Displaying publications 1 - 20 of 26 in total

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  1. Recent advances in simultaneous thermostability-activity improvement of industrial enzymes through structure modification
    Nezhad NG, Rahman RNZRA, Normi YM, Oslan SN, Shariff FM, Leow TC
    Int J Biol Macromol, 2023 Mar 31;232:123440.
    PMID: 36708895 DOI: 10.1016/j.ijbiomac.2023.123440
    Engineered thermostable microbial enzymes are widely employed to catalyze chemical reactions in numerous industrial sectors. Although high thermostability is a prerequisite of industrial applications, enzyme activity is usually sacrificed during thermostability improvement. Therefore, it is vital to select the common and compatible strategies between thermostability and activity improvement to reduce mutants̕ libraries and screening time. Three functional protein engineering approaches, including directed evolution, rational design, and semi-rational design, are employed to manipulate protein structure on a genetic basis. From a structural standpoint, integrative strategies such as increasing substrate affinity; introducing electrostatic interaction; removing steric hindrance; increasing flexibility of the active site; N- and C-terminal engineering; and increasing intramolecular and intermolecular hydrophobic interactions are well-known to improve simultaneous activity and thermostability. The current review aims to analyze relevant strategies to improve thermostability and activity simultaneously to circumvent the thermostability and activity trade-off of industrial enzymes.
    Matched MeSH terms: Protein Engineering*
  2. Current achievements, strategies, obstacles, and overcoming the challenges of the protein engineering in Pichia pastoris expression system
    Eskandari A, Nezhad NG, Leow TC, Rahman MBA, Oslan SN
    World J Microbiol Biotechnol, 2023 Dec 08;40(1):39.
    PMID: 38062216 DOI: 10.1007/s11274-023-03851-6
    Yeasts serve as exceptional hosts in the manufacturing of functional protein engineering and possess industrial or medical utilities. Considerable focus has been directed towards yeast owing to its inherent benefits and recent advancements in this particular cellular host. The Pichia pastoris expression system is widely recognized as a prominent and widely accepted instrument in molecular biology for the purpose of generating recombinant proteins. The advantages of utilizing the P. pastoris system for protein production encompass the proper folding process occurring within the endoplasmic reticulum (ER), as well as the subsequent secretion mediated by Kex2 as a signal peptidase, ultimately leading to the release of recombinant proteins into the extracellular environment of the cell. In addition, within the P. pastoris expression system, the ease of purifying recombinant protein arises from its restricted synthesis of endogenous secretory proteins. Despite its achievements, scientists often encounter persistent challenges when attempting to utilize yeast for the production of recombinant proteins. This review is dedicated to discussing the current achievements in the usage of P. pastoris as an expression host. Furthermore, it sheds light on the strategies employed in the expression system and the optimization and development of the fermentative process of this yeast. Finally, the impediments (such as identifying high expression strains, improving secretion efficiency, and decreasing hyperglycosylation) and successful resolution of certain difficulties are put forth and deliberated upon in order to assist and promote the expression of complex proteins in this prevalent recombinant host.
    Matched MeSH terms: Protein Engineering
  3. Fulltext Development of a pyrG mutant of Aspergillus oryzae strain S1 as a host for the production of heterologous proteins
    Ling SO, Storms R, Zheng Y, Rodzi MR, Mahadi NM, Illias RM, et al.
    ScientificWorldJournal, 2013;2013:634317.
    PMID: 24381522 DOI: 10.1155/2013/634317
    The ease with which auxotrophic strains and genes that complement them can be manipulated, as well as the stability of auxotrophic selection systems, are amongst the advantages of using auxotrophic markers to produce heterologous proteins. Most auxotrophic markers in Aspergillus oryzae originate from chemical or physical mutagenesis that may yield undesirable mutations along with the mutation of interest. An auxotrophic A. oryzae strain S1 was generated by deleting the orotidine-5'-monophosphate decarboxylase gene (pyrG) by targeted gene replacement. The uridine requirement of the resulting strain GR6 pyrGΔ0 was complemented by plasmids carrying a pyrG gene from either Aspergillus nidulans or A. oryzae. β -Galactosidase expression by strain GR6 pyrGΔ0 transformed with an A. niger plasmid encoding a heterologous β -galactosidase was at least 150 times more than that obtained with the untransformed strain. Targeted gene replacement is thus an efficient way of developing auxotrophic mutants in A. oryzae and the auxotrophic strain GR6 pyrGΔ0 facilitated the production of a heterologous protein in this fungus.
    Matched MeSH terms: Protein Engineering/methods*
  4. Expression of a thermostable xylanase gene from Bacillus coagulans ST-6 in Lactococcus lactis
    Raha AR, Chang LY, Sipat A, Yusoff K, Haryanti T
    Lett Appl Microbiol, 2006 Mar;42(3):210-4.
    PMID: 16478506
    The aim of the study is to evaluate whether xylanase can be used as a potential reporter gene for cloning and expression studies in Lactococcus.
    Matched MeSH terms: Protein Engineering*
  5. Protein engineering of GH11 xylanase from Aspergillus fumigatus RT-1 for catalytic efficiency improvement on kenaf biomass hydrolysis
    Damis SIR, Murad AMA, Diba Abu Bakar F, Rashid SA, Jaafar NR, Illias RM
    Enzyme Microb Technol, 2019 Dec;131:109383.
    PMID: 31615675 DOI: 10.1016/j.enzmictec.2019.109383
    Enzyme hydrolysis faces a bottleneck due to the recalcitrance of the lignocellulose biomass. The protein engineering of GH11 xylanase from Aspergillus fumigatus RT-1 was performed near the active site and at the N-terminal region to improve its catalytic efficiency towards pretreated kenaf (Hibiscus cannabinus) hydrolysis. Five mutants were constructed by combined approaches of error-prone PCR, site-saturation and site-directed mutagenesis. The double mutant c168 t/Q192H showed the most effective hydrolysis reaction with a 13.9-fold increase in catalytic efficiency, followed by mutants Y7L and c168 t/Q192 H/Y7L with a 1.6-fold increase, respectively. The enhanced catalytic efficiency evoked an increase in sugar yield of up to 28% from pretreated kenaf. In addition, mutant c168 t/Q192 H/Y7L improved the thermostability at higher temperature and acid stability. This finding shows that mutations at distances less than 15 Å from the active site and at putative secondary binding sites affect xylanase catalytic efficiency towards insoluble substrates hydrolysis.
    Matched MeSH terms: Protein Engineering*
  6. Humanizing glycosylation pathways in eukaryotic expression systems
    Khan AH, Bayat H, Rajabibazl M, Sabri S, Rahimpour A
    World J Microbiol Biotechnol, 2017 Jan;33(1):4.
    PMID: 27837408
    Glycosylation represents the most widespread posttranslational modifications, found in a broad spectrum of natural and therapeutic recombinant proteins. It highly affects bioactivity, site-specificity, stability, solubility, immunogenicity, and serum half-life of glycoproteins. Numerous expression hosts including yeasts, insect cells, transgenic plants, and mammalian cells have been explored for synthesizing therapeutic glycoproteins. However, glycosylation profile of eukaryotic expression systems differs from human. Glycosylation strategies have been proposed for humanizing the glycosylation pathways in expression hosts which is the main theme of this review. Besides, we also highlighted the glycosylation potential of protozoan parasites by emphasizing on the mammalian-like glycosylation potential of Leishmania tarentolae known as Leishmania expression system.
    Matched MeSH terms: Protein Engineering/methods*
  7. Laccase enzymes: purification, structure to catalysis and tailoring
    Moin SF, Omar MN
    Protein Pept Lett, 2014;21(8):707-13.
    PMID: 23855667
    Laccases belong to the multicopper binding protein family that catalysis the reduction of oxygen molecule to produce water. These enzymes are glycosylated proteins and have been isolated and purified from fungi, bacteria, plant, insects and lichens. The variety of commercial and industrial application of laccases has attracted much attention towards the research addressing different aspects of the protein characterization, production and fit for purpose molecule. Here we briefly discuss the purification, catalytic mechanism in light of available understanding of structure-function relationship and the tailoring side of the protein, which has been the subject of recent research. Purification strategy of laccases is a method of choice and is facilitated by increased production of the enzyme. The structure-function relationship has given insights to unfold the catalytic mechanism. Site directed mutagenesis and other modification at C-terminal end or surrounding environment of copper centres have shown promising results to fit for purpose aspect, with a lot remains to be explored in glycosylation status and its alteration.
    Matched MeSH terms: Protein Engineering
  8. Minireview: applied structural bioinformatics in proteomics
    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.
    Matched MeSH terms: Protein Engineering
  9. Fulltext Thermal stability engineering of Glomerella cingulata cutinase
    Chin IS, Abdul Murad AM, Mahadi NM, Nathan S, Abu Bakar FD
    Protein Eng. Des. Sel., 2013 May;26(5):369-75.
    PMID: 23468570 DOI: 10.1093/protein/gzt007
    Cutinase has been ascertained as a biocatalyst for biotechnological and industrial bioprocesses. The Glomerella cingulata cutinase was genetically modified to enhance its enzymatic performance to fulfill industrial requirements. Two sites were selected for mutagenesis with the aim of altering the surface electrostatics as well as removing a potentially deamidation-prone asparagine residue. The N177D cutinase variant was affirmed to be more resilient to temperature increase with a 2.7-fold increase in half-life at 50°C as compared with wild-type enzyme, while, the activity at 25°C is not compromised. Furthermore, the increase in thermal tolerance of this variant is accompanied by an increase in optimal temperature. Another variant, the L172K, however, exhibited higher enzymatic performance towards phenyl ester substrates of longer carbon chain length, yet its thermal stability is inversely affected. In order to restore the thermal stability of L172K, we constructed a L172K/N177D double variant and showed that these two mutations yield an improved variant with enhanced activity towards phenyl ester substrates and enhanced thermal stability. Taken together, our study may provide valuable information for enhancing catalytic performance and thermal stability in future engineering endeavors.
    Matched MeSH terms: Protein Engineering*
  10. Modified enzymes for reactions in organic solvents
    Salleh AB, Basri M, Taib M, Jasmani H, Rahman RN, Rahman MB, et al.
    Appl Biochem Biotechnol, 2002 10 25;102-103(1-6):349-57.
    PMID: 12396136
    Recent studies on biocatalysis in water-organic solvent biphasic systems have shown that many enzymes retain their catalytic activities in the presence of high concentrations of organic solvents. However, not all enzymes are organic solvent tolerant, and most have limited and selective tolerance to particular organic solvents. Protein modification or protein tailoring is an approach to alter the characteristics of enzymes, including solubility in organic solvents. Particular amino acids may play pivotal roles in the catalytic ability of the protein. Attaching soluble modifiers to the protein molecule may alter its conformation and the overall polarity of the molecule. Enzymes, in particular lipases, have been chemically modified by attachment of aldehydes, polyethylene glycols, and imidoesters. These modifications alter the hydrophobicity and conformation of the enzymes, resulting in changes in the microenvironment of the enzymes. By these modifications, newly acquired properties such as enhancement of activity and stability and changes in specificity and solubility in organic solvents are obtained. Modified lipases were found to be more active and stable in organic solvents. The optimum water activity (a(w)) for reaction was also shifted by using modified enzymes. Changes in enantioselective behavior were also observed.
    Matched MeSH terms: Protein Engineering/methods
  11. Enhanced secretory production of hemolysin-mediated cyclodextrin glucanotransferase in Escherichia coli by random mutagenesis of the ABC transporter system
    Low KO, Mahadi NM, Abdul Rahim R, Rabu A, Abu Bakar FD, Abdul Murad AM, et al.
    J Biotechnol, 2010 Dec;150(4):453-9.
    PMID: 20959127 DOI: 10.1016/j.jbiotec.2010.10.001
    The hemolysin transport system was found to mediate the release of cyclodextrin glucanotransferase (CGTase) into the extracellular medium when it was fused to the C-terminal 61 amino acids of HlyA (HlyAs(61)). To produce an improved-secretion variant, the hly components (hlyAs, hlyB and hlyD) were engineered by directed evolution using error-prone PCR. Hly mutants were screened on solid LB-starch plate for halo zone larger than the parent strain. Through screening of about 1 × 10(4) Escherichia coli BL21(DE3) transformants, we succeeded in isolating five mutants that showed a 35-217% increase in the secretion level of CGTase-HlyAs(61) relative to the wild-type strain. The mutation sites of each mutant were located at HlyB, primarily along the transmembrane domain, implying that the corresponding region was important for the improved secretion of the target protein. In this study we describe the finding of novel site(s) of HlyB responsible for enhancing secretion of CGTase in E. coli.
    Matched MeSH terms: Protein Engineering/methods
  12. Use of Megaprimer and Overlapping Extension PCR (OE-PCR) to Mutagenize and Enhance Cyclodextrin Glucosyltransferase (CGTase) Function
    Goh KM, Liew KJ, Chai KP, Illias RM
    Methods Mol Biol, 2017;1498:385-396.
    PMID: 27709591
    Protein engineering is a very useful tool for probing structure-function relationships in proteins. Specifically, site-directed mutagenized proteins can provide useful insights into structural, binding and catalytic mechanisms of a protein, particularly when coupled with crystallization. In this chapter, we describe two protocols for performing site-directed mutagenesis of any protein-coding sequence, namely, megaprimer PCR and overlapping extension PCR (OE-PCR). We use as an example how these two SDM methods enhanced the function of a cyclodextrin glucosyltransferase (CGTase) from Bacillus lehensis strain G1.
    Matched MeSH terms: Protein Engineering/methods
  13. Fulltext Engineering the production of major catechins by Escherichia coli carrying metabolite genes of Camellia sinensis
    Umar KM, Abdulkarim SM, Radu S, Abdul Hamid A, Saari N
    ScientificWorldJournal, 2012;2012:529031.
    PMID: 22645428 DOI: 10.1100/2012/529031
    A mimicked biosynthetic pathway of catechin metabolite genes from C. sinensis, consisting of flavanone 3 hydroxylase (F3H), dihydroflavonol reductase (DFR), and leucoanthocyanidin reductase (LCR), was designed and arranged in two sets of constructs: (a) single promoter in front of F3H and ribosome-binding sequences both in front of DFR and LCR; (b) three different promoters with each in the front of the three genes and ribosome-binding sequences at appropriate positions. Recombinant E. coli BL (DE3) harbouring the constructs were cultivated for 65 h at 26 °C in M9 medium consisting of 40 g/L glucose, 1 mM IPTG, and 3 mM eriodictyol. Compounds produced were extracted in ethyl acetate in alkaline conditions after 1 h at room temperature and identified by HPLC. Two of the four major catechins, namely, (-)-epicatechin (0.01) and (-)-epicatechin gallate (0.36 mg/L), and two other types ((+)-catechin hydrate (0.13 mg/L) and (-)-catechin gallate (0.04 mg/L)) were successfully produced.
    Matched MeSH terms: Protein Engineering/methods*
  14. Fulltext Functional expression of a novel α-amylase from Antarctic psychrotolerant fungus for baking industry and its magnetic immobilization
    He L, Mao Y, Zhang L, Wang H, Alias SA, Gao B, et al.
    BMC Biotechnol, 2017 02 28;17(1):22.
    PMID: 28245836 DOI: 10.1186/s12896-017-0343-8
    BACKGROUND: α-Amylase plays a pivotal role in a broad range of industrial processes. To meet increasing demands of biocatalytic tasks, considerable efforts have been made to isolate enzymes produced by extremophiles. However, the relevant data of α-amylases from cold-adapted fungi are still insufficient. In addition, bread quality presents a particular interest due to its high consummation. Thus developing amylases to improve textural properties could combine health benefits with good sensory properties. Furthermore, iron oxide nanoparticles provide an economical and convenient method for separation of biomacromolecules. In order to maximize the catalytic efficiency of α-amylase and support further applications, a comprehensive characterization of magnetic immobilization of α-amylase is crucial and needed.

    RESULTS: A novel α-amylase (AmyA1) containing an open reading frame of 1482 bp was cloned from Antarctic psychrotolerant fungus G. pannorum and then expressed in the newly constructed Aspergillus oryzae system. The purified recombinant AmyA1 was approximate 52 kDa. AmyA1 was optimally active at pH 5.0 and 40 °C, and retained over 20% of maximal activity at 0-20 °C. The K m and V max values toward soluble starch were 2.51 mg/mL and 8.24 × 10-2 mg/(mL min) respectively, with specific activity of 12.8 × 103 U/mg. AmyA1 presented broad substrate specificity, and the main hydrolysis products were glucose, maltose, and maltotetraose. The influence of AmyA1 on the quality of bread was further investigated. The application study shows a 26% increase in specific volume, 14.5% increase in cohesiveness and 14.1% decrease in gumminess in comparison with the control. AmyA1 was immobilized on magnetic nanoparticles and characterized. The immobilized enzyme showed improved thermostability and enhanced pH tolerance under neutral conditions. Also, magnetically immobilized AmyA1 can be easily recovered and reused for maximum utilization.

    CONCLUSIONS: A novel α-amylase (AmyA1) from Antarctic psychrotolerant fungus was cloned, heterologous expression in Aspergillus oryzae, and characterized. The detailed report of the enzymatic properties of AmyA1 gives new insights into fungal cold-adapted amylase. Application study showed potential value of AmyA1 in the food and starch fields. In addition, AmyA1 was immobilized on magnetic nanoparticles and characterized. The improved stability and longer service life of AmyA1 could potentially benefit industrial applications.

    Matched MeSH terms: Protein Engineering/methods
  15. Fulltext Heterologous, Expression, and Characterization of Thermostable Glucoamylase Derived from Aspergillus flavus NSH9 in Pichia pastoris
    Karim KM, Husaini A, Hossain MA, Sing NN, Mohd Sinang F, Hussain MH, et al.
    Biomed Res Int, 2016;2016:5962028.
    PMID: 27504454 DOI: 10.1155/2016/5962028
    A novel thermostable glucoamylase cDNA without starch binding domain (SBD) of Aspergillus flavus NSH9 was successfully identified, isolated, and overexpressed in Pichia pastoris GS115. The complete open reading frame of glucoamylase from Aspergillus flavus NSH9 was identified by employing PCR that encodes 493 amino acids lacking in the SBD. The first 17 amino acids were presumed to be a signal peptide. The cDNA was cloned into Pichia pastoris and the highest expression of recombinant glucoamylase (rGA) was observed after 8 days of incubation period with 1% methanol. The molecular weight of the purified rGA was about 78 kDa and exhibited optimum catalytic activity at pH 5.0 and temperature of 70°C. The enzyme was stable at higher temperature with 50% of residual activity observed after 20 min at 90°C and 100°C. Low concentration of metal (Mg(++), Fe(++), Zn(++), Cu(++), and Pb(++)) had positive effect on rGA activity. This rGA has the potential for use and application in the saccharification steps, due to its thermostability, in the starch processing industries.
    Matched MeSH terms: Protein Engineering/methods*
  16. PHA synthase (PhaC): interpreting the functions of bioplastic-producing enzyme from a structural perspective
    Chek MF, Hiroe A, Hakoshima T, Sudesh K, Taguchi S
    Appl Microbiol Biotechnol, 2019 Feb;103(3):1131-1141.
    PMID: 30511262 DOI: 10.1007/s00253-018-9538-8
    Polyhydroxyalkanoates (PHAs) are biopolymers synthesized by a wide range of bacteria, which serve as a promising candidate in replacing some conventional petrochemical-based plastics. PHA synthase (PhaC) is the key enzyme in the polymerization of PHA, and the crystal structures were successfully determined using the catalytic domain of PhaC from Cupriavidus necator (PhaCCn-CAT) and Chromobacterium sp. USM2 (PhaCCs-CAT). Here, we review the beneficial mutations discovered in PhaCs from a structural perspective. The structural comparison of the residues involved in beneficial mutation reveals that the residues are near to the catalytic triad, but not inside the catalytic pocket. For instance, Ala510 of PhaCCn is near catalytic His508 and may be involved in the open-close regulation, which presumably play an important role in substrate specificity and activity. In the class II PhaC1 from Pseudomonas sp. 61-3 (PhaC1Ps), Ser325 stabilizes the catalytic cysteine through hydrogen bonding. Another residue, Gln508 of PhaC1Ps is located in a conserved hydrophobic pocket which is next to the catalytic Asp and His. A class I, II-conserved Phe420 of PhaCCn is one of the residues involved in dimerization and its mutation to serine greatly reduced the lag phase. The current structural analysis shows that the Phe362 and Phe518 of PhaC from Aeromonas caviae (PhaCAc) are assisting the dimer formation and maintaining the integrity of the core beta-sheet, respectively. The structure-function relationship of PhaCs discussed in this review will serve as valuable reference for future protein engineering works to enhance the performance of PhaCs and to produce novel biopolymers.
    Matched MeSH terms: Protein Engineering
  17. Fulltext Native to designed: microbial -amylases for industrial applications
    Lim SJ, Oslan SN
    PeerJ, 2021;9:e11315.
    PMID: 34046253 DOI: 10.7717/peerj.11315
    Background: -amylases catalyze the endo-hydrolysis of -1,4-D-glycosidic bonds in starch into smaller moieties. While industrial processes are usually performed at harsh conditions, -amylases from mainly the bacteria, fungi and yeasts are preferred for their stabilities (thermal, pH and oxidative) and specificities (substrate and product). Microbial -amylases can be purified and characterized for industrial applications. While exploring novel enzymes with these properties in the nature is time-costly, the advancements in protein engineering techniques including rational design, directed evolution and others have privileged their modifications to exhibit industrially ideal traits. However, the commentary on the strategies and preferably mutated residues are lacking, hindering the design of new mutants especially for enhanced substrate specificity and oxidative stability. Thus, our review ensures wider accessibility of the previously reported experimental findings to facilitate the future engineering work.

    Survey methodology and objectives: A traditional review approach was taken to focus on the engineering of microbial -amylases to enhance industrially favoured characteristics. The action mechanisms of - and -amylases were compared to avoid any bias in the research background. This review aimed to discuss the advances in modifying microbial -amylases via protein engineering to achieve longer half-life in high temperature, improved resistance (acidic, alkaline and oxidative) and enhanced specificities (substrate and product). Captivating results were discussed in depth, including the extended half-life at 100C, pH 3.5 and 10, 1.8 M hydrogen peroxide as well as enhanced substrate (65.3%) and product (42.4%) specificities. These shed light to the future microbial -amylase engineering in achieving paramount biochemical traits ameliorations to apt in the industries.

    Conclusions: Microbial -amylases can be tailored for specific industrial applications through protein engineering (rational design and directed evolution). While the critical mutation points are dependent on respective enzymes, formation of disulfide bridge between cysteine residues after mutations is crucial for elevated thermostability. Amino acids conversion to basic residues was reported for enhanced acidic resistance while hydrophobic interaction resulted from mutated hydrophobic residues in carbohydrate-binding module or surface-binding sites is pivotal for improved substrate specificity. Substitution of oxidation-prone methionine residues with non-polar residues increases the enzyme oxidative stability. Hence, this review provides conceptual advances for the future microbial -amylases designs to exhibit industrially significant characteristics. However, more attention is needed to enhance substrate specificity and oxidative stability since they are least reported.

    Matched MeSH terms: Protein Engineering
  18. Rational Design of Thermostable Carbonic Anhydrase Mutants Using Molecular Dynamics Simulations
    Parra-Cruz R, Jäger CM, Lau PL, Gomes RL, Pordea A
    J Phys Chem B, 2018 09 13;122(36):8526-8536.
    PMID: 30114369 DOI: 10.1021/acs.jpcb.8b05926
    The stability of enzymes is critical for their application in industrial processes, which generally require different conditions from the natural enzyme environment. Both rational and random protein engineering approaches have been used to increase stability, with the latter requiring extensive experimental effort for the screening of variants. Moreover, some general rules addressing the molecular origin of protein thermostability have been established. Herein, we demonstrate the use of molecular dynamics simulations to gain molecular level understanding of protein thermostability and to engineer stabilizing mutations. Carbonic anhydrase (CA) is an enzyme with a high potential for biotechnological carbon capture applications, provided it can be engineered to withstand the high temperature process environments, inevitable in most gas treatment units. In this study, we used molecular dynamics simulations at 343, 353, and 363 K to study the relationship between structure flexibility and thermostability in bacterial α-CAs and applied this knowledge to the design of mutants with increased stability. The most thermostable α-CA known, TaCA from Thermovibrio ammonificans, had the most rigid structure during molecular dynamics simulations, but also showed regions with high flexibility. The most flexible amino acids in these regions were identified from root mean square fluctuation (RMSF) studies, and stabilizing point mutations were predicted based on their capacity to improve the calculated free energy of unfolding. Disulfide bonds were also designed at sites with suitable geometries and selected based on their location at flexible sites, assessed by B-factor calculation. Molecular dynamics simulations allowed the identification of five mutants with lower RMSF of the overall structure at 400 K, compared to wild-type TaCA. Comparison of free-energy landscapes between wild-type TaCA and the most promising mutants, Pro165Cys-Gln170Cys and Asn140Gly, showed an increased conformational stability of the mutants at 400 K.
    Matched MeSH terms: Protein Engineering
  19. Fulltext Protein engineering of selected residues from conserved sequence regions of a novel Anoxybacillus α-amylase
    Ranjani V, Janeček S, Chai KP, Shahir S, Abdul Rahman RN, Chan KG, et al.
    Sci Rep, 2014 Jul 28;4:5850.
    PMID: 25069018 DOI: 10.1038/srep05850
    The α-amylases from Anoxybacillus species (ASKA and ADTA), Bacillus aquimaris (BaqA) and Geobacillus thermoleovorans (GTA, Pizzo and GtamyII) were proposed as a novel group of the α-amylase family GH13. An ASKA yielding a high percentage of maltose upon its reaction on starch was chosen as a model to study the residues responsible for the biochemical properties. Four residues from conserved sequence regions (CSRs) were thus selected, and the mutants F113V (CSR-I), Y187F and L189I (CSR-II) and A161D (CSR-V) were characterised. Few changes in the optimum reaction temperature and pH were observed for all mutants. Whereas the Y187F (t1/2 43 h) and L189I (t1/2 36 h) mutants had a lower thermostability at 65°C than the native ASKA (t1/2 48 h), the mutants F113V and A161D exhibited an improved t1/2 of 51 h and 53 h, respectively. Among the mutants, only the A161D had a specific activity, k(cat) and k(cat)/K(m) higher (1.23-, 1.17- and 2.88-times, respectively) than the values determined for the ASKA. The replacement of the Ala-161 in the CSR-V with an aspartic acid also caused a significant reduction in the ratio of maltose formed. This finding suggests the Ala-161 may contribute to the high maltose production of the ASKA.
    Matched MeSH terms: Protein Engineering*
  20. Purification of the extra-cellular domain of Nipah virus glycoprotein produced in Escherichia coli and possible application in diagnosis
    Eshaghi M, Tan WS, Chin WK, Yusoff K
    J Biotechnol, 2005 Mar 30;116(3):221-6.
    PMID: 15707682
    The glycoprotein (G) of Nipah virus (NiV) is important for virus infectivity and induction of the protective immunity. In this study, the extra-cellular domain of NiV G protein was fused with hexahistidine residues at its N-terminal end and expressed in Escherichia coli. The expression under transcriptional regulation of T7 promoter yielded insoluble protein aggregates in the form of inclusion bodies. The inclusion bodies were solubilized with 8 M urea and the protein was purified to homogeneity under denaturing conditions using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The denatured protein was renatured by gradual removal of the urea. Light scattering analysis of the purified protein showed primarily monodispersity. The purified protein showed significant reactivity with the antibodies present in the sera of NiV-infected swine, as demonstrated in Western blot analysis and enzyme-linked immunosorbent assay (ELISA). Taken together, the data indicate the potential usefulness of the purified G protein for structural or functional studies and the development of immunoassay for detection of the NiV antibodies.
    Matched MeSH terms: Protein Engineering/methods*
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