Protein-Solvent Interaction of Thermostable Lipase for Biocatalysis in Organic Solvents

A high expression of a solvent stable enzyme enables characterization of it solvent stability profiles which could aid in many aspects of structural studies in understanding protein-solvent interaction. A thermostable and solvent stable lipase (Lip 42) gene previously isolated from Bacillus sp. strain 42 was subcloned into pET-51b. High expression was achieved using this vector which employs T7 promoter, using E. coli host strain BL21(DE3)pLysS. Expression was achieved at 160 U/mg protein, after 25 hour incubation at 37°C using 0.5 mM isopropyl β-d-thiogalactopyranoside (IPTG). Lip 42 was purified using Strep-tag affinity chromatography and it molecular weight size is 43 kDa by SDS-PAGE. Studies on purified lipase in solvent with different polarities (log P values) showed that it was generally stable in water miscible solvents such as DMSO, DMF, ethanol, and methanol; and in apolar solvent such as n-hexane. The enzyme retained high residual activities in 45 to 60% v/v hydrophilic solvents, and also some enhancements (> 100%) were observed in low DMSO compositions (15-30% v/v). Stability preference in water miscible solvents would make Lip 42 lipase a suitable enzyme to be employed in bio-diesel production. Structural characterizations of the enzyme in different solvent compositions were carried out using fluorescence spectroscopy, and also by using both far- and near- UV circular dichroisms (CD). Both secondary and tertiary structures were retained in low solvent ( 60% v/v, the tertiary structure was perturbed accompanied by the formation of molten globule (MG), or an expanded helical structure state. Far-UV CD studies in methanol indicated the conserved secondary structure with an increase in α-helices, and decreased in β-sheets. Near UV-CD spectra in low methanol and DMSO compositions (30 - 45% v/v) resembled the native protein. At solvent > 60% v/v, the distinct tertiary structure perturbation was observed each in methanol and DMSO. Intrinsic fluorescence spectra in both solvents showed blue shifts at 0-45% v/v indicating a buried Tryptophan, and at > 60% v/v showed red shift indicating an exposed Tryptophan. Extrinsic fluorescence studies showed the possible formation of inactive molten globule (MG) at > 60% v/v solvent. In this state, the collapse of tertiary structure with an intact secondary structure was manifested by the loss in biological function. Based on solvent stability profiles, molecular dynamic (MD) simulations were run in the presence of water, 60% v/v DMSO + 40% v/v water, and 100% v/v DMSO.Structural (RMSD) and flexibility (RMSF) changes indicated that the major changes in the lid involving two helix-loop-helix motif loops. In 60% v/v DMSO, the gap between the loops was narrower and there was a collapse of a nearby hydrophobic cluster. However, the cluster was still seen in water and neat (100% v/v) DMSO. Consequently, the H-bond interaction and hydrophobic cluster region are important elements in protein solvent interaction. A site-directed mutation on the lid region (V171S) with residue Ser 171 replacing Val (hydrophobic to polar) impaired the enhancement in low solvent compositions. This effect was more pronounced at higher pre-incubation temperature (50°C), showing 120% reduction from the amount achievable by the native enzyme. This indicated the crucial role of the hydrophobic residue on helix-loop-helix motif in providing the hydrophobic effect pre-requisite for interfacial activation mechanism. In conclusions, these studies provide better understanding in protein solvent interaction and suggest a suitable parameter in rational design strategy for a better non-aqueous catalysis

Lactococcus lactis strain FA1, FA2, FA3 and FA4 partial sequence of 16S ribosomal RNA gene isolated from Black tip shark (Carcahinus Limbatus)

This is a collection of partial sequence of 16S ribosomal RNA gene for Lactococcus lactis strain FA1, FA2, FA3 and FA4 isolated from Black tip shark (Carcahinus Limbatus). the sequences was submitted into NCBI genbank database and registered with accession numbers

Investigation on plasmid DNA separation under vacuum suction using cellulose generated and polyethersulfone ultra filters

E. coli DH5α harbouring pET28a+ plasmid vector was lysed by alkaline lysis and the clarified plasmid solutions were subjected to ultrafiltration experiments using two types of ultrafiltration membrane; i.e., cellulose generated (Ultracel) and polyethersulfome PES (Biomax). Transmembrane pressures (TMP) through vacuum suction of 0.1 to 0.7 bar were applied to the plasmid sample, and the corresponding flow rates and fluxes for both filters were investigated. Even though, these two filters showed a slight different in the flux, a marked different in DNA transmission were observed. DNA transmission were generally higher with cellulose generated filters, whereby DNA transmission by Ultracel filter was at44.3, 63.9, 74.1, 55.6 and 54.5% at TMP of 0.1, 0.18, 0.39, 0.6 and 0.7 bar, respectively. Meanwhile, for PES filters the DNA transmission was at 22.3, 38.1, 39.1, 38.0 and 37.5% at TMP of 0.1, 0.3, 0.4, 0.6 and 0.7 bar, respectively. With the cellulose generated filter, DNA transmission reached an optimum (~70%) at about 0.6 bar after which the transmission depleted at higher TMP of 0.7 bar. Throughout all of the TMP, DNA transmissions observed were generally lower with PES filter. The properties of the filter material could have contributed to the differences in DNA permeatio

Manfaat bioinformatik percepat rungkai, cari penawar virus

Melalui sampel seorang pesakit di Wuhan, saintis menerusi kaedah penjujukan generasi baharu memperoleh jujukan lengkap asid deoksiribonukleik (DNA) genom virus SARS-Cov-2. Maklumat ini mendedahkan SARS-Cov-2 hampir menyamai virus terdapat pada kelawar (96 peratus) dan tenggiling (90 peratus). Berdasarkan maklumat protein pepaku (spike), SARS-CoV-2 dikatakan menggunakan tenggiling sebagai perumah perantara

Keharmonian kerajaan, saintis bendung COVID-19

KETIKA wabak COVID-19, banyak negara mengalami fasa perintah berkurung atau Perintah Kawalan Pergerakan (PKP) sebagai langkah membendung penyebaran. Walaupun PKP menjejaskan banyak kehidupan, keselamatan rakyat menjadi isu utama jika wabak sudah tersebar. Cabaran pemimpin sesebuah negara adalah ketika membuat keputusan sama ada mendahulukan kepentingan ekonomi atau keselamatan rakyat. Sebarang bentuk kesilapan akan mengakibatkan kebanjiran pesakit di institusi kesihatan yang kelengkapan dan kakitangannya mungkin terhad. Keharmonian terbentuk antara pemimpin politik dengan saintis amat penting dalam menentukan tindak balas sesebuah negara ketika krisis pandemik. Misalnya, Kerajaan China dikatakan cuba untuk menafikan kewujudan coronavirus baharu yang mula disedari di Wuhan. Sememangnya dari awal lagi, dikatakan sudah wujud semacam amaran saintis di China mengenai virus ini. Akhirnya kerajaan terpaksa akur dan sekarang COVID-19 menjadi pandemik. Di Amerika Syarikat (AS) pula, Presiden Donald Trump begitu tegas mengumumkan pengurangan dana penyelidikan sains dan sumbangan kepada Pertubuhan Kesihatan Sedunia (WHO). Tindakan Trump dilihat sebahagian langkah politiknya untuk mengembalikan 'kegemilangan' AS. Trump dilihat menganggap ringan pandangan pakar penyakit berjangkit, Dr Anthony Fauci. Trump seolah memperkecilkan COVID-19 dan secara kontroversi menggelarnya 'virus China'. Dinamika hubungan Trump-Dr Fauci menjadi genting, terutama di ambang COVID-19 melanda AS. Sekarang, AS merekodkan sejuta jangkitan dengan lebihi 60,000 kematian. Di Korea Selatan, satu-satunya negara yang menjadi pusat tular pertama COVID-19 di luar China, Presiden Moon Jae-in dikatakan berjaya membendung wabak itu. Kunci utama dalam mengawal pandemik ini adalah kemampuan menyaring mereka yang asimtomatik. Tanpa melalui fasa PKP, Korea Selatan melakukan hampir 350,000 saringan dan ini dibantu infrastruktur serta pembangunan sains negara itu yang kukuh. Korea Selatan membelanjakan kira-kira 5 peratus daripada Keluaran Dalam Negara Kasar (KDNK) dalam sektor penyelidikan dan pembangunan (R&D), sekali gus melebihi AS yang mencatatkan 2.7 peratus dan China (2 peratus). Di samping itu, kerajaannya dikatakan bertindak secara telus dan berkongsi segala fakta pandemik kepada orang awam. Di Eropah, Jerman juga dikatakan begitu berjaya dalam mengekang penularan COVID-19. Tatkala negara Eropah lain berdepan dengan situasi darurat, Jerman merekodkan jumlah kematian yang agak kecil. Dengan jumlah jangkitan melebihi 159,000 orang, Jerman melakukan saringan secara skala besar. Canselor Jerman, Angela Merkel dilihat begitu berjaya dalam mengendalikan COVID-19. Ia turut dikaitkan dengan latar belakangnya dalam bidang kimia kuantum dan sentiasa terbuka terhadap pandangan saintis Jerman dari semasa ke semasa. Di New Zealand, Perdana Menteri, Jacinda Ardern mengumumkan negara itu berjaya membendung COVID-19 sepenuhnya pada 27 April lalu. Dalam kalangan pemimpin Barat, Ardern dilihat berjaya menonjolkan ciri kepemimpinan yang proaktif dengan mengambil berat setiap pandangan saintis, pakar mahupun rakyat. Pandemik ini menunjukkan ketelusan kerajaan amat penting. Sebagai contoh, Indonesia dengan lebih 250 juta orang penduduk mempunyai jumlah kematian tertinggi di Asia tenggara. Pada awalnya, kadar jangkitan dilihat agak rendah berbanding Malaysia, Singapura atau Filipina. Sehinggakan WHO kurang yakin dengan angka yang dilaporkan pihak Indonesia. Pakar tempatan juga mendesak agar kerajaan Jokowi bertindak lebih telus. Pihak kerajaan dikatakan bertindak lewat dan mula memesan kit ujian saringan. Kita tidak pasti jika senario yang sama berlaku di berapa negara di Asia Selatan yang lain. Apa yang terjadi di beberapa negara menunjukkan kejayaan membendung pandemik bukan sahaja terletak pada infrastruktur canggih dan kemajuan sesebuah negara, ia juga bergantung kepada keharmonian hubungan antara pucuk pimpinan dengan saintis atau pakar

Kepentingan memahami struktur protein dalam memerangi dan mengawal sesuatu wabak.

Enzim terlibat dalam pelbagai rangkaian tindak balas metabolisma yang terdapat pada organisma hidup. Enzim kebanyakannya terdiri daripada protein. Tanpa pengetahuan tentang struktur protein, fungsi sesuatu protein seperti enzim tidak dapat difahami. Pengetahuan mengenai struktur dan fungsi enzim ini banyak membantu dalam mencegah atau memerangi wabak berjangkit seperti denggi, tuberkulosis, HIV, taun serta sebarang penyakit berkait metabolisma tubuh seperti anteroskelerosis, anemia, fenilketonuria, diabetis dan lain-lain. Sebagai contoh, dalam wabak Covid19 yang telah menjadi pandemik pada awal 2020, sementara menunggu vaksin tiba, ubat berbentuk anti-viral tersedia menjadi pilihan untuk memulihkan pesakit. Untuk menguji ubat tersebut, pengetahuan mengenai struktur protein amat diperlukan. Protein pancang (spikes) pada permukaan virus telah menjadi fokus dalam kalangan para penyelidik kerana Virus Covid 19 memerlukan protein ini untuk berpaut pada penerima (reseptor) protein yang terdapat di permukaan sel-sel tisu epidermal salur pernafasan manusia (Rajah 1). Pengetahuan mengenai struktur protein pancang dan struktur protein reseptor manusia menjadi kunci kepada kejayaan membangunkan ubat atau vaksin. Kaedah pengkomputeran bioinformatik digunakan untuk mensimulasikan saling-tindak antara bahan bakal ubat dengan protein reseptor manusia. Sebatian yang berpotensi jika ianya berjaya, dapat menghalang proses pemautan di antara virus dan sel manusia. Oleh itu, boleh dikatakan tanpa pengetahuan struktur protein, proses pembangunan ubat atau terapeutik tidak dapat dilakukan. Bagaimana pula struktur protein diperolehi atau diketahui? Dalam kajian biologi struktur terdapat 3 teknik utama yang digunakan untuk mempelajari protein. Teknik-teknik ini termasuklah

Kolumnis: politik populis ancaman tangani COVID-19

Selepas melanda Wuhan di China, wabak COVID-19 mula menular ke Korea Selatan, Iran, beberapa negara Asia lain sebelum tiba ke Eropah dan Amerika Syarikat (AS). Ironinya, negara maju yang lengkap dengan infrastruktur kesihatan tercanggih dan terkehadapan kelihatan langsung tidak dapat mengawalnya. Pada masa sama, perubahan politik sedang terjadi di hampir segenap pelosok dunia memberi kesan terhadap penularan pandemik

Pigmented pseudoalteromonas sp. isolated from marine sponge with anti-microbial activities against selected human pathogens

Marine sponges have been the potential source of bioactive compounds with potent antimicrobial properties. Sponge associated microbes significantly provide the route of biosynthesis of some of these compounds. In this work, a total of 100 bacterial colonies were screened from a marine sponge from Class Demospongiae, which has been collected from Merambong Island, the state of Johor, Malaysia. In disk diffusion assay, only 2 out of 100 isolates; namely C40 and C52, were able to demonstrate active inhibitions against selected human pathogens (Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, except for Staphylococcus aureus). Isolates C40 and C52 were characterized to be Gram negative short rods, non-spore formers and catalase positive. Unlike the majority of other isolates from sponge which were Gram positive rods, Isolate C40 and C52 are Gram negative rods which grew in yellow pigmented colonies. Genotypic characterization using 16S ribosomal RNA sequencing were carried out on each isolate (accession number for C40 and C52 is MT645493 and MT645494, respectively). The 16S ribosomal RNA sequences revealed that these strains belonged to genus Pseudoalteromonas sp. with 97-98% similarities. Inhibitions studies showed that this sponge associated microorganisms potentially produce anti-microbial compounds useful for biotechnologies