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Home > Gene Cloning, Characterization and Thermodynamic Studies of Highly Thermostable Celluloytic Enzymes from Genus Thermotoga

Gene Cloning, Characterization and Thermodynamic Studies of Highly Thermostable Celluloytic Enzymes from Genus Thermotoga

Thesis Info

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Author

Fatima Akram

Program

PhD

Institute

Government College University

City

Lahore

Province

Punjab

Country

Pakistan

Thesis Completing Year

2017

Thesis Completion Status

Completed

Subject

Biotechnology

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/12926/1/Fatima_Akram_Biotechnology_HSR_2017_GCU_Lahore_23.01.2018.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676726252768

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With a paradigm shift in industry, moving from natural fuels to alternative renewable resource utilization, concisely the growing demands of bioenergy has led to the emphasis on novel cellulolytic enzymes to improve efficiency of bioconversion process of lignocellulosic plant biomass. Currently, biodegradation is an area of extensive research, the cynosure of biofuel industry is on the utilization of non-edible lignocellulosic biomass (feedstock, agricultural and municipal residues) as an exploitable, inexpensive and potential source of alternative renewable energy in the form of bioethanol, thus the need of efficient thermostable cellulases are expected to increase in the future. β-glucosidase is an essential member of cellulase enzyme system that plays a critical role in cellulosic substrate hydrolysis and in many other biological processes. Therefore, the present study describes cloning of two novel highly thermostable cellulolytic enzymes β-1,4-glucosidases (TnBglA and TnBglB) from a bacterium Thermotoga naphthophila RKU-10T and overexpressed in Escherichia coli BL21 CodonPlus (DE3)-RIPL. Purification and biochemical characterizations together with kinetic and thermodynamic analysis give insights about the thermostability of both enzymes. Various cultivation and induction strategies were applied to enhance the production of engineered host cells density and expression of highly efficient thermotolerant TnBglA and TnBglB, induced individually with IPTG and an alternative inducer lactose. Culture conditions and other parameters including media compositions, pre-induction optical density, agitation, inducer concentrations, temperature and time of induction were optimized to achieve maximum yield of heterologous proteins. Genomic DNA of T. naphthophila was used as template to amplify two cellulolytic genes TnbglA and TnbglB (ADA66698.1 and ADA66752.1) of 1.341 and 2.166 kb, which encoded β-1,4-glucosidase proteins of 446 and 721 amino acid residues, respectively. Amplicons of genes were cloned initially in pTZ57R/T vector by employing dA×dT tailing technique and consequently subjected to sequence analysis. Sequence homology analysis demonstrated that TnBglA and TnBglB belong to glycoside hydrolase family 1 (GH1) and family 3 (GH3), respectively. Further, both genes were sub-cloned in pET-21a(+) vector and over-expressed in E. coli BL21 under the control of inducible T7 lac promoter. Initially, LB medium was used for the production and optimization of various cultivation parameters to get the high level expression of both recombinant enzymes. However, optimal expression and activity of both enzymes were observed when culture induced with 0.5 mM IPTG after heat shock treatment (42°C, 1 h) at 0.6 pre-induction optical density (OD600nm) followed by incubation at 22°C for 72 h in a shaking incubator at 200 rpm. Both extracellular TnBglA and TnBglB with a molecular weight of 51.50 and 81.14 kDa, respectively were purified to homogeneity by ion-exchange and hydrophobic interaction chromatography after heat treatment at 70°C for 1 h. Purified enzymes TnBglA and TnBglB displayed optimal activity at pH 7.0 (95°C) and pH 5.0 (85°C temperature), respectively. Both enzymes were quite stable over a broad range of pH (6.0-8.5) and temperature (60- 90°C), fairly stable up to 8 h at 80°C. GH1 TnBglA activity was stimulated in the presence of glucose and xylose, with a Ki value of 1200 and 1300 mM, respectively. Whereas, GH3 TnBglB revealed Ki value of 150 mM for glucose and 200 mM for xylose, both enzymes displayed affinity towards p-nitrophenyl substrates and cellobiose. Km, Vmax and kcat values of TnBglA, using pNPG as substrate, were 1.5 mM, 297 mmol mg- 1min-1, and 1527778 s-1, respectively. Whereas, TnBglB showed Km, Vmax and kcat values of 0.45 mM, 153 mmol mg-1min-1 and kcat 1214285.7 s-1, respectively using pNPG as substrate. Thermodynamic parameters as ΔH*, ΔG* and ΔS* for pNPG hydrolysis by TnBglA were calculated at 95°C as 25.7 kJ mol-1, 47.24 kJ mol-1 and -58.6 J mol-1 K-1, respectively. Thermodynamic parameters for pNPG hydrolysis by TnBglB like ΔH*, ΔG* and ΔS* were calculated at 85°C as 24.1 kJ mol-1, 46.55 kJ mol-1 and -62.74 J mol-1 K-1, respectively. TnBglA displayed a half-life (t1/2) of 5.21 min at 97°C with denaturation parameters of enzyme including ΔH*D, ΔG*D and ΔS*D were 662.04 kJ mol-1, 110.10 kJ mol-1 and 1.491 kJ mol-1 K-1, respectively. TnBglB showed a half-life (t1/2) of 4.44 min at 94°C with denaturation parameters of enzyme including ΔH*D, ΔG*D and ΔS*D were 283.78 kJ mol-1, 108.7 kJ mol-1 and 0.477 kJ mol-1 K-1, respectively. Generally, inadvertently preparing medium and unintentional induction of engineered E. coli BL21, give poor or variable yields of heterologous proteins. Therefore, to enhance the activity and production of an industrially relevant cloned enzymes through various cultivation and induction strategies. High-cell-density and optimal heterologous proteins expression were obtained in 4×ZB medium after 72 h inducement at 22°C when culture gave heat shock (at 42°C for 1 h) at 0.6 OD600nm and induced either with 0.5 mM IPTG/150 mM lactose. TnBglA and TnBglB activities were enhanced 3.8 and 0.096 fold in 4×ZB medium with 150 mM lactose, respectively under optimal conditions. However, considerably greater dry cell weight of TnBglA and TnBglB cultures were 11.30 g DCW L-1 and 11.08 g DCW L-1 in 4×ZB medium, respectively induced with 150 mM lactose. Use of the inexpensive and non-toxic inducer lactose, and an effective process strategy is essential to achieve a high level of enzyme expression. The expression and purification scheme, presented here, has a potential of scaling up to obtain pure and active enzymes, relatively economical for further studies and other applications. Finally, this is a report on enhanced production of highly heat active cloned β-glucosidases from T. naphthophila (TnBglA and TnBglB) with high catalytic efficiency and low product inhibition, which have excellent tolerance against glucose and xylose, and also exhibit independence of detergents, chemical inhibitors and metal cations. All these significant features make both TnBglA and TnBglB appropriate candidates for biotechnological and industrial applications.
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۔ خاندان مراتب اور ادب

خاندان مراتب اور ادب

                سیّد مراتب علی اختر کے اجداد سے لے کر اب تک گیلانی سادات ادب کے پودے کی آبیاری میں مصروف ہیں۔ یوں تو تصوف اور ادب کا چولی دامن کا ساتھ ہے اس لئے اہل تصوف کا ادب شناس اور صاحب ذوق ہونا لازمی امر ہے مگر اس خاندان کی ہستیاں جنھوں نے خود شعر کہے ان کا ذکر کچھ یوں ہے۔

حضرت شیخ غوث محمد اُچی

                آپ سادات شیخو کے جدّامجد ہیں آپ صاحبِ دیوان شاعر تھے اور آپ کا دیوان ’’دیوانِ قادری‘‘ کے نام سے موجود ہے۔(۱)پروفیسر ڈاکٹر محمد جمیل قلندر اس پر تحقیق کر رہے ہیں۔آپ مشاہیر و اکابرسادات حسنی ہیں حضرت غوث ِاعظمؒ سے نسبت آبائی ہے۔ صاحبِ عظمت و کرامت، واقفِ منقول ومعقول تھے۔ عبادت و ریاضت اور زہدوورعمیں یکتائے روزگار تھے۔

سیّد اصغر علی گیلانی صاحبِ شجرۃ الانوار رقم طراز ہیں کہ سیّد محمد کے بزرگوں میں سے اوّل سید ابوالعباس بن سید صفی الدین سیدعبدالقادر جیلانیؒ اپنے چھوٹے بھائی سید ابو سلیمان کے ساتھ 656ہجری میں ہلاکو خاں تاتاری کے حملہ بغداد اور قتل و غارت کے وقت بغداد سے نکل کر روم آگئے۔ پھر جب کچھ امن و امان ہوا تو حلب میں آکر اقامت گزین ہوگئے۔ سید محمد غوث یہیں پیدا ہوئے۔ تعلیم و تربیت اپنے والد سے حاصل کی۔ عنفوان شباب میں پدر بزرگوار کی اجازت سے مختلف ممالک اسلامیہ کی سیر و سیاحت کو نکلے ، حرمین الشریفین کی حج و زیارت سے مشرف ہوئے۔ عراق، عرب، ایران، خراسان، ترکستان اور سندھ و ہند کی طویل سیاحت کی۔ یہاں کے اکابر علماء وفضلا اور مشائخ و صوفیا سے ملاقاتیں کیں۔ لاہور بھی تشریف لائے۔چندے یہاں قیام کیا پھر ناگور چلے گئے۔ یہاں ایک مسجد تعمیر کی۔...

Conflict Between Religions a Study of Modern Approaches in Sῑrah Writing

The world has been changing ever since its creation, yet the pace of change in the last one hundred years or so has been the most rapid ever. The effects of these changes were beyond the limitations of time and region and therefore they directly affected the Muslim world as well. Muslim scholars did not ignore these changes and realized their responsibilities and wrote books of Sīrah which provided guidance in connection with these rapid changes and conditions. This research has highlight an important issue of the conflict between religions, its inception, history and primarily focused on the opinions of the authors of selected books of Sῑrah written in recent past. This research has also elaborated the modern approaches in Sῑrah writing. The study has mainly focused on significant Sῑrah books of three languages i.e. Arabic, English and Urdu. These books include Fiqh Al Sῑrah by Muḥammad Sa’īd Ramaḍān Al Būtī, Fiqh Al Sῑrah by Muḥammad Al Ghazālī, Madnī Mu‘āshrah by Akram Ḍīā Al ‘Umrī, The Life and the Work of the Prophet by Dr. Muḥammad Ḥamīdullāh, The Spirit of Islam by Sayīd Amīr ‘Alī, Muhammad A Biography of Prophet by Karen Armstrong, Sīrah Al Nabī by ‘Allāmah Shiblī Nu’mānī, Aṣaḥ Al Sῑyar by ‘Abdul Raūf Dānāpūrī, Ḍīā Al Nabī by Pīr Muḥammad Karam Shāh, Sīrati Sarwari ‘Alam by Abūl A‘lā Mūdūdī, Raḥmatullil'ālamīn by Qāḍī Muhammad Sulymān Manṣūrpūrī and Muḥammad Rasūlallāh by Sayīd Muḥammad Mīyān.

Study on Glucan Producing Potential of Members of Oral Viridans Streptococci

Viridans group streptococci (VGS), normal inhabitant of oral cavity, are involved in a number of oral and extra-oral diseases. Among oral diseases, dental caries is the most common public health problem throughout the world. The cariogenicity of oral streptococci is attributed to their acidogenic potential and glucan (extracellular polysaccharide) production. For the study, 552 subjects were selected at random from different localities of Karachi, Pakistan. The subjects were categorized as carious (29.5%, 163) and non-carious (70.5%, 389) with respect to prevalence of dental caries. The carious and non-carious subjects were further categorized with respect to sex and sociodemographic characteristics i.e. age (<20, 21-40, 41-60, 61-80 and 81-100 years), socioeconomic status (SES) based on monthly income (low, middle and high), marital status (married and unmarried), dietary habits [daily consumption of vegetable, meat, fruit, mixed diet (vegetable and meat), milk and milk products], use of tobacco products, use of betel quid products, chewing habits, use of tea and oral hygiene practices. Overall, the prevalence of dental caries was found higher in males (58.9%, 96/163) as compared to females (41.1%, 67/163). The same higher trend of prevalence of dental caries was observed for all categories with respect to sociodemographic characteristics except age where prevalence of dental caries was higher in female of 61-80 years age group. In the present study, 525 isolates belonging to 09 different species of VGS were obtained from oral cavity of 552 subjects. Streptococcus anginosus was the most abundant (53.5%) followed by S. mutans (14.5%), S. mitis (11.4%), S. uberis (6.5%), S. intermedius (4.6%), S. sanguinis (3.8%), S. oralis (3.4%), S. salivarius (1.3%) and S. acidominimus (1%). A preliminary screening to determine the glucan producing potential of all isolates of VGS was performed. Out of 525 isolates, 41.5% were glucan producers while 58.5% were glucan non-producers. Species-wise comparison revealed the highest frequency of S. mutans (80.3%) exhibiting glucan producing potential followed by S. oralis (61.1%), S. intermedius (50%), S. anginosus (36.7%), S. mitis (35%) and S. salivarius (14.9%). None of the isolates of S. uberis and S. acidominimus showed the ability to produce glucan. The frequency of isolates having glucan producing potential was observed higher from carious subjects (46.3%) as compared to non-carious subjects (39.1%). Whereas, species-wise distribution of isolates indicated that the prominent glucan producing species were S. sanguinis (100%, 3/3) and S. mutans (76%, 22/29) from carious and S. mutans (83%, 39/47) from non-carious subjects. The glucan producing potential of VGS was also estimated quantitatively. S. sanguinis produced largest quantity (276.2 mg mean, 206.2-324.6 range) followed by S. mutans (143.5 mg mean, 43.5-521.1 range). The antibiotic susceptibility pattern of 525 isolates of VGS was also evaluated against 24 antibiotics viz., penicillin, amoxicillin, cephalothin, cefazolin, cefotaxime, ceftazidime, imipenem, chloramphenicol, clindamycin, trimethoprim, azithromycin, erythromycin, clarithromycin, ciprofloxacin, levofloxacin, doxycycline, tetracycline, gentamicin, streptomycin, tobramycin, teicoplanin, vancomycin, rifampin and linezolid. The highest incidence of resistance (48.4%) was observed against erythromycin. Overall, 39.2% isolates were susceptible to all the antibiotics used for the study. The emergence of multi-drug resistance (MDR) was noted among remaining 60.8% isolates. The resistance rate was observed as 5.5% isolatesresistant to 1 antibiotic, 4% isolates to 2 antibiotics, 6.3% to 3, 3.2% to 4, 2.5% to 5, 5% to 6, 2.9% to 7, 2% to 8, 4.2% to 9, 1.7% to 10, 2.1% each to 11 and 16, 2.7% to 12, 6% to 13, 2.9% to 14, 4% to 15, 2% to 17, 0.4% each to 18, 21 and 24, 0.8% to 19 and 0.2% to 20 antibiotics. In the present study, the in vitro antibacterial activities of oil, aqueous infusion and aqueous decoction of clove buds (Eugenia caryophyllata) and aqueous infusion and aqueous decoction of miswak (Salvadora persica) were determined against isolates having glucan producing potential viz., S. anginosus (19), S. mutans (10), S. mitis (14), S. intermedius (10), S. sanguinis (10), S. oralis (10) and S. salivarius (07). The highest antibacterial activity was noted for clove oil as all the isolates were found susceptible. It exhibited strong antibacterial activity against S. mutans (20.2mm mean zone of inhibition±3.4SD) from carious subjects and S. sanguinis (17.7mm mean zone of inhibition ±1.1SD) from non-carious subjects. The aqueous decoction of clove buds exhibited the highest zone of inhibition against S. mutans obtained from carious subjects (16.8mm±3.4SD) followed by non-carious subjects (14.3mm±2.3SD). The aqueous infusion of clove buds showed the highest zone of inhibition against S. mutans isolated from carious (13.2mm±3.2SD) and non-carious (13.6mm±4.2SD) subjects. Aqueous infusion and aqueous decoction of miswak failed to inhibit the tested VGS. The MICs and MBCs of the clove oil, aqueous infusion and decoction of buds of clove against VGS was recorded as 5 – 0.625%, 5% and 5 – 2.5%, respectively. The effect of clove oil and aqueous infusion of miswak was also evaluated on glucan production. The VGS isolates producing large amount of glucan were selected for the study. The effects of different concentrations of clove oil (0.5%, 0.25%, 0.125%, 0.0625% and 0.0313%) and aqueous infusion of miswak (10%, 8%, 5%, 2.5%, 1.25% and 0.5%) was assessed for the synthesis of glucan. The different concentrations of herbs preparations exhibited varying degree of reduction in glucan production. Clove oil was more effective and reduced the formation of glucan to approximately 80% in a dose dependent manner.