Pencirian enzim ekstraselular protease daripada bakteria Alkalophilic Bacillus lehensis G1 dari Malaysia telah dikaji. Enzim protease yang dirembeskan diuji pada agar susu skim 2%. Keputusan menunjukkan protease ekstraselular mampu mengekalkan aktiviti sehingga suhu 60°C di dalam julat pH yang luas iaitu 3 hingga 11 dengan suhu optimum pada 40°C dan pH optimum pada 7.0. Aktiviti enzim juga diperhatikan akan meningkat dengan penambahan beberapa ion iaitu Mn2+, Fe2+, Cu2+, Mg2+ dan Co2+. Manakala aktiviti protease didapati sedikit direncat dengan kehadiran ion Ca2+, K+ dan Ni2+ dengan baki aktiviti sebanyak 85%, 81% dan 75%. Protease ekstraselular juga didapati serasi dengan beberapa cecair detergen komersial dari Malaysia, yang menunjukkan protease ini boleh dimanfaatkan sebagai pembersih kotoran pada pakaian. Selain itu, potensi kegunaan protease yang dihasilkan oleh B. lehensis G1 ke atas penguraian gelatin dari filem X-ray yang telah digunakan juga telah dilakukan di dalam kajian ini.
Protease is an enzyme that catalysed the hydrolysis of protein into peptide. Application of protease in industry has been linked with cost effective substrates and complex of enzyme-substrate stability. Molecular docking approach has identified casein as a preference substrates. However, lack of data on casein mode of binding to protease and enzyme stability represents a limitation for its production and structural optimization. In this study, we have used a molecular dynamic (MD) to examine the stability of complex enzyme-substrate of protease from Bacillus lehensis G1. The 3D structure of protease (BleG1_1979) was docked with substrate casein using AutoDock Vina. Structural analysis of the substrate-binding cleft revealed a binding site of casein was predominantly at the hydrophobic region of BleG1_1979. The MD of complex BleG1_1979-casein was tested with two temperatures; 298 K and 310 K using GROMACS v5.1.4. MD simulation showed a stable behaviour of BleG1_1979 over the 20 ns simulation period. The molecular docking and MD simulation suggested that the production of protease from B. lehensis G1 by utilization of casein and the stability of complex protease-casein could be a potential application to generate a cost effective enzyme to be develop for industrial use.
Effective and rapid methods for RNA extraction from conidia, germinating conidia and appressoria of the fungal plant pathogen, Colletotrichum gloeosporioides is reported in this study. The procedure for the RNA extraction from conidia and germinating conidia was carried out using TRI REAGENT® solution (Molecular Research Center, USA) and can be completed in less than one and a half hours. The procedure for RNA extraction from appressoria was carried out using a modified protocol employing guanidine isothiocyanate and mechanical cell disruption by glass beads. The efficiency of the RNA extraction procedures was evaluated by several measures to determine RNA integrity, purity and applicability in RT-PCR. RNA integrity was assessed by observing the integrity of the major RNA species (18S and 28S rRNA) on denaturing agarose gel electrophoresis. The ethidium bromide-staining pattern of intact total RNA extracted from the three fungal morphogenetic cells showed clearly defined 28S and 18S rRNA bands and no genomic DNA contamination. Spectrophotometric assessment of RNA from each sample indicated relatively high purity and absence of carbohydrate contamination. Finally, we have demonstrated that the methods used for RNA extraction of conidia, germinating conidia and appressoria produced RNA of sufficient quality suitable for RT-PCR in detecting the expression of protein kinase A regulatory subunit gene in C. gloeosporioides.
Public perceptions, understanding and acceptance of modern biotechnology can both promote and hamper their commercial introduction and adoption. Various studies have shown that consumer acceptance of modern biotechnology tend to be conditional and dependent on several factors. Public perceptions of biotechnology have received extensive attention in recent years in most Western countries such as Europe, USA and Canada but there have been limited similar surveys in developing countries. Most of the earlier studies used uni-dimensional or bi-dimensional instrument with multi-items or the most is four dimensions with single item. In this study, public attitude towards genetically modified (GM) soybean that is already available in the Malaysian market. A survey was carried out on 577 general public respondents in the Klang Valley region. In order to detect the structure of attitude amongst the expert group in the Klang Valley region, structural equation modeling (SEM) using AMOS version 5.1 was carried out. Result of the survey has confirmed that attitude towards complex issues such as biotechnology should be seen as multi-faceted/ multidimensional process. The most important factors predicting encouragement of GM soybean are the specific application-linked perceptions about the benefits and acceptance of risk while moral concern, risk and familiarity are significant predictors of intermediate factors. Researchers, policy makers and industries interested in developing and marketing GM products in Malaysia should consider the various factors mentioned in this in order to gain public approval.
The ability of a locally isolated clinical strain of C. albicans to adapt and response towards oxidative stress were investigated in this study. Treatment of C. albicans with hydrogen peroxide (H2O2) will exert an oxidative stress to C. albicans cells and affect the growth rate of this opportunistic pathogen. Cells that were grown in Yeast Peptone Dextrose (YPD) medium with the presence of 4.0 mM H2O2 gave a doubling time of 51 min/generation compared to 44 min/generation for those grown in 0.4 mM H2O2 and without H2O2. In order to determine the resistance level of C. albicans towards oxidative stress, cells were exposed to different concentration of H2O2. Results showed that percentage of cells viability decreased with the increased concentration of H2O2. These data indicated that C. albicans could overcome oxidative stress to a certain level before they were killed. To determine whether this strain exhibited a stress induce protection, cells were treated with mild oxidative stress for one hour before exposed to a stronger oxidative stress. Data obtained shows that cells that were pre-treated with mild oxidative stress showed higher percentage of survival compared to non pre-treated cells. This strongly suggested that stress induce protection was present in this C. albicans. Finally, in order to determine whether oxidative stress can induce yeast to hyphal morphogenesis in C. albicans, cells were then exposed to 0.4 mM H2O2 at 37 ºC and the number of hyphal formed were compared to hyphal formation when the cells were grown at 37 ºC only. However the results showed that oxidative stress failed to induce yeast to hyphal morphogenesis in this clinically isolated C. albicans strain.
Keywords: Candida albicans; oxidative stress
In this study, the pyrG gene which encodes for orotidine 5-monophosphate decarboxylase (OMP decarboxylase) of Aspergillus oryzae strain S1 was cloned and analysed. This 1.8kb A. oryzae pyrG encompasses the 5’-regulatory flanking region (465 bp), open reading frame (899 bp) and 3’-regulatory region (475 bp). The pyrG contained one intron at position 623-687 bp based on the AUGUSTUS and FGENESH (SoftBerry) analysis corresponding to the intron present in the pyrG of A. oryzae (Accession Number: Y13811). In silico analysis showed that the enzyme encoded by the A. oryzae S1 pyrG gene has a theoretical molecular weight of 30.28 kDa and theoretical pI value of 5.92. This enzyme is hydrophilic, located in a region outside of the transmembrane and it functions in the cytoplasm. Five motives such as N-glycosylation site, protein kinase C (PKC) phosphorylation site, casein kinase II (CK-2) phosphorylation site, N-myristolation site and orotidine 5-monophoshate decarboxylase active site have been identified in the pyrG amino acid sequence. The three dimensional structure of this enzyme generated via protein homology modeling using the bioinformatic software, Swiss Model, shows that OMP decarboxylase is a protein with an α/ß barrel structure possessing 8 ß-strands surrounded by 9 α-helices. The amino acid residues involved in the active site have been identified and it is located on one of the ß-strands. The pyrG DNA sequence will be used for the complementation of a pyrG auxotroph mutant of A. oryzae.
Stem cells, also known as mother cells are capable of undergoing both cell division and differentiation. The most primitive stem cells are totipotent cells which are capable of producing a complete organism from one cell. There are two types of haemopoietic stem cells depending on their developmental stages known as embryo and adult haemopoietic stem cells. Studies showed that only 0.01-0.05% of total bone marrow cell population consists of haemopoietic stem cells. This small population of stem cells exists in three different sizes with different characteristics. In addition, the microenvironment which contains various regulatory molecules plays an important role in the differentiation of stem cells into specific adult cells.
[Sel stem juga dikenali sebagai sel induk berupaya untuk menjalani kedua-dua proses pembahagian dan pembezaan sel. Sel stem yang paling primitif iaitu sel totipoten berupaya untuk membentuk satu organisma lengkap daripada satu sel. Sel stem hemopoietik terdiri daripada dua jenis bergantung kepada peringkat perkembangan individu iaitu sel stem hemopoietik embrio dan dewasa. Kajian mendapati hanya 0.01-0.05% daripada keseluruhan populasi sel sumsum tulang berupaya bertindak sebagai sel stem hemopoietik. Daripada julat yang kecil ini sel stem hemopoietik wujud dalam tiga saiz yang mempunyai ciri yang berbeza. Selain daripada itu mikrosekitaran yang mempunyai molekul-molekul regulatori yang berbeza-beza juga memainkan peranan yang penting dalam pembezaan sel stem kepada sel-sel matang yang spesifik].
Mekanisme pengambilan dan penghasilan asid amino bagi mikroorganisma psikrofil yang bermandiri dan berpoliferasi
pada persekitaran sejuk melampau masih belum difahami sepenuhnya. Objektif kajian ini ialah untuk mengenal pasti
gen yang terlibat dalam penjanaan asid amino bagi yis psikrofil, Glaciozyma antarctica serta menentukan pengekspresan
gen tersebut semasa kehadiran dan kekurangan asid amino dalam medium pertumbuhan. Pengenalpastian gen telah
dilakukan melalui penjanaan penanda jujukan terekspres (ESTs) daripada dua perpustakaan cDNA yang dibina daripada
sel yang dikultur dalam medium pertumbuhan kompleks dan medium pertumbuhan minimum tanpa asid amino. Sebanyak
3552 klon cDNA daripada setiap perpustakaan dipilih secara rawak untuk dijujuk menghasilkan 1492 transkrip unik
(medium kompleks) dan 1928 transkrip unik (medium minimum). Analisis pemadanan telah mengenl pasti gen mengekod
protein yang terlibat di dalam pengambilan asid amino bebas, biosintesis asid amino serta gen yang terlibat dengan
kitar semula asid amino berdasarkan tapak jalan yang digunakan oleh yis model, Saccharomyces cerevisiae. Analisis
pengekspresan gen menggunakan kaedah RT-qPCR menunjukkan pengekspresan gen mengekod protein yang terlibat di
dalam pengambilan asid amino bebas iaitu permease adalah tinggi pada medium kompleks manakala pengekspresan
kebanyakan gen mengekod protein yang terlibat dalam kitar semula dan biosintesis asid amino adalah tinggi di dalam
medium minimum. Kesimpulannya, gen yang terlibat dalam penjanaan dan pengambilan asid amino bagi mikroorganisma
psikrofil adalah terpulihara seperti mikroorganisma mesofil dan pengekspresan gen-gen ini adalah diaruh oleh kehadiran
atau ketiadaan asid amino bebas pada persekitaran.
Yeasts with GRAS (Generally Regarded as Safe) status are commonly used as hosts for heterologous protein production. Yeasts are suitable expression hosts as they have been extensively characterised genetically. The objective of this project was to isolate yeasts from Malaysian food sources and subsequently to develop these as alternative hosts for heterologous protein production. Yeasts were isolated from Malaysian traditional fermented food namely ‘tapai’, ‘tuak’ and ‘ragi’. A total of 23 isolates were obtained and subjected to molecular identification by amplification and sequencing of the universally conserved ribosomal internal transcribed spacer (ITS), 26S rDNA and 18S rDNA sequences. We identified three species of yeasts, Saccharomyces cerevisiae, Hanseniaspora guilliermondii and Pichia anomala, which have a long tradition of usage in food production and have no adverse effects on humans. To test the feasibility of the yeasts as heterologous expression hosts, we have constructed an integrative vector, p1926Zeo containing the yeast 26S rDNA and Zeocin® resistance cassette. The p1926Zeo vector was linearised and transformed into both P. anomala and H. guilliermondii isolates via electroporation. Both hosts were successfully transformed at a relatively high efficiency. The transformants obtained had a growth profile similar to the respective wild type, indicating that integration of the plasmids into the host chromosome did not affect growth. These transformants were stable as they exhibited resistance to Zeocin even after 20 generations. Thus, both P. anomala and H. guilliermondii isolates exhibited the potential to be further developed as alternative heterologous protein expression hosts.
Kitin merupakan polisakarida struktur yang dapat dicurai oleh enzim kitinolisis kepada pelbagai terbitan yang boleh digunakan dalam bidang perubatan, pertanian dan rawatan air. Pengenalpastian dan pencirian gen-gen Trichoderma virens UKM1 mengekod enzim terlibat dalam pencuraian kitin krustasea telah dilakukan melalui penjanaan penanda jujukan terekspres (ESTs) dan analisis pengekspresan gen menggunakan mikroatur DNA. Sebanyak tiga perpustakaan cDNA T. virens UKM1 yang masing-masing diaruh oleh kitin, glukosamina dan kitosan telah dibina. Sejumlah 1536 klon cDNA telah dijujuk dan sebanyak 1033 ESTs berkualiti telah dijana. Seterusnya, perbezaan pengekspresan gen apabila pertumbuhan kulat diaruh dengan kehadiran kitin krustasea dan tanpa kitin pada hari ketiga dan kelima telah ditentukan. Sebanyak 1824 klon cDNA telah dititik ke atas slaid kaca dan dihibrid bersama dengan cDNA terlabel Cy3 atau Cy5 yang disintesis daripada mRNA yang dipencil daripada kulat yang ditumbuhkan dalam medium mengandungi kitin krustasea atau glukosa (kawalan). Sebanyak 91 dan 61 gen, masing-masing bagi hari ketiga dan kelima didapati terekspres melebihi dua gandaan apabila kulat menggunakan kitin krustasea sebagai sumber karbon. Beberapa gen mengekod kitinase seperti ech1 dan cht3 (endokitinase), nag1 (eksokitinase) dan nagB (glukosamina 6-P-deaminase) didapati terekspres dengan tinggi pada kedua-dua hari. Selain daripada itu, gen mengekod protein hidrofobin, protease serina dan beberapa protein hipotetik juga terekspres dengan tinggi dengan kehadiran kitin krustasea. Protein-protein ini dijangka memainkan peranan penting dalam membantu pencuraian kitin krustasea.