Displaying publications 1 - 20 of 122 in total

Abstract:
Sort:
  1. Abd Rahman NH, Jaafar NR, Abdul Murad AM, Abu Bakar FD, Shamsul Annuar NA, Md Illias R
    Int J Biol Macromol, 2020 Sep 15;159:577-589.
    PMID: 32380107 DOI: 10.1016/j.ijbiomac.2020.04.262
    Short-chain fructooligosaccharides (scFOSs) can be produced from the levan hydrolysis using levanase. Levanase from Bacillus lehensis G1 (rlevblg1) is an enzyme that specifically converts levan to scFOSs. However, the use of free levanase presents a lack of stability and reusability, thus hindering the synthesis of scFOSs for continuous reactions. Here, CLEAs for rlevblg1 were prepared and characterized. Cross-linked levanase aggregates using glutaraldehyde (CLLAs-ga) and bovine albumin serum (CLLAs-ga-bsa) showed the best activity recovery of 92.8% and 121.2%, respectively. The optimum temperature of CLLAs-ga and CLLAs-ga-bsa was increased to 35 °C and 40 °C, respectively, from its free rlevblg1 (30 °C). At high temperature (50 °C), the half-life of CLLAs-ga-bsa was higher than that of free rlevblg1 and CLLAs-ga. Both CLLAs exhibited higher stability at pH 9 and pH 10. Hyperactivation of CLLAs-ga-bsa was achieved with an effectiveness factor of more than 1 and with improved catalytic efficiency. After 3 h reaction, CLLAs-ga-bsa produced the highest total scFOSs yield of 35.4% and total sugar of 60.4% per gram levan. Finally, the reusability of CLLAs for 8 cycles with more than 50% activity retained makes them as a potential synthetic catalyst to be explored for scFOSs synthesis.
    Matched MeSH terms: Recombinant Proteins/metabolism
  2. Abd-Aziz N, Tan BC, Rejab NA, Othman RY, Khalid N
    Mol Biotechnol, 2020 Apr;62(4):240-251.
    PMID: 32108286 DOI: 10.1007/s12033-020-00242-2
    In the past decade, interest in the production of recombinant pharmaceutical proteins in plants has tremendously progressed because plants do not harbor mammalian viruses, are economically competitive, easily scalable, and capable of carrying out complex post-translational modifications required for recombinant pharmaceutical proteins. Mucuna bracteata is an essential perennial cover crop species widely planted as an underground cover in oil palm and rubber plantations. As a legume, they have high biomass, thrive in its habitat, and can fix nitrogen. Thus, M. bracteata is a cost-efficient crop that shows ideal characteristics as a platform for mass production of recombinant protein. In this study, we established a new platform for the transient production of a recombinant protein in M. bracteata via vacuum-assisted agro-infiltration. Five-week-old M. bracteata plants were vacuum infiltrated with Agrobacterium tumefaciens harboring a plasmid that encodes for an anti-toxoplasma immunoglobulin (IgG) under different parameters, including trifoliate leaf positional effects, days to harvest post-infiltration, and the Agrobacterium strain used. Our results showed that vacuum infiltration of M. bracteata plant with A. tumefaciens strain GV3101 produced the highest concentration of heterologous protein in its bottom trifoliate leaf at 2 days post-infiltration. The purified anti-toxoplasma IgG was then analyzed using Western blot and ELISA. It was demonstrated that, while structural heterogeneity existed in the purified anti-toxoplasma IgG from M. bracteata, its transient expression level was two-fold higher than the model platform, Nicotiana benthamiana. This study has laid the foundation towards establishing M. bracteata as a potential platform for the production of recombinant pharmaceutical protein.
    Matched MeSH terms: Recombinant Proteins/metabolism
  3. Abdul Wahab R, Basri M, Raja Abdul Rahman RN, Salleh AB, Abdul Rahman MB, Leow TC
    Enzyme Microb Technol, 2016 Nov;93-94:174-181.
    PMID: 27702478 DOI: 10.1016/j.enzmictec.2016.08.020
    Site-directed mutagenesis of the oxyanion-containing amino acid Q114 in the recombinant thermophilic T1 lipase previously isolated from Geobacillus zalihae was performed to elucidate its role in the enzyme's enantioselectivity and reactivity. Substitution of Q114 with a hydrophobic methionine to yield mutant Q114M increased enantioselectivity (3.2-fold) and marginally improved reactivity (1.4-fold) of the lipase in catalysing esterification of ibuprofen with oleyl alcohol. The improved catalytic efficiency of Q114L was concomitant with reduced flexibility in the active site while the decreased enantioselectivity of Q114L could be directly attributed to diminished electrostatic repulsion of the substrate carboxylate ion that rendered partial loss in steric hindrance and thus enantioselectivity. The highest E-values for both Q114L (E-value 14.6) and Q114M (E-value 48.5) mutant lipases were attained at 50°C, after 12-16h, with a molar ratio of oleyl alcohol to ibuprofen of 1.5:1 and at 2.0% (w/v) enzyme load without addition of molecular sieves. Pertinently, site-directed mutagenesis on the Q114 oxyanion of T1 resulted in improved enantioselectivity and such approach may be applicable to other lipases of the same family. We demonstrated that electrostatic repulsion phenomena could affect flexibility/rigidity of the enzyme-substrate complex, aspects vital for enzyme activity and enantioselectivity of T1.
    Matched MeSH terms: Recombinant Proteins/metabolism
  4. Abu ML, Nooh HM, Oslan SN, Salleh AB
    BMC Biotechnol, 2017 Nov 10;17(1):78.
    PMID: 29126403 DOI: 10.1186/s12896-017-0397-7
    BACKGROUND: Pichia guilliermondii was found capable of expressing the recombinant thermostable lipase without methanol under the control of methanol dependent alcohol oxidase 1 promoter (AOXp 1). In this study, statistical approaches were employed for the screening and optimisation of physical conditions for T1 lipase production in P. guilliermondii.

    RESULT: The screening of six physical conditions by Plackett-Burman Design has identified pH, inoculum size and incubation time as exerting significant effects on lipase production. These three conditions were further optimised using, Box-Behnken Design of Response Surface Methodology, which predicted an optimum medium comprising pH 6, 24 h incubation time and 2% inoculum size. T1 lipase activity of 2.0 U/mL was produced with a biomass of OD600 23.0.

    CONCLUSION: The process of using RSM for optimisation yielded a 3-fold increase of T1 lipase over medium before optimisation. Therefore, this result has proven that T1 lipase can be produced at a higher yield in P. guilliermondii.

    Matched MeSH terms: Recombinant Proteins/metabolism*
  5. Abubakar MB, Aini I, Omar AR, Hair-Bejo M
    J Biomed Biotechnol, 2011;2011:414198.
    PMID: 21541235 DOI: 10.1155/2011/414198
    Avian influenza (AI) is a highly contagious and rapidly evolving pathogen of major concern to the poultry industry and human health. Rapid and accurate detection of avian influenza virus is a necessary tool for control of outbreaks and surveillance. The AI virus A/Chicken/Malaysia/5858/2004 (H5N1) was used as a template to produce DNA clones of the full-length NS1 genes via reverse transcriptase synthesis of cDNA by PCR amplification of the NS1 region. Products were cloned into pCR2.0 TOPO TA plasmid and subsequently subcloned into pPICZαA vector to construct a recombinant plasmid. Recombinant plasmid designated as pPICZαA-NS1 gene was confirmed by PCR colony screening, restriction enzyme digestion, and nucleotide sequence analysis. The recombinant plasmid was transformed into Pichia pastoris GS115 strain by electroporation, and expressed protein was identified by SDS-PAGE and western blotting. A recombinant protein of approximately ~28 kDa was produced. The expressed protein was able to bind a rabbit polyclonal antibody of nonstructural protein (NS1) avian influenza virus H5N1. The result of the western blotting and solid-phase ELISA assay using H5N1 antibody indicated that the recombinant protein produced retained its antigenicity. This further indicates that Pichia pastoris could be an efficient expression system for a avian influenza virus nonstructural (NS1).
    Matched MeSH terms: Recombinant Proteins/metabolism
  6. Ali MS, Yun CC, Chor AL, Rahman RN, Basri M, Salleh AB
    Protein J, 2012 Mar;31(3):229-37.
    PMID: 22350313 DOI: 10.1007/s10930-012-9395-8
    A mutant of the lipase from Geobacillus sp. strain T1 with a phenylalanine to leucine substitution at position 16 was overexpressed in Escherichia coli strain BL21(De3)pLysS. The crude enzyme was purified by two-step affinity chromatography with a final recovery and specific activity of 47.4 and 6,315.8 U/mg, respectively. The molecular weight of the purified F16L lipase was approximately 43 kDa by 12% SDS-PAGE analysis. The F16L lipase was demonstrated to be a thermophilic enzyme due its optimum temperature at 70 °C and showed stability over a temperature range of 40-60 °C. The enzyme exhibited an optimum pH 7 in phosphate buffer and was relatively stable at an alkaline pH 8-9. Metal ions such as Ca(2+), Mn(2+), Na(+), and K(+) enhanced the lipase activity, but Mg(2+), Zn(2+), and Fe(2+) inhibited the lipase. All surfactants tested, including Tween 20, 40, 60, 80, Triton X-100, and SDS, significantly inhibited the lipolytic action of the lipase. A high hydrolytic rate was observed on long-chain natural oils and triglycerides, with a notable preference for olive oil (C18:1; natural oil) and triolein (C18:1; triglyceride). The F16L lipase was deduced to be a metalloenzyme because it was strongly inhibited by 5 mM EDTA. Moderate inhibition was observed in the presence of PMSF at a similar concentration, indicating that serine residues are involved in its catalytic action. Further, the activity was not impaired by water-miscible solvents, including methanol, ethanol, and acetone.
    Matched MeSH terms: Recombinant Proteins/metabolism
  7. Ali MS, Ganasen M, Rahman RN, Chor AL, Salleh AB, Basri M
    Protein J, 2013 Apr;32(4):317-25.
    PMID: 23645400 DOI: 10.1007/s10930-013-9488-z
    A new strain of psychrophilic bacteria (designated strain AMS8) from Antarctic soil was screened for extracellular lipolytic activity and further analyzed using molecular approach. Analysis of 16S rDNA showed that strain AMS8 was similar to Pseudomonas sp. A lipase gene named lipAMS8 was successfully isolated from strain AMS8, cloned, sequenced and overexpressed in Escherichia coli. Sequence analysis revealed that lipAMS8 consist of 1,431 bp nucleotides that encoded a polypeptide consisting of 476 amino acids. It lacked an N-terminal signal peptide and contained a glycine- and aspartate-rich nonapeptide sequence at the C-terminus, which are known to be the characteristics of repeats-in-toxin bacterial lipases. Furthermore, the substrate binding site of lipAMS8 was identified as S(207), D(255) and H(313), based on homology modeling and multiple sequence alignment. Crude lipase exhibited maximum activity at 20 °C and retained almost 50 % of its activity at 10 °C. The molecular weight of lipAMS8 was estimated to be 50 kDa via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal expression level was attained using the recombinant plasmid pET32b/BL21(DE3) expressed at 15 °C for 8 h, induced by 0.1 mM isopropyl β-D thiogalactoside (IPTG) at E. coli growth optimal density of 0.5.
    Matched MeSH terms: Recombinant Proteins/metabolism
  8. Amerizadeh A, Khoo BY, Teh AY, Golkar M, Abdul Karim IZ, Osman S, et al.
    BMC Infect Dis, 2013;13:287.
    PMID: 23800344 DOI: 10.1186/1471-2334-13-287
    Toxoplasma gondii is an obligate intracellular zoonotic parasite of the phylum Apicomplexa which infects a wide range of warm-blooded animals, including humans. In this study in-vivo induced antigens of this parasite was investigated using in-vivo induced antigen technology (IVIAT) and pooled sera from patients with serological evidence of acute infection.
    Matched MeSH terms: Recombinant Proteins/metabolism
  9. Ang SS, Salleh AB, Chor LT, Normi YM, Tejo BA, Rahman MBA, et al.
    Protein J, 2018 04;37(2):180-193.
    PMID: 29508210 DOI: 10.1007/s10930-018-9764-z
    The bioconversion of vitamin D3 catalyzed by cytochrome P450 (CYP) requires 25-hydroxylation and subsequent 1α-hydroxylation to produce the hormonal activated 1α,25-dihydroxyvitamin D3. Vitamin D3 25-hydroxylase catalyses the first step in the vitamin D3 biosynthetic pathway, essential in the de novo activation of vitamin D3. A CYP known as CYP107CB2 has been identified as a novel vitamin D hydroxylase in Bacillus lehensis G1. In order to deepen the understanding of this bacterial origin CYP107CB2, its detailed biological functions as well as biochemical characteristics were defined. CYP107CB2 was characterized through the absorption spectral analysis and accordingly, the enzyme was assayed for vitamin D3 hydroxylation activity. CYP-ligand characterization and catalysis optimization were conducted to increase the turnover of hydroxylated products in an NADPH-regenerating system. Results revealed that the over-expressed CYP107CB2 protein was dominantly cytosolic and the purified fraction showed a protein band at approximately 62 kDa on SDS-PAGE, indicative of CYP107CB2. Spectral analysis indicated that CYP107CB2 protein was properly folded and it was in the active form to catalyze vitamin D3 reaction at C25. HPLC and MS analysis from a reconstituted enzymatic reaction confirmed the hydroxylated products were 25-hydroxyitamin D3 and 1α,25-dihydroxyvitamin D3 when the substrates vitamin D3 and 1α-hydroxyvitamin D3 were used. Biochemical characterization shows that CYP107CB2 performed hydroxylation activity at 25 °C in pH 8 and successfully increased the production of 1α,25-dihydroxyvitamin D3 up to four fold. These findings show that CYP107CB2 has a biologically relevant vitamin D3 25-hydroxylase activity and further suggest the contribution of CYP family to the metabolism of vitamin D3.
    Matched MeSH terms: Recombinant Proteins/metabolism
  10. Arifin N, Basuni M, Lan CA, Yahya AR, Noordin R
    Protein J, 2010 Oct;29(7):509-15.
    PMID: 20845068 DOI: 10.1007/s10930-010-9281-1
    This paper describes a refinement in the purification step that facilitated the downstream recovery of high purity BmR1 recombinant protein, which is a protein used as a test reagent in the commercialized rapid tests for detection of lymphac filariasis i.e. Brugia Rapid™ and panLF rapid™. Purification was performed by immobilized metal affinity chromatography (IMAC), followed by ion exchange chromatography (IEX). Results showed that a total of 10.27 mg of BmR1 was obtained when IMAC was performed using 20 mM of imidazole and 5 column volume of wash buffer containing 500 mM of NaCl. Purity of the target protein was enhanced when buffer at pH 5.8 was used during the IEX. Two proteins that recurrently appeared below the BmR1 recombinant protein were identified by mass-spectrometry analysis as the same protein, thus they were probably degradation products of BmR1. These strategies improve purity of the target protein to be used in applications such as production of aptamers and monoclonal antibodies.
    Matched MeSH terms: Recombinant Proteins/metabolism
  11. Armugam A, Earnest L, Chung MC, Gopalakrishnakone P, Tan CH, Tan NH, et al.
    Toxicon, 1997 Jan;35(1):27-37.
    PMID: 9028006
    cDNAs encoding three phospholipase A2 (PLA2) isoforms in Naja naja sputatrix were cloned and characterized. One of them encoded an acidic PLA2 (APLA) while the others encoded neutral PLA2 (NPLA-1 and NPLA-2). The specific characteristics of APLA and NPLA were attributed to mutations at nt139 and nt328 from G to C and G to A, respectively, resulting in amino acid substitutions from Asp20 and 83 in APLA to His20 and Asn83 in NPLA. Amino acid sequencing of purified protein also showed the presence of this Asp20 and His20 in APLA and NPLA, respectively. The cDNA encoding one of the PLA2 (NAJPLA-2A), when expressed in Escherichia coli, yielded a protein that exhibited PLA2 activity.
    Matched MeSH terms: Recombinant Proteins/metabolism
  12. Arockiaraj J, Easwvaran S, Vanaraja P, Singh A, Othman RY, Bhassu S
    Fish Shellfish Immunol, 2012 Jul;33(1):121-9.
    PMID: 22565019 DOI: 10.1016/j.fsi.2012.04.010
    In this study, we have reported a full length of peroxiredoxin (designated MrPrdx) gene, identified from the transcriptome of freshwater prawn Macrobrachium rosenbergii. The complete gene sequence of the MrPrdx is 940 base pairs in length, and encodes 186 amino acids. MrPrdx contains a long thioredoxin domain in the amino acid sequence between 34 and 186. The gene expressions of MrPrdx in healthy and the infectious hypodermal and hematopoietic necrosis virus (IHHNV) challenged M. rosenbergii were examined using quantitative real time polymerase chain reaction. MrPrdx is highly expressed in all the other tissues of M. rosenbergii considered for analysis and the highest in gills. The expression is strongly up-regulated in gills after IHHNV infection. To understand MrPrdx functional properties, the recombinant MrPrdx protein was expressed in Escherichia coli BL21 (DE3) and purified. A peroxidise activity assay was conducted using recombinant MrPrdx protein at different concentrations. This peroxidises activity showed that the recombinant MrPrdx is a thiol-dependant protein. Additionally, this result showed that recombinant MrPrdx protein, as a secretory protein can remove H₂O₂ and protect DNA damage. This finding leads a possible way to propose the recombinant MrPrdx protein as an effective medicine for reactive oxygen species (ROS) related diseases.
    Matched MeSH terms: Recombinant Proteins/metabolism
  13. Arockiaraj J, Vanaraja P, Easwvaran S, Singh A, Othman RY, Bhassu S
    Mol Biol Rep, 2012 Jun;39(6):6671-82.
    PMID: 22290288 DOI: 10.1007/s11033-012-1473-7
    In this study, we have reported a full length of small heat shock protein 37 (designated MrHSP37) gene, identified from the transcriptome database of freshwater prawn Macrobrachium rosenbergii. The complete gene sequence of the MrHSP37 is 2,425 base pairs in length, and encodes 338 amino acids. MrHSP37 contains a long heat shock protein family profile in the amino acid sequence between 205 and 288. The mRNA expressions of MrHSP37 in healthy and the infectious hypodermal and hematopoietic necrosis virus (IHHNV) challenged M. rosenbergii were examined using quantitative real time polymerase chain reaction (qRT-PCR). MrHSP37 is highly expressed in hepatopancreas and all the other tissues (walking leg, gills, muscle, stomach, haemocyte, intestine, pleopods, brain and eye stalk) of M. rosenbergii taken for analysis. The expression is strongly up-regulated after IHHNV challenge. To understand its biological activity, the recombinant MrHSP37 gene was constructed and expressed in Escherichia coli BL21 (DE3). The results of ATPase assay showed that the recombinant MrHSP37 protein exhibited apparent ATPase activity which increased with the concentration of the protein. And also the purified recombinant MrHSP37 protein was used for thermal aggregation assay (chaperone activity). It showed that the recombinant MrHSP37 protein is an active chaperone in this assay. Taken together, these results suggest that MrHSP37 is potentially involved in the immune responses against IHHNV challenge in M. rosenbergii.
    Matched MeSH terms: Recombinant Proteins/metabolism
  14. Arockiaraj J, Easwvaran S, Vanaraja P, Singh A, Othman RY, Bhassu S
    Fish Shellfish Immunol, 2012 May;32(5):670-82.
    PMID: 22293093 DOI: 10.1016/j.fsi.2012.01.013
    In this study, we reported a full length of catalase gene (designated as MrCat), identified from the transcriptome database of freshwater prawn Macrobrachium rosenbergii. The complete gene sequence of the MrCat is 2504 base pairs in length, and encodes 516 amino acids. The MrCat protein contains three domains such as catalase 1 (catalase proximal heme-ligand signature) at 350-358, catalase 2 (catalase proximal active site signature) at 60-76 and catalase 3 (catalase family profile) at 20-499. The mRNA expressions of MrCat in healthy and the infectious hypodermal and hematopoietic necrosis virus (IHHNV) challenged M. rosenbergii were examined using quantitative real time polymerase chain reaction (qRT-PCR). The MrCat is highly expressed in digestive tract and all the other tissues (walking leg, gills, muscle, hemocyte, hepatopancreas, pleopods, brain and eye stalk) of M. rosenbergii taken for analysis. The expression is strongly up-regulated in digestive tract after IHHNV challenge. To understand its biological activity, the recombinant MrCat gene was constructed and expressed in Escherichia coli BL21 (DE3). The recombinant MrCat existed in high thermal stability and broad spectrum of pH, which showed over 95% enzyme activity between pH 5 and 10.5, and was stable from 40 °C to 70 °C, and exhibited 85-100% enzyme activity from 30 °C to 40 °C.
    Matched MeSH terms: Recombinant Proteins/metabolism
  15. Arockiaraj J, Easwvaran S, Vanaraja P, Singh A, Othman RY, Bhassu S
    Fish Shellfish Immunol, 2012 Jan;32(1):161-9.
    PMID: 22119573 DOI: 10.1016/j.fsi.2011.11.006
    Caspase 3c (MrCasp3c) was sequenced from the freshwater giant prawn Macrobrachium rosenbergii using Illumina Solexa Genome Analyzer Technique. MrCasp3c consisted of 2080 bp nucleotide encoded 521 polypeptide with an estimated molecular mass of 59 kDa. MrCasp3c sequence contains caspase family p20 domain profile and caspase family p10 domain profile at 236-367 and 378-468 respectively. The quantitative real time PCR analysis revealed a broad expression of MrCasp3c with the highest expression in haemocyte and the lowest in stomach. The expression of MrCasp3c after challenge with the infectious hypodermal and haematopoietic necrosis virus (IHHNV) was tested in haemocyte. In addition, MrCasp3c was expressed in Escherichia coli by prokaryotic expression plasmid pMAL-c2x. The enzyme activity of MrCasp3c was also found to be up-regulated by IHHNV in haemocyte and hepatopancreas tissues. This study suggested that MrCasp3c may be an effector caspase associated with the induction of apoptosis which is potentially involved in the immune defence of M. rosenbergii.
    Matched MeSH terms: Recombinant Proteins/metabolism
  16. Arshad NM, In LL, Soh TL, Azmi MN, Ibrahim H, Awang K, et al.
    Oncotarget, 2015 Jun 30;6(18):16151-67.
    PMID: 26158863
    Previous in vitro and in vivo studies have reported that 1'-S-1'-acetoxychavicol acetate (ACA) isolated from rhizomes of the Malaysian ethno-medicinal plant Alpinia conchigera Griff (Zingiberaceae) induces apoptosis-mediated cell death in tumour cells via dysregulation of the NF-κB pathway. However there were some clinical development drawbacks such as poor in vivo solubility, depreciation of biological activity upon exposure to an aqueous environment and non-specific targeting of tumour cells. In the present study, all the problems above were addressed using the novel drug complex formulation involving recombinant human alpha fetoprotein (rhAFP) and ACA.
    Matched MeSH terms: Recombinant Proteins/metabolism*
  17. Atago Y, Shimodaira J, Araki N, Bin Othman N, Zakaria Z, Fukuda M, et al.
    Biosci Biotechnol Biochem, 2016 May;80(5):1012-9.
    PMID: 26828632 DOI: 10.1080/09168451.2015.1127134
    Rhodococcus jostii RHA1 (RHA1) degrades polychlorinated biphenyl (PCB) via co-metabolism with biphenyl. To identify the novel open reading frames (ORFs) that contribute to PCB/biphenyl metabolism in RHA1, we compared chromatin immunoprecipitation chip and transcriptomic data. Six novel ORFs involved in PCB/biphenyl metabolism were identified. Gene deletion mutants of these 6 ORFs were made and were tested for their ability to grow on biphenyl. Interestingly, only the ro10225 deletion mutant showed deficient growth on biphenyl. Analysis of Ro10225 protein function showed that growth of the ro10225 deletion mutant on biphenyl was recovered when exogenous recombinant Ro10225 protein was added to the culture medium. Although Ro10225 protein has no putative secretion signal sequence, partially degraded Ro10225 protein was detected in conditioned medium from wild-type RHA1 grown on biphenyl. This Ro10225 fragment appeared to form a complex with another PCB/biphenyl oxidation enzyme. These results indicated that Ro10225 protein is essential for the formation of the PCB/biphenyl dioxygenase complex in RHA1.
    Matched MeSH terms: Recombinant Proteins/metabolism
  18. Bakri MM, Rich AM, Cannon RD, Holmes AR
    Mol Oral Microbiol, 2015 Feb;30(1):27-38.
    PMID: 24975985 DOI: 10.1111/omi.12064
    Alcohol consumption is a risk factor for oral cancer, possibly via its conversion to acetaldehyde, a known carcinogen. The oral commensal yeast Candida albicans may be one of the agents responsible for this conversion intra-orally. The alcohol dehydrogenase (Adh) family of enzymes are involved in acetaldehyde metabolism in yeast but, for C. albicans it is not known which family member is responsible for the conversion of ethanol to acetaldehyde. In this study we determined the expression of mRNAs from three C. albicans Adh genes (CaADH1, CaADH2 and CaCDH3) for cells grown in different culture media at different growth phases by Northern blot analysis and quantitative reverse transcription polymerase chain reaction. CaADH1 was constitutively expressed under all growth conditions but there was differential expression of CaADH2. CaADH3 expression was not detected. To investigate whether CaAdh1p or CaAdh2p can contribute to alcohol catabolism in C. albicans, each gene from the reference strain C. albicans SC5314 was expressed in Saccharomyces cerevisiae. Cell extracts from an CaAdh1p-expressing S. cerevisiae recombinant, but not an CaAdh2p-expressing recombinant, or an empty vector control strain, possessed ethanol-utilizing Adh activity above endogenous S. cerevisiae activity. Furthermore, expression of C. albicans Adh1p in a recombinant S. cerevisiae strain in which the endogenous ScADH2 gene (known to convert ethanol to acetaldehyde in this yeast) had been deleted, conferred an NAD-dependent ethanol-utilizing, and so acetaldehyde-producing, Adh activity. We conclude that CaAdh1p is the enzyme responsible for ethanol use under in vitro growth conditions, and may contribute to the intra-oral production of acetaldehyde.
    Matched MeSH terms: Recombinant Proteins/metabolism
  19. Batumalaie K, Khalili E, Mahat NA, Huyop F, Wahab RA
    Biochimie, 2018 Sep;152:198-210.
    PMID: 30036604 DOI: 10.1016/j.biochi.2018.07.011
    Spectroscopic and calorimetric methods were employed to assess the stability and the folding aspect of a novel recombinant alkaline-stable lipase KV1 from Acinetobacter haemolyticus under varying pH and temperature. Data on far ultraviolet-circular dichroism of recombinant lipase KV1 under two alkaline conditions (pH 8.0 and 12.0) at 40 °C reveal strong negative ellipticities at 208, 217, 222 nm, implying its secondary structure belonging to a α + β class with 47.3 and 39.0% ellipticity, respectively. Results demonstrate that lipase KV1 adopts its most stable conformation at pH 8.0 and 40 °C. Conversely, the protein assumes a random coil structure at pH 4.0 and 80 °C, evident from a strong negative peak at ∼ 200 nm. This blue shift suggests a general decline in enzyme activity in conjunction with the partially or fully unfolded state that invariably exposed more hydrophobic surfaces of the lipase protein. The maximum emission at ∼335 nm for pH 8.0 and 40 °C indicates the adoption of a favorable protein conformation with a high number of buried tryptophan residues, reducing solvent exposure. Appearance of an intense Amide I absorption band at pH 8.0 corroborates an intact secondary structure. A lower enthalpy value for pH 4.0 over pH 8.0 and 12.0 in the differential scanning calorimetric data corroborates the stability of the lipase at alkaline conditions, while a low Km (0.68 ± 0.03 mM) for tributyrin verifies the high affinity of lipase KV1 for the substrate. The data, herein offer useful insights into future structure-based tunable catalytic activity of lipase KV1.
    Matched MeSH terms: Recombinant Proteins/metabolism
  20. Bayat O, Baradaran A, Ariff A, Mohamad R, Rahim RA
    Biotechnol Lett, 2014 Mar;36(3):581-5.
    PMID: 24185903 DOI: 10.1007/s10529-013-1390-4
    Human interferon alpha (IFN-α) was expressed in two strains of Lactococcus lactis by aid of two promoters (P32 and Pnis) giving rise to two recombinant strains: MG:IFN and NZ:IFN, respectively. The expression of IFN was confirmed by ELISA and western blotting. Highest production was achieved using glucose for growth of both recombinant strains with nisin, used for induction of the recombinant strain with Pnis promoter, at 30 ng/ml. The optimum time for MG:IFN was 9 h and for NZ:IFN was 4.5 h. The highest productions by MG:IFN and NZ:IFN were 1.9 and 2.4 μg IFN/l, respectively. Both of the expressed IFNs showed bioactivities of 1.9 × 10(6) IU/mg that were acceptable for further clinical studies.
    Matched MeSH terms: Recombinant Proteins/metabolism
Filters
Contact Us

Please provide feedback to Administrator (afdal@afpm.org.my)

External Links