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Search Results: 1 - 10 of 2160 matches for " Raja Noor Zaliha Raja; "
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Engineering catalytic efficiency of thermophilic lipase from Geobacillus zalihae by hydrophobic residue mutation near the catalytic pocket  [PDF]
Roswanira Abdul Wahab, Mahiran Basri, Mohd Basyaruddin Abdul Rahman, Raja Noor Zaliha Raja Abdul Rahman, Abu Bakar Salleh, Leow Thean Chor
Advances in Bioscience and Biotechnology (ABB) , 2012, DOI: 10.4236/abb.2012.32024
Abstract: In-silico and experimental investigations were conducted to explore the effects of substituting hydrophobic residues; Val, Met, Leu, Ile, Trp and Phe into the oxyanion Q114 of T1 lipase. We hypothesized that the oxyanion Q114, involved in substrate binding is also associated with modulation of conformational stability and in conferring specific enzyme attributes. The insilico investigations accurately predicted the quality of the protein packing in some of the variants. Our study found by altering the hydrophobicity of the oxyanion 114, remarkably altered enzyme conformational stability and catalytic attributes. Substitution with Leu resulted improvements in four out of the six tested characteristics. The hydrophobic Leu might have improved local structure folding and increased hydrophobic interactions with other residues in the vicinity of the mutation. The Met variant showed higher activity over the wild-type in hydrolyzing a wider range of natural oils. The bulky amino acids, Phe and Trp negatively affected T1 lipase and resulted in the largest disruption of protein stability and inferior enzyme characteristics. We have successfully illustrated that a single point residue changes at oxyanion 114 could result in a myriad of enzyme attributes, which implied there was some interplay between hydrophobicity and conformation for lipase catalytic functions.
Role of α-Helical Structure in Organic Solvent-Activated Homodimer of Elastase Strain K
Raja Noor Zaliha Raja Abd. Rahman,Abu Bakar Salleh,Mahiran Basri,Chee Fah Wong
International Journal of Molecular Sciences , 2011, DOI: 10.3390/ijms12095797
Abstract: Recombinant elastase strain K overexpressed from E. coli KRX/pCon2(3) was purified to homogeneity by a combination of hydrophobic interaction chromatography and ion exchange chromatography, with a final yield of 48% and a 25-fold increase in specific activity. The purified protein had exhibited a first ever reported homodimer size of 65 kDa by SDS-PAGE and MALDI-TOF, a size which is totally distinct from that of typically reported 33 kDa monomer from P. aeruginosa. The organic solvent stability experiment had demonstrated a stability pattern which completely opposed the rules laid out in previous reports in which activity stability and enhancement were observed in hydrophilic organic solvents such as DMSO, methanol, ethanol and 1-propanol. The high stability and enhancement of the enzyme in hydrophilic solvents were explained from the view of alteration in secondary structures. Elastinolytic activation and stability were observed in 25 and 50% of methanol, respectively, despite slight reduction in α-helical structure caused upon the addition of the solvent. Further characterization experiments had postulated great stability and enhancement of elastase strain K in broad range of temperatures, pHs, metal ions, surfactants, denaturing agents and substrate specificity, indicating its potential application in detergent formulation.
A Newly Isolated Thermostable Lipase from Bacillus sp.
Fairolniza Mohd Shariff,Raja Noor Zaliha Raja Abd. Rahman,Mahiran Basri,Abu Bakar Salleh
International Journal of Molecular Sciences , 2011, DOI: 10.3390/ijms12052917
Abstract: A thermophilic lipolytic bacterium identified as Bacillus sp. L2 via 16S rDNA was previously isolated from a hot spring in Perak, Malaysia. Bacillus sp. L2 was confirmed to be in Group 5 of bacterial classification, a phylogenically and phenotypically coherent group of thermophilic bacilli displaying very high similarity among their 16S rRNA sequences (98.5–99.2%). Polymerase chain reaction (PCR) cloning of L2 lipase gene was conducted by using five different primers. Sequence analysis of the L2 lipase gene revealed an open reading frame (ORF) of 1251 bp that codes for 417 amino acids. The signal peptides consist of 28 amino acids. The mature protein is made of 388 amino acid residues. Recombinant lipase was successfully overexpressed with a 178-fold increase in activity compared to crude native L2 lipase. The recombinant L2 lipase (43.2 kDa) was purified to homogeneity in a single chromatography step. The purified lipase was found to be reactive at a temperature range of 55–80 °C and at a pH of 6–10. The L2 lipase had a melting temperature (Tm) of 59.04 °C when analyzed by circular dichroism (CD) spectroscopy studies. The optimum activity was found to be at 70 °C and pH 9. Lipase L2 was strongly inhibited by ethylenediaminetetraacetic acid (EDTA) (100%), whereas phenylmethylsulfonyl fluoride (PMSF), pepstatin-A, 2-mercaptoethanol and dithiothreitol (DTT) inhibited the enzyme by over 40%. The CD spectra of secondary structure analysis showed that the L2 lipase structure contained 38.6% α-helices, 2.2% ?-strands, 23.6% turns and 35.6% random conformations.
3D Structure Elucidation of Thermostable L2 Lipase from Thermophilic Bacillus sp. L2
Raja Noor Zaliha Raja Abd. Rahman,Fairolniza Mohd Shariff,Mahiran Basri,Abu Bakar Salleh
International Journal of Molecular Sciences , 2012, DOI: 10.3390/ijms13079207
Abstract: The crystallization of proteins makes it possible to determine their structure by X-ray crystallography, and is therefore important for the analysis of protein structure-function relationships. L2 lipase was crystallized by using the J-tube counter diffusion method. A crystallization consisting of 20% PEG 6000, 50 mM MES pH 6.5 and 50 mM NaCl was found to be the best condition to produce crystals with good shape and size (0.5 × 0.1 × 0.2 mm). The protein concentration used for the crystallization was 3 mg/mL. L2 lipase crystal has two crystal forms, Shape 1 and Shape 2. Shape 2 L2 lipase crystal was diffracted at 1.5 ? and the crystal belongs to the orthorhombic space group P2 12 12 1, with unit-cell parameters a = 72.0, b = 81.8, c = 83.4 ?, α = β = γ = 90°. There is one molecule per asymmetric unit and the solvent content of the crystals is 56.9%, with a Matthew’s coefficient of 2.85 ? Da ?1. The 3D structure of L2 lipase revealed topological organization of α/β-hydrolase fold consisting of 11 β-strands and 13 α-helices. Ser-113, His-358 and Asp-317 were assigned as catalytic triad residues. One Ca 2+ and one Zn 2+ were found in the L2 lipase molecule.
Enzymatic Properties and Mutational Studies of Chalcone Synthase from Physcomitrella patens
Raja Noor Zaliha Raja Abdul Rahman,Iffah Izzati Zakaria,Abu Bakar Salleh,Mahiran Basri
International Journal of Molecular Sciences , 2012, DOI: 10.3390/ijms13089673
Abstract: PpCHS is a member of the type III polyketide synthase family and catalyses the synthesis of the flavonoid precursor naringenin chalcone from p-coumaroyl-CoA. Recent research reports the production of pyrone derivatives using either hexanoyl-CoA or butyryl-CoA as starter molecule. The Cys-His-Asn catalytic triad found in other plant chalcone synthase predicted polypeptides is conserved in PpCHS. Site directed mutagenesis involving these amino acids residing in the active-site cavity revealed that the cavity volume of the active-site plays a significant role in the selection of starter molecules as well as product formation. Substitutions of Cys 170 with Arg and Ser amino acids decreased the ability of the PpCHS to utilize hexanoyl-CoA as a starter molecule, which directly effected the production of pyrone derivatives (products). These substitutions are believed to have a restricted number of elongations of the growing polypeptide chain due to the smaller cavity volume of the mutant’s active site.
Molecular Modeling of a Predominant β-CGTase G1 and Analysis of Ionic Interaction in CGTase
Kian Mau Goh,Nor Muhammad Mahadi,Osman Hassan,Raja Noor Zaliha Raja Abdul Rahman
Biotechnology , 2008,
Abstract: The protein 3D structure for CGTase G1 was determined by homology modeling and a good structure was generated after three rounds of energy minimization process. The ERRAT and Verify3D scores for the predicted structure were determined as 98.07 and 90.991%, respectively. The presences of ionic interactions inside the CGTase G1 structure were compared with five CGTases crystal-structures. Mesophilic CGTases have lesser numbers of ionic pairs (average of 72.3 pairs) than of the thermophilic CGTases (average of 78.6 pairs). Most of the interactions in CGTases were involved in the form of networking. The average number for networking ionic pairs in thermostable and mesophilic CGTases is 69.3 and 62.7, respectively. Most of the ionic interactions in CGTases were found in Domain A and the most complex ionic networking was located in this catalytic domain as well. These charged-residues generate interlinking networking that covers a huge area that surrounds the active site groove. A few numbers of secondary structures strands were interlinked by the ionic interactions and this creates a natural protection for the catalytic TIM-barrel structure (Domain A) against heat. Most of the residues involved are consensus, however, slight variations were found. The triad Asp181-Arg185-Asp175 might plays an important factor in the networking which causes the half life of CGTase G1 to be slightly higher compared to other CGTases originally produced by mesophilic strains.
Improvement of Thermal Stability via Outer-Loop Ion Pair Interaction of Mutated T1 Lipase from Geobacillus zalihae Strain T1
Rudzanna Ruslan,Raja Noor Zaliha Raja Abd. Rahman,Thean Chor Leow,Mohd Shukuri Mohamad Ali,Mahiran Basri,Abu Bakar Salleh
International Journal of Molecular Sciences , 2012, DOI: 10.3390/ijms13010943
Abstract: Mutant D311E and K344R were constructed using site-directed mutagenesis to introduce an additional ion pair at the inter-loop and the intra-loop, respectively, to determine the effect of ion pairs on the stability of T1 lipase isolated from Geobacillus zalihae. A series of purification steps was applied, and the pure lipases of T1, D311E and K344R were obtained. The wild-type and mutant lipases were analyzed using circular dichroism. The Tm for T1 lipase, D311E lipase and K344R lipase were approximately 68.52 °C, 70.59 °C and 68.54 °C, respectively. Mutation at D311 increases the stability of T1 lipase and exhibited higher Tm as compared to the wild-type and K344R. Based on the above, D311E lipase was chosen for further study. D311E lipase was successfully crystallized using the sitting drop vapor diffusion method. The crystal was diffracted at 2.1 ? using an in-house X-ray beam and belonged to the monoclinic space group C2 with the unit cell parameters a = 117.32 ?, b = 81.16 ? and c = 100.14 ?. Structural analysis showed the existence of an additional ion pair around E311 in the structure of D311E. The additional ion pair in D311E may regulate the stability of this mutant lipase at high temperatures as predicted in silico and spectroscopically.
Expression of an Organic Solvent Stable Lipase from Staphylococcus epidermidis AT2
Raja Noor Zaliha Raja Abd. Rahman,Nor Hafizah Ahmad Kamarudin,Jalimah Yunus,Abu Bakar Salleh,Mahiran Basri
International Journal of Molecular Sciences , 2010, DOI: 10.3390/ijms11093195
Abstract: An organic solvent tolerant lipase gene from Staphylococcus epidermidis AT2 was successfully cloned and expressed with pTrcHis2 in E. coli TOP10. Sequence analysis revealed an open reading frame (ORF) of 1,933 bp in length which coded for a polypeptide of 643 amino acid residues. The polypeptide comprised of a signal peptide (37 amino acids), pro-peptide and a mature protein of 390 amino acids. Expression of AT2 lipase resulted in an 18-fold increase in activity, upon the induction of 0.6?mM?IPTG after a 10?h incubation period. Interestingly, this lipase was stable in various organic solvents (25% (v/v), mainly toluene, octanol, p-xylene and n-hexane). Literature shows that most of the organic solvent stable bacterial lipases were produced by Pseudomonas sp. and Bacillus sp., but very few from Staphylococcus sp. This lipase demonstrates great potential to be employed in various industrial applications.
Lipase production and growth modeling of a novel thermophilic bacterium: Aneurinibacillus thermoaerophilus strain AFNA
Ebrahimpour,Afshin; Rahman,Raja Noor Zaliha Raja Abd; Kamarudin,Nor Hafizah Ahmad; Basri,Mahiran; Salleh,Abu Bakar;
Electronic Journal of Biotechnology , 2011,
Abstract: aneurinibacillus thermoaerophilus strain afna as a novel isolated extracellular thermostable organic solvent tolerant lipase producing bacterium was employed in the present study. the lipase production of strain afna and its correlation with bacterial growth was studied via a modeling assessment by response surface methodology (rsm) and artificial neural network (ann) techniques. the best achieved models were multilayer full feed forward incremental back propagation network and modified cubic response surface model (mrsm) using backward elimination. the highest lipase specific activity (13.1 umg-1) and bacterial growth (od600 = 3.0) were obtained at technically similar: growth temperature (53 and 53oc), inoculum size (2.6 and 3.0%), agitation rate (118 and 115 rpm) and initial ph (7.0 and 7.2) but different medium volume (139 and 87 ml) and incubation period (48 and 38 hrs), respectively. in addition, the importance of effective parameters on the bacterial growth and lipase production was studied where ph and inoculum size were the most and the least effective factors, respectively. significant correlation between lipase production and bacterial growth was observed when bivariate correlation was employed to analyse the data. as a conclusion, lipase production was the result of a synergistic combination of effective parameters interactions and these parameters were in equilibrium.
Combination of Oxyanion Gln114 Mutation and Medium Engineering to Influence the Enantioselectivity of Thermophilic Lipase from Geobacillus zalihae
Roswanira Abdul Wahab,Mahiran Basri,Mohd Basyaruddin Abdul Rahman,Raja Noor Zaliha Raja Abdul Rahman,Abu Bakar Salleh,Thean Chor Leow
International Journal of Molecular Sciences , 2012, DOI: 10.3390/ijms130911666
Abstract: The substitution of the oxyanion Q114 with Met and Leu was carried out to investigate the role of Q114 in imparting enantioselectivity on T1 lipase. The mutation improved enantioselectivity in Q114M over the wild-type, while enantioselectivity in Q114L was reduced. The enantioselectivity of the thermophilic lipases, T1, Q114L and Q114M correlated better with log p as compared to the dielectric constant and dipole moment of the solvents. Enzyme activity was good in solvents with log p < 3.5, with the exception of hexane which deviated substantially. Isooctane was found to be the best solvent for the esterification of ( R, S)-ibuprofen with oleyl alcohol for lipases Q114M and Q114L, to afford E values of 53.7 and 12.2, respectively. Selectivity of T1 was highest in tetradecane with E value 49.2. Solvents with low log p reduced overall lipase activity and dimethyl sulfoxide ( DMSO) completely inhibited the lipases. Ester conversions, however, were still low. Molecular sieves employed as desiccant were found to adversely affect catalysis in the lipase variants, particularly in Q114M. The higher desiccant loading also increased viscosity in the reaction and further reduced the efficiency of the lipase-catalyzed esterifications.
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