Introduction to Candida antarctica lipase B (CalB)
Candida antarctica lipase B (CalB), an E.C 3.1.1.3 hydrolase enzyme, consists of a polypeptide chain with 317 amino acids and a molecular weight of 33,273 Da Its unique amino acid sequence lacks significant homology to other lipases, with the traditional GxSxG consensus sequence around the active site serine replaced by TWSQG (TxSxG) CalB features a catalytic triad composed of Ser105, Asp187, and His224, maintaining the typical sequential order of catalytic residues found in other lipases The crystal structure and gene sequence of CalB are illustrated in Figure 1.1.
Figure 1.1 (A) The optimized CalB gene sequence and corresponding primary polypeptide [2] (B) the crystal structure of CalB (PDB code: 1TCA) [1].
CalB exhibits remarkable substrate selectivity, demonstrating both regio-selectivity and enantioselectivity, along with stability in organic solvents These valuable characteristics have garnered significant interest from scientists and engineers for various research and industrial applications Notably, in 1999, Shimada et al discovered the potential of Novozyme 435, further highlighting CalB's significance in biocatalysis.
(commercial form of immobilized CalB) efficiently converted vegetable oils up to 98.4% to biodiesel [5] In 2004, Houde et al mentioned production of isopropyl myristate, a component for cosmetic production, using Novozyme
Novozyme 435 has been utilized as a biocatalyst in various chemical processes, including the synthesis of glycerol carbonate from glycerol and dimethyl carbonate in 2007, and the enantioselective production of DD-polylactide from lactide in 2009 Despite its effectiveness, the industrial application of Novozyme 435 remains limited due to the high cost of immobilized enzymes, such as Novozyme 435 and Chirazyme L-2 To enhance its industrial viability, there is a critical need to reduce enzyme costs and improve their properties.
Introduction to Serum paraoxonase 1 (PON1)
Serum paraoxonase 1 (PON1) is a mammalian enzyme classified under EC 3.1.1.2, and it is part of the EC 3.1 hydrolase enzyme family The PON1 polypeptide consists of 355 amino acid residues, resulting in a molecular weight of 40,531.23 Da The enzyme's stability and activity are significantly influenced by two calcium ions located in its active site, with one acting as the catalytic calcium.
-5- the bottom of protein was proposed as structural calcium [10, 11] The gene sequence and crystal structure of PON1 are seen in Figure 1.2
PON1 demonstrates a remarkable ability to catalyze the hydrolysis of various organophosphates (OPs), including pesticide metabolites like diethyl-paraoxon, diazinon-oxon, and chloropyrifos-oxon, as well as nerve agents such as sarin, soman, and VX This enzyme is a promising candidate for treating individuals exposed to OPs and for environmental decontamination However, the catalytic efficiency of the wild-type PON1 for most OPs remains low, highlighting the need for enhanced catalytic efficiency to improve its practical applications.
PON1 not only hydrolyzes organophosphates (OPs) but also catalyzes the hydrolysis of lactones, including the formation of compounds such as γ-butyrolactone, γ-hexalactone, and mevalonic lactone.
Gamma-valerolactone (GVL) is an emerging platform molecule for synthesizing various chemicals and fuels, including valeric acid esters (biodiesel), C8+ alkanes (jet fuel), and 2-methyl tetrahydrofuran (a fuel additive) Despite its potential, the mechanisms behind PON1-catalyzed lactonase and lactonization remain poorly understood Understanding these processes is essential for the effective production of target lactones.
PON1 and CalB, enhanced for better performance, are crucial for the synthesis of eco-friendly products such as biodiesel, polylactic acids, glycerol carbonate, and γ-valerolactone Additionally, they play a significant role in detoxifying harmful compounds like paraoxons, chloropyrifos-oxon, and diazinon-oxon, effectively addressing accidental exposure to these toxic substances.
Figure 1.2 (A) The optimized gene sequence of G3C9 paraoxonase 1 synthesized by Script (USA) and its corresponding primary polypeptide (B) the crystal structure of PON1 (PDB code: 3SRG) [9]
Protein engineering to improve characteristics of enzymes
Protein engineering serves as a powerful method for enhancing enzyme characteristics, including thermal stability, regioselectivity, and enantioselectivity Two widely utilized techniques in this field are directed evolution and rational protein design.
Directed evolution utilizes molecular biology techniques, such as error-prone polymerase chain reaction (epPCR) and DNA shuffling, to create extensive libraries of gene variants, allowing for the selection of mutants with enhanced characteristics This approach does not require detailed structural data or knowledge of the relationship between amino acid sequences and enzyme function However, as the size of the enzyme and the number of simultaneously altered amino acids increase, the number of potential variants grows exponentially— for instance, a small protein with 200 amino acids can theoretically yield over nine billion variants with just three substitutions Consequently, an efficient screening system is crucial for managing this vast array of enzyme variants.
Rational protein design differs from directed evolution by requiring not only the protein structure but also an understanding of the sequence-structure and mechanism-function relationships This approach begins with computational simulations to identify potential residues that can enhance target characteristics, followed by site-directed mutagenesis to create the desired mutants The success of this method heavily relies on the selection of residues identified through simulations Various computational strategies are employed based on the specific traits sought in the improved enzymes, such as thermal stability, solvent resistance, and substrate specificity.
-8- factor, Rosetta design and, MODIP were used for improving thermal stability of enzyme [22, 23]
Scheme 1.1 The principal diagram for directed evolution and rational protein design to improve stability of protein (Adapted from [21])
Enzyme immobilization
Immobilized enzymes serve as effective biocatalysts for industrial applications due to their ability to be recovered and reused, making them suitable for various process formats, including continuous and batch reactors These enzymes also demonstrate enhanced stability compared to their soluble counterparts Various techniques for enzyme immobilization exist, including absorption, covalent binding, and entrapment, with adsorption being one of the most significant methods The physical interactions, such as van der Waals forces, ionic interactions, and hydrogen bonding, facilitate the localization of the enzyme on the supporting carrier An example of this is the physical absorption of Candida antarctica lipase B (CalB).
The Lewattit VPOC 1600, a commercial form of Novozym 435, utilizes covalent bonds for enzyme immobilization, which relies on the specific interactions of amino acid side chains such as arginine, aspartic acid, and histidine with functional groups like imidazole and indolyl on carriers Alternatively, enzymes can be entrapped within a cross-linked matrix, such as cellulose triacetate fibers, or within insoluble beads or microspheres, such as silicate sol-gel glasses.
Objectives
The objectives of this study are development of Candida antarctica lipase
B (CalB) exhibits high thermal stability due to rational protein design and enzyme immobilization, while serum paraoxonase 1 (PON1) demonstrates exceptional catalytic efficiency in the hydrolysis of organophosphates, also achieved through rational protein design This article explores the mechanistic insights into PON1-catalyzed lactonase activity, particularly focusing on its interaction with 5- substrates.
Our research focuses on enhancing the thermal stability of CalB and the catalytic efficiency of PON1 through innovative strategies We developed a rational protein design approach that leverages residual flexibility to identify key residues that can improve CalB's thermal stability Additionally, we employed various immobilization techniques to further stabilize CalB For PON1, we utilized molecular docking to pinpoint selective pockets for organophosphate interaction, enhancing its catalytic efficiency A combination of experimental methods and simulation techniques was used to clarify the mechanism of PON1-catalyzed lactonase reactions The comprehensive development process for both CalB and PON1 is illustrated in Scheme 1.2.
Scheme 1.2 Overall procedure for development of Candida antarctica lipase B and serum paraoxonase 1
1 Prediction of possible residue pairs to form disulfide bond: MODIP and disulfide by design v1.2
2 Selection of potential residue pairs to improve thermal stability of CalB:
Analysis of residual flexibility (i.e., B- factor of residues and FRODAN dynamics)
Study 4: Elucidation of PON1-catalyzed lactonase reaction by PON1 through kinetic and QM/MM studies
Study 3: Rational protein design of PON1 to efficient hydrolysis of organophosphates
Study 2: immobilization of CalB: physical absorption and R1-salaffin mediated biosilicification
Study 1: Rational protein design of disulfide bond of CalB
Development of thermal stability of CalB Development of PON1
1 Site-directed mutagenesis to make mutants
3 Analyzing thermal stability of CalB mutants
Verification of the change at molecular level:
1 Molecular dynamic simulations of the
CalB mutants at room temperature and
Synthesis of CalB fused R1 silaffin (CalB-R1) enzyme:
Verifying thermal stability of immobilized CalB-R1:
1 Analyzing thermal stability of free and immobilized CalB- R1
1 Determination of selective pocket toward two paraoxons: Autodock4.2
2 Estimating the effect of mutation
1 Kinetic study for PON1- catalyzed hydrolysis of two - lactones
2 Calculating reaction Gibbs free energy for each lactone
1 Setup and verification of the Enzyme-substrate complex system
3 QM/MM Potential energy surface scan for two lactones with two QM subsystems at HF/3-21G(d,p) method
4 Further QM/MM optimization at higher level (i.e., HF/6- 31G(d,p) for transition state areas
5 Single point energy for maxima and minima points at higher level (i.e., MP2 level) to get better reaction activation energy
1 Site-directed mutagenesis to make mutants
3 Analyzing activity of PON1 mutants for hydrolysis of diethyl-paraoxon (EPO)
4 Kinetics study for the evolved mutants
5 Expansion of the evolved PON1 mutants for hydrolysis of dimethyl- paraoxon
6 Analyzing thermal stability of the evolved PON1 mutants
Validation the change at molecular level:
1 Molecular Docking of the evolved PON1 mutants with EPO
1 Physical absorption of CalB- R1 on VPOC 1600 macroporous bead
2 Biosilicification of VPOC 1600-immobilzed CalB-R1
3 Determination of silica content in immobilized CalB-R1
References
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[3] Kirk O., Christensen W M Lipases from Candida atarctica: unique biocatalysts from a unique origin Org Process Res Dev 2002, 6, 446-451
[4] Anderson E M., Larsson K M., Kirk O., One biocatalyst-many applications: The use of Candida antarctica B-lipase in organic synthesis, Biocat Biotransform 1998, 16, 181-204
[5] Shimada Y., Watanabe Y., Samukawa T., Sugihara A., Noda H., Fukuda H., and Tominaga Y., Conversion of vegetable oil to biodiesel using immobilized candida antarctica lipase, J Am Oil Chem Soc 1999, 76, 789-793.]
[6] Houde A., Kademi A., Leblanc D Lipases and their industrial applications,
[7] Kim S C., Kim Y H., Lee H., Yoon D Y., Song B K Lipase- catalyzed synthesis of glycerol carbonate from renewable glycerol and dimethyl carbonate through transesterification, J Mol Cat B: Enzym 2007 , 49, 75–78
[8] Hans M., Keul H., Moeller M Ring-Opening polymerization of DD-lactide catalyzed by novozyme 435 Macromol Biosci 2009, 9, 239–247
[9] Ben-David M., Elias M., Filippi J.-J., Duủach E., Silman I., Sussman J L., Tawfik D S Catalytic versatility and backups in enzyme active sites: the case of serum paraoxonase 1 J Mol Boil 2012, 418, 181-196
The serum paraoxonase family of enzymes plays a crucial role in detoxification and combating atherosclerosis, as detailed by Harel et al in their study published in Nature Structural & Molecular Biology Their research highlights the structure and evolutionary aspects of these important enzymes, which are vital for maintaining cardiovascular health.
[11] Kuo, C.; La Du B N Calcium binding by human and rabbit serum paraoxonases structural stability and enzymatic activity, Drug Metab Dispos
[12] Peterson M W., Fairchild S Z., Otto T C., Mohtashemi M., Cerasoli D M., Chang W E VX hydrolysis by human serum paraoxonase1: A comparison of experimental and computational results Plos One 2011, 6, 1-7
[13] Eckerson H W., Wyte C M., La Du B N The human Serum paraoxonase/Arylesterase polymorphism Am J Hum Gen 1983, 35, 1126-
[14] Stevens R C., Suzuki S M., Cole T B., Park S S., Richter R J., Furlong
C E Engineered recombinant human paraoxonase 1 (rHuPON1) purified from
Escherichia coli protects against organophosphates poisoning Proc Natl Acad Sci U S A 2008, 105, 12780-12784
[15] Li W F., Furlong C E., Costa L G Paraoxonase protects against chlorpyrifos toxicity in mice Toxicol Lett 1995, 76, 219-226
[16] Draganov D I., La Du B N Pharmacogenetics of paraoxonases: a brief review Naunyun-Schmeisderg’s Arch Pharmacol 2004, 369, 78-88
[17] Teiber, J F.; Draganov, D I.; La Du, B N Lactonase and lactonizing activities of human serum paraoxonase (PON1) and rabbit serum PON3,
[18] Martin, C H.; Wu, D.; Jones Prather, K L Integrated Bioprocessing for the pH-Dependent Production of 4-Valerolactone from Levulinate in Pseudomonas putida KT2440, Appl Environ Microbiol 2010, 76, 417–424
[19] Alonso D M., Wettstein S G., Dumesic J A Gamma-valerolactone, a sustainable platform molecule derived from lignocellulosic biomass Green Chem 2013, 15, 584-595
[20] Bornscheuer U T directed evolution of enzymes Angew Chem Int Ed
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[22] Kim H S., Le Q A T., Kim Y H Development of thermostable lipase B from Candida antarctica (CalB) through in silico design employing B-factor and RosettaDesign Enzyme Microb Technol 2010, 47, 1-5
[23] Siadat O R., Lougarre A., Lamouroux L., Ladurantie C., and Fournier D The effect of engineered disulfide bonds on the stability of Drosophila melanogaster acetylcholinesterase, BMC Biochemistry 2006, 7-12
[24] Dicosimo R., McAuliffe J., Poulose A J Bohlmann G Industrial use of immobilized enzymes Chem Sov Rev 2013, 42, 6437-6474.
[25] Jesionowski T., Zdarta J., Krajewska B Enzyme immobilization by adsorption: a review Adsorption 2004, 20, 801-821
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[27] Chen B., Hu J., Miller E M., Xie W., Cai M., Gross R A Candida antarctica Lipase B Chemically Immobilized on Epoxy-Activated Micro- and
Nanobeads: Catalysts for Polyester Synthesis Biomacromolecules 2008, 9,
Development of thermal stability of Candida antarctica lipase B (CalB) through in-silico design of disulfide bridge
Introduction
Candida antarctica lipase B is a very versatile enzyme with catalytic triad
The enzyme CalB, characterized by the residues Ser105, His224, and Asp187, exhibits remarkable properties such as high regioselectivity, enantioselectivity, and stability in organic solvents It has been effectively utilized in the resolution of racemic alcohols, amines, and acids, as well as in the synthesis of optically pure compounds Additionally, CalB is recognized as a robust biocatalyst for biodiesel production.
Enzymes play a crucial role in industrial applications, particularly when considering their thermal stability, which is essential for maintaining optimal reaction rates as temperature rises This increase in temperature not only enhances the enzymatic reaction rate but also improves the solubility of reactants and reduces their viscosity, facilitating more efficient processes Additionally, compounds like DD-polylactide from lactide and glycerol carbonate serve as important multifunctional agents in cosmetics and chemical intermediates.
CalB exhibits relative thermal stability; however, our previous study indicated that the conversion of glycerol to glycerol carbonate using Novozym 435 decreased at temperatures above 60 °C due to thermal inactivation Similarly, the alcoholysis of cottonseed oil for biodiesel production using Novozym 435 was adversely impacted at temperatures exceeding 50 °C Consequently, enhancing the thermal stability of CalB is essential to meet industrial standards.
Protein engineering has been effectively utilized to enhance the thermal stability of various proteins, including CalB and xylanase Techniques such as error-prone polymerase chain reaction (PCR) and rational protein design, which considers the flexibility of amino acid residues (B-factor values) and employs Rosetta Design, have been instrumental in improving CalB's thermal stability in prior research.
Despite attempts to enhance the thermal stability of CalB, significant improvements were not achieved Consequently, advancing the thermostability of CalB remains crucial for its effective use in industrial applications.
Engineering disulfide bonds in proteins is an effective approach in rational protein design, successfully enhancing the thermostability of various proteins, including T4 lysozyme, subtilisin, and Escherichia coli ribonuclease H.
Trichoderma reesei endo-1, 4-β-xylanase II relies on disulfide bonds, which are covalent linkages that significantly enhance the protein's stability by reducing the entropy of its unfolded state These bonds also decrease the unfolding rate of irreversibly denatured proteins The potential for residue pairs to form disulfide bonds can be predicted using computational tools like MODIP and DbD MODIP assesses stereo-chemical parameters, such as dihedral angles and S-S bond distances, to identify residue pairs that are geometrically favorable for disulfide bond formation This approach has been effectively utilized to enhance the stability of enzymes like indole glycerol phosphate synthase.
The computational tool DbD analyzes potential disulfide bonds in Drosophila melanogaster acetylcholinesterase by utilizing fixed bond lengths of Cβ-Sγ (1.81 Å) and Sγ-Sγ (2.04 Å), along with a bond angle of Cβ-Sγ-Sγ (104.15°) It predicts residue pairs within a torsion angle tolerance of ±30° from set values of +100° and -80° for χ3 Additionally, the energy of each predicted disulfide bond is calculated, providing a criterion to evaluate the likelihood of residue pairs forming these bonds, with lower energy values indicating a higher probability of bond formation.
The probability of forming disulfide bonds is higher in certain proteins, as demonstrated by the successful use of DbD to enhance the thermal stability of xylanase from Bacillus stearothermophilus and Rhizomucor miehei lipase While computational tools like MODIP and DbD effectively predict potential disulfide bonds, they do not assess the impact of these bonds on protein stability To engineer disulfide bonds for improved thermal stability, predicted residue pairs must be refined by aligning target proteins with their mesophilic and thermophilic relatives to identify conserved cysteines, as well as excluding pairs near catalytic residues or those leading to short-range bonds However, for proteins with limited family information or excessive potential residue pairs, these selection methods can be inadequate Thus, a more effective and generalized tool is necessary to identify promising residue pairs for enhancing protein thermal stability.
This study presents the development of thermally stable CalB using an innovative approach that combines MODIP and DbD to identify potential residue pairs for disulfide bond formation Additionally, a novel selection tool based on residual flexibility analysis was introduced to assess the impact of these newly formed disulfide bonds on enhancing protein stability.
Figure 2.1 The disulfide bonds from two cysteines (adapted from [27])
Materials and Methods
The optimized CalB gene, synthesized and donated by Professor Seongsoon Park from Sungshin Women’s University, was utilized in this study Essential materials included restriction enzymes EcoRI and NotI from Takara (Japan), a QIAquick TM PCR purification kit from Qiagen (Germany), and E coli DH5α competent cells from RBC (Taiwan) Additionally, the Pichia pastoris X-33 strain and pPICZαA plasmid were sourced from Invitrogen (USA), while para-nitrophenyl palmitate (pNPP) was obtained from Sigma-Aldrich (USA) All PCR primers were synthesized by Cosmo Genetech (Korea), and all materials were used without further purification.
2.2.2 Cloning and expression of CalB variants
Site-directed mutagenesis of the CalB gene involved substituting specific residues with cysteine through overlap extension PCR The primers used for this mutagenesis are detailed in Table 2.1 Both the wild-type CalB gene and its mutants were subsequently cloned into the pPICZαA plasmid and transformed into host cells.
The Pichia pastoris X-33 strain was transformed via electroporation and subsequently inoculated into 50 ml of buffered methanol-complex medium (BMMY), which contains 1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 0.00004% biotin, and 0.5% methanol The culture was incubated at 28 °C for 96 hours with shaking at 200 rpm, during which protein expression was induced by the addition of 100% methanol to achieve a final concentration of 0.5% every 24 hours After fermentation, CalB enzymes were harvested by centrifugation at 4000 rpm and 4 °C for 10 minutes to separate the cells from the supernatant, which was then buffer-exchanged using an Amicon 15 ultra membrane with a specific molecular weight cutoff.
Table 2.1 Primers for overlap PCR extension for site-directed mutagenesis
Primer name Sequence a (from 5’ to 3’)
EcoRI_ CalB_F CCGG GAATT CTT GCC GTC AGG TTC TGA CCC
NotI_ CalB_R CCGG GCGGCCGC TCA TGG CGT AAC GAT ACC
Q156C_MF CCT AGT GTC TGG TGT CAG ACG ACC
Q156C_MR GGT CGT CTG ACA CCA GAC ACT AGG
L163C_MF GGT TCC GCG TGT ACG ACC GCC
L163C_MR GGC GGT CGT ACA CGC GGA ACC
N169C_MF GCC CTC CGG TGT GCA GGT GG
N169C_MR CC ACC TGC ACA CCG GAG GGC
F304C_MF GCC CGT CCG TGT GCG GTC GGC
F304C_MR GCC GAC CGC ACA CGG ACG GGC
A162C_MF CG ACC GGT TCC TGT CTG ACG ACC GCC
A162C_MR GGC GGT CGT ACA GGA ACC GGT CG
K308C_MF GCG GTC GGC TGT CGT ACT TGC TCA GG
K308C_MR CC TGA GCA AGT ACG ACA GCC GAC CGC
S50C_MF AGC TTT GAT TGT AAT TGG ATT
S50C_MR AAT CCA ATT ACA ATC AAA GCT
A273C_MF A CAA AAG GTA TGT GCT GCG GCT T
A273C_MR A AGC CGC AGC ACA TAC CTT TTG T
S239C_MF C GTC GGT CGA TGT GCC CTG CGC T
S239C_MR A GCG CAG GGC ACA TCG ACC GAC G
D252C_MF T CGT AGT GCA TGT TAT GGC ATTA
The reverse primers for the D252C_MR T AAT GCC ATA ACA TGC ACT ACG A sequence were designed to be complementary to the forward primers, with the mutagenized nucleotides highlighted Additionally, the bold characters indicate the restriction sites for E.coRI (GAATTC) and NotI (GCGGCCGC).
2.2.3 Assay of lipase activity pNPP (Sigma-Aldrich, USA) was used to compare the activity of the CalB variants One unit of enzyme activity was defined as the amount of enzyme needed to liberate 1 àmol of para-nitro phenol per minute The extinction coefficient for para-nitro phenol was 18.45 mM −1 cm −1 at 405 nm, pH 8.0 [33] The assay employed 10 mM pNPP dissolved in a solution of acetonitrile (1%), ethanol (4%) and 50 mM Tris-HCl buffer (pH 8.0) (95%) Ten microliters of the enzyme solution (50 ug/ml) was added to 3 ml of the above substrate solution, and the reaction was performed at 45 °C for 30 minutes [34]
2.2.4 Analysis of thermal stability and melting temperature of CalB
To evaluate the thermal stability of CalB variants, enzyme solutions were incubated at temperatures ranging from 40 to 70 °C for one hour, followed by a 10-minute cooling period on ice The enzyme activity was subsequently assessed according to the methodology outlined in Section 2.2.3.
The melting temperature (Tm) of CalB variants was determined using differential scanning fluorimetry (DSF) with the CFX96 Real-Time PCR Detection System (Bio-Rad, USA) Purified CalB variants were obtained through Fast Protein Liquid Chromatography (FPLC) using a Superose TM 6 10/300 GL size exclusion column (GE Healthcare, USA) A 96-well thin-wall PCR plate (Bio-Rad, USA) was prepared, with each well containing 1 μM protein samples, 1X SYPRO Orange dye (Invitrogen, USA), 150 mM NaCl, and 10 mM HEPES buffer (pH 7.0), alongside a negative control well with deionized H2O The assay recorded fluorescence variations between 30–70 °C, increasing at a rate of 0.5 °C every 30 seconds, with excitation and emission wavelengths set at 490 nm and 575 nm, respectively The Tm was calculated as the transition midpoint value between the start and maximum points using the manufacturer's software.
Thermal deactivation of the CalB variants was based on the following kinetic model:
Where N, I, and kd are the native state, thermal deactivated state, and thermal deactivation rate constant, respectively
The CalB variants were incubated at 50 °C for different time intervals from
The enzyme activities were assessed after incubation for 0 to 90 minutes, followed by a 10-minute cooling period on ice The data were analyzed using first-order kinetics, with the first-order rate constants (kd) determined through linear regression of the natural logarithm of residual activity against incubation time Additionally, the half-life (t1/2) of the CalB variants at 50 °C was calculated using the formula t1/2 = ln2 / kd.
Enzyme assays with 10 μl of the CalB variants were carried out in Tris-HCl buffer, pH 8.0 (95%), ethanol (4%) with increasing concentration of pNPP from
10 μM to 100 μM Lineweaver-Burk plots were plotted to determine Km and Vmax of the CalB variants [36]
2.2.7 Molecular dynamics (MD) simulation for flexibility analysis
The crystal structure of CalB wild type (PDB code: 1tca) and its mutants, designed using DbD version 1.20, underwent energy minimization optimization through the conjugate gradient algorithm in Discovery Studio 2.5.
Accelrys in the USA utilized the CHARMm force-field alongside the Momany-Rone partial charge for atom type assignments An implicit solvent model was implemented, with dielectric constants for the protein and solvent set to 1 and 80, respectively Additionally, default values were maintained for other parameters, while the maximum steps and root mean square (RMS) gradient values were configured to 10,000 and 0.001, respectively.
To assess the flexibility of CalB variants at room temperature, their minimized structures were analyzed using the FIRST 6.2 server This process generated 1,000 conformers through FRODAN dynamics The changes in flexibility were evaluated by calculating the residual root mean square deviation (RMSD) among these conformers.
To evaluate the flexibility of CalB variants at high temperatures (600 K), their minimized structures were analyzed using Discovery Studio 2.5 A standard dynamic cascade protocol facilitated the molecular dynamics (MD) simulations, maintaining consistent force field parameters, partial charges, and dielectric constants from the initial energy minimization The MD simulation included two energy minimization phases, heating, equilibration, and a production stage The steepest descent and conjugate gradient algorithms were applied sequentially for energy minimization, with maximum steps set to 10,000 and RMS gradient values of 0.2 for steepest descent, and 10,000 and 0.0001 for conjugate gradient The heating phase involved increasing the temperature from 50 to 300 K over 2,000 steps, while structures were equilibrated at 300 K for 1,000 steps The production stage was conducted in the NVT ensemble for 1 ns at 600 K, comprising 1,000,000 steps, with trajectory data saved every 20,000 steps.
(20 ps) and 50 trajectories were used for analyzing the structural changes of the
CalB variants The RMSD values of 50 trajectories were calculated to investigate flexibility changes at high temperature All molecular structures were visualized by Pymol software (http://www.pymol.org)
Results and Discussions
2.3.1 Selection of target residues for protein engineering
To enhance the thermal stability of the enzyme, engineering disulfide bridges through site-directed mutagenesis requires careful selection of specific residue pairs This study utilized two computational tools, MODIP and DbD V1.20, to predict potential residue pairs for disulfide bond formation Subsequently, we identified promising residue pairs aimed at improving the thermal stability of the CalB wild type using a novel selection tool focused on residual flexibility.
Through MODIP, we identified ninety-nine residue pairs for potential disulfide bonds, categorized from grades A to D Among these, the grade A pairs, which have the highest likelihood of forming disulfide bonds, led to the selection of twenty-one residue pairs from the CalB wild type for further analysis Additionally, the second computational tool, DbD V1.20, provided further predictions.
43 possible residue pairs for formation of disulfide bonds in CalB (Figure 2.2)
To enhance the reliability of predicted residue pairs for potential disulfide bond formation, we compared the pairs identified by DbD with those classified as grade A in MODIP A total of seventeen residue pairs were consistently predicted by both computational tools, highlighted in the yellow circle (Figure 2.2) However, due to the excessive number of residue pairs for experimental validation, we proposed additional criteria We hypothesized that introducing disulfide bonds from residue pairs exhibiting high flexibility could significantly enhance thermostability Thus, the flexibility of the residues was a key factor in our analysis.
In evaluating CalB mutants, 28 residue pairs, known as the B-factor of residue pairs, were analyzed alongside the flexibility of residues near newly introduced disulfide bonds The B-factor indicates the extent of atomic electron density smearing due to thermal motion and positional disorder To enhance the protein's thermal stability, residues with high B-factors were replaced with more suitable alternatives.
The flexibility of residue pairs in CalB was assessed by summing the B-factors of 17 suggested pairs, with the exception of three pairs involved in native disulfide bonds The B-factors, which reflect static state flexibility, were ranked, revealing four pairs with the highest B-factors and one with the lowest, selected for further analysis of disulfide bond effects on mutant flexibility using FRODAN dynamics Notably, three double mutants (N169C-F304C, A162C-K308C, and Q156C-L163C) exhibited reduced flexibility near the engineered disulfide bonds, indicating enhanced rigidity and positioning them as promising candidates for site-directed mutagenesis aimed at improving CalB's thermal stability Additionally, two other mutants (S239C-D252C and S50C-A273C) were created to evaluate their impact on the enzyme's thermostability.
Figure 2.2 illustrates the alignment of potential residue pairs for disulfide bridges, comparing data from grade A of MODIP and DbD V1.20 The common residue pairs identified by both DbD and grade A of MODIP are highlighted in yellow.
Table 2.2 Ranking from the highest to the lowest B-factor of the residue pairs from MODIP and DbD V1.20 for possible disulfide bridge
Ranking Residue i Residue j Sum of B- factor of residue i and residue j
Table 2.3 presents a ranking of CalB variants based on the degree of flexibility change induced by the introduction of new disulfide bonds at room temperature, with all calculations conducted using the FIRST server.
Average residual RMSD within 5 Å from the disulfide bond [Å ]
2.3.2 Thermal stability of CalB variants
The thermal stability of CalB variants is significantly affected by temperature, as illustrated in Figure 2.3 At 40 °C, most CalB variants maintained approximately 70-90% of their original enzyme activity, with the exception of CalB Q156C/L163C, which only retained 50% A notable decline in enzyme activity was observed at 50 °C, where CalB A162C/K308C exhibited 1.5-fold greater residual activity compared to the wild type (60% vs 40%) In contrast, other CalB mutants showed drastic reductions in activity, with values dropping to as low as 5% At temperatures between 60-70 °C, CalB A162C/K308C demonstrated significantly higher residual activity than both the wild type and other mutants, confirming its superior thermal stability Conversely, mutants such as S50C/A273C, Q156C/L163C, S239C/D252C, and N169C/F304C did not exhibit enhanced thermal stability.
Table 2.4 presents data indicating that the T50 60 of the CalB A162C/K308C mutant is 8.5 °C higher than that of its wild type, while other CalB mutants did not exhibit any significant improvements in T50 60 compared to their wild type.
CalB wild type CalB A162C/K308C CalB S50C/A273C CalB Q156C/L163C CalB N169C/F304C CalB S239C-D252C
Figure 2.3 The effect of preheating treatment temperature from 40-70 °C for
60 minutes on the enzyme activity of the CalB variants
The study investigated the impact of preheating time at 50 °C on the thermal stability of CalB variants Notably, CalB A162/K308C demonstrated significantly higher thermal stability compared to the wild type and other CalB mutants throughout the preheating duration After 150 minutes, CalB A162C/K308C exhibited residual activity that was 3.2 to 22 times greater than that of the wild type and various mutants, including S50C/A273C and Q156C/L163C Additionally, the half-life (t1/2) of CalB variants, derived from the inactivation constant k d, revealed that CalB A162C/K308C had a half-life 4.5 times longer than the wild type, with t1/2 values of approximately 49 minutes for the wild type and 220 minutes for A162C/K308C at 50 °C.
CalB wild type CalB A162C/K308C CalB S50C/A273C CalB Q156C/L163C CalB N169C/F304C CalB S239C/D252C
Figure 2.4 Effect of preheating temperature at 50 °C with different incubation time intervals on the enzyme activity of the CalB variants
Ln ( re sidua l ac tiv ity U/ml )
CalB Wild type CalB A162C/K308C CalB S50C/A273C CalB Q156C/L163C CalB N169C/F304C CalB S239C/D252C
Figure 2.5 Thermal deactivation of the CalB variants at 50 °C
The thermal deactivation of CalB mutants Q156C/L163C and N169C/F304C revealed unexpected results in flexibility analysis at room temperature To assess the thermal impact on the conformational stability of these mutants, the melting temperature (Tm) of the CalB variants was determined using a Differential Scanning Fluorimetry (DSF) assay This technique measures the increase in fluorescence intensity of a dye that binds to hydrophobic regions of the protein, which become exposed during the unfolding process As the protein undergoes thermal melting, its hydrophobic domains are gradually revealed.
The transition midpoint value (Tm) between the starting and maximum points is detailed in Table 2.4, revealing that mutants CalB N169C/F304C and A162C/K308C exhibit higher Tm values than the wild type, by 1.5 °C and 1.1 °C, respectively In contrast, CalB Q156C/L163C shows a significant decrease in Tm, being 8.7 °C lower than the wild type Additionally, a flexibility analysis of these three mutants, conducted through molecular dynamics (MD) simulation at elevated temperatures, is discussed in Section 2.3.4 to clarify the impact of the newly introduced disulfide bonds on their thermal stability.
The thermal stability analysis of CalB variants demonstrated that CalB A162C/K308C exhibited enhanced thermal stability in both conformation and thermal deactivation compared to the wild type In contrast, CalB N169C/F304C showed improved conformation stability, but its thermal deactivation was reduced relative to the wild type The disulfide bonds introduced in CalB N169C/F304C may have affected its catalytic sites However, the thermal stability of other CalB mutants was found to be lower than that of the wild type Previous studies have indicated that the introduction of additional disulfide bonds can destabilize protein stability in certain mutants, such as those in Drosophila melanogaster acetylcholinesterase and Bacillus amyloliquefaciens subtilisin.
Figure 2.6 illustrates the relationship between thermal stability and flexibility in CalB variants As the flexibility within 5 Å of the newly introduced disulfide bonds in the CalB mutants decreased from the wild type, the thermal stability parameters of these variants increased This observation supports our hypothesis that introducing new disulfide bonds from highly flexible residue pairs enhances the thermal stability of CalB.
Table 2.4 Thermal parameters of the CalB variants and time required for residual activity to be reduced to half (t 1/2 ) of the CalB variants at 50 °C
N.D.: Not determined t 1/2 = ln2 k d a k d (first order rate constant of inactivation) was determined based on Figure 2.5
Residual RMSDmutant-Residula RMSDwild-type (A 0
-0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 log (T 50 60 of C alB v ar ian ts)
Lo g ( t 1/2 of C alB v ar ian ts)
1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 log (T m of C alB v ar ian ts)
The relationship between the flexibility reduction within 5 Å of newly introduced disulfide bonds in CalB mutants compared to the wild type is illustrated, highlighting its impact on the thermal stability of CalB variants Specifically, the thermal stability metrics, including the T50, half-life (t1/2), and melting temperature (T m), correlate with the difference in root mean square deviation (RMSD) between the mutants and the wild type.
Conclusions
We utilized two computational tools, MODIP and DbD v1.20, to predict potential disulfide bonds in proteins Subsequently, we introduced a novel selection tool based on the B-factor values of residue pairs in the CalB wild type, along with the residual flexibility analysis of both the CalB wild type and its in-silico mutants using the FIRST server This approach enabled us to identify promising mutants with enhanced thermostability.
Our proposed procedure led to the identification of CalB A162C/K308C, which demonstrated significantly improved thermal stability compared to its wild type Notably, the T50 60 value, melting temperature (Tm), and half-life of CalB A162C/K308C were 8.5 °C, 1.1 °C, and 4.5 times greater than those of the wild type, respectively.
The MD simulation-based residual flexibility analysis revealed that the rigidity of the CalB A162C/K308C mutant enhanced the stability of surrounding residues and the overall structure This rigidity of the disulfide bond residue pair was linked to the thermal stability of the CalB variants Additionally, our innovative selection tool, which evaluates the flexibility of residue pairs before and after the introduction of disulfide bonds, has proven effective in identifying promising candidates for enhancing thermal stability in other proteins through disulfide bond engineering.
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Development of thermal stable of Candida antarctica lipase B
Introduction
Candida antarctica lipase B (CalB) is a prominent enzyme for biodiesel production, DD-polylactide synthesis, and glycerol carbonate formation According to chapter 2, the biotransformations facilitated by Novozym 435, an immobilized form of CalB, showed reduced final conversion rates of glycerol carbonate at 60 °C and adversely affected biodiesel synthesis at elevated temperatures.
Lipase-catalyzed biodiesel production is inefficient at low temperatures, particularly due to the high viscosity of various triacyl glycerols To enhance the economic feasibility of biotransformation processes, the development of highly thermostable CalB is essential for reducing biocatalyst costs A recent study on rational protein design revealed that the CalB A162C/K308C mutant demonstrates significantly improved thermal stability, with a T 50 60 value of 54.5 °C, which is 8.5 °C higher than the wild type.
Engineering our thermal stable CalB A162C/K308C through protein engineering presents significant challenges Therefore, exploring alternative strategies and integrating them with our thermal stable mutant is a more effective approach.
Novozym 435 has been reported to experience physical desorption of CalB during reactions, potentially impacting its thermal stability To enhance the thermal stability of conventional immobilized CalB, developing efficient enzyme immobilization strategies is essential Enzyme immobilization has proven effective in improving thermal stability, and sol-gel entrapment is a widely used method due to its mild conditions.
Various methods have been proposed for sol-gel formation to build nanostructures and immobilize enzymes However, many reagents used in silica sol-gel processes necessitate acidic or alkaline pH conditions, which can compromise protein structures during the formation process.
Silaffins, polycationic peptides derived from the diatom Cylindrotheca fusiformis, can rapidly precipitate silica when introduced to a silicic acid solution The native Silaffin-1A, characterized by its zwitterionic structure, includes polyamine moieties and phosphate groups modified in lysine and serine residues, facilitating silaffin self-assembly—a crucial step in biosilica formation Seven silaffin polypeptides, ranging from R1 to R7, have been isolated from diatoms, with R1 silaffin demonstrating the highest efficacy as a fusion partner for protein biosilicification Utilizing silaffin peptides for enzyme immobilization through biosilicification offers a promising alternative to the harsh conditions typically associated with sol-gel processes.
This study focuses on the fusion of CalB with R1 silaffin to create CalB-R1, which was immobilized in a two-step process Initially, the CalB-R1 fusion protein was physically adsorbed onto macroporous polyacrylate carriers, followed by biosilicification using TMOS as a precursor The final product is referred to as CalB-R1.
“double-immobilized CalB-R1”) was tested to measure its thermal tolerance
Figure 3.1 (A): primary structure of polypeptide sil1p with seven repeating unit from silaffin R1 to R7 domains (Adapted from [9]) (B) Schematic chemical structure of native silaffin-1A1 at pH 5.0 (Adapted from [10])
Material and methods
The CalB gene (NCBI gi: 515791 or Z30645) was synthesized by GenScript (USA), while the QIAquick TM PCR purification kit and DH5α competent cells were sourced from Qiagen (USA) and RBC Bioscience (Taiwan), respectively The Pichia pastoris X-33 strain and pPICZαA plasmid were obtained from Invitrogen (USA), and the macroporous polyacrylate carrier (Lewatit VPOC 1600) was purchased from Bayer (Germany) Additionally, para-nitrophenyl butyrate (pNPB) and tetramethyl orthosilicate (TMOS) were supplied by Sigma–Aldrich (USA), with all PCR primers synthesized by Cosmo Genetech (South Korea) All materials were utilized without further purification.
3.2.2 Cloning, expression, and purification of CalB-R1
The fusion of gene encoding for R1 silaffin domain to C- terminal of CalB gene was performed by PCR, which is similar to previous work of our group
The His6-tag was incorporated at the N-terminal of the CalB gene to facilitate enzyme purification via an affinity column (Ni-NTA) PCR primers for the synthesis of the CalB-R1 fusion gene are detailed in Table 3.1, with the PCR conducted three times to attach all three fractions of the R1 silaffin domain to the CalB gene The sequence of the CalB-R1 gene was verified at Cosmo Gentech in Seoul, Korea Subsequently, the CalB-R1 gene was introduced into Pichia pastoris X-33 cells through electroporation The transformed cells were then cultured in 50 mL of buffered minimal glycerol-complex (BMGY) media, consisting of 1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer (pH 6.0), 1.34% yeast nitrogen base (YNB), 4 x 10^-5% biotin, and 1% glycerol, and incubated at 30 °C for 16 to 18 hours.
250 rpm to obtain seed culture The seed culture was then transferred to 2 L of
-60- basal salt medium (BSM: 54 mL of 85% H3PO4, 1.86 g of CaSO4, 36.4 g of
In a BioFlo 310 fermenter, a mixture of K2SO4, 29.8 g of MgSO4•7H2O, 8.26 g of KOH, 80 g of glycerol, and 2 L of distilled water was cultivated at 28 °C and pH 5.0 for 96 hours Dissolved oxygen levels were kept at or above 30% saturation through agitation (500–1100 rpm), along with air (2–4 L/min) and pure oxygen (0.2–0.6 L/min) supply The induction of CalB-R1 protein expression was achieved by adding 100% methanol to reach a final concentration of 0.5% every 24 hours, following the manufacturer's guidelines from the EasySelect Pichia Expression kit by Invitrogen Dry cell mass during fermentation was measured by filtering culture samples through a 0.45-μm filter, washing the cells with distilled water, and drying them at 95 °C.
After 24 hours, minimal changes in weight were observed The CalB-R1 enzyme was harvested through centrifugation at 4,500×g and 4 °C for 10 minutes, followed by the removal of cells Subsequently, the purification of the CalB-R1 enzyme in the supernatant was conducted in accordance with prior research methods.
[13] The pH of the supernatant was firstly adjusted to pH 8.0 by using NaOH
The supernatant was centrifuged at 4,500×g and 4 °C for 10 minutes to eliminate the precipitate Following this, the supernatant was incubated with Ni-NTA agarose beads at 4 °C for one hour before being loaded onto the column Purification of CalB-R1 was achieved by washing the Ni-NTA column multiple times with a washing buffer consisting of 50 mM NaH2PO4, 20 mM imidazole, and 300 mM NaCl at pH 8.0, followed by elution using an elution buffer of 50 mM NaH2PO4, 250 mM imidazole, and 300 mM NaCl at pH 8.0 The concentration of the expressed CalB-R1 was then determined using Bradford reagent according to the manufacturer's instructions.
Table 3.1 Primers for synthesis of CalB-R1 gene
(*) The sequences underlined are nucleotides encoding for silafin R1 fused to CalB gene; bold characters for restriction sites: EcoRI (GAATTC) and NotI (GCGGCCGC)
3.2.3 Physical absorption and biosilicification of CalB-R1
The immobilization and biosilicification of CalB-R1 are illustrated in Scheme 3.1 The supernatant from fed-batch fermentation was buffer-exchanged using a 50 mM Tris-HCl buffer at pH 8.0 with an Amicon Ultra Filter (10,000 molecular-weight cutoff) For immobilization, 50 mg of CalB-R1 was combined with 1.25 g of ethanol-pre-wetted Lewatit VPOC 1600 and incubated at 25 °C for 48 hours, resulting in a loading content of 40 μg of CalB-R1 per mg of supporting carrier Both VPOC-immobilized CalB-R1 and Novozym 435 were utilized for biosilicification with TMOS, where the immobilized enzymes were mixed together for the process.
In this study, 2 mL of phosphate-citrate buffer at pH levels 5.0, 6.0, and 7.0 was prepared at room temperature, while TMOS was hydrolyzed using 1 mM HCl The pre-hydrolyzed TMOS was then combined with immobilized CalBs at a final concentration of 100 mM and incubated at room temperature for 15 minutes Following this, the biosilicificated enzymes were collected through filtration and washed twice with the corresponding buffer for each pH Finally, the wet biosilicificated enzymes were dried in a desiccator at 4 °C for 24 hours.
Scheme 3.1 Procedure for double immobilization of CalB-R1
3.2.4 Analysis of silica deposition on the supporting carrier
To verify the presence of silica in double immobilized CalBs, SEM-EDX with a focused ion beam (FIB) was employed The analysis utilized a Quanta 3D FIB/field emission gun equipped with an energy-dispersive X-ray spectroscopy (EDX) detector from FEI Company, USA For image scanning, an accelerating voltage of 5 kV was applied, while a voltage of 15 kV was used to obtain signals during the EDX analysis.
3.2.5 Enzyme assay pNPB (Sigma-Adrich, USA) was used to measure enzyme activity of free and immobilized CalB-R1s One unit of enzyme activity was defined as the amount of enzyme needed to hydrolyze 1 μmol of pNPB per minute at 30 °C and pH 8.0) The releasing product, para-nitrophenol, was measured in UV/VIS spectroscopy at 405 nm The assay was perfomed in 50 mM Tris-HCl buffer (pH 8.0) containing 0.1 mM pNPP [15, 16] The enzyme (i.e., 5–10 μg of the free CalB-R1 or 5 mg of each immobilized CalB-R1 or Novozym 435) was added to 15 mL of the above substrate solution, and the reaction was performed at 30 °C for 30 minutes
This study investigates the thermal inactivation and stability of free CalB-R1, immobilized CalB-R1, and Novozym 435 enzymes The analysis involves using 5–10 μg of free CalB-R1 and 5 mg of each immobilized CalB-R1 and Novozym 435 for evaluation.
In a PCR tube, 100 μL of 50 mM Tris-HCl buffer (pH 8.0) was added, and each sample was incubated at temperatures ranging from 30 to 90 °C for 1 hour, followed by a 10-minute cooling period on ice The enzyme activity was assessed according to the methodology outlined in Section 3.2.5, with T50 60 being defined in this context.
The temperature at which half of the enzyme activity is preserved after one hour of incubation is 65 °C, compared to the activity observed after one hour at 30 °C All experimental results presented are the average of three measurements.
The thermal inactivation of free and immobilized CalB-R1 was evaluated by incubating the enzymes at 60 °C for varying durations from 0 to 60 minutes, followed by a 10-minute cooling period on ice Enzyme activities were subsequently measured as outlined in Section 3.2.5 The thermal inactivation rate constant (k d) and half-life (t 1/2), which indicates the time required for the enzyme to retain 50% of its initial activity, were determined as described in Section 2.2.5.
The reusability of immobilized enzymes was assessed through multiple reaction cycles After each cycle, the enzymes were recovered from the reaction mixture by filtering the solution using Whatman paper with a pore size of 0.2 μm, followed by washing the immobilized enzymes on the filter with 10 ml of buffer solution.
50 mM Tris-HCl buffer (pH 8.0) The immobilized enzymes which recovered in the previous cycle were again used for another cycle in a fresh reaction solution.
Results and discussion
3.3.1 Expression and purification of CalB-R1
The successful expression of CalB fused with R1 silaffin was achieved in the Pichia pastoris X-33 system Notably, lipase activity for the hydrolysis of pNPB began to significantly increase after 48 hours of cultivation, reaching approximately 2.5 U/mL The impact of cultivation time on the expression level of CalB-R1 was demonstrated through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), as illustrated in Figure 3.3 (A).
The purification of CalB-R1 is illustrated in Figure 3.3 (B), where the gel reveals a single band at approximately 37 kDa The specific activity of the purified CalB-R1 is measured at 20 U/mg, which is comparable to the 30 U/mg reported for CalB in prior research [17] Notably, the fusion of the R1 silafin to the C-terminal of CalB has only a minor effect on its lipase activity.
Figure 3.2.Effect of the incubation time on the cell density and enzyme activity of Pichia pastoris X-33 containing CalB-R1: (a) dry cell weight (), optical density at 600 nm (), and lipase activity ().
The expression and purification of CalB-R1 were analyzed using SDS-PAGE In the expression analysis, protein markers were indicated in Lane M, while Lanes 1 to 5 displayed Bovine serum albumin concentrations of 10, 25, 50, 100, and 200 μg/ml Lanes 6 to 10 represented CalB-R1 samples collected after 48, 60, 72, 84, and 96 hours of expression For the purification analysis, Lane M again showed protein markers, with Lane ① containing the CalB-R1 culture supernatant and Lane ② displaying the purified CalB-R1.
3.3.2 Double immobilization and analysis of silica deposition on the supporting carrier
The protein quantification and analysis of CalB-R1 in the aqueous immobilization medium demonstrated that 85% of the free enzyme (42.5 mg) was successfully adsorbed onto the Lewatit VPOC 1600 resin Subsequently, the VPOC-immobilized CalB-R1 underwent biosilicification using 100 mM TMOS Analysis of the presence of synthesized silica revealed that it was located exclusively inside the supporting carrier VPOC 1600, as illustrated in Figure 3.4, with no silicon atoms detected on the surface of the carrier.
The study highlights the use of highly macroporous material, specifically with a diameter exceeding 100 nm, which allows for effective adsorption of CalB-R1 on its surface The R1 silaffin polypeptide facilitates the formation of a silica matrix from prehydrolyzed TMOS within the pores of the supporting carrier Unlike conventional sol-gel processes that often lead to excessive silica buildup around enzymes, resulting in significant mass transfer resistance, the biosilicification method employed in this research minimizes this issue by applying a very thin layer of silica, as illustrated in Figure 3.4.
The analysis of silica deposition in the supporting carrier was conducted using SEM-EDX combined with FIB techniques The study includes a SEM image showcasing the surface of the supporting carrier post-biosilicification, alongside the elemental composition of this surface Additionally, a cross-sectional SEM image of the supporting carrier after biosilicification is presented, along with its corresponding elemental composition.
3.3.3 Influence of biosilicification on the thermostability of CalB-R1
The thermal stability of free and immobilized CalB is significantly affected by temperature, as illustrated in Figure 3.5 Notably, the physical absorption of CalB onto macroporous VPOC 1600 enhances its thermal stability compared to free CalB-R1, with T50 values of 45 °C for free CalB-R1, 58 °C for VPOC-immobilized CalB-R1, 62 °C for Novozym 435, and higher for double immobilized CalB-R1 Novozym 435 is produced through the physical absorption of free CalB onto VPOC, further demonstrating the benefits of immobilization in improving enzyme stability.
1600 [4], had slightly higher thermal stability compared to the VPOC- immobilized CalB-R1 in term of T50 60
The thermostability of free CalB used in Novozym 435 appears to be slightly higher than that of free CalB-R1 However, a direct comparison of thermostability between these two free CalB enzymes is challenging due to the difficulty in obtaining free CalB from Novozym 435.
The double immobilized CalB-R1, created through R1 silaffin-mediated biosification of VPOC-immobilized CalB-R1, demonstrated significantly enhanced thermal stability compared to free CalB-R1, VPOC-immobilized CalB-R1, and Novozym 435, with a T 50 60 value reaching 72 °C.
The double immobilized CalB-R1 exhibited a remarkable thermal stability, with a temperature tolerance of 27, 14, and 10 °C higher than that of free CalB-R1, VPOC-immobilized CalB-R1, and Novozym 435, respectively After incubation at 70 °C, the double immobilized CalB-R1 retained approximately 58% of its activity, in stark contrast to the 18%, 32%, and significantly lower retention rates observed for free CalB-R1, VPOC-immobilized CalB-R1, and Novozym 435.
24 %, respectively The biosilicification -mediated by R1 silaffin part in the immobilized CalB-R1 significantly improved the thermal stability of the single immobilized CalB-R1 To verify the effect of R1 silaffin on biosilicification,
The thermostability of Novozym 435 and TMOS-treated Novozym 435 was found to be comparable, indicating that the treatment with 100 mM TMOS did not effectively biosilicificate Novozym 435 under the experimental conditions used, which may account for the minor differences observed in their thermal stability.
CALB-R1 immobilized on VP OC
Figure 3.5 Influence of immobilization on thermostability
The thermal inactivation of free and immobilized CalB-R1 at 60 °C reveals significant differences in stability, as shown in Figure 3.6 The thermal inactivation rate constants (kd) were derived from the logarithmic plot of activity versus time Notably, the half-life (t 1/2) of double-immobilized CalB-R1 was found to be 31.6-, 7.6-, 8.3-, and 6.6-fold longer than that of free CalB-R1, VPOC-immobilized CalB-R1, Novozym 435, and TMOS-treated Novozym 435, respectively (Table 3.2) This indicates that double-immobilized CalB-R1 exhibits enhanced thermal stability at 60 °C compared to its free and single-immobilized counterparts, as well as the commercial enzyme Novozym 435 Enzyme immobilization effectively limits detrimental motion at elevated temperatures, reducing the unfolding rate and aggregation likelihood, thereby improving thermostability The combination of physical adsorption and biosilicification used in this study strengthens the immobilized CalB-R1, as physical adsorption restricts local structural motion while biosilicification encapsulates the protein with silica In contrast, single immobilization techniques, such as adsorption or sol-gel entrapment, often face challenges related to enzyme leakage from the supporting carrier.
The example of 435 during the reaction highlights the limitations of single immobilization techniques Utilizing silaffin R1-mediated biosilicification for single-immobilized CalB-R1 serves as an effective model to enhance the thermal stability of other single-immobilized enzymes that lack sufficient thermal resilience, as it prevents the physical desorption of the absorbed enzymes.
Table 3.2 Thermal inactivation parameters at 60°C
Novozym 435 treated with TMOS 0.012 58.3 a t1/2= ln2/kd, (kd : first-order rate constant of inactivation)
Free CALB-R1 CALB-R1 immobilized on VP OC Double-immobilized CALB-R1 Novozym 435
Figure 3.6.Thermal inactivation of free and immobilized CalB-R1 at 60 °C Residual activity was measured at 30 °C, pH 8.0.
3.3.4 Influence of pH on biosilicification
The activity of CalB was optimal at pH 8.0, but the silaffin-mediated synthesis of the silica matrix was significantly affected by pH levels Our investigation into pH conditions ranging from 5.0 to 7.0 revealed that biosilicification resulted in increased silica concentration as pH decreased, peaking at pH 5.0 Notably, the double-immobilized CalB-R1 synthesized at this pH exhibited the highest thermal stability, with a T50 value of approximately 72 °C The correlation between synthesized silica concentration and thermal stability indicates that biosilicification at pH 5.0 enhances the protective silica matrix around CalB-R1, preventing enzyme leakage during reactions This study underscores the importance of pH in silaffin-mediated silica synthesis and its role in improving the thermostability of CalB-R1.
Table 3.3 Si content of a cross section of the supporting carrier according to the pH used for biosilicification pH for biosilicification
[°C] pH 5.0 12.46 72 pH 6.0 9.90 50 pH 7.0 5.58 46 aThe Si content was determined SEM-EDX after removal of the supporting carrier using a FIB
3.3.5 The reusability of the immobilized CalB-R1
Immobilized enzymes that maintain their characteristics after repeated use are crucial for industrial applications This study examined the reusability of immobilized CalB variants, revealing that both Novozym 435 and VPOC-immobilized CalB-R1 demonstrated similar reusability However, the two physically adsorbed CalB enzymes retained less than 30% of their initial activity after five cycles, indicating that the hydrolysis of pNPP catalyzed by these physically immobilized enzymes was not detrimental to their performance.
Conclusions
The thermostability of CalB-R1 was significantly enhanced through a combined immobilization method that involved physical adsorption on VPOC 1600 macroporous material and biosilicification mediated by R1 silaffin fused CalB The double immobilized CalB-R1 demonstrated superior thermal stability compared to free CalB-R1, VPOC-immobilized CalB-R1, and Novozym 435 Notably, the T50 60 of the double immobilized CalB-R1 was markedly improved.
The VPOC-immobilized CalB-R1 demonstrated a thermal stability increase of 27, 14, and 10 °C compared to free CalB-R1 and Novozym 435, respectively The silica matrix, facilitated by the R1 silaffin part in CalB-R1, effectively trapped the enzyme on the macroporous polyacrylate carrier, preventing its inactivation at elevated temperatures SEM-EDX analysis confirmed the presence of the synthesized silica matrix surrounding CalB-R1 on the support, with silica content correlating to the enhanced thermal stability observed in double-immobilized CalB-R1 This biosilicification process, mediated by the R1 silaffin in CalB, serves as a promising template for improving the thermal stability of other physically absorbed enzymes that lack sufficient thermal resilience.
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