Jeong Chan Joo

Thermostabilization of Bacillus circulans xylanase: Computational optimization of unstable residues based on thermal fluctuation analysis

Low thermostability often hampers the applications of xylanases in industrial processes operated at high temperature, such as degradation of biomass or pulp bleaching. Thermostability of enzymes can be improved by the optimization of unstable residues via protein engineering. In this study, computational modeling instead of random mutagenesis was used to optimize unstable residues of Bacillus circulans xylanase (Bcx). The thermal fluctuations of unstable residues known as important to the thermal unfolding of Bcx were investigated by the molecular dynamics (MD) simulations at 300 K and 330 K to identify promising residues. The N52 site in unstable regions showed the highest thermal fluctuations. Subsequently, computational design was conducted to predict the optimal sequences of unstable residues. Five optimal single mutants were predicted by the computational design, and the N52Y mutation showed the thermostabilization effect. The N52 residue is conserved in Bacillus species xylanases and the structure analysis revealed that the N52Y mutation introduced more hydrophobic clusters for thermostability, as well as a more favorable aromatic stacking environment for substrate binding. We confirm that flexible residues at high temperature in unstable regions can be promising targets to improve thermostability of enzymes.

Development of thermostable Candida antarctica lipase B through novel in silico design of disulfide bridge

Lipase B from Candida antarctica (CalB) is a versatile biocatalyst for various bioconversions. In this study, the thermostability of CalB was improved through the introduction of a new disulfide bridge. Analysis of the B‐factors of residue pairs in CalB wild type (CalB‐WT) followed by simple flexibility analysis of residues in CalB‐WT and its designated mutants using FIRST server were newly proposed to enhance the selective power of two computational tools (MODIP and DbD v1.20) to predict the possible disulfide bonds in proteins for the enhancement of thermostability. Five residue pairs (A162‐K308, N169‐F304, Q156‐L163, S50‐A273, and S239C‐D252C) were chosen and the respective amino acid residues were mutated to cysteine. In the results, CalB A162C‐K308C showed greatly improved thermostability while maintaining its catalytic efficiency compared to that of CalB‐WT. Remarkably, the temperature at which 50% of its activity remained after 60‐min incubation (T  5060 ) of CalB A162C_K308C was increased by 8.5°C compared to that of CalB‐WT (55 and 46.5°C, respectively). Additionally, the half‐life at 50°C of CalB A162C‐K308C was 4.5‐fold higher than that of CalB‐WT (220 and 49 min, respectively). The improvement of thermostability of CalB A162C‐K308C was elucidated at the molecular level by molecular dynamics (MD) simulation. Biotechnol. Bioeng. 2012; 109:867–876.

Recent advances in development of biomass pretreatment technologies used in biorefinery for the production of bio-based fuels, chemicals and polymers

Biochemical conversion of biomass into biofuels, biochemicals, and biopolymers has attracted much interest throughout the world in terms of biorefineries. Lignocellulosic biomass is one of the most plentifully available biomass resources on the earth. It is composed of three main biopolymers - cellulose, hemicelluloses, and lignin, all of which are cross-linked to each other to resist degradation by enzymes and microorganisms resulting in so-called biomass recalcitrance. The biorefinery process typically consists of three steps: pretreatment, hydrolysis, and fermentation. Energy and cost efficiency of biorefinery is predominantly dependent on how to produce inexpensive sugars from complex cell wall component of lignocellulosic biomass by overcoming biomass recalcitrance. There have been tremendous efforts to develop effective biomass pretreatment technologies for obtaining the highest yield of fermentable sugars from biomass feedstocks at the lowest cost. The present review discusses various pretreatment technologies to understand how to effectively break down biomass into fermentable sugars that are eventually used for microbial fermentation to produce biomass-based fuels, chemicals, and polymers.

Thermostabilization of Bacillus circulans xylanase via computational design of a flexible surface cavity.

Despite recent advances in our understanding of the importance of protein-surface properties for protein thermostability, to date many rational designs have been focused instead on protein-core characteristics such as core packing and cavity filling. Rational strategies to design protein surfaces to improve protein thermostability have not yet been well investigated. Here, an efficient rational design of a surface cavity for improving protein thermostability without reducing enzyme activity is suggested. Bacillus circulans xylanase (Bcx) was used as a model enzyme. Two structural features related to protein thermostability, protein cavities and flexibility were considered to identify thermo-labile residues. Residues with flexible motions in surface cavities were selected and redesigned for xylanase thermostabilization using a computational method to stabilize the local interactions of the surface cavities. Three thermostable single mutants (F48Y, T50V, and T147L) were experimentally identified, and combination of the single mutants resulted in a more thermostable triple mutant (F48Y/T50V/T147L). The thermostability and the catalytic efficiency of the triple mutant were 15 times and 1.3 times higher than wild-type Bcx, respectively. Our surface-cavity design strategy showed that flexible surface residues tolerant to mutations are valid targets for thermostabilization with no reduction in catalytic activity, and that local-interaction stabilization of cavity-lining residues using the computational method can be an effective alternative to the conventional cavity-filling method. This strategy can be used as a practical approach to increase protein thermostability.

Stabilization of Candida antarctica lipase B in hydrophilic organic solvent by rational design of hydrogen bond

Enzymatic reactions conducted in organic solvents have many advantages. However, organic solvent molecules may replace water molecules at the protein surface and penetrate into the enzyme, which could lead to the denaturation of the enzyme or changes in its reaction kinetics and substrate specificity. Thus, it is important to enhance the stability of enzymes in organic solvents. To date, there has been no efficient rational approach developed to enhance enzyme stability in hydrophilic solvents. We developed a rational approach to enzyme design. The design rules were established by investigating stable mutants from previous studies of directed evolution. Candida antarctica lipase B (CalB) was used as a target enzyme due to its versatile applications in organic solvents. The N97Q, N264Q, and D265E mutants of CalB showed higher organic solvent stability than the wild type.

The development of a thermostable CiP (Coprinus cinereus peroxidase) through in silico design

Protein thermostability is a crucial issue in the practical application of enzymes, such as inorganic synthesis and enzymatic polymerization of phenol derivatives. Much attention has been focused on the enhancement and numerous successes have been achieved through protein engineering methods. Despite fruitful results based on random mutagenesis, it was still necessary to develop a novel strategy that can reduce the time and effort involved in this process. In this study, a rapid and effective strategy is described for increasing the thermal stability of a protein. Instead of random mutagenesis, a rational strategy was adopted to theoretically stabilize the thermo labile residues of a protein using computational methods. Protein residues with high flexibility can be thermo labile due to their large range of movement. Here, residue B factor values were used to identify putatively thermo labile residues and the RosettaDesign program was applied to search for stable sequences. Coprinus cinereus (CiP) heme peroxidase was selected as a model protein for its importance in commercial applications, such as the polymerization of phenolic compounds. Eleven CiP residues with the highest B factor values were chosen as target mutation sites for thermostabilization, and then redesigned using RosettaDesign to identify sequences. Eight mutants based on the redesigns, were produced as functional enzymes and two of these (S323Y and E328D) showed increased thermal stability over the wild‐type in addition to conserved catalytic activity. Thus, this strategy can be used as a rapid and effective in silico design tool for obtaining thermostable proteins.

Prediction of the solvent affecting site and the computational design of stable Candida antarctica lipase B in a hydrophilic organic solvent

Enzyme reactions in organic solvent such as for organic synthesis have great industrial potential. However, enzymes lose their stability in hydrophilic organic solvents due to the deformation of the enzyme by the solvent. It is thus important to enhance the stability of enzymes in hydrophilic organic solvents. Previous approaches have not considered on the interaction between enzymes and solvents due to the lack of information. In this study, the structural motions of the enzyme in methanol cosolvent and the interaction between the enzyme surface and the solvent molecule were investigated using molecular dynamics simulation (MD). By analyzing the MD simulation results, the surface residues of Candida antarctica lipase B (CalB) with higher root mean square deviation (RMSD) in a methanol solvent were considered as methanol affecting site and selected for site-directed mutagenesis. The methanol affecting site was computationally redesigned by lowering the RMSD. Among the candidate mutants, the A8T, A92E, N97Q and T245S mutants showed higher organic solvent stability at various methanol concentrations. The rational approach developed in this study could be applied to the stabilization of other industrial enzymes used in organic solvents.

Hydrophobic interaction network analysis for thermostabilization of a mesophilic xylanase

One widely known drawback of enzymes is their instability in diverse conditions. The thermostability of enzymes is particularly relevant for industrial applications because operation at high temperatures has the advantage of a faster reaction rate. Protein stability is mainly determined in this study by intra-molecular hydrophobic interactions that have a collective and 3-dimensional clustering effect. To interpret the thermostability of enzymes, network analysis was introduced into the protein structure, and a network parameter of structural hierarchy, k of k-clique, was used to discern more developed hydrophobic interaction clusters in the protein structure. The favorable clustering conformations of hydrophobic residues, which seemed to be important for protein thermostability, were discovered by the application of a network analysis to hydrophobic interactions of GH11 xylanases. Coordinating higher k-clique hydrophobic interaction clusters through the site-directed mutagenesis of the model enzyme, Bacillus circulans xylanase, stabilized the local structure and thus improved thermostability, such that the enzyme half-life and melting temperature increased by 78 fold and 8.8 °C, respectively. This study highlights the advantages of interpreting collective hydrophobic interaction patterns and their structural hierarchy and the possibility of applying network analysis to the thermostabilization of enzymes.

Biosynthesis of poly(2-hydroxybutyrate-co-lactate) in metabolically engineered Escherichia coli

We have previously reported in vivo biosynthesis of polyhydroxyalkanoates containing 2-hydroxyacid monomers such as lactate and 2-hydroxybutyrate in recombinant Escherichia coli strains by the expression of evolved Clostridium propionicum propionyl-CoA transferase (PctCp) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1Ps6-19). Here, we report the biosynthesis of poly(2-hydroxybutyrate-co-lactate)[P(2HB-co-LA)] by direct fermentation of metabolically engineered E. coli strain. Among E. coli strains WL3110, XL1-Blue, and BL21(DE3), recombinant E. coli XL1-Blue strain expressing PhaC1437 and Pct540 produced P(76.4mol%2HB-co-23.6mol%LA) to the highest content of 88 wt% when it was cultured in a chemically defined medium containing 20 g/L of glucose and 2 g/L of sodium 2-hydroxybutyrate. When recombinant E. coli XL1-Blue strain expressing PhaC1437 and Pct540 was cultured in a chemically defined medium containing 20 g/L of glucose and varying concentration of sodium 2-hydroxybutyrate, 2HB monomer fraction in P(2HB-co-LA) increased proportional to the concentration of sodium 2-hydroxybutyrate added to the culture medium. P(2HB-co-LA)] could also be produced from glucose as a sole carbon source without sodium 2-hydroxybutyrate into the culture medium. Recombinant E. coli XL1-Blue strain expressing the phaC1437, pct540, cimA3.7, and leuBCD genes together with the L. lactis Il1403 panE gene, successfully produced P(23.5mol%2HB-co-76.5mol%LA)] to the polymer content of 19.4 wt% when it cultured in a chemically defined medium containing 20 g/L of glucose. The metabolic engineering strategy reported here should be useful for the production of novel copolymer P(2HB-co-LA)].

Shifting the optimum pH of Bacillus circulans xylanase towards acidic side by introducing arginine

Electrostatic interactions are important in protein folding, binding, flexibility, stability and function. The pH at which the enzyme is maximally active is determined by the pKas of the active site residues, which are modulated by several factors including the change in electrostatics in its vicinity. As the acidic xylanases are important in food and animal feed industries, electrostatic interactions are introduced in Bacillus circulans xylanase to shift their pH optima towards the acidic side. Arg substitutions are made to modulate the pKas of the active site residues. Neutral residues are substituted by Arg in such a way that the substituted residue can make direct interaction with the catalytic residues. However, the mutations with other titratable residues (Asp, Arg, Lys, His, Tyr, and Ser) present in between the catalytic sites and the substituted sites are avoided. Site directed mutagenesis was conducted to confirm the strategy. The results show the shift in pH optima of the mutants towards the acidic side by 0.5–1.5 unit. Molecular dynamics simulation of the mutant V37R reveals that the decrease in activity is due to the increase in distance between the substrate oxygen atoms and catalytic glutamates.