High level expression and characterization of a novel thermostable, organic solvent tolerant, 1,3-regioselective lipase from Geobacillus sp. strain ARM

Ebrahimpour, Afshin; Rahman, Raja Noor Zaliha Raja Abdul; Basri, Mahiran; Salleh, Abu Bakar

doi:10.1016/j.biortech.2011.03.083

Cited by 74 publications

(42 citation statements)

References 28 publications

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“…The recombinant plasmid pTrcHis/ARM harboring ARM lipase gene was extracted from Escherichia coli Top 10 (pTrcHis/ARM) [11] cloned previously grew on Luria-Bertani (LB) agar containing 50 μg/ ml ampicillin.…”

Section: Organismmentioning

confidence: 99%

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The Role of Arg157Ser in Improving the Compactness and Stability of ARM Lipase

Salleh¹

2012

JCSB

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Consensus approach is an efficient strategy to identify hot residue important for compactness and stability of protein. Structure of ARM lipase was modeled to explore the possible effect of critical point mutation towards structure and function. The significant difference of amino acid at position 157 between ARM lipase (Arg157) and other thermostable lipases (Ser157) was targeted as a critical residue. Using YASARA software, Arg157 was substituted to Ser and subsequently the energy minimized. Both ARM and R157S lipases were analyzed by MD simulations at different temperatures (50ºC,60ºC, and 70ºC). MD simulation result showed that R157S lipase had lower value of RMSD, RMSF, solvent accessible surface area (SASA) and radius of gyration than native ARM lipase. It indicated that R157S lipase had higher compactness in the structure leading to enhanced stability. To validate the computational data, the substitution of Arg157 to Ser has been conducted using site-directed mutagenesis experimentally. The catalytic efficiency (k cat /K M ) of R157S lipase was refold better than ARM lipase of 70°C. Circular dichorism study revealed that R157S lipase had increased thermostability with higher T m value (71.6°C) than its wildtype (63.9°C) indicating a better compactness as revealed by spectrofluorocence study. Thus the rational design of substituting Arg157 with Ser improved the protein folding of mutant lipase as shown in MD simulations and subsequently increased the catalytic effectiveness and thermodynamic stability. during evolution. The substitution of critical residue was analyzed using in silico to avoid unnecessary conformational distortion in engineered lipase and validate with kinetic study and thermodynamic study. Methods Preparation of 3D structures of ARM and R157S lipasesThe 3D structure of ARM lipase was predicted by comparative modeling. The first step in comparative modeling is the assignment of the unknown ARM lipase structure to a protein family with thousands of sequences already stored in protein databases (PDB). The structure of ARM lipase was modeled using available software packages such as HOMOLOGY and MODELLER, Accelrys, San Diego (http://www. accelrys.com). The unknown ARM lipase structure (target protein) was built from the known 3D structure of T1 lipase (2DSN) as a template. The final model of ARM lipase was evaluated using Ramachandran plot. Using YASARA, the three-dimensional structure of the mutant (R157S lipase) was modeled by substituting Arg157 to Ser and energy minimization was used to release bad contact. MD simulations of ARM and R157S lipasesMD simulations were performed on ARM lipase and R157S lipase structures using Amber03 force field in YASARA package. The periodic box was filled with explicit water molecules to a density of 0.98320 g/l. The whole system was neutralized by adding counter ions and all ionizable protein groups were protonated according to their tabulated pKa values at pH 7 of the medium. Water molecules were relaxed by simulated annealing procedure. Minimiza...

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Section: Organismmentioning

confidence: 99%

“…In the present investigation, recombinant ARM lipase from a newly isolated Geobacillus sp. was used [11]. ARM lipase has comparatively low thermostability compared to other homologous thermostable lipases despite sharing a high similarity in amino acid sequences (~80%).…”

Section: Introductionmentioning

confidence: 99%

The Role of Arg157Ser in Improving the Compactness and Stability of ARM Lipase

Salleh¹

2012

JCSB

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“…However, VCO, known as a functional oil or mineral oil was produced in mild conditions (Chen et al, 2007) in order to maintain the bioactive compounds. As indicated by khoramnia et al (2011) and Ebrahimpour et al (2011), the mineral contents of the cultures play an essential role in the function of lipases. Generally, as indicated by Ribeiro et al (2009), the increased U2S content in the MoO/PO and MoO/VCO blends produced by enzymatic interesterification lead to greater potential for applying these interesterified blends in foods.…”

Section: Effect Of Enzymatic Interesterifi Cation Onmentioning

confidence: 89%

Enzymatic interesterification on the physicochemical properties of <em>Moringa oleifera</em> seed oil blended with palm olein and virgin coconut oil

Ghazali¹,

Dollah²,

Abdulkarim³

et al. 2015

Grasas y Aceites

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SUMMARY:The enzymatic interesterification (IE) of palm olein (PO) and virgin coconut oil (VCO) with the high oleic acid (86%) Moringa oleifera seed oil (MoO) could yield a good source of oleic acid fat stock that may contain desirable nutritional and physical properties. Lipozyme RMIM resulted in different functionalities for the MoO/PO and MoO/VCO blends due to inherent changes in triacylglycerol (TAG) compositions which, in turn, led to different trends in DSC thermograms and solid fat contents (SFC). The enzymatic IE of MoO/VCO increased U 2 S and S 2 U (up to 20% medium and long chain, MLCT) while it decreased U 3 (triunsaturated) and S 3 (trisaturated) TAGs. The IE of the MoO/PO blends increased U 2 S and S 3 (MMP, myristic, myristic, palmitic) and decreased S 2 U, resulting in a lowering of melting points and SFC for MoO/VCO, while showing an increase in them for MoO/PO. A 2.55% increase in S3 after 24 h MoO/PO 30:70 IE revealed a 6.5% harder oil at 10 °C which may imply a wider application compared to the original liquid oils. Novel MLCTs with improved nutritional and physical properties were generated in the MoO/VCO blends after IE due to the incorporation of oleic acid and medium chain fatty acids. MoO/PO 50:50 and 70:30 w/w after 12 h IE and MoO/VCO 30:70 are suitable for incorporation into the fat phase in ice-cream formulations while, the spreadability and plasticity of MoO/VCO 70:30 improved at low temperatures. Both interesterified blends could be used as high oleic acid frying oils. KEYWORDS: Interesterification; Lipozyme RMIM; Moringa oleifera seed oil; Physicochemical properties; Structured lipids RESUMEN:Interesterificación enzimática sobre las propiedades físico-químicas de las mezclas de aceites de semillas de Moringa oleifera con oleína de palma y aceite de coco virgen. La interesterificación enzimática (IE) de la oleína de palma (PO), aceite de coco virgen (VCO) con alto contenido en ácido oleico (86%) y aceites de semilla de Moringa oleífera (MOO) podría ser una buena fuente de ácido oleico con propiedades nutricionales y físicas deseables. La lipozyme RMIM produce diferentes funcionalidades para las mezclas MoO/PO y MoO/ VCO debido a los cambios inherentes en la composición de triacilgliceroles (TAG) que, a su vez, dieron lugar a diferentes perfiles en los termogramas de DSC y en los contenidos de grasa sólida (SFC). La IE de MoO/VCO aumentó los TAGs, U 2 S y S 2 U (hasta un 20% de cadena media y larga, MLCT), mientras que disminuyó los TAGs triinsaturados (U 3 ) y trisaturados (S 3 ). La IE de mezclas MoO/PO, aumentó los TAGs U 2 S y S 3 (MMP, mirístico, mirístico, palmítico) y disminuyó S 2 U, lo que da lugar a una reducción del punto de fusión y de SFC para el MoO/VCO mientras que estos parámetros aumentan para MoO/PO. El aumento de un 2.55% en S 3 después de 24 h de IE de MoO/PO 30:70 da lugar a aceites 6,5% más duros a10 °C, que pueden tener unas aplicaciones más amplias en comparación con los aceites líquidos originales. Nuevos MLCTs con mejores propiedades

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“…Metal ions may bind to some of the amino acid side chains of protein, therefore influencing the structure and function of enzymes and altering the enzyme activity and stability (Ebrahimpour et al 2011). In general, all metal ions had similar effects on the lipase and endoglucanase activities of parent enzymes and chimera Lip-EG1CD.…”

Section: Effects Of Metal Ions and Chemical Reagents On Activitymentioning

confidence: 99%

The Enzymatic Deinking of Waste Papers by Engineered Bifunctional Chimeric Neutral Lipase – Endoglucanase

Liu¹,

Long²,

Wu³

et al. 2017

BioResources

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Endoglucanase and lipase showed good deinking efficiency for waste papers. The performances could be greatly improved further by the combined use of the two enzymes. To reduce the enzyme production cost and enhance synergistic action of endoglucanase and lipase on laser-printed paper and newspaper, a chimeric enzyme with endoglucanase and lipase activity was constructed and expressed in Pichia pastoris. The data indicated that the chimera Lip-EG1CD improved the ink removal efficiencies and sheet brightness better than a single enzyme or a mixture of two enzymes. The chimera Lip-EG1CD demonstrated an 89% removal of toner on both papers and 91% ISO and 60% ISO sheet brightness for laser-printed paper and newspaper, respectively. Handsheet strength was also clearly improved. It revealed that the combined deinking of endoglucanase and lipase on waste papers could be strengthened by constructing proper chimera due to intramolecular synergistic action. This would be useful for developing an economical process for waste paper recycling.

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High level expression and characterization of a novel thermostable, organic solvent tolerant, 1,3-regioselective lipase from Geobacillus sp. strain ARM

Cited by 74 publications

References 28 publications

The Role of Arg157Ser in Improving the Compactness and Stability of ARM Lipase

The Role of Arg157Ser in Improving the Compactness and Stability of ARM Lipase

Enzymatic interesterification on the physicochemical properties of <em>Moringa oleifera</em> seed oil blended with palm olein and virgin coconut oil

The Enzymatic Deinking of Waste Papers by Engineered Bifunctional Chimeric Neutral Lipase – Endoglucanase

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