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Kinetics and Thermodynamics of Proteases from Bacillus Sp.

Thesis Info

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Author

Younas Butt, Kashif

Program

PhD

Institute

Bahauddin Zakariya University

City

Multan

Province

KPK

Country

Pakistan

Thesis Completing Year

2018

Thesis Completion Status

Completed

Subject

Chemistry

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/12434/1/Kashif%20younaz%20butt%20chemistry%202018%20bzu%20not%20same.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676726569522

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Present project focuses on the study of protease from the indigenously isolated strain Bacillus. Effects of different substrate levels, ionic concentrations (MgCl2, CaCl2, K2HPO4, (NH4)2SO4, Urea), cane molasses and yeast sludge on the production of proteases by Bacillus subtilis. Various conditions like incubation period, mode of fermentation, pH, temperature etc. were optimized. After optimization of conditions, the proteases were produced on large scale. The proteases thus produced were subjected to the purification through various steps like ammonium sulphate precipitation, ion exchange chromatography and gel filtration. Kinetic study of proteases was also carried out in order to calculate Km and Vmax of the enzymes. Thermal inactivation was studied in order to calculate the Kd, energy of activation (Ea), Gibbs free energy (∆G), Enthalpy (∆H) and entropy (∆S). Activation or inhibition effect of different metal ions and chelating agents like EDTA, EGTA and PMSF on the protease was examined. Maximum activity of the crude proteases ( 502 U) was obtained at pH 7.5, temperature 45 °C after 48 hours of continuous shaking at 220 rpm using Bacillus subtilis cultured on skim milk (2%) in the optimized medium containing MgCl2 ( 0.01%), (NH4)2SO4 (0.3%) , CaCl2 ( 0.03%), K2HPO4 ( 0.5%), yeast sludge (300 μL) and cane molasses (0.03%). Specific activity of the purified protease was (80 U/mg), yield is 3.18 % and 3 folds increase in activity. Kinetic investigation revealed that that Km and Vmax were 0.2mg/mL and 60 U/mg. Energy of activation Ea , Free energy of activation ∆G , Activaton Enthalpy ∆H, Activation entropy (∆S) were -403 KJ/mol, 91.4 KJ/mol, -403.37 KJ/mol and -407.80 J/mol. Thermo stabilization of proteases is mostly accompanied by a decrease in ∆H and ∆S. A metal ions effect on the activity of the proteases was also studied. PMSF inhibited the enzyme activity, but EDTA and EGTA would not had much pronounced effect on the activity of enzyme. That proved the class of proteases to be the serine due to the inhibition of PMSF. It was concluded that Bacillus subtilis had significant potential for the production of proteases. With respect to the kinetic study of proteases, small vales of Km than the substrate concentration showed large affinity of the enzyme with the substrate and found good source of for the production on proteases on large scale. The nature of the reaction mechanism was found to be first order because [S] was much less than Km. Activation enthalpy had negative values indicates that the enzyme was of endothermic nature. Economically cheaper metals enhanced the enzyme activity. So the Bacillus subtilis was found good catalytic agent for the production of proteases.
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غزل

دل سے اک شخص کی یادوں کو رہا کرتے ہوئے
مر ہی جاوں نہ کہیں اس کو قضا کرتے ہوئے
تو کہ معبود سے نگران بھی سکتا ہے
میں نے سوچا ہی نہ تھا تجھ کو خدا کرتے ہوئے
ہاتھ اٹھاتے ہوئے وہم و گماں میں بھی نہ تھا
لب پہ اک نام جو آیا ہے دعا کرتے ہوئے
ہر کہانی کے قلم کار نے کیوں مار دیے
سارے کردار محبت میں وفا کرتے ہوئے
ہاتھ میں رنگ رکھا  ، باد صبا پر آنکھیں
پھول سے پھول کی خوشبو کو جدا کرتے ہوئے
شہر کا شہر ہی ویران نہ ہو جائے کہیں
میرے اک خواب کو تعبیر نما کرتے ہوئے
اب تو میں لوٹ کے جنگل میں نہیں جا سکتا
کھو گیا ہوں میں ترے گھر کا پتا کرتے ہوئے
تو کہے تو میں ترے دل سے نکل جاتا ہوں
میں نے جنت بھی چھوڑی تھی خطا کرتے ہوئے
آندھیوں سے تو مری جنگ نہیں ہے کوئی
شمع تو بجھنے نہ دی ذکر ہوا کرتے ہوئے
میں نے خیرات میں بھی پیار ہی مانگا ہے عدید
سوچتے کیا ہے سخی مجھ پہ عطا کرتے ہوئے

أفكار أرنولد توينبي عن الحضارة الإسلامية ومدى تأثره برؤية ابن خلدون: دراسة وصفية و تحليلية

Ibn e Khaldun (1406 C.E.) has been an imminent scholar and well known for his work in the study of civilization. His vision regarding Civilization holds the significant place according to the philosophers of history. Arnold J. Toynbee (1975 C.E.) is one such prominent thinker who not only applauded the thoughts of Ibn e Khaldun but was influenced by Ibn e Khaldun’s views as it can be seen in Toynbee’s book: “A Study of History”. As a philosopher of history, he has much contribution in the field; He interlinks History with civilization. He presented a thoughtful book surrounding his civilizational vision; which explains the causes of world’s ups and downs. Although he presented a quality research about the division of the civilizations in the light of religion, many aspects of his work need to be reassessed. As per his understanding of world civilizations, he represents twenty-one civilizations, but with the passage of time, the number reduces and now only five are left in the contemporary epoch. According to him, religion has played significant role in the rise and fall of civilizations through their various stages. This research will highlight his thought about Islam through a comparison between Toynbee and Ibn e Khaldun’s Islamic civilizational thoughts. The study will also mention several problems in his approach to the Islamic Civilization. Furthermore, along with due importance of both scholars in the subject of history, their authoritative status will be stated. This research aims to discuss some misconceptions of the West that are based on Toynbee's understating of Islamic civilization and history. And, consequently, it intends to improve relations between people of the west and east.

Microfacies, Diagenesis and Depositional Environments of Samana Suk Formation Middle Jurassic Carbonates Exposed in South East Hazara and Samana Range

This document presents the results of detailed work on the microfacies analysis, depositional environments and diagenetic history of the Middle Jurassic carbonates system, (The Samana Suk Formation) investigated in the three measured outcrop sections (total thickness 785m) in the Hazara and Kohat area. The three sections (Sanghar Gali, Bagh and Samana Range) lie along the northern rim of the Upper Indus Basin of Pakistan. Rocks exposed in the three above mentioned localities were investigated in the field for facies and sedimentary structures. 800 samples were collected and each sample was further investigated on polished slab, and 850 thin-sections were prepared for detailed petrographic analysis. All the data generated was interpreted for depositional environments and diagenetic imprints. This work is also supported by selected SEM photographs, forty two oxygen and carbon isotope analyses and fourteen trace element analyses. The data from the three studied sections is presented semi-quantitatively in Sedimentary Synthesis Logs and Diagenetic Feature Logs. The results are also supported by biostratigraphic analysis of selected shales and limestone samples. All the results of these studies were integrated to propose a likely depositional or sequence stratigraphic model of the Samana Suk Formation. Benthic foraminiferal data suggest that carbonate sedimentation started in Bajocian (Middle Jurassic) and might have continued until Callovian time. The data has also confirmed the Bathonian age from the middle part of the formation in the Samana Range Section. On the whole, the faunal/floral assemblage suggest sedimentation of the Samana Suk Formation occurred on the shallow to very shallow marine setting typical of gently dipping shelf/ramp., The assemblages suggest various episodes of sea level rise and fall. Thirty microfacies have been identified that were deposited in variety of environments. Generally the formation is dominated by oolitic grainstone, that have deposited in shallow marine subtidal to intertidal environments as an oolitic shoal complex on a gently inclined carbonate ramp/platform which might have attained slightly rimed shape in a later stage. Within the whole succession about 19, 20 and 31 shallowing upward cycles have been interpreted in the Sanghar Gali, Bagh and Samana Range Sections respectively. These cycles vary in numbers and thickness in the three studied sections and do not correlate individually across the study area. Such cycles normally start from mudstone (M2) and/or wackestone (W1 or W2) and terminate at oolitic grainstone while occasionally complete cycles go up to pelletal pack-grainstone (PG1) and/or unfossiliferous mudstone (M1), representing the tidal flat facies. These cycles are parasequences which started from subtidal environments and terminate in intertidal environments and rarely end in supratidal environment. Twenty six different diagenetic features have been identified in the Samana Suk Formation. Overall, diagenetic sequence ranges from marine phreatic to deep burial. However dominant influences of meteoric phreatic and mixed meteoric marine environments during shallow burial have also been observed. Dolomite, though minor component relative to limestone, is present in several forms in the Samana Suk Formation such as layers and thin beds, dolomitized burrows, grain selective and cement/matrix selective dolomite, molds and fracture filling dolomite, dolomite developed along sutured seams and stylolites, and pervasive dolomite. Dolomite in the Sanghar Gali and Bagh Sections is common, while in the Samana Range Section it is rare. Dolomitized burrows and pervasive dolomite in the upper 35m of the Bagh Section are volumetrically important. Dolomite layers, beds and pervasive dolomite were developed in meteoric marine mixing zone. Bio-pumping together with bacterial activities in the burrow network, related to the burrowing organisms, was probably responsible for the preferential dolomitization of burrow fillings. The grainstone horizons originally had good primary intergranular/ interparticle porosity, which was first reduced by early mechanical compaction and later on occluded during diagenesis by seven different types of calcite cements. Three phases of dissolution and four phases of fracturing have been identified which have generated secondary intergranular porosity (i. e., mouldic, vuggy and fracture) which has also been occluded. Two sets of fractures are filled with medium to coarse blocky calcite, 3rd set is filled with internal sediments whereas 4th set is filled with dolomite. The high resolution microfacies and cyclicity, biostratigraphic abundance data has been used to interpret the significant depositional surfaces such as flooding surfaces (transgressions) and sequence boundaries (regressions). Four flooding surfaces and four sequence boundaries have been interpreted which divides the whole Samana Suk Formation into four 3rd order sequences. These sequences have only highstand and transgressive system tracts. This interpretation is based on the most striking contrast in the microfacies. The proposed model shows proximal facies (mainly oolitic shoal and back shoal facies) towards the east-southeast while distal or open marine facies (mainly mudstone/marl) towards the west-northwest. This also implies that the lowstand system tract is probably preserved further west of the Samana Range section.