Displaying publications 1 - 20 of 30 in total

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  1. Kamarudin NH, Rahman RN, Ali MS, Leow TC, Basri M, Salleh AB
    Protein J, 2014 Jun;33(3):296-307.
    PMID: 24777627 DOI: 10.1007/s10930-014-9560-3
    The gene encoding a cold-adapted, organic solvent stable lipase from a local soil-isolate, mesophilic Staphylococcus epidermidis AT2 was expressed in a prokaryotic system. A two-step purification of AT2 lipase was achieved using butyl sepharose and DEAE sepharose column chromatography. The final recovery and purification fold were 47.09 % and 3.45, respectively. The molecular mass of the purified lipase was estimated to be 43 kDa. AT2 lipase was found to be optimally active at pH 8 and stable at pH 6-9. Interestingly, this enzyme demonstrated remarkable stability at cold temperature (<30 °C) and exhibited optimal activity at a temperature of 25 °C. A significant enhancement of the lipolytic activity was observed in the presence of Ca(2+), Tween 60 and Tween 80. Phenylmethylsulfonylfluoride, a well known serine inhibitor did not cause complete inhibition of the enzymatic activity. AT2 lipase exhibited excellent preferences towards long chain triglycerides and natural oils. The lipolytic activity was stimulated by dimethylsulfoxide and diethyl ether, while more than 50 % of its activity was retained in methanol, ethanol, acetone, toluene, and n-hexane. Taken together, AT2 lipase revealed highly attractive biochemical properties especially because of its stability at low temperature and in organic solvents.
  2. Tan MS, Teh YH, Ho KL, Stanslas J
    Protein J, 2020 02;39(1):54-61.
    PMID: 31620959 DOI: 10.1007/s10930-019-09872-1
    Being an important regulator of cell growth and survival, a point mutation at glycine-12 residue of Kras4B to valine (V), renders Kras4BG12V oncogenic. Kras4B recombinant protein is used as a bait to fish its potential ligands in the attempt of drugging this oncoprotein and to validate its pharmacologically relevant ligand in protein-ligand interaction studies. Nevertheless, synthesis of Kras4B recombinant protein is challenging as it was reported being susceptible to aggregation into inclusion bodies in the bacterial host, resulting in a poor yield of recombinant protein. Here, we describe a novel method to produce native Kras4BG12V protein by using pET SUMO protein expression system as a solution to the formation of inclusion bodies. Kras4BG12V oncogene was cloned into pET SUMO vector, followed by a 12 h chemically induced protein expression in Escherichia coli at 20 °C. Native Kras4BG12V protein was produced in a series of protein purification steps involving immobilised nickel ion-affinity column chromatography, SUMO fusion protein and polyhistidine tag removal, and size exclusion column chromatography. The identity of the purified Kras4BG12V protein was validated by immunoblot analysis. The purified protein exhibited self-dimerising, indicating that the purified protein structurally resembles Kras4B. Its physical interaction with 4,6-dichloro-2-methyl-3-aminoethyl-indole (DCAI), a known binder of Kras4B, confirms the identity of the purified protein as Kras4BG12V. The native Kras4BG12V protein was successfully purified in a substantial amount by using the pET SUMO protein expression system.
  3. Lau BYC, Othman A, Ramli US
    Protein J, 2018 12;37(6):473-499.
    PMID: 30367348 DOI: 10.1007/s10930-018-9802-x
    Proteomics technologies were first applied in the oil palm research back in 2008. Since proteins are the gene products that are directly correspond to phenotypic traits, proteomic tools hold a strong advantage above other molecular tools to comprehend the biological and molecular mechanisms in the oil palm system. These emerging technologies have been used as non-overlapping tools to link genome-wide transcriptomics and metabolomics-based studies to enhance the oil palm yield and quality through sustainable plant breeding. Many efforts have also been made using the proteomics technologies to address the oil palm's Ganoderma disease; the cause and management. At present, the high-throughput screening technologies are being applied to identify potential biomarkers involved in metabolism and cellular development through determination of protein expression changes that correlate with oil production and disease. This review highlights key elements in proteomics pipeline, challenges and some examples of their implementations in plant studies in the context of oil palm in particular. We foresee that the proteomics technologies will play more significant role to address diverse issues related to the oil palm in the effort to improve the oil crop.
  4. Lau BY, Clerens S, Morton JD, Dyer JM, Deb-Choudhury S, Ramli US
    Protein J, 2016 Apr;35(2):163-70.
    PMID: 26993480 DOI: 10.1007/s10930-016-9655-0
    The details of plant lipid metabolism are relatively well known but the regulation of fatty acid production at the protein level is still not understood. Hence this study explores the importance of phosphorylation as a mechanism to control the activity of fatty acid biosynthetic enzymes using low and high oleic acid mesocarps of oil palm fruit (Elaeis guineensis variety of Tenera). Adaptation of neutral loss-triggered tandem mass spectrometry and selected reaction monitoring to detect the neutral loss of phosphoric acid successfully found several phosphoamino acid-containing peptides. These peptides corresponded to the peptides from acetyl-CoA carboxylase and 3-enoyl-acyl carrier protein reductase as identified by their precursor ion masses. These findings suggest that these enzymes were phosphorylated at 20th week after anthesis. Phosphorylation could have reduce their activities towards the end of fatty acid biosynthesis at ripening stage. Implication of phosphorylation in the regulation of fatty acid biosynthesis at protein level has never been reported.
  5. 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.
  6. Kalhori N, Nulit R, Go R
    Protein J, 2013 Oct;32(7):551-9.
    PMID: 24132392 DOI: 10.1007/s10930-013-9516-z
    Pentose phosphate pathway (PPP) composed of two functionally-connected phases, the oxidative and non-oxidative phase. Both phases catalysed by a series of enzymes. Transketolase is one of key enzymes of non-oxidative phase in which transfer two carbon units from fructose-6-phosphate to erythrose-4-phosphate and convert glyceraldehyde-3-phosphate to xylulose-5-phosphate. In plant, erythrose-4-phosphate enters the shikimate pathway which is produces many secondary metabolites such as aromatic amino acids, flavonoids, lignin. Although transketolase in plant system is important, study of this enzyme is still limited. Until to date, TKT genes had been isolated only from seven plants species, thus, the aim of present study to isolate, study the similarity and phylogeny of transketolase from sugarcane. Unlike bacteria, fungal and animal, PPP is complete in the cytosol and all enzymes are found cytosolic. However, in plant, the oxidative phase found localised in the cytosol but the sub localisation for non-oxidative phase might be restricted to plastid. Thus, this study was conducted to determine subcellular localization of sugarcane transketolase. The isolation of sugarcane TKT was done by reverse transcription polymerase chain reaction, followed by cloning into pJET1.2 vector and sequencing. This study has isolated 2,327 bp length of sugarcane TKT. The molecular phylogenetic tree analysis found that transketolase from sugarcane and Zea mays in one group. Classification analysis found that both plants showed closer relationship due to both plants in the same taxon i.e. family Poaceae. Target P 1.1 and Chloro P predicted that the compartmentation of sugarcane transketolase is localised in the chloroplast which is 85 amino acids are plant plastid target sequence. This led to conclusion that the PPP is incomplete in the cytosol of sugarcane. This study also found that the similarity sequence of sugarcane TKT closely related with the taxonomy plants.
  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.
  8. Chin CF, Teoh EY, Chee MJY, Al-Obaidi JR, Rahmad N, Lawson T
    Protein J, 2019 12;38(6):704-715.
    PMID: 31552579 DOI: 10.1007/s10930-019-09868-x
    Mango (Mangifera indica L.) is an economically important fruit. However, the marketability of mango is affected by the perishable nature and short shelf-life of the fruit. Therefore, a better understanding of the mango ripening process is of great importance towards extending its postharvest shelf life. Proteomics is a powerful tool that can be used to elucidate the complex ripening process at the cellular and molecular levels. This study utilized 2-dimensional gel electrophoresis (2D-GE) coupled with MALDI-TOF/TOF to identify differentially abundant proteins during the ripening process of the two varieties of tropical mango, Mangifera indica cv. 'Chokanan' and Mangifera indica cv 'Golden Phoenix'. The comparative analysis between the ripe and unripe stages of mango fruit mesocarp revealed that the differentially abundant proteins identified could be grouped into the three categories namely, ethylene synthesis and aromatic volatiles, cell wall degradation and stress-response proteins. There was an additional category for differential proteins identified from the 'Chokanan' variety namely, energy and carbohydrate metabolism. However, of all the differential proteins identified, only methionine gamma-lyase was found in both 'Chokanan' and 'Golden Phoenix' varieties. Six differential proteins were selected from each variety for validation by analysing their respective transcript expression using reverse transcription-quantitative PCR (RT-qPCR). The results revealed that two genes namely, glutathione S-transferase (GST) and alpha-1,4 glucan phosphorylase (AGP) were found to express in concordant with protein abundant. The findings will provide an insight into the fruit ripening process of different varieties of mango fruits, which is important for postharvest management.
  9. Al-Obaidi JR, Saidi NB, Usuldin SR, Hussin SN, Yusoff NM, Idris AS
    Protein J, 2016 Apr;35(2):100-6.
    PMID: 27016942 DOI: 10.1007/s10930-016-9656-z
    Ganoderma species are a group of fungi that have the ability to degrade lignin polymers and cause severe diseases such as stem and root rot and can infect economically important plants and perennial crops such as oil palm, especially in tropical countries such as Malaysia. Unfortunately, very little is known about the complex interplay between oil palm and Ganoderma in the pathogenesis of the diseases. Proteomic technologies are simple yet powerful tools in comparing protein profile and have been widely used to study plant-fungus interaction. A critical step to perform a good proteome research is to establish a method that gives the best quality and a wide coverage of total proteins. Despite the availability of various protein extraction protocols from pathogenic fungi in the literature, no single extraction method was found suitable for all types of pathogenic fungi. To develop an optimized protein extraction protocol for 2-DE gel analysis of Ganoderma spp., three previously reported protein extraction protocols were compared: trichloroacetic acid, sucrose and phenol/ammonium acetate in methanol. The third method was found to give the most reproducible gels and highest protein concentration. Using the later method, a total of 10 protein spots (5 from each species) were successfully identified. Hence, the results from this study propose phenol/ammonium acetate in methanol as the most effective protein extraction method for 2-DE proteomic studies of Ganoderma spp.
  10. Shehu D, Alias Z
    Protein J, 2018 06;37(3):261-269.
    PMID: 29779193 DOI: 10.1007/s10930-018-9774-x
    Glutathione S-transferases (GSTs) are a family of enzymes that function in the detoxification of variety of electrophilic substrates. In the present work, we report a novel zeta-like GST (designated as KKSG9) from the biphenyl/polychlorobiphenyl degrading organism Acidovorax sp. KKS102. KKSG9 possessed low sequence similarity but similar biochemical properties to zeta class GSTs. Functional analysis showed that the enzyme exhibits wider substrate specificity compared to most zeta class GSTs by reacting with 1-chloro-2,4-dinitrobenzene (CDNB), p-nitrobenzyl chloride (NBC), ethacrynic acid (EA), hydrogen peroxide, and cumene hydroperoxide. The enzyme also displayed dehalogenation function against dichloroacetate, permethrin, and dieldrin. The functional role of Tyr12 was also investigated by site-directed mutagenesis. The mutant (Y12C) displayed low catalytic activity and dehalogenation function against all the substrates when compared with the wild type. Kinetic analysis using NBC and GSH as substrates showed that the mutant (Y12C) displayed a higher affinity for NBC when compared with the wild type, however, no significant change in GSH affinity was observed. These findings suggest that the presence of tyrosine residue in the motif might represent an evolutionary trend toward improving the catalytic activity of the enzyme. The enzyme as well could be useful in the bioremediation of various types of organochlorine pollutants.
  11. Pan Y, Abd-Rashid BA, Ismail Z, Ismail R, Mak JW, Ong CE
    Protein J, 2011 Dec;30(8):581-91.
    PMID: 22001938 DOI: 10.1007/s10930-011-9365-6
    This study aimed to express two major drug-metabolizing human hepatic cytochromes P450 (CYPs), CYP2D6 and CYP3A4, together with NADPH-cytochrome P450 oxidoreductase (OxR) in Escherichia coli and to evaluate their catalytic activities. Full length cDNA clones of both isoforms in which the N-terminus was modified to incorporate bovine CYP17α sequence were inserted into a pCWori(+) vector. The modified CYP cDNAs were subsequently expressed individually, each together with OxR by means of separate, compatible plasmids with different antibiotic selection markers. The expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. Enzyme activities were examined using high performance liquid chromatography (HPLC) assays with probe substrates dextromethorphan and testosterone for CYP2D6 and CYP3A4, respectively. Results from immunoblotting demonstrated the presence of both CYP proteins in bacterial membranes and reduced CO difference spectra of the cell preparations exhibited the characteristic absorbance peak at 450 nm. Co-expressed OxR also demonstrated an activity level comparable to literature values. Kinetic parameters, K(m) and V(max) values determined from the HPLC assays also agreed well with literature values. As a conclusion, the procedures described in this study provide a relatively convenient and reliable means of producing catalytically active CYP isoforms suitable for drug metabolism and interaction studies.
  12. Budiman C, Lindang HU, Cheong BE, Rodrigues KF
    Protein J, 2018 06;37(3):270-279.
    PMID: 29761378 DOI: 10.1007/s10930-018-9772-z
    SIB1 FKBP22 is a peptidyl prolyl cis-trans isomerase (PPIase) member from a psychrotrophic bacterium, Shewanella sp. SIB1, consisting of N- and C-domains responsible for dimerization and catalytic PPIase activity, respectively. This protein was assumed to be involved in cold adaptation of SIB1 cells through its dual activity of PPIase activity and chaperone like-function. Nevertheless, the catalytic inhibition by FK506 and its substrate specificity remain unknown. Besides, ability of SIB1 FKBP22 to inhibit phosphatase activity of calcinuerin is also interesting to be studied since it may reflect wider cellular functions of SIB1 FKBP22. In this study, we found that wild type (WT) SIB1 FKBP22 bound to FK506 with IC50 of 77.55 nM. This value is comparable to that of monomeric mutants (NNC-FKBP22, C-domain+ and V37R/L41R mutants), yet significantly higher than that of active site mutant (R142A). In addition, WT SIB1 FKBP22 and monomeric variants were found to prefer hydrophobic residues preceding proline. Meanwhile, R142A mutant has wider preferences on bulkier hydrophobic residues due to increasing hydrophobicity and binding pocket space. Surprisingly, in the absence of FK506, SIB1 FKBP22 and its variants inhibited, with the exception of N-domain, calcineurin phosphatase activity, albeit low. The inhibition of SIB1 FKBP22 by FK506 is dramatically increased in the presence of FK506. Altogether, we proposed that local structure at substrate binding pocket of C-domain plays crucial role for the binding of FK506 and peptide substrate preferences. In addition, C-domain is essential for inhibition, while dimerization state is important for optimum inhibition through efficient binding to calcineurin.
  13. Avicor SW, Wajidi MF, El-Garj FM, Jaal Z, Yahaya ZS
    Protein J, 2014 Oct;33(5):457-64.
    PMID: 25199940 DOI: 10.1007/s10930-014-9580-z
    Mosquito coils are insecticides commonly used for protection against mosquitoes due to their toxic effects on mosquito populations. These effects on mosquitoes could induce the expression of metabolic enzymes in exposed populations as a counteractive measure. Cytochrome P450 family 4 (CYP4) are metabolic enzymes associated with a wide range of biological activities including insecticide resistance. In this study, the efficacies of three commercial mosquito coils with different pyrethroid active ingredients were assessed and their potential to induce the expression of CYP4 genes in Aedes albopictus analyzed by real-time quantitative PCR. Coils containing 0.3 % D-allethrin and 0.005 % metofluthrin exacted profound toxic effects on Ae. albopictus, inducing high mortalities (≥90 %) compared to the 0.2 % D-allethrin reference coil. CYP4H42 and CYP4H43 expressions were significantly higher in 0.3 % D-allethrin treated mosquitoes compared to the other treated populations. Short-term (KT50) exposure to mosquito coils induced significantly higher expression of both genes in 0.005 % metofluthrin exposed mosquitoes. These results suggest the evaluated products provided better protection than the reference coil; however, they also induced the expression of metabolic genes which could impact negatively on personal protection against mosquito.
  14. Zeeshan F, Tabbassum M, Kesharwani P
    Protein J, 2019 10;38(5):551-564.
    PMID: 31054037 DOI: 10.1007/s10930-019-09837-4
    Protein drugs are important therapeutic agents however; they may degrade during formulation processing. The objective of this study was to investigate the correlation between secondary structure alterations and the retentions of biological activity of protein upon the application of thermal stress. Catalase, horseradish peroxidase and α- chymotrypsin were employed as model proteins. Each protein was heated in a solid and solution state at a temperature of 70 °C for 1 h. Attenuated total reflectance Fourier transform infrared spectroscopy, size-exclusion chromatography and biological activity assay were performed. Results showed that heat-exposure of protein solids at 70 °C caused minimum changes in secondary structure and biological activity was almost retained. However, thermal exposure of protein aqueous solution induced significant changes in the secondary structure indicated by area overlap values and caused considerable reduction in the biological activity. The changes in secondary structures were found to be in full alignment with the loss of biological activity for both protein solids as well as aqueous solutions. Catalase lost entire biological activity upon heating in the solution state. In conclusion, the findings of the present study indicate a direct correlation between protein secondary structure alterations and the retention of biological activity which can be taken into account during the development and delivery of protein drugs formulations.
  15. Choong YS, Tye GJ, Lim TS
    Protein J, 2013 Oct;32(7):505-11.
    PMID: 24096348 DOI: 10.1007/s10930-013-9514-1
    The limited sequence similarity of protein sequences with known structures has led to an indispensable need for computational technology to predict their structures. Structural bioinformatics (SB) has become integral in elucidating the sequence-structure-function relationship of a protein. This report focuses on the applications of SB within the context of protein engineering including its limitation and future challenges.
  16. Abedi Karjiban R, Lim WZ, Basri M, Abdul Rahman MB
    Protein J, 2014 Aug;33(4):369-76.
    PMID: 24871480 DOI: 10.1007/s10930-014-9568-8
    Lipases are known for their versatility in addition to their ability to digest fat. They can be used for the formulation of detergents, as food ingredients and as biocatalysts in many industrial processes. Because conventional enzymes are frangible at high temperatures, the replacement of conventional chemical routes with biochemical processes that utilize thermostable lipases is vital in the industrial setting. Recent theoretical studies on enzymes have provided numerous fundamental insights into the structures, folding mechanisms and stabilities of these proteins. The studies corroborate the experimental results and provide additional information regarding the structures that were determined experimentally. In this paper, we review the computational studies that have described how temperature affects the structure and dynamics of thermoenzymes, including the thermoalkalophilic L1 lipase derived from Bacillus stearothermophilus. We will also discuss the potential of using pressure for the analysis of the stability of thermoenzymes because high pressure is also important for the processing and preservation of foods.
  17. Srikumar PS, Rohini K, Rajesh PK
    Protein J, 2014 Jun;33(3):289-95.
    PMID: 24770803 DOI: 10.1007/s10930-014-9561-2
    Mutations in human laforin lead to an autosomal neurodegenerative disorder Lafora disease. In N-terminal carbohydrate binding domain of laforin, two mutations W32G and K87A are reported as highly disease causing laforin mutants. Experimental studies reported that mutations are responsible for the abolishment of glycogen binding which is a critical function of laforin. Our current computational study focused on the role of conformational changes in human laforin structure due to existing single mutation W32G and prepared double mutation W32G/K87A related to loss of glycogen binding. We performed 10 ns molecular dynamics (MD) simulation studies in the Gromacs package for both mutations and analyzed the trajectories. From the results, the global properties like root mean square deviation, root mean square fluctuation, radius of gyration, solvent accessible surface area and hydrogen bonds showed structural changes in atomic level observed in W32G and W32G/K87A laforin mutants. The conformational change induced by mutants influenced the loss of the overall stability of the native laforin. Moreover, the change in overall motion of protein was analyzed by principal component analysis and results showed protein clusters expanded more than native and also change in direction in case of double mutant in conformational space. Overall, our report provides theoretical information on loss of structure-function relationship due to flexible nature of laforin mutants. In conclusion, comparative MD simulation studies support the experimental data on W32G and W32G/K87A related to the lafora disease mechanism on glycogen binding.
  18. Abedi Karjiban R, Abdul Rahman MB, Basri M, Salleh AB, Jacobs D, Abdul Wahab H
    Protein J, 2009 Jan;28(1):14-23.
    PMID: 19130194 DOI: 10.1007/s10930-008-9159-7
    Molecular Dynamics (MD) simulations have been used to understand how protein structure, dynamics, and flexibility are affected by adaptation to high temperature for several years. We report here the results of the high temperature MD simulations of Bacillus stearothermophilus L1 (L1 lipase). We found that the N-terminal moiety of the enzyme showed a high flexibility and dynamics during high temperature simulations which preceded and followed by clear structural changes in two specific regions; the small domain and the main catalytic domain or core domain of the enzyme. These two domains interact with each other through a Zn(2+)-binding coordination with Asp-61 and Asp-238 from the core domain and His-81 and His-87 from the small domain. Interestingly, the His-81 and His-87 were among the highly fluctuated and mobile residues at high temperatures. The results appear to suggest that tight interactions of Zn(2+)-binding coordination with specified residues became weak at high temperature which suggests the contribution of this region to the thermostability of the enzyme.
  19. Hamid TH, Rahman RN, Salleh AB, Basri M
    Protein J, 2010 May;29(4):290-7.
    PMID: 20509044 DOI: 10.1007/s10930-010-9251-7
    The use of lipase in hydrophilic solvent is usually hampered by inactivation. The solvent stability of a recombinant solvent stable lipase isolated from thermostable Bacillus sp. strain 42 (Lip 42), in DMSO and methanol were studied at different solvent-water compositions. The enzymatic activities were retained in up to 45% v/v solvent compositions. The near-UV CD spectra indicated that tertiary structures were perturbed at 60% v/v and above. Far-UV CD in methanol indicated the secondary structure in Lip 42 was retained throughout all solvent compositions. Fluorescence studies indicated formations of molten globules in solvent compositions of 60% v/v and above. The enzyme was able to retain its secondary structures in the presence of methanol; however, there was a general reduction in beta-sheet and an increase in alpha-helix contents. The H-bonding arrangements triggered in methanol and DMSO, respectively, caused different forms of tertiary structure perturbations on Lip 42, despite both showing partial denaturation with molten globule formations.
  20. Daim LD, Ooi TE, Yusof HM, Majid NA, Karsani SA
    Protein J, 2015 Aug;34(4):304-12.
    PMID: 26263918 DOI: 10.1007/s10930-015-9626-x
    Oil palm (Elaeis guineensis) is an important economic crop cultivated for its nutritional palm oil. A significant amount of effort has been undertaken to understand oil palm growth and physiology at the molecular level, particularly in genomics and transcriptomics. Recently, proteomics studies have begun to garner interest. However, this effort is impeded by technical challenges. Plant sample preparation for proteomics analysis is plagued with technical challenges due to the presence of polysaccharides, secondary metabolites and other interfering compounds. Although protein extraction methods for plant tissues exist, none work universally on all sample types. Therefore, this study aims to compare and optimize different protein extraction protocols for use with two-dimensional gel electrophoresis of young and mature leaves from the oil palm. Four protein extraction methods were evaluated: phenol-guanidine isothiocyanate, trichloroacetic acid-acetone precipitation, sucrose and trichloroacetic acid-acetone-phenol. Of these four protocols, the trichloroacetic acid-acetone-phenol method was found to give the highest resolution and most reproducible gel. The results from this study can be used in sample preparations of oil palm tissue for proteomics work.
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