Effects of Structural Modification on the Thermostability of F1 Protease from Bacillus Stearothermophilus F1

A thermophilic Bacillus stearothermophilus F1 was found to produce an extremely thermostable serine protease. The F1 protease sequence was modeled onto the crystal structure of thermitase with 61% sequence identity. The F1 protease contains a catalytic triad comprising of Asp39, His72 and Ser226. The predicted structure of F1 protease comprised 10 α-helices and 10 β-sheets arranged in a single domain. Comparison of the predicted 3D structure of F1 protease with the crystal structure of serine proteases from mesophilic bacteria and archaea, led to the identification of features related to protein stabilization. Higher themostability was found to be correlated with an increased number of residues involved in ion pairs or networks of ion pair. In order to investigate F1 protease stability, two mutated (W200R and D58S) F1 protease were designed. The analysis of molecular dynamics simulations of W200R mutant revealed that an additional three new ion pairs between Arg200 and Asp202, however there was no ion pair interaction at position Ser58. To confirm the role of ion pair, site-directed mutagenesis was carried out. Both mutated F1 proteases were designed, cloned into pGEX-4T1 and expressed in E. coli BL21 (DE3) pLysS. The optimum expression level for the wild type F1 protease, W200R and D58S mutants were 94 U/mL, 112 U/mL and 68 U/mL, respectively. The wild type F1 protease, W200R mutant and D58S mutant were purified by affinity chromatography and heat-treatment. A single band was visible at SDS-PAGE at approximately 33.5 kDa. The purified wild type, W200R mutant and D58S mutant showed 95%, 115% and 64% recovery with purification fold of 21.7, 33.8, and 17.2, respectively. In the presence of 2 mM CaCl2, the wild type had half-lives of 60 min and 7 min at 85 ºC and 90 ºC. Meanwhile, the W200R mutant had half-lives of 75 min and 12 min at 85 °C and 90 ºC, which was more stable than wild type. The stability of W200R mutant was 1.25 times higher than that of the F1 protease. The enhanced thermostability can be correlated to the increase in the number of residues involving ion pairs and ion pairs networks. In contrast, the D58S mutant showed half-life of 45 min at 85 ºC but there was no enzymatic activity at 90 ºC. Thus, the D58S mutant was less thermostable than that wild type which could be due to the removal of ion pairs on this mutated F1 protease (computational work). Far-UV CD at 221 nm was used to detect the denatured proteins. As the temperature was increased from 50 °C to 90 °C, the change in ellipticity at 221 nm revealed a sigmoidal monophasic transition curve of mutant’s proteins which indicated unfolding of a protein.The melting point at pH 8.0 of the wild type F1 protease, W200R mutant and D58S mutant were 70 °C, 72 °C and 63 °C, respectively. Far-UV CD measurements studies indicated the overall features of the secondary structure of protein. The wild type contains 25.5% of α-helix, 17.7% of β-sheet, 22.5% of turn and 34.0% of random coil. There was some loss of β-sheet in W200R but a 3.8% and 1.7% increase in an α-helix and a random coil, respectively, indicated that Arg200 stabilized the α-helix content. The β-sheet was reduced to less than 4% and random coil rose up to more than 8%. A small decrease in the α-helix was observed in D58S mutant. This could be due to the substitution of Asp58 to Ser causing disruption of four ion pairs between Asp58 and Arg103, located at β-sheet. The results obtained confirmed the important role of intermolecular ion pairs in the stability of the whole structure of F1 protease. These results also showed that simulation procedures and molecular biology techniques can be used together to direct protein engineering and/ or site-directed mutagenesis

Factors Influencing The Purchased Of Products Or Services Through The Internet

Internet shopping has received considerable attention in the popular issue as the future of in-home shopping. The Decision to attend for shopping behavior is a complex and many factors will significantly influence the consumer choice between a retail store and lnternet shopping mall. The probability of adopting Web retailing by private lnternet users may depend on, not only the characteristics of the products and services being marketed, but also the other characteristics of the consumers who see the Web as a viable option for their retailing activity. In this paper, the main aim of the study is to investigate what the factors influencing the purchased of product or service through the lnternet. The multistage factors that requires the consumer to consider such factors as shopping experience, time consideration, price perception, desire for comfort, risk perception and safety and security concern. A total of 160 consumer from various level in the Kuala Lumpur were selected as a respondents of the study. Correlation analysis were used to seek for the significant relationship between the choice decision the variables and also to seek for the significant relationship between the demographic profile and the choice decision to purchased product or services through internet. The Descriptive analysis also used to see the percentage and standard deviation for each factors. The result showed that all of the factors such as shopping experience, time consideration, price perception, desire for comfort, risk perception and safety and security concern are significant at the consumer choice decision to purchased product or services through lnternet. The finding of this study in general will be beneficial to consumer to make a choice for purchased product or services either through lnternet or in-store based on the factors above

Intellectual property rights and agro-based natural product: Malaysian legal perspective

Malaysia is a country which has been blessed with plethora of natural resources and product such as plants and animals. It is an established fact that the agriculture sector part of our natural product contributed substantially to the growth and development of the Malaysian economy. At the same time, it falls as part of the property which must be protected to ensure its sustainability. This paper will look into the right and protection of agro-based natural product specifically to the plant varieties and geographical indications from Intellectual Property Rights perspective in Malaysia

Memahami Reka Bentuk Sejagat: Kajian Kes Di Taman Permainan Mudah Akses, Taman Perbandaran Pulau Pinang

Kajian ini bertujuan untuk memberi pendedahan tentang Reka Bentuk Sejagat (RBS) kepada para pelajar Tahun Dua (semester ketiga) di dalam bidang Rekabentuk Produk di dalam subjek Ergonomik. Kefahaman mereka tentang topik RBS ini akan dinilai diakhir kursus.Kajian literatur berkaitan dengan RBS dan kepentingannya dalam mereka bentuk produk di persekitaran awam serta Orang Kurang Upaya (OKU) dan taman permainan turut dikenalpasti. Kajian lapangan ini mengaplikasikan pendekatan kajian secara kualitatif dengan menggunakan kaedah Pemerhatian Turut Serta dan Temubual Kumpulan Fokus yang melibatkan seorang OKU Buta, dua orang OKU fizikal yang berkerusi roda, dan dua orang kanak-kanak normal, pereka bentuk dari syarikat pemaju dan Presiden Persatuan Sukan dan Rekreasi OKU Negeri (PESRON) Pulau Pinan

The effect of human resource practices on organizational citizenship behavior

Many researchers conducted study in the area of organizational citizenship behaviour but there is little research and less focus on service organizations, particularly educational institution. The study is a replication of Veldhoven and Meijman (1996), Johnson (1996), Lytle, Hom and Mokwa (1998), Weer (2006), Podsakoff and Mckenzie (1994) which adapting 25 item instruments on participation in decision making, training and development and career development that will impact on the exhibition of organizational citizenship behaviour among administrative staff in the university. The study aims to understand the relationship of human resource practices on organizational citizenship behaviour within the context of university as educational institution. Based on the analysis it was found that career management and training made significant contributions to organizational citizenship behaviour. Recommendations and implications for future research and practice were also discusse

Flexural strength of concrete prism strengthened using CFRP with modified mortar

Reinforced concrete (RC) is a good durable material that commonly used in construction compared to others. However, reinforced concrete structures may be damaged and are suffering from various deteriorations because of overloading and an inadequacy of design which may be attributed to cracks, spalling, large deflection, and more. Hence, it is essential to find repair and strengthening techniques of deteriorated concrete structures not only suitable in terms of the economy, but also to use them for their intended service life

Kontrak e-dagang: perbandingan aqad (kontrak) dalam muamalah Islam

Secara umumnya aktiviti jual beli secara e-dagang merupakan suatu instrumen jual beli yang sangat diperlukan oleh masyarakat zaman globalisasi kini. Ini kerana jual beli edagang lebih kepada menginterpretasikan jual beli dalam bentuk yang lebih moden dan kompleks. Pengiktirafan undang-undang di Malaysia tentang keesahan mesej elektonik dalam urusan perdagangan dan keusahawanan bagi memenuhi tuntutan undang-undang dan untuk memudahkan urusniaga perdagangan melalui penggunaan media elektronik telah diperuntukkan di bawah seksyen 6 Akta Perdagangan Elektronik 2006. Justeru itu, artikel ini akan membincangkan teori caqad (kontrak) dalam muamalah Islam dan konvensional. Perbincangan akan difokuskan kepada konsep kontrak yang diamalkan dalam transaksi jual beli e-dagang dan caqad jual beli dalam transaksi muamalah Islam dengan membuat kajian perbandingan antara kedua-duanya. Penilaian secara kritis terhadap keseluruhan elemen dalam kontrak e-dagang akan dilakukan bagi memastikan sama ada ia bertepatan atau tidak dengan kontrak yang dikehendaki oleh syarak. Justeru itu diharapkan artikel ini akan memberi keyakinan kepada umat Islam tentang kepentingan jual beli transaksi muamalah semasa melalui kontrak e-dagang dalam usaha mempertingkatkan lagi perkembangan muamalah Islam di Malaysia

Kementerian Tambah 200 Tempat Pengajian Perubatan

Kementerian Pengajian Tinggi akan menambah 200 tempat bagi mengikuti pengajian perubatan di sembilan institusi pengajian tinggi awam (IPTA) yang menawarkan program itu sebagai langkah menyelesaikan masalah kekurangan doktor di negara ini, mulai tahun depan

Personal representative cum trustee in estate administration: conflict of duties and obligations

The office of personal representative in the administration of a deceased’s estate requires a personal representative who must at the utmost ensure himself to have a solemn understanding of his duties and obligations. The personal representative must act with the highest degree of trustworthiness and good faith in discharging his office. However, in the process of estate administration, one question that has nagged the office of personal representative is that what should be the two distinct capacities of personal representative and trustee since the expression of trustee under the Malaysian Trustee Act 1949 includes the duties incidental to the office of a personal representative. The question is relevant because the role of a personal representative is quite distinct from that of a trustee and is subject to different legal framework and liability. Hence, this paper seeks to examine the legal position of a personal representative and to identify the line separating the duties of a personal representative from his duties as a trustee. For this purpose, the paper analyses relevant statutory provisions as provided by the Probate and Administration Act 1959 and the Trustee Act 1949. An analysis of the decided cases is also made to identify the distinction between the offices of a personal representative to that of a trustee in the administration of the deceased’s estate. The paper concludes that there is no objective test as to when and how a personal representative becomes a trustee since a person may be a personal representative in respects of certain assets and trustee in respect of others. What matter most is that they must carry out their job diligently and reasonably in which failure to do so will result in their facing legal action by the beneficiary or other interested parties