Endo-inulinase Stabilization by Pyridoxal Phosphate Modification: A Kinetics, Thermodynamics, and Simulation Approach

Abstract

The structural and storage and functional thermostabilization of endo-inulinase (EC 3.2.1.7) through semi-rational modification of surface accessible lysine residues by pyridoxal-5'-phosphate (PLP) and ascorbate reduction have been explored. Improved stability was observed on modifications in the absence or presence of inulin, which indicates storage or functional thermostabilization, respectively. Comparisons have been made between non-modified and modified enzyme by the determination of Tm as an indicator of structural stability, temperature-dependent half-lives (t1/2), energy barrier of the inactivation process, and thermodynamic parameters (ΔH, ΔG, and ΔS) in a storage thermostability approach. These parameters coincided well with the observed stabilization of the engineered enzyme. Moreover, relative activities with sucrose and inulin were determined for non-modified and modified endo-inulinases at different temperatures. A comparison of the sucrose-to-inulin ratios of the initial rate of hydrolysis as an indicator of substrate specificity revealed about twofold improvement in inulinase versus sucrose activity by enzyme modification. Molecular dynamics simulations and molecular docking approaches were employed to explain the observed structural and functional thermostabilization of endo-inulinase upon modification. We hypothesize the establishment of intramolecular interactions between the covalently attached PLP-Lys381 and Arg526 and Ser376 residues as a representative of modification-originated intramolecular contacts in the modified enzyme.

Immobilization of inulinase from Aspergillus niger on octadecyl substituted nanoporous silica: Inulin hydrolysis in a continuous mode operation

“…make the simulation of the PLP-modified inulinase, possible which proves the formation of the new contacts between covalently attached PLP-Lys381 with guanidinium group of Arg526 and the hydroxyl group of Ser376 as a result of modification which are considered to be the reason for improved thermostability of the plpmodified enzyme. In addition, the results of intramolecular interactions analysis of inulinase gained by docking approach after PLP-modification at the Lys381 using the LIGPLOT program proves the formation of new hydrogen bonds between the phosphate group of PLP and Arg526 and Ser376 as well (Torabizadeh et al, 2011). A significant improvement in the thermal stability of the enzyme after immobilization has already been reported (Catana et al, 2007;Bajpal & Margaritis, 1985;Kim et al, 1982;Wenling et al, 1999) which was so remarkable in the chemically modified inulinase compared to the native one.…”

“…The observed peak at 1720 cm -1 could represent the capture of CO2 on the sample surface which is mostly seen in functionalized silica particles (Kim et al, 2015) Specific activities of the inulinases, loaded on the nanoporous silica support were 1.42±0.21, 1.61±0.24, 3.52±0.15 and 3.78± 0.28 μmol.min -1 .mg -1 for free native, free modified, immobilized native and immobilized modified inulinases, respectively. Improvement in the specific activity following the adsimmobilization has also been reported by Nabati et al, 2011 andNorouzi et al, 2010. Preferential chemical modification of the non-catalytic domain of endo-inulinase to enhance the thermostability was done through semi-rational chemical modification of the accessible lysine residues by adding PLP to form Schiff bases, then reducing them by ascorbate (Torabizadeh et al, 2010;Torabizadeh et al, 2011). We have previously reported supporting results on the inulinase modification, including the increase in α-helix content and the melting temperature (Tm) using spectropolarimetric (CD) and calorimetric (DSC) methods in details (Torabizadeh et al, 2011;Shapiro et al, 1968).…”

“…Improvement in the specific activity following the adsimmobilization has also been reported by Nabati et al, 2011 andNorouzi et al, 2010. Preferential chemical modification of the non-catalytic domain of endo-inulinase to enhance the thermostability was done through semi-rational chemical modification of the accessible lysine residues by adding PLP to form Schiff bases, then reducing them by ascorbate (Torabizadeh et al, 2010;Torabizadeh et al, 2011). We have previously reported supporting results on the inulinase modification, including the increase in α-helix content and the melting temperature (Tm) using spectropolarimetric (CD) and calorimetric (DSC) methods in details (Torabizadeh et al, 2011;Shapiro et al, 1968). However, to ensure the modification procedure, intrinsic fluorescence after excitation at 280 nm and also endo-inulinase thermal stability analysis were done, the results of which are presented in Fig.…”

“…The half-life of an enzyme which is the time required to lose 50 percent of the enzyme initial activity was calculated according to the equation t1/2=ln2/kin (Torabizadeh et al, 2011). Also, the reusability analysis of the immobilized inulinases (native and modified) was investigated for 10 hydrolysis cycles, while each cycle lasted 8 hours in a continuous operation mode.…”

Bio-Catalysis and Biomedical Perspectives of Magnetic Nanoparticles as Versatile Carriers

“…Inulinase (EC:3.2.1.7) is well-known hydrolytic biocatalyst that can produce a high level of pure fructose syrup from inulin by a one-step enzymatic process in contrast to uneconomical and hazardous acid hydrolysis, as well as, multi-step enzymatic starch breakdown by glucoamylase, glucose isomerase, α-amylase, and pullulanase [54,55]. Due to lower solubility and high microbial contamination of inulin in the water at room temperature, industrial-scale inulin hydrolysis needs to be executed at elevated temperatures for higher inulin substrate utilization due to the increased solubility [56]. Therefore, thermostable inulinases are desirable biocatalysts for the chemical and food industry.…”