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Molecular Characterization of Thermo Stable Proteases from Thermophilic Bacterial Strains

Thesis Info

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Author

Majeed, Tanveer

Program

PhD

Institute

Pakistan Institute of Engineering and Applied Sciences

City

Islamabad

Province

Islamabad.

Country

Pakistan

Thesis Completing Year

2016

Thesis Completion Status

Completed

Subject

Biotechnology

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/13207/1/Tanveer_Majeed_HSR_Biotechnology_2016_PIEAS_08.08.2016.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676726692446

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Thermophilic microorganisms have gained world-wide importance due to their tremendous potential to produce thermostable enzymes that have wide applications in pharmaceuticals and industries. Proteases are such enzymes which account for nearly 60% of the total world-wide enzyme sales. Thermostable proteases are of greater advantage in applications because they do not only denature at high temperatures, but they also remain active at such temperatures. This project explores the production and biochemical and molecular characterization of the thermostable proteases from thermophilic bacterial strains. The growth conditions of Bacillus subtilis strains in submerged cultivation have been investigated to understand fermentation behavior of the microorganism and the production pattern of the thermostable enzyme. The optimum protease production (178 U/mL) was observed with 1% casein substrate after 48 h of cultivation at 60°C from Bacillus subtilis BSP in shake flasks study. The optimization of various factors affecting the protease production was statistically determined by Box Behnken design based on response surface methodology (RSM). The simultaneous effects of four test interacting factors on the protease production were monitored and conditions were optimized for maximum enzyme production. Optimized cultivation media formulation included: initial pH 8, casein concentration 2% (w/v) and inoculum density 1% when fermentation was carried out for 48 h. The enzyme was 9.8 fold purified in a 2-step procedure involving ammonium sulfate precipitation, DEAE cellulose and Sephadex G-200 gel permeation chromatography. The enzyme was shown to have a molecular weight of 36 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). It was most active at 50°C, pH 8, with casein as substrate. This enzyme was 100% stable at 50°C and retained its 82% activity at 60°C after 30 minutes of incubation. The enzyme became completely inactivated after incubation at 80°C for 3h. It was strongly activated by metal ions such as Ca2+ and Mn+2. Enzyme activity was inhibited strongly by ethylene diamine tetra acetic acid (EDTA) confirming its identity as a metalloprotease. Protease was not only stable in the presence of organic solvents and surfactants studied but it also exhibited a higher activity than in the absence of some surfactants. Consequently, the DNA encoding the thermostable protease of Bacillus subtilis BSP has been identified. The gene contained an open reading frame of 1638 bp and encoded for a mature peptide sequence of 318 amino acid residues. The protease gene was cloned and expressed in E.coli expression system and the recombinant protease was purified. Sequence analysis showed that this protease has close homology with thermolysin class of proteases. Primary structure analysis of thermostable protease showed 35% of its content to be alpha helix making it stable for three dimensional structure modeling. Homology model of thermolysin has been constructed using swiss model as the workspace. The model was validated by ProSA and RMSD. The results showed the final refined model is reliable. It has 0.06 Å as RMSD and has -2.19 as Z-score. For expression in Bacillus subtilis 1A751 two integration vectors (pDR111 and pSG1154) were compared in order to get the higher extracellular protease yield. 1A751/pDR-BSP-MprT transformant was found to be secreted higher amount of thermostable protease in the culture medium when compared to 1A751/pSG-BSP-MprT strain. The recombinant enzyme was undergone for further studies to check its stability under native enzyme conditions and it was found to be as stable as native protease BSP-MprT. The thermostability of Bacillus subtilis BSP protease was improved by two rounds of error prone PCR mutagenesis. A random mutant library of Bacillus subtilis BSP protease was generated by ep-PCR. The generated mutants have been successfully expressed in E. coli. The mutant proteases obtained in this mutant library were screened for increased protease thermostability. The SDS-PAGE pattern of enzyme was exhibiting a well-defined band (36 kDa). In this study, the thermostability was enhanced from 60°C to 80°C by the single mutation Gly347Cys which has a stabilizing effect on the irreversible thermal inactivation. The BSPmutant has exhibited increase in thermal stability and 2.7 folds half-life at 80°C when compared to wild type protease. Sequence analysis and comparison of both wt-BSPMprT and Mutant-BSP-MprT showed that wt-BSP-MprT had only one cysteine residue at Cys518 position while another cysteine has substituted Gly347 in MutantBSP-MprT. Introduction of another cysteine resulted in the formation of disulfide bridge between the two cysteine residues which play important role in the stability of protein tertiary structure. The inter subunit disulfide bond was estimated to be one of the factors for thermal stability. Another putative alkaline serine thermostable protease gene (aprE) from the thermophilic bacterium Coprothermobacter proteolyticus was cloned and expressed in Bacillus subtilis. The enzyme was determined to be a serine protease based on inhibition by PMSF. Biochemical characterization demonstrated that the enzyme had optimal activity under alkaline conditions (pH 8–10). In addition, the enzyme had an elevated optimum temperature (60°C). The protease was also stable in the presence of many surfactants and oxidant. Thus, the C. proteolyticus protease has potential applications in industries such as the detergent market.
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مولانا انعام الحسن کاندھلوی

مولانا انعام الحسن کاندھلوی مرحوم
امیرالتبلیغ مولانا انعام الحسن کاندھلوی شنبہ ۱۰؍ جون بروز عاشورہ محرم اپنے مالک حقیقی سے جاملے، اناﷲ وانا الیہ راجعون۔
اس دور قحط الرجال میں ان کی وفات قوم و ملت کا بڑا جانکاہ حادثہ ہے، وہ کاندھلہ ضلع مظفر نگر کی مردم خیز بستی کے صدیقی شیوخ کے اس مشہور خاندان سے تعلق رکھتے تھے جس میں کئی پشتوں سے اہل علم و فضل اور اصحاب رشد و ہدایت پیدا ہوتے رہے ہیں، شاہ عبدالعزیز محدث دہلوی کے نامور و محبوب شاگرد مفتی الہٰی بخش اسی خاندان کے جد امجد تھے، اس خانوادے کے افراد حضرت سید احمد شہیدؒ کی تحریک جہاد و احیائے اسلام میں بھی پیش پیش رہ چکے ہیں، علم و عمل کی اسی جامعیت اور بلند نظر و علوئے ہمت کی خاندانی روایت نے اس خاندان کو مولانا محمد الیاسؒ اور ان کے صاحبزادے مولانا محمد یوسفؒ کے سے داعیان حق بخشے جو اسلام کی تبلیغ و اشاعت، خلوص و ﷲیت اور تقویٰ و بے نفسی میں نمونہ سلف صالحین تھے۔
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Isolation and Characterization of Cytolytic Protein Gene from Local Isolates of Bacillus Thuringiensis

Chemical insecticides are widely used to control the insects, these insecticides are not environmentally healthy as they are not biodegradeable and hence are biomagnified. These insecticides are also not host specific; they also kill the beneficial insects. So there is a need to search for a control agent which should not harm the environment and also to human beings. Several different methods have been used in recent past to control the insects which include use of pheromones for trapping or disruption of mating behavior, insect growth regulators that interfere with larval development, parasitoids, fungi, viruses and bacteria, which debilitate or cause death in the infected insect. One of the most successful biological control organisms is a naturally occurring bacterial pathogen, Bacillus thuringiensis generally known as B.t. Formulations based on B.t. have been used for decades as biological insecticides for agriculture and forestry, as well as for vector control against mosquitoes and black flies. Interest in B.t. proteins has increased during the last two decades because of their unique qualities which are unmatched by any conventional insecticide. Of the 297 genes known to encode B.t. proteins, some share a high degree of homology, while others have diverse nucleotide sequences. Because of the interest in B.t., the list of new B.t. subspecies is growing as is the group of economically important target insects. B.t. produces crystal proteins during sporulation. These crystal proteins are of two types Cry and Cyt. Both of these types of proteins are different in their mode of action. Cytolytic proteins have an additional property of having cytolytic activity against different cells and also against mammalian erythrocytes. These proteins especially Cyt proteins are active against mosquito larvae. Recent studies reported the development of resistance in mosquitoes against Cry toxins. Researchers tried different methods to overcome this resistance and they found that when Cyt proteins are used in combination with Cry proteins they greatly reduced the resistance of mosquitoes against B.t. toxins. This indicated that Cyt proteins work synergistically with Cry proteins. In the present study, soil samples collected from different areas of Lahore, Kasur and Faisalabad. A total of 50 soil samples were collected, these soils were rich in organic manure. B.t. like bacteria were isolated from these soil samples using differential medium containing sodium acetate buffer. These isolates were then subjected to biochemical characterization by performing biochemical tests. The expected B.t. like isolates were screened for the presence of cyt genes. After confirmation of presence of cyt 2B gene, mosquitocidal activity of these isolates were checked by using B.t. spores and total B.t. cell proteins against 3rd instar larvae. From the bioassays, it was found that NB B.t.4 was found to be most toxic with LC50 value of 400±1.15 μg/ml and 68±0.46 μg/ml for its spores and total cell protein, respectively. After bioassays, six most toxic B.t. isolates were then selected for further study. Ribotyping of these isolates was done to amplifying 16S rRNA gene to identify these isolates. Protein profile of these isolates was checked to confirm the presence of 29 kDa protein band. Full length cyt 2B gene was amplified, cloned in pTZ57RT cloning vector, and pET22b vector was used for expression. IPTG induction of 1 mM was found good for expression ranging incubation time of 4-6 hours. Expressed protein was then purified by anion exchange chromatography. Bioassays were performed using recombinant organism (E. coli transformed with cyt 2B gene), expressed crude protein and purified protein. It was found that the purified protein was most toxic with LC50 Value of 50±1.68 μg/ml.