assortment to isolate S. avermitilis. About 50 isolates of Streptomyces species were attained through selective prescreening procedures. All of these isolates were studied for production of the secondary metabolite, avermectin. Different test like soluble pigment color and melanin formation were used for identification. Biochemical characterizations of those isolates closely resembling the control in morphological characteristics, soluble pigment color and melanin formation tests were performed. Results: The 10 selected isolates were identified as the avermectin-producing strain by fermentation and characterized on ISP2 medium for aerial and reverse side mycelia color, soluble pigment color and melanin formation, in comparison with S. avermitilis DSM 41445. The best avermectin-producing isolate S1-C (10.15 mg/L) showed similar result as S. avermitilis DSM 41445, when subjected for culture characteristics analysis in different media along with biochemical characterization. Conclusions: From the results, it was concluded that agricultural lands around Pakistan Council of Scientific and Industrial Research (PCSIR) Campus Lahore were rich sources of industrially important Streptomyces, especially S. avermitilis.

Response surface methodology-based optimization of glucose acylation bio-catalyzed by immobilized lipase

Abstract This paper describes in detail the selection and optimization of immobilized lipases for enhanced regioselective acylation of glucose into glucose monolaurate (GlcML). Initially, nature of biocatalyst, immobilization approach, reaction media, glucose, and lauric acid concentration were screened out. Finally, lipases from Rhizopus arrhizus immobilized on dead mycelia were investigated under various reaction conditions (Temperature, shaking speed, enzyme dose, and water content) following a fully rotatable central composite design (FRCCD) to optimize the activity of lipases. The immobilized lipases-based biocatalysts in the presence of polar solvents (tertiary alcohols) and higher concentrations of substrates i.e. glucose and lauric acid (100 and 300 mmol L−1, respectively) offered conversion rate of 1.5 mmolmin−1 L−1. Moreover, optimization of reaction conditions revealed that 162.5 lipase units/100mL at 31.25 °C, 3% water content, and 105 RPM shaking speed enhanced the conversion rate by 0.5 mmolmin−1 L−1 rendering the reaction more economical. Hence, lipases-based immobilized biocatalysts may provide an intelligent and green choice for commercial scale synthesis of GlcML for food and pharmaceutical industries.

Chromatographic resolution of angiotensin II receptor antagonists (sartans)

First time a simple, sensitive and unified quantification method has been developed to analyze the complete class of angiotensin II receptor antagonists which are used in the treatment of hypertension either alone or in combination with some other drugs. The most important advantage of developed method was that the eight separate drugs can be determined on a single chromatographic system without modifications in detection wavelength and mobile phase. The drugs were separated on a Purospher Star 4.6mm×25cm, 5μm, C18 column maintained at 40°C with 1mLmin(-1) flow rate using ultra violet detection at 254nm. Good separation (Rs>2.0) was achieved in a short analysis allowing simultaneous determination of all eight sartans. The effect of variation in flow rate, detection wavelength and column oven temperature was also studied. The proposed method was statistically validated in terms of precision, accuracy, linearity, specificity and robustness. The newly developed method proved to be specific, robust and accurate for the quantification of eight sartans in commercial pharmaceutical formulations.