Displaying publications 1 - 20 of 21 in total

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  1. Che Hussian CHA, Raja Abd Rahman RNZ, Thean Chor AL, Salleh AB, Mohamad Ali MS
    PeerJ, 2018;6:e5833.
    PMID: 30479887 DOI: 10.7717/peerj.5833
    T1 Lipase is a thermostable secretary protein of Geobacillus zalihae strain previously expressed in a prokaryotic system and purified using three-step purification: affinity 1, affinity 2, and ion exchange chromatography (IEX). This approach is time consuming and offers low purity and recovery yield. In order to enhance the purification strategy of T1 lipase, affinity 2 was removed so that after affinity 1, the cleaved Glutathione S-transferase (GST) and matured T1 lipase could be directly separated through IEX. Therefore, a rational design of GST isoelectric point (pI) was implemented by prediction using ExPASy software in order to enhance the differences of pI values between GST and matured T1 lipase. Site-directed mutagenesis at two locations flanking the downstream region of GST sequences (H215R and G213R) was successfully performed. Double point mutations changed the charge on GST from 6.10 to 6.53. The purified lipase from the new construct GST tag mutant-T1 was successfully purified using two steps of purification with 6,849 U/mg of lipase specific activity, 33% yield, and a 44-fold increase in purification. Hence, the increment of the pI values in the GST tag fusion T1 lipase resulted in a successful direct separation through IEX and lead to successful purification.
  2. Garba L, Mohamad Yussoff MA, Abd Halim KB, Ishak SNH, Mohamad Ali MS, Oslan SN, et al.
    PeerJ, 2018;6:e4347.
    PMID: 29576935 DOI: 10.7717/peerj.4347
    Membrane-bound fatty acid desaturases perform oxygenated desaturation reactions to insert double bonds within fatty acyl chains in regioselective and stereoselective manners. The Δ9-fatty acid desaturase strictly creates the first double bond between C9 and 10 positions of most saturated substrates. As the three-dimensional structures of the bacterial membrane fatty acid desaturases are not available, relevant information about the enzymes are derived from their amino acid sequences, site-directed mutagenesis and domain swapping in similar membrane-bound desaturases. The cold-tolerantPseudomonassp. AMS8 was found to produce high amount of monounsaturated fatty acids at low temperature. Subsequently, an active Δ9-fatty acid desaturase was isolated and functionally expressed inEscherichia coli. In this paper we report homology modeling and docking studies of a Δ9-fatty acid desaturase from a Cold-tolerantPseudomonassp. AMS8 for the first time to the best of our knowledge. Three dimensional structure of the enzyme was built using MODELLER version 9.18 using a suitable template. The protein model contained the three conserved-histidine residues typical for all membrane-bound desaturase catalytic activity. The structure was subjected to energy minimization and checked for correctness using Ramachandran plots and ERRAT, which showed a good quality model of 91.6 and 65.0%, respectively. The protein model was used to preform MD simulation and docking of palmitic acid using CHARMM36 force field in GROMACS Version 5 and Autodock tool Version 4.2, respectively. The docking simulation with the lowest binding energy, -6.8 kcal/mol had a number of residues in close contact with the docked palmitic acid namely, Ile26, Tyr95, Val179, Gly180, Pro64, Glu203, His34, His206, His71, Arg182, Thr85, Lys98 and His177. Interestingly, among the binding residues are His34, His71 and His206 from the first, second, and third conserved histidine motif, respectively, which constitute the active site of the enzyme. The results obtained are in compliance with thein vivoactivity of the Δ9-fatty acid desaturase on the membrane phospholipids.
  3. Garba L, Mohamad Ali MS, Oslan SN, Rahman RN
    PLoS One, 2016 Aug 5;11(8):e0160681.
    PMID: 27494717 DOI: 10.1371/journal.pone.0160681
    Fatty acid desaturase enzymes play an essential role in the synthesis of unsaturated fatty acids. Pseudomonas sp. A3 was found to produce a large amount of palmitoleic and oleic acids after incubation at low temperatures. Using polymerase Chain Reaction (PCR), a novel Δ9- fatty acid desaturase gene was isolated, cloned, and successfully expressed in Escherichia coli. The gene was designated as PA3FAD9 and has an open reading frame of 1,185 bp which codes for 394 amino acids with a predicted molecular weight of 45 kDa. The activity of the gene product was confirmed via GCMS, which showed a functional putative Δ9-fatty acid desaturase capable of increasing the total amount of cellular unsaturated fatty acids of the E. coli cells expressing the gene. The results demonstrate that the cellular palmitoleic acids have increased two-fold upon expression at 15°C using only 0.1 mM IPTG. Therefore, PA3FAD9 from Pseudomonas sp.A3 codes for a Δ9-fatty acid desaturase-like protein which was actively expressed in E. coli.
  4. Khusaini MS, Rahman RN, Mohamad Ali MS, Leow TC, Basri M, Salleh AB
    PMID: 21393852 DOI: 10.1107/S1744309111002028
    An organic solvent-tolerant lipase from Bacillus sp. strain 42 was crystallized using the capillary-tube method. The purpose of studying this enzyme was in order to better understand its folding and to characterize its properties in organic solvents. By initially solving its structure in the native state, further studies on protein-solvent interactions could be performed. X-ray data were collected at 2.0 Å resolution using an in-house diffractometer. The estimated crystal dimensions were 0.09×0.19×0.08 mm. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a=117.41, b=80.85, c=99.44 Å, β=96.40°.
  5. Latip W, Raja Abd Rahman RNZ, Chor Leow AT, Mohd Shariff F, Mohamad Ali MS
    PeerJ, 2016;4:e2420.
    PMID: 27781152 DOI: 10.7717/peerj.2420
    A gene encoding a thermotolerant lipase with broad pH was isolated from an Antarctic Pseudomonas strain AMS3. The recombinant lipase AMS3 was purified by single-step purification using affinity chromatography, yielding a purification fold of approximately 1.52 and a recovery of 50%. The molecular weight was approximately ∼60 kDa including the strep and affinity tags. Interestingly, the purified Antarctic AMS3 lipase exhibited broad temperature profile from 10-70 °C and stable over a broad pH range from 5.0 to pH 10.0. Various mono and divalent metal ions increased the activity of the AMS3 lipase, but Ni(2+) decreased its activity. The purified lipase exhibited the highest activity in the presence of sunflower oil. In addition, the enzyme activity in 25% v/v solvents at 50 °C particularly to n-hexane, DMSO and methanol could be useful for catalysis reaction in organic solvent and at broad temperature.
  6. Latip W, Raja Abd Rahman RNZ, Leow ATC, Mohd Shariff F, Kamarudin NHA, Mohamad Ali MS
    Int J Mol Sci, 2018 Feb 13;19(2).
    PMID: 29438291 DOI: 10.3390/ijms19020560
    Lipase plays an important role in industrial and biotechnological applications. Lipases have been subject to modification at the N and C terminals, allowing better understanding of lipase stability and the discovery of novel properties. A thermotolerant lipase has been isolated from Antarctic Pseudomonas sp. The purified Antarctic AMS3 lipase (native) was found to be stable across a broad range of temperatures and pH levels. The lipase has a partial Glutathione-S-transferase type C (GST-C) domain at the N-terminal not found in other lipases. To understand the influence of N-terminal GST-C domain on the biochemical and structural features of the native lipase, the deletion of the GST-C domain was carried out. The truncated protein was successfully expressed in E. coli BL21(DE3). The molecular weight of truncated AMS3 lipase was approximately ~45 kDa. The number of truncated AMS3 lipase purification folds was higher than native lipase. Various mono and divalent metal ions increased the activity of the AMS3 lipase. The truncated AMS3 lipase demonstrated a similarly broad temperature range, with the pH profile exhibiting higher activity under alkaline conditions. The purified lipase showed a substrate preference for a long carbon chain substrate. In addition, the enzyme activity in organic solvents was enhanced, especially for toluene, Dimethylsulfoxide (DMSO), chloroform and xylene. Molecular simulation revealed that the truncated lipase had increased structural compactness and rigidity as compared to native lipase. Removal of the N terminal GST-C generally improved the lipase biochemical characteristics. This enzyme may be utilized for industrial purposes.
  7. Lim SJ, Mohamad Ali MS, Sabri S, Muhd Noor ND, Salleh AB, Oslan SN
    Med Mycol, 2021 Dec 03;59(12):1127-1144.
    PMID: 34506621 DOI: 10.1093/mmy/myab053
    Candidiasis is a fungal infection caused by Candida spp. especially Candida albicans, C. glabrata, C. parapsilosis and C. tropicalis. Although the medicinal therapeutic strategies have rapidly improved, the mortality rate as candidiasis has continuously increased. The secreted and membrane-bound virulence factors (VFs) are responsible for fungal invasion, damage and translocation through the host enterocytes besides the evasion from host immune system. VFs such as agglutinin-like sequences (Als), heat shock protein 70, phospholipases, secreted aspartyl proteinases (Sap), lipases, enolases and phytases are mostly hydrolases which degrade or interact with the enterocyte membrane components. Candidalysin, however, acts as a peptide toxin to induce necrotic cell lysis. To date, structural studies of the VFs remain underexplored, hindering their functional analyses. Among the VFs, only Sap and Als have their structures deposited in Protein Data Bank (PDB). Therefore, this review scrutinizes the mechanisms of these VFs by discussing the VF-deficient studies of several Candida spp. and their abilities to produce these VFs. Nonetheless, their latest reported sequential and structural analyses are discussed to impart a wider perception of the host-pathogen interactions and potential vaccine or antifungal drug targets. This review signifies that more VFs structural investigations and mining in the emerging Candida spp. are required to decipher their pathogenicity and virulence mechanisms compared to the prominent C. albicans.

    LAY SUMMARY: Candida virulence factors (VFs) including mainly enzymes and proteins play vital roles in breaching the human intestinal barrier and causing deadly invasive candidiasis. Limited VFs' structural studies hinder deeper comprehension of their mechanisms and thus the design of vaccines and antifungal drugs against fungal infections.

  8. Lim SJ, Muhd Noor ND, Sabri S, Mohamad Ali MS, Salleh AB, Oslan SN
    J Biomol Struct Dyn, 2024 Jan 08.
    PMID: 38189364 DOI: 10.1080/07391102.2023.2300757
    Meyerozyma guilliermondii is a rare yeast pathogen contributing to the deadly invasive candidiasis. M. guilliermondii strain SO, as a promising protein expression host, showed 99% proteome similarity with the clinically isolated ATCC 6260 (type strain) in a recent comparative genomic analysis. However, their in vitro virulence features and in vivo pathogenicity were uncharacterized. This study aimed to characterize the in vitro and in vivo pathogenicity of M. guilliermondii strain SO and analyze its Als proteins (MgAls) via comprehensive bioinformatics approaches. M. guilliermondii strain SO showed lower and higher sensitivity towards β-mercaptoethanol and lithium, respectively than the avirulent S. cerevisiae but exhibited the same tolerance towards cell wall-perturbing Congo Red with C. albicans. With 7.5× higher biofilm mass, M. guilliermondii strain SO also demonstrated 75% higher mortality rate in the zebrafish embryos with a thicker biofilm layer on the chorion compared to the avirulent S. cerevisiae. Being one of the most important Candida adhesins, sequence and structural analyses of four statistically identified MgAls showed that MgAls1056 was predicted to exhibit the most conserved amyloid-forming regions, tandem repeat domain and peptide binding cavity (PBC) compared to C. albicans Als3. Favoured from the predicted largest ligand binding site and druggable pockets, it showed the highest affinity towards hepta-threonine. Non-PBC druggable pockets in the most potent virulence contributing MgAls1056 provide new insights into developing antifungal drugs targeting non-albicans Candida spp. Virtual screening of available synthetic or natural bioactive compounds and MgAls1056 deletion from the fungal genome should be further performed and validated experimentally.Communicated by Ramaswamy H. Sarma.
  9. Lim SJ, Muhd Noor ND, Sabri S, Mohamad Ali MS, Salleh AB, Oslan SN
    Med Mycol, 2024 Jan 09;62(1).
    PMID: 38061839 DOI: 10.1093/mmy/myad126
    Invasive candidiasis caused by the pathogenic Candida yeast species has resulted in elevating global mortality. The pathogenicity of Candida spp. is not only originated from its primary invasive yeast-to-hyphal transition; virulence factors (transcription factors, adhesins, invasins, and enzymes), biofilm, antifungal drug resistance, stress tolerance, and metabolic adaptation have also contributed to a greater clinical burden. However, the current research theme in fungal pathogenicity could hardly be delineated with the increasing research output. Therefore, our study analysed the research trends in Candida pathogenesis over the past 37 years via a bibliometric approach against the Scopus and Web of Science databases. Based on the 3993 unique documents retrieved, significant international collaborations among researchers were observed, especially between Germany (Bernhard Hube) and the UK (Julian Naglik), whose focuses are on Candida proteinases, adhesins, and candidalysin. The prominent researchers (Neils Gow, Alistair Brown, and Frank Odds) at the University of Exeter and the University of Aberdeen (second top performing affiliation) UK contribute significantly to the mechanisms of Candida adaptation, tolerance, and stress response. However, the science mapping of co-citation analysis performed herein could not identify a hub representative of subsequent work since the clusters were semi-redundant. The co-word analysis that was otherwise adopted, revealed three research clusters; the cluster-based thematic analyses indicated the severeness of Candida biofilm and antifungal resistance as well as the elevating trend on molecular mechanism elucidation for drug screening and repurposing. Importantly, the in vivo pathogen adaptation and interactions with hosts are crucial for potential vaccine development.
  10. Maiangwa J, Hamdan SH, Mohamad Ali MS, Salleh AB, Zaliha Raja Abd Rahman RN, Shariff FM, et al.
    J Mol Graph Model, 2021 06;105:107897.
    PMID: 33770705 DOI: 10.1016/j.jmgm.2021.107897
    Critical to the applications of proteins in non-aqueous enzymatic processes is their structural dynamics in relation to solvent polarity. A pool of mutants derived from Geobacillus zalihae T1 lipase was screened in organic solvents (methanol, ethanol, propanol, butanol and pentanol) resulting in the selection of six mutants at initial screening (A83D/K251E, R21C, G35D/S195 N, K84R/R103C/M121I/T272 M and R106H/G327S). Site-directed mutagenesis further yielded quadruple mutants A83D/M121I/K251E/G327S and A83D/M121I/S195 N/T272 M, both of which had improved activity after incubation in methanol. The km and kcat values of these mutants vary marginally with the wild-type enzyme in the methanol/substrate mixture. Thermally induced unfolding of mutants was accompanied with some loss of secondary structure content. The root mean square deviations (RMSD) and B-factors revealed that changes in the structural organization are intertwined with an interplay of the protein backbone with organic solvents. Spatially exposed charged residues showed correlations between the solvation dynamics of the methanol solvent and the hydrophobicity of the residues. The short distances of the radial distribution function provided the required distances for hydrogen bond formation and hydrophobic interactions. These dynamic changes demonstrate newly formed structural interactions could be targeted and incorporated experimentally on the basis of solvent mobility and mutant residues.
  11. Maiangwa J, Mohamad Ali MS, Salleh AB, Rahman RNZRA, Normi YM, Mohd Shariff F, et al.
    PeerJ, 2017;5:e3341.
    PMID: 28533982 DOI: 10.7717/peerj.3341
    The dynamics and conformational landscape of proteins in organic solvents are events of potential interest in nonaqueous process catalysis. Conformational changes, folding transitions, and stability often correspond to structural rearrangements that alter contacts between solvent molecules and amino acid residues. However, in nonaqueous enzymology, organic solvents limit stability and further application of proteins. In the present study, molecular dynamics (MD) of a thermostable Geobacillus zalihae T1 lipase was performed in different chain length polar organic solvents (methanol, ethanol, propanol, butanol, and pentanol) and water mixture systems to a concentration of 50%. On the basis of the MD results, the structural deviations of the backbone atoms elucidated the dynamic effects of water/organic solvent mixtures on the equilibrium state of the protein simulations in decreasing solvent polarity. The results show that the solvent mixture gives rise to deviations in enzyme structure from the native one simulated in water. The drop in the flexibility in H2O, MtOH, EtOH and PrOH simulation mixtures shows that greater motions of residues were influenced in BtOH and PtOH simulation mixtures. Comparing the root mean square fluctuations value with the accessible solvent area (SASA) for every residue showed an almost correspondingly high SASA value of residues to high flexibility and low SASA value to low flexibility. The study further revealed that the organic solvents influenced the formation of more hydrogen bonds in MtOH, EtOH and PrOH and thus, it is assumed that increased intraprotein hydrogen bonding is ultimately correlated to the stability of the protein. However, the solvent accessibility analysis showed that in all solvent systems, hydrophobic residues were exposed and polar residues tended to be buried away from the solvent. Distance variation of the tetrahedral intermediate packing of the active pocket was not conserved in organic solvent systems, which could lead to weaknesses in the catalytic H-bond network and most likely a drop in catalytic activity. The conformational variation of the lid domain caused by the solvent molecules influenced its gradual opening. Formation of additional hydrogen bonds and hydrophobic interactions indicates that the contribution of the cooperative network of interactions could retain the stability of the protein in some solvent systems. Time-correlated atomic motions were used to characterize the correlations between the motions of the atoms from atomic coordinates. The resulting cross-correlation map revealed that the organic solvent mixtures performed functional, concerted, correlated motions in regions of residues of the lid domain to other residues. These observations suggest that varying lengths of polar organic solvents play a significant role in introducing dynamic conformational diversity in proteins in a decreasing order of polarity.
  12. Mohamad Ali MS, Mohd Fuzi SF, Ganasen M, Abdul Rahman RN, Basri M, Salleh AB
    Biomed Res Int, 2013;2013:925373.
    PMID: 23738333 DOI: 10.1155/2013/925373
    The psychrophilic enzyme is an interesting subject to study due to its special ability to adapt to extreme temperatures, unlike typical enzymes. Utilizing computer-aided software, the predicted structure and function of the enzyme lipase AMS8 (LipAMS8) (isolated from the psychrophilic Pseudomonas sp., obtained from the Antarctic soil) are studied. The enzyme shows significant sequence similarities with lipases from Pseudomonas sp. MIS38 and Serratia marcescens. These similarities aid in the prediction of the 3D molecular structure of the enzyme. In this study, 12 ns MD simulation is performed at different temperatures for structural flexibility and stability analysis. The results show that the enzyme is most stable at 0°C and 5°C. In terms of stability and flexibility, the catalytic domain (N-terminus) maintained its stability more than the noncatalytic domain (C-terminus), but the non-catalytic domain showed higher flexibility than the catalytic domain. The analysis of the structure and function of LipAMS8 provides new insights into the structural adaptation of this protein at low temperatures. The information obtained could be a useful tool for low temperature industrial applications and molecular engineering purposes, in the near future.
  13. Mohamad Aris SN, Thean Chor AL, Mohamad Ali MS, Basri M, Salleh AB, Raja Abd Rahman RN
    Biomed Res Int, 2014;2014:904381.
    PMID: 24516857 DOI: 10.1155/2014/904381
    Three-dimensional structure of thermostable lipase is much sought after nowadays as it is important for industrial application mainly found in the food, detergent, and pharmaceutical sectors. Crystallization utilizing the counter diffusion method in space was performed with the aim to obtain high resolution diffracting crystals with better internal order to improve the accuracy of the structure. Thermostable T1 lipase enzyme has been crystallized in laboratory on earth and also under microgravity condition aboard Progress spacecraft to the ISS in collaboration with JAXA (Japanese Aerospace Exploration Agency). This study is conducted with the aims of improving crystal packing and structure resolution. The diffraction data set for ground grown crystal was collected to 1.3 Å resolution and belonged to monoclinic C2 space group with unit cell parameters a = 117.40 Å, b = 80.95 Å, and c = 99.81 Å, whereas the diffraction data set for space grown crystal was collected to 1.1 Å resolution and belonged to monoclinic C2 space group with unit cell parameters a = 117.31 Å, b = 80.85 Å, and c = 99.81 Å. The major difference between the two crystal growth systems is the lack of convection and sedimentation in microgravity environment resulted in the growth of much higher quality crystals of T1 lipase.
  14. Mohd Azrin NA, Mohamad Ali MS, Raja Abd Rahman RNZ, Mohd Shariff F, Ahmad Kamarudin NH, Muhd Noor ND
    J Biomol Struct Dyn, 2023 Aug 22.
    PMID: 37608543 DOI: 10.1080/07391102.2023.2249105
    Rand protease is a serine protease that shared common characteristics with members of the MEROPS S8 subtilisin family. It is thermostable, highly stable in organic solvent and broad in specificity. Many structures of homologous protein solved by X-ray crystallography and NMR have been deposited to Protein Data Bank (PDB) which allowed this study to rely on structure prediction by deep learning to build three-dimensional (3D) structure of full length and mature Rand protease (flRP and mRP). In silico cysteine mutation to 7 predicted high affinity Ca2+ coordinating residues were introduced, and the mutants were subjected to molecular dynamics simulation to study its effect on flRP and mRP. MD simulation showed a marked increase in flexibility of the pro-peptide segment indicating the impact of single cysteine substitution at high affinity Ca2+ coordinating residues to autolysis of flRP. MD simulation for mRP reaffirmed the role of Ca2+ coordinating sites in providing stability to Rand protease. In addition, these residues also affect the autolysis, folding and hydrophobicity of RP. Essential dynamics observed large contribution of the first few eigenvectors of flRP, mRP and their high affinity Ca2+ coordinating residues mutants to the TMSF values which indicates that these values account for a large portion of the overall atomic fluctuations. These results have given a more comprehensive understanding on the role of cysteine substituted Ca2+ coordinating surface loop to the structure of flRP and mRP which are important in contributing to the structural stability of subtilisin.Communicated by Ramaswamy H. Sarma.
  15. Mohtar NS, Abdul Rahman MB, Mustafa S, Mohamad Ali MS, Raja Abd Rahman RNZ
    PeerJ, 2019;7:e6880.
    PMID: 31183251 DOI: 10.7717/peerj.6880
    Sago starch is traditionally used as food especially in Southeast Asia. Generally, sago is safe for consumption, biodegradable, easily available and inexpensive. Therefore, this research was done to expand the potential of sago by using it as a support for enzyme immobilization. In this study, ARM lipase, which was isolated from Geobacillus sp. strain ARM, was overexpressed in Escherichia coli system and then purified using affinity chromatography. The specific activity of the pure enzyme was 650 U/mg, increased 7 folds from the cell lysate. The purified enzyme was immobilized in gelatinized sago and spray-dried by entrapment technique in order to enhance the enzyme operational stability for handling at high temperature and also for storage. The morphology of the gelatinized sago and immobilized enzyme was studied by scanning electron microscopy. The results showed that the spray-dried gelatinized sago was shrunken and became irregular in structure as compared to untreated sago powder. The surface areas and porosities of spray-dried gelatinized sago with and without the enzyme were analyzed using BET and BJH method and have shown an increase in surface area and decrease in pore size. The immobilized ARM lipase showed good performance at 60-80  °C, with a half-life of 4 h and in a pH range 6-9. The immobilized enzyme could be stored at 10 °C with the half-life for 9 months. Collectively, the spray-dried immobilized lipase shows promising capability for industrial uses, especially in food processing.
  16. Salwoom L, Raja Abd Rahman RNZ, Salleh AB, Mohd Shariff F, Convey P, Pearce D, et al.
    Molecules, 2019 Feb 16;24(4).
    PMID: 30781467 DOI: 10.3390/molecules24040715
    In recent years, studies on psychrophilic lipases have been an emerging area of research in the field of enzymology. This study focuses on bacterial strains isolated from anthropogenically-influenced soil samples collected around Signy Island Research Station (South Orkney Islands, maritime Antarctic). Limited information on lipase activities from bacteria isolated from Signy station is currently available. The presence of lipase genes was determined using real time quantification PCR (qPCR) in samples obtained from three different locations on Signy Island. Twenty strains from the location with highest lipase gene detection were screened for lipolytic activities at a temperature of 4 °C, and from this one strain was selected for further examination based on the highest enzymatic activities obtained. Analysis of 16S rRNA sequence data of this strain showed the highest level of sequence similarity (98%) to a Pseudomonas sp. strain also isolated from Antarctica. In order to increase lipase production of this psychrophilic strain, optimisation of different parameters of physical and nutritional factors were investigated. Optimal production was obtained at 10 °C and pH 7.0, at 150 rev/min shaking rate over 36 h incubation.
  17. Salwoom L, Raja Abd Rahman RNZ, Salleh AB, Mohd Shariff F, Convey P, Mohamad Ali MS
    Int J Mol Sci, 2019 Mar 13;20(6).
    PMID: 30871178 DOI: 10.3390/ijms20061264
    In recent years, studies on psychrophilic lipases have become an emerging area of research in the field of enzymology. The study described here focuses on the cold-adapted organic solvent tolerant lipase strain Pseudomonas sp. LSK25 isolated from Signy Station, South Orkney Islands, maritime Antarctic. Strain LSK25 lipase was successfully cloned, sequenced, and over-expressed in an Escherichia coli system. Sequence analysis revealed that the lipase gene of Pseudomonas sp. LSK25 consists of 1432 bp, lacks an N-terminal signal peptide and encodes a mature protein consisting of 476 amino acids. The recombinant LSK25 lipase was purified by single-step purification using Ni-Sepharose affinity chromatography and had a molecular mass of approximately 65 kDa. The final recovery and purification fold were 44% and 1.3, respectively. The LSK25 lipase was optimally active at 30 °C and at pH 6. Stable lipolytic activity was reported between temperatures of 5⁻30 °C and at pH 6⁻8. A significant enhancement of lipolytic activity was observed in the presence of Ca2+ ions, the organic lipids of rice bran oil and coconut oil, a synthetic C12 ester and a wide range of water immiscible organic solvents. Overall, lipase strain LSK25 is a potentially desirable candidate for biotechnological application, due to its stability at low temperatures, across a range of pH and in organic solvents.
  18. Veno J, Ahmad Kamarudin NH, Mohamad Ali MS, Masomian M, Raja Abd Rahman RNZ
    Int J Mol Sci, 2017 Nov 04;18(11).
    PMID: 29113034 DOI: 10.3390/ijms18112202
    In the industrial processes, lipases are expected to operate at temperatures above 45 °C and could retain activity in organic solvents. Hence, a C-terminal truncated lipase from Staphylococcus epidermis AT2 (rT-M386) was engineered by directed evolution. A mutant with glycine-to-cysteine substitution (G210C) demonstrated a remarkable improvement of thermostability, whereby the mutation enhanced the activity five-fold when compared to the rT-M386 at 50 °C. The rT-M386 and G210C lipases were purified concurrently using GST-affinity chromatography. The biochemical and biophysical properties of both enzymes were investigated. The G210C lipase showed a higher optimum temperature (45 °C) and displayed a more prolonged half-life in the range of 40-60 °C as compared to rT-M386. Both lipases exhibited optimal activity and stability at pH 8. The G210C showed the highest stability in the presence of polar organic solvents at 50 °C compared to the rT-M386. Denatured protein analysis presented a significant change in the molecular ellipticity value above 60 °C, which verified the experimental result on the temperature and thermostability profile of G210C.
  19. Yaacob N, Mohamad Ali MS, Salleh AB, Abdul Rahman NA
    PeerJ, 2016;4:e1751.
    PMID: 26989608 DOI: 10.7717/peerj.1751
    Background. Not all yeast alcohol dehydrogenase 2 (ADH2) are repressed by glucose, as reported in Saccharomyces cerevisiae. Pichia stipitis ADH2 is regulated by oxygen instead of glucose, whereas Kluyveromyces marxianus ADH2 is regulated by neither glucose nor ethanol. For this reason, ADH2 regulation of yeasts may be species dependent, leading to a different type of expression and fermentation efficiency. Lachancea fermentati is a highly efficient ethanol producer, fast-growing cells and adapted to fermentation-related stresses such as ethanol and organic acid, but the metabolic information regarding the regulation of glucose and ethanol production is still lacking. Methods. Our investigation started with the stimulation of ADH2 activity from S. cerevisiae and L. fermentati by glucose and ethanol induction in a glucose-repressed medium. The study also embarked on the retrospective analysis of ADH2 genomic and protein level through direct sequencing and sites identification. Based on the sequence generated, we demonstrated ADH2 gene expression highlighting the conserved NAD(P)-binding domain in the context of glucose fermentation and ethanol production. Results. An increase of ADH2 activity was observed in starved L. fermentati (LfeADH2) and S. cerevisiae (SceADH2) in response to 2% (w/v) glucose induction. These suggest that in the presence of glucose, ADH2 activity was activated instead of being repressed. An induction of 0.5% (v/v) ethanol also increased LfeADH2 activity, promoting ethanol resistance, whereas accumulating acetic acid at a later stage of fermentation stimulated ADH2 activity and enhanced glucose consumption rates. The lack in upper stream activating sequence (UAS) and TATA elements hindered the possibility of Adr1 binding to LfeADH2. Transcription factors such as SP1 and RAP1 observed in LfeADH2 sequence have been implicated in the regulation of many genes including ADH2. In glucose fermentation, L. fermentati exhibited a bell-shaped ADH2 expression, showing the highest expression when glucose was depleted and ethanol-acetic acid was increased. Meanwhile, S. cerevisiae showed a constitutive ADH2 expression throughout the fermentation process. Discussion. ADH2 expression in L. fermentati may be subjected to changes in the presence of non-fermentative carbon source. The nucleotide sequence showed that ADH2 transcription could be influenced by other transcription genes of glycolysis oriented due to the lack of specific activation sites for Adr1. Our study suggests that if Adr1 is not capable of promoting LfeADH2 activation, the transcription can be controlled by Rap1 and Sp1 due to their inherent roles. Therefore in future, it is interesting to observe ADH2 gene being highly regulated by these potential transcription factors and functioned as a promoter for yeast under high volume of ethanol and organic acids.
  20. Yaacob N, Mohamad Ali MS, Salleh AB, Rahman RNZRA, Leow ATC
    J Mol Graph Model, 2016 07;68:224-235.
    PMID: 27474867 DOI: 10.1016/j.jmgm.2016.07.003
    The utilization of cold active lipases in organic solvents proves an excellent approach for chiral synthesis and modification of fats and oil due to the inherent flexibility of lipases under low water conditions. In order to verify whether this lipase can function as a valuable synthetic catalyst, the mechanism concerning activation of the lid and interacting solvent residues in the presence of organic solvent must be well understood. A new alkaline cold-adapted lipase, AMS8, from Pseudomonas fluorescens was studied for its structural adaptation and flexibility prior to its exposure to non-polar, polar aprotic and protic solvents. Solvents such as ethanol, toluene, DMSO and 2-propanol showed to have good interactions with active sites. Asparagine (Asn) and tyrosine (Tyr) were key residues attracted to solvents because they could form hydrogen bonds. Unlike in other solvents, Phe-18, Tyr-236 and Tyr-318 were predicted to have aromatic-aromatic side-chain interactions with toluene. Non-polar solvent also was found to possess highest energy binding compared to polar solvents. Due to this circumstance, the interaction of toluene and AMS8 lipase was primarily based on hydrophobicity and molecular recognition. The molecular dynamic simulation showed that lid 2 (residues 148-167) was very flexible in toluene and Ca(2+). As a result, lid 2 moves away from the catalytic areas, leaving an opening for better substrate accessibility which promotes protein activation. Only a single lid (lid 2) showed the movement following interactions with toluene, although AMS8 lipase displayed double lids. The secondary conformation of AMS8 lipase that was affected by toluene observed a reduction of helical strands and increased coil structure. Overall, this work shows that cold active lipase, AMS8 exhibits distinguish interfacial activation and stability in the presence of polar and non-polar solvents.
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