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Home > Studies on the Submerged Fermentation of Invertase by Saccharomyces Cerevisiae

Studies on the Submerged Fermentation of Invertase by Saccharomyces Cerevisiae

Thesis Info

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Author

Mian Muhammad Aslam

Program

PhD

Institute

Government College University

City

Lahore

Province

Punjab

Country

Pakistan

Thesis Completing Year

2006

Thesis Completion Status

Completed

Subject

Applied Sciences

Language

English

Link

http://prr.hec.gov.pk/jspui/handle/123456789/1941

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676727355318

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In the present study, eighty six strains of Saccharomyces cerevisiae were isolated from different samples of fruits and soil by serial dilution method. The strain IS-66 gave maximum extracellular invertase production (1.10 U/ml). The enzyme activity reached to 5.6 U/ml when incubation time (48 h), sucrose concentration (5 g/l) and pH 5.5 were optimized. The wild strain IS-66 was exposed to ultraviolet (UV) radiations to obtain a mutant with improved enzyme activity. UV induced mutagenesis did not produce any stable mutant and almost all of the mutants produced relatively lesser invertase than the parental strain. Strain IS-66 was further subjected to chemical mutagenesis using nitrous acid and ethyl methane sulphonate (EMS). After extensive screening, two mutants were developed with increased enzyme activity NA-45 (20.74 U/ml) and EMS-42 (34.2 U/ml) from the wild-culture (IS-66). The mutant strain EMS-42 was cultured on the medium containing 2-deoxy-D-glucose (2dg) and its stability in invertase production was determined at different concentrations of 2dg. The concentration of 0.04 mg/ml was found optimal, as at this concentration EMS-42 showed consistent enzyme activity. Six media were evaluated for the production of invertase in shake flasks. M1 medium (g/l) containing yeast extract 3, peptone 5 and sucrose 30 g/l gave better production of invertase (25.28 U/ml) after 48 h of inoculation. Different sugars such as sucrose, glucose, fructose, lactose, galactose, maltose, raffinose and molasses were investigated on the enzyme production. Of these, sucrose was found to be best (44.03 U/ml) after optimizing the concentration at 10 g/l. Incubation temperature (30oC), inoculum size (2.0 %, v/v) and volume of the medium (50 ml/250 ml Erlenmeyer flask) were optimized. The effect of different additional nitrogen sources such as organic, inorganic and agriculture byproducts were also tested. Peptone at the concentration of 6 g/l gave maximum production of invertase (50 U/ml). The addition of inorganic nitrogen sources and agricultural byproducts nitrogen were not found to have any impact on the enhancement in enzyme production rather it was decreased from the control especially in case of agricultural byproducts. In stirred fermentor (7.5 L), the scale up studies for invertase production was carried out. The enzyme production (65.12 U/ml) was obtained after 24 h of incubation. The overall increase in enzyme activity (15 U/ml) and fermentation time was shortened by 24 h while scaling up enzyme production from shake flask to fermentor. The maximum enzyme activity (80.06 U/ml) was achieved after xvioptimization of cultural conditions such as sucrose (15 %, w/v), pH (4.5), inoculum size (7.5 %, v/v), agitation intensity (240 rpm) and aeration rate (1 vvm, 10 % DO). The intracellular enzyme activity was also determined by sonication. The maximium enzyme activity (57 U/ml) was found in mutant strain of EMS-42 after 24 h fermentation in the fermentor. During sonication, the maximum specific activity of 106 U/mg of protein was obtained with 0.5 duty cycle of impulses at amplitude of 40 % and pH 5 for 60 min. The calcium alginate entrapment technique was used for immobilization of whole cells of S. cerevisiae EMS-42 to form inverted syrup. It was noticed that maximum sucrose hydrolysis (63.40 %) was achieved after 18 h of incubation time. By optimization of cultural conditions for sucrose hydrolysis, the maximum hydrolysis percentage (76.3 %) was obtained at 50oC, pH 5.5 using sucrose (60 %) as substrate. An extracellular invertase was purified to homogeneity by two step purification i.e., ammonium sulfate precipitation and DEAE-Sephadex A-50. The enzyme was present in the supernatant of 85 % saturation being glycoprotein in nature. DEAE column chromatography eluted the enzyme as single active fraction at 0.2 M NaCI. The enzyme was purified by 15 fold with recovery of 38 %. The molecular mass of 110 kDa was determined after SDS-PAGE. The carbohydrate content was found to be 48 %. The intracellular invertase contains both forms of glycosylated (large) and non-glycosylated (small). The same procedure was applied for glycosylated intracellular invertase (L- form) while three purification steps were performed for non-glycosylated invertase (S- form). The L-form was purified by 19 fold with recovery of 32 %. Like extracellular invertase, the molecular weight (110 kDa) for L-form was found. Ammonium sulfate precipitation separated the enzyme (S-form) as insoluble fraction. This form of enzyme was eluted at 0.3 M NaCl using DEAE-Sephadex. A single band of molecular weight (55 kDa) was estimated after Sephadex G-50 with purification (16 fold) and recovery of 17 %. For both purified non-glycosylated and glycosylated invertase the optimum pH (5) was same whereas optimal temperature, MnCl 2 and the values of the K m and V max were found to be as 50 and 60oC, 109 and 111 %, 1.2 and 1.8 mM, 909 and 1429 U/ml/min, respectively.
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پیار نوں لاویں ہن نہ لیک

پیار نوں لاویں نہ ہن لیک
سجناں دے ہو رہو نزدیک
تیرے حال دی منگدے خیر
میرے حال نیں بالکل ٹھیک
کدی تاں یار ملن نوں آسی
رکھدے اسیں نِت اڈیک
عشق دی رمزاں عاشق جانے
نقطے نیں ایہہ بہت باریک
جتھے آس امید نہ ہووے
اوتھے کی منگنا ایں بھیک
پاک نبیؐ دا در نہ چھڈیں
ایتھوں ملسی رحم دی بھیک
عشق نبیؐ دا جے نہ ہووے
سمجھ لے دنیا دل تاریک

Development Discourse and Womens Empowerment in Sindh

This research paper investigates and introduces the idea of development discourse and women empowerment question in Sindh. Based on the investigation and analysis of the writings by Sindhi intelligentsia in the Sindhi print media and book publications that present the discourse of development, progress, prosperity, and social change in Sindh, in this paper we engage with and call attention to the question of women empowerment. By integrating women empowerment and rights in this paper, we aim to set the research agenda and draw attention to gender and development theory and practice. In a way, this paper takes up and supports the gender-sensitive research approach to development discourse, public policy, and planning in Pakistan. It prioritizes gender and women's empowerment framework in research, teaching, and development practice.

Study of Physiological and Biochemical Parameters of Farm Raised Fish With Toxicants

Keenjhar lake of Sindh, Pakistan, second largest natural freshwater lake had a rich flora of submerged, floating and emergent aquatic plants and it was also rich in the fish on which live hood of local communities mainly depended on the resources. The lake is also very important for the Karachi Water and Sewerage Board, as it is the only source that provides drinking water to 1.8 million people of the city. Besides being the main water supply source, the lake is also considered to be an important site for breeding and a passageway for water birds that migrate to Pakistan in winter from Russian Siberia and other cold countries. The highly contaminated industrial effluent, which comprises of very toxic waste from hundreds of industrial units of Kotri and Noori abad, is being discharged in the lake without proper treatment. It is responsible to increases different type of organic and inorganic contaminates, that persistent in the lake and cause serious damage in the physico-chemical parameters of lake water. The pollution of lake in 2011 was also addressed by supreme court of Pakistan due to severe contaminates burden. This research project comprises an analysis of some important biophysical and biochemical parameters of fish species Notopterus notopterus collected in the duration of three years to check the toxicity level of fluoride contaminate and their effects on fish community. Fluoride was selected as a major inorganic persistent contaminate of aquatic resources, univalent poisonous gaseous halogen, the most chemically reactive and electronegative of all the elements of Periodic Table, readily oxidizes the many organic compounds. Fish were collected in the months of February and March from 2008-2010.Weight and length of the fish was recorded after the collection from the lake. After collection fish were kept in tap water (receiving from the same lake in Karachi city for drinking purpose) in laboratory for two months and two doses (1.5 and 3 g /70 L in each aquarium) of fluoride was administrated in two separate fish aquariums while one was taken as non-treated (control). The weight and length of the fish were monitored at two different dose (1.5 & 3.0 g/70 L) of NaF. It showed significant decline during the time interval of 24,48, and 72h with different mortality rate in each year. The time interval of 24h, 48h and 72h wereselected after analysis of different parameters at variable time intervals, it was observed that marked effect on biochemical constituents were observed significantly in this duration and after this no significant changes showed that initial administration of fluoride concentration was crucial. All physical parameters of the site of collection including temperature of the water, humidity of the air were noted. An astonishing thing was observed related to that of temperature and humidity at Lake Site that the temperature of the lake was high and humidity was low during the same months of collection in year 2009 and 2010 with reduced length and weight of same fish under investigation as compared to 2008. Physioco-morphological variations were observed related to applied dose on whole fish body.The skin becomes black with turned scale, weakness in movement of fish, eye vision was affected, and mouth of fish was stuck. Dissection of the fish showed that all main organs were dissolved while heart, liver, kidney were not visualized in the body at the time of mortality in fluoride treated fish which was different for each year. The lowest time of mortality was observed in the fish collected in year 2010 (45 d) showed burden of contaminant in lake while it was maximum (90d) for 2008 collections at both doses.As a matter of fact that approximately all chemical reaction in a living cell requires enzymes in order to occur at speed enough for sustaining life. Results showed that enzyme activity was a good environmental biomarker of pollution pressure. Alterations in enzymes profile are important pollution indices. Alteration in enzymes activity may be related with the muscles morphology and physiology.Cell membrane permeability lets the enzymes leaching or decrease membrane permeability allows it to accumulate in cell. Cellular damage is another major cause of decrease or increase in enzyme activity result in the inhibition of carbohydrate- protein metabolism. Three key enzymes like Alkaline Phosphatase (ALP), Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) of protein– carbohydrate metabolism were observed in the three consecutive years, viz 2008-2010 at 24h, 48h and 72h. Results of activity of enzyme like ALP, AST and ALT were examined in the three important tissues like muscles, liver and gills. Year wise study revealed that in year 2010 non-treated fish showed very high enzymes activity which was clearly showed the pollution burden inthe lake followed with temperature variation. It reflects that toxicity factor is temperature, time and dose dependent phenomena. Enzymes activity of treated fish showed overall increase in activity of carbohydrate metabolism which may be due the denaturing of site of interaction or to overcome the stress. This may be related to the morphology and physiology of fish which certainly associated with structural damage. The interruption of biochemical and physiological integrity is computable by the changes in the enzyme activities in liver, muscles and gills. Transamination is one of the fundamental pathways for the biosynthesis and deamination of amino acids, allowing carbohydrate and protein metabolism during variable energy demands of the fish under different adaptive situations. Elevated transaminase levels showed liver damage/ hepatitis, related liver morphology and physiology affected by fluoride results in the failure of liver function. Alteration in enzymes activity was significantly highlighted on carbohydrate metabolism in gills, liver and muscles where overall increase in concentration of carbohydrate from 2008-2010 was observed. A slight variation in 2008 at both applied dose of fluoride (p < 05) while non-significant variation in protein in three important tissues in 2008 reflects less pollution burden while investigation of 2010 showed sharp decline in proteins contents of fish. It was revealed that the lipid was the major source of energy under stress in the three year period of study due to non-utilization of carbohydrate or inhibition of oxidation of carbohydrate to produce energy. In control fish lipids content were higher in three years. They may be the provider of energy for survival under fluoride contamination. Decline in lipid content during three years of investigation support the above hypothesis. It was observed that sub lethal physiological effects of fluoride on macro and micro metals residue accumulation in functional organ showed high concentration of essential(Na, K, Ca, Mg) and non-essential metals(Pb,Hg,Co,Cd). This may be due to fluoride induced permeability in gills, liver and muscles tissues which results in mineral nutrients unbalancing owing to free movement of non-essential metals (Pb, Hg, Co and Cd) in organs of fish under studied. Higher concentration of toxic metal in fish may be related with the altered enzymes and protein glucose metabolism which in turn related with permeability of gills and liver epithelium and dissolution of muscles tissues, which significantly affects the scale loss due to fluoride mobility with increase in metalaccumulation in tissues of fish. Fluoride caused serious pathological liver conditions including shrinkage of liver cells characterized by the dissolution of the liver, blood clots, necrosis and other degenerative changes. Heavy metals may change the cell membrane permeability and due to inactive or denatured enzymes cause dysenzymia. Inhibition of both transaminase suggested that fluoride affect the intermediary metabolism which in turn decrease the production of energy in term of ATP through TCA cycle. The measurement of enzymes ALT, AST and ALP in muscles indicate metabolic disorders including diabetes mellitus, renal failure etc might be related to increase in glucose concentration. While decrease in protein concentration may be related to conversion of protein to energy production via gluconeogenesis to overcome the stress for fish survival. The decrease in glucose level in muscles may be also related to perturb carbohydrate metabolism which was insufficient for energy demand of fish for survival in stress. It was suggested that higher electronegativity of fluoride play a crucial rule in oxidation and degradation of tissues and also reaction of fluoride with oxygen suppresses the oxygen absorption by which energy demand of fish cannot meet by oxidation of carbohydrate therefore concentration of carbohydrate increase with time due to non-utilization in energy production. The depletion in protein content of muscles by fluoride due to inhibition of amino acids metabolism, preventing cells from synthesizing protein or depletion may be of its utilization in energy production. The decrease in protein in muscles may be due degradation and possible utilization for metabolic purposes. The decrease in total lipids may be due to inhibition of lipids synthesis by fluoride as well as increase utilization of stored lipids as a source of energy to conduct regular metabolic function." xml:lang="en_US