Site Saturation Research Articles

Copper-saturated chitosan beads (CuSCBs) for Enhanced phosphate removal

This study investigates the sequential use of non-cross-linked chitosan beads (CBs) for copper and phosphate adsorption, emphasizing copper site saturation and selective phosphate binding. Initially, CBs were loaded with copper until saturation was achieved, where all available sites were occupied by copper at an initial concentration of 100 mg/L. These copper-saturated chitosan beads (CuSCBs) were subsequently applied for phosphate adsorption, achieving a maximum adsorption capacity of 85.89 mg/g at pH 5. X-ray Photoelectron Spectroscopy (XPS) analysis revealed a shift in the Cu2O binding energy to lower values upon phosphate adsorption, indicating that phosphate ions interact primarily with the copper ions intercalated on the CBs rather than the chitosan matrix. Additionally, the nitrogen spectra showed no shift, confirming that the amine groups of chitosan do not participate in phosphate binding. The findings suggest a 1:1 interaction ratio between copper and phosphate ions, with each phosphate ion binding to one copper ion, creating a stable and selective adsorption site. The adsorption data fit the Langmuir isotherm and pseudo-second-order kinetic models, suggesting a homogeneous monolayer adsorption mechanism driven by chemisorption, where each copper site on the chitosan surface selectively binds one phosphate ion. Furthermore, the CuSCBs demonstrated minimal copper leaching and retained 90 % of their phosphate adsorption capacity over multiple adsorption–desorption cycles, highlighting their potential as effective and reusable adsorbents for water treatment applications targeting copper and phosphate removal.

Development of over 30-years of high spatiotemporal resolution air pollution models and surfaces for California

California’s diverse geography and meteorological conditions necessitate models capturing fine-grained patterns of air pollution distribution. This study presents the development of high-resolution (100 m) daily land use regression (LUR) models spanning 1989–2021 for nitrogen dioxide (NO2), fine particulate matter (PM2.5), and ozone (O3) across California. These machine learning LUR algorithms integrated comprehensive data sources, including traffic, land use, land cover, meteorological conditions, vegetation dynamics, and satellite data. The modeling process incorporated historical air quality observations utilizing continuous regulatory, fixed site saturation, and Google Streetcar mobile monitoring data. The model performance (adjusted R2) for NO2, PM2.5, and O3 was 84 %, 65 %, and 92 %, respectively.Over the years, NO2 concentrations showed a consistent decline, attributed to regulatory efforts and reduced human activities on weekends. Traffic density and weather conditions significantly influenced NO2 levels. PM2.5 concentrations also decreased over time, influenced by aerosol optical depth (AOD), traffic density, weather, and land use patterns, such as developed open spaces and vegetation. Industrial activities and residential areas contributed to higher PM2.5 concentrations. O3 concentrations exhibited no significant annual trend, with higher levels observed on weekends and lower levels associated with traffic density due to the scavenger effect. Weather conditions and land use, such as commercial areas and water bodies, influenced O3 concentrations.To extend the prediction of daily NO2, PM2.5, and O3 to 1989, models were developed for predictors such as daily road traffic, normalized difference vegetation index (NDVI), Ozone Monitoring Instrument (OMI)–NO2, monthly AOD, and OMI-O3. These models enabled effective estimation for any period with known daily weather conditions.Longitudinal analysis revealed a consistent NO2 decline, regulatory-driven PM2.5 decreases countered by wildfire impacts, and spatially variable O3 concentrations with no long-term trend. This study enhances understanding of air pollution trends, aiding in identifying lifetime exposure for statewide populations and supporting informed policy decisions and environmental justice advocacy.

Biocatalytic Synthesis of Ruxolitinib Intermediate via Engineered Imine Reductase.

An enzyme catalyzed strategy for the synthesis of a chiral hydrazine from 3-cyclopentyl-3-oxopropanenitrile 5 and hydrazine hydrate 2 is presented. An imine reductase (IRED) from Streptosporangium roseum was identified to catalyze the reaction between 3-cyclopentyl-3-oxopropanenitrile 5 and hydrazine hydrate 2 to produce trace amounts of (R)-3-cyclopentyl-3-hydrazineylpropanenitrile 4. We employed a 2-fold approach to optimize the catalytic performance of this enzyme. First, a transition state analogue (TSA) model was constructed to illuminate the enzyme-substrate interactions. Subsequently, the Enzyme_design and Funclib methods were utilized to predict mutants for experimental evaluation. Through three rounds of site-directed mutagenesis, site saturation mutagenesis, and combinatorial mutagenesis, we obtained mutant M6 with a yield of 98% and an enantiomeric excess (ee) of 99%. This study presents an effective method for constructing a hydrazine derivative via IRED-catalyzed reductive amination of ketone and hydrazine. Furthermore, it provides a general approach for constructing suitable enzymes, starting from nonreactive enzymes and gradually enhancing their catalytic activity through active site modifications.

Cotransport of Thallium(I) and kaolinite colloids in quartz sand media containing sodium humate: Ionic strength, pH and kaolinite colloid concentration

In real soil environments, humus, colloids and other components significantly affect pollutant migration behavior. Investigating Tl(I) and kaolinite colloids’ cotransport in quartz sand media containing sodium humate (HA-Na) is vital for comprehending Tl(I) migration underground. This study examined the migration of Tl(I) and kaolinite colloids across varying pH levels (5, 7), ionic strengths (ISs) (1, 5, 50 mmol/L), and kaolinite colloid concentrations. Results indicate that lower IS and pH promote Tl(I) migration when transported alone. In cotransport system, kaolinite promotes Tl(I) migration under acidic conditions but inhibits it under neutral conditions, except at high kaolinite concentrations, where the effect shifts from inhibition to promotion. This is primarily due to changes in the zeta potential of quartz sand, HA-Na, and kaolinite, as well as Tl(I) adsorption after HA-Na and kaolinite occupy binding sites. Competitive adsorption between cations and Tl(I) also plays a significant role. Conversely, in individual system, higher IS and pH inhibit kaolinite migration, while increased kaolinite concentration promotes it. In cotransport system, Tl(I) promotes kaolinite migration under acidic conditions but inhibits it under neutral conditions, except at low kaolinite concentrations. This relates to changes in the zeta potential between kaolinite and the medium, as well as the retention of HA-Na in the column and its adsorption onto kaolinite. Competitive adsorption and binding site saturation also have an impact. This study enhances understanding of Tl(I) migration by revealing the dual effect of kaolinite colloids under different environmental conditions, contributing to better knowledge of Tl(I) fate and transport in natural environments.

Enhancing the amination activity of meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum by modifying the crucial residue His154 for deamination

During the deamination and amination processes of meso-diaminopimelate dehydrogenase (meso-DAPDH) from Symbiobacterium thermophilum (StDAPDH), residue R71 was observed to display distinct functions. H154 has been proposed as a basic residue that facilitates water molecules to attack the D-chiral carbon of meso-DAP during deamination. Inspired by the phenomenon of R71, the effects of H154 during deamination and amination were investigated in this study with the goal of enhancing the amination activities of StDAPDH. Single site saturation mutagenesis indicated that almost all of the H154 mutants completely lost their deamination activity towards meso-DAP. However, some H154 variants showed enhanced kcat/Km values towards pyruvic acid and other bulky 2-keto acids, such as 2-oxovaleric acid, 4-methyl-2-oxopentanoic acid, 2-ketobutyric acid, and 3-methyl-2-oxobutanoic acid. When combined with the previously reported W121L/H227I mutant, triple mutants with significantly improved kcat/Km values (2.4-, 2.5-, 2.5-, and 4.0-fold) towards these 2-keto acids were obtained. Despite previous attempts, mutations at the H154 site did not yield the desired results. Moreover, this study not only recognizes the distinctive impact of H154 on both the deamination and amination reactions, but also provides guidance for further high-throughput screening in protein engineering and understanding the catalytic mechanism of StDAPDH.

Directed Evolution of Acoustic Reporter Genes Using High-Throughput Acoustic Screening.

A major challenge in the fields of biological imaging and synthetic biology is noninvasively visualizing the functions of natural and engineered cells inside opaque samples such as living animals. One promising technology that addresses this limitation is ultrasound (US), with its penetration depth of several cm and spatial resolution on the order of 100 μm. Within the past decade, reporter genes for US have been introduced and engineered to link cellular functions to US signals via heterologous expression in commensal bacteria and mammalian cells. These acoustic reporter genes (ARGs) represent a novel class of genetically encoded US contrast agent, and are based on air-filled protein nanostructures called gas vesicles (GVs). Just as the discovery of fluorescent proteins was followed by the improvement and diversification of their optical properties through directed evolution, here we describe the evolution of GVs as acoustic reporters. To accomplish this task, we establish high-throughput, semiautomated acoustic screening of ARGs in bacterial cultures and use it to screen mutant libraries for variants with increased nonlinear US scattering. Starting with scanning site saturation libraries for two homologues of the primary GV structural protein, GvpA/B, two rounds of evolution resulted in GV variants with 5- and 14-fold stronger acoustic signals than the parent proteins. We anticipate that this and similar approaches will help high-throughput protein engineering play as large a role in the development of acoustic biomolecules as it has for their fluorescent counterparts.

Micro-Inclusion Engineering via Sc Incompatibility for Luminescence and Photoconversion Control in Ce3+-Doped Tb3Al5-xScxO12 Garnet.

Aluminum garnets display exceptional adaptability in incorporating mismatching elements, thereby facilitating the synthesis of novel materials with tailored properties. This study explored Ce3+-doped Tb3Al5-xScxO12 crystals (where x ranges from 0.5 to 3.0), revealing a novel approach to control luminescence and photoconversion through atomic size mismatch engineering. Raman spectroscopy confirmed the coexistence of garnet and perovskite phases, with Sc substitution significantly influencing the garnet lattice and induced A1g mode softening up to Sc concentration x = 2.0. The Sc atoms controlled sub-eutectic inclusion formation, creating efficient light scattering centers and unveiling a compositional threshold for octahedral site saturation. This modulation enabled the control of energy transfer dynamics between Ce3+ and Tb3+ ions, enhancing luminescence and mitigating quenching. The Sc admixing process regulated luminous efficacy (LE), color rendering index (CRI), and correlated color temperature (CCT), with adjustments in CRI from 68 to 84 and CCT from 3545 K to 12,958 K. The Ce3+-doped Tb3Al5-xScxO12 crystal (where x = 2.0) achieved the highest LE of 114.6 lm/W and emitted light at a CCT of 4942 K, similar to daylight white. This approach enables the design and development of functional materials with tailored optical properties applicable to lighting technology, persistent phosphors, scintillators, and storage phosphors.

Improving the inhibitory resistance of xylanase FgXyn11C from Fusarium graminearum to SyXIP-I by site-directed mutagenesis

The aim of this study was to improve the inhibitory resistance of xylanase FgXyn11C from Fusarium graminearum to XIP in cereal flour. Site saturation mutagenesis was performed using computer-aided redesign. Firstly, based on multiple primary structure alignments, the amino acid residues in the active site architecture were identified, and specific residue T144 in the thumb region of FgXyn11C was selected for site-saturation mutagenesis. After screening, FgXyn11CT144F was selected as the best mutant, as it displayed the highest enzymatic activity and resistance simultaneously compared to other mutants. The specific activity of FgXyn11CT144F was 208.8 U/mg and it exhibited complete resistance to SyXIP-I. Compared with the wild-type, FgXyn11CT144F displayed similar activity and the most resistant against SyXIP-I. The optimal temperature and pH of the wild-type and purified FgXyn11CT144F were similar at pH 5.0 and 30 °C. Our findings provided preliminary insight into how the specific residue at position 144 in the thumb region of FgXyn11C influenced the enzymatic properties and interacted with SyXIP-I. The inhibition sensitivity of FgXyn11C was reduced through directed evolution, leading to creation of the mutant enzyme FgXyn11CT144F. The FgXyn11CT144F resistance to SyXIP-I has potential application and can also provide references for engineering other resistant xylanases of the GHF11.

Preparation of (S)-epichlorohydrin using a novel halohydrin dehalogenase by selective conformation adjustment.

Chiral epichlorohydrin (ECH) is an attractive intermediate for chiral pharmaceuticals and chemicals preparation. The asymmetric synthesis of chiral ECH using 1,3-dicholoro-2-propanol (1,3-DCP) catalyzed by a haloalcohol dehalogenase (HHDH) was considered as a feasible approach. However, the reverse ring opening reaction caused low optical purity of chiral ECH, thus severely restricts the industrial application of HHDHs. In the present study, a novel selective conformation adjustment strategy was developed with an engineered HheCPS to regulate the kinetic parameters of the forward and reverse reactions, based on site saturation mutation and molecular simulation analysis. The HheCPS mutant E85P was constructed with a markable change in the conformation of (S)-ECH in the substrate pocket and a slight impact on the interaction between 1,3-DCP and the enzyme, which resulted in the kinetic deceleration of the reverse reactions. Compared with HheCPS, the catalytic efficiency (kcat(S)-ECH/Km(S)-ECH) of the reversed reaction dropped to 0.23-fold (from 0.13 to 0.03mM-1s-1), while the catalytic efficiency (kcat(1,3-DCP)/Km(1,3-DCP)) of the forward reaction only reduced from 0.83 to 0.71mM-1s-1. With 40mM 1,3-DCP as substrate, HheCPS E85P catalyzed the synthesis of (S)-ECH with the yield up to 55.35% and the e.e. increased from 92.54 to >99%. Our work provided an effective approach for understanding the stereoselective catalytic mechanism as well as the green manufacturing of chiral epoxides.

Identification and structure-based engineering of a dipeptidase CpPepD from Clostridium perfringens for the synthesis of l-carnosine

l-Carnosine (l-Car), an endogenous dipeptide presents in muscle and brain tissues of various vertebrates, has a wide range of application values. The enzymatic preparation of l-Car is a promising synthetic method because it avoids the protection and deprotection steps. In the present study, a dipeptidase gene (CpPepD) from Clostridium perfringens with high l-Car synthetic activity was cloned and characterized. In an effort to improve the performance of this enzyme, we carried out site saturation mutagenesis using CpPepD as the template. By the o-phthalaldehyde (OPA)-derived high throughput screening method, mutant A171S was obtained with 2.2-fold enhanced synthetic activity. The enzymatic properties of CpPepD and mutant A171S were investigated. Under the optimized conditions, 63.94 mM (14.46 g L−1) or 67.02 mM (15.16 g L−1) l-Car was produced at the substrate concentrations of 6 M β-Ala and 0.2 M l-His using wild-type or mutant A171S enzyme, respectively. Although the mutation enhanced the enzyme activity, the reaction equilibrium was barely affected.

High-throughput visualization mutation screening technology to enhance the specificity of CadR based whole-cell cadmium biosensor

As a heavy metal pollutant, Cd2+ often enters the human body through the food chain causing great harm to human health. Whole-cell biosensor is an emerging technology for rapid on-site detection of heavy metals with the advantages of inexpensive, fast to mass-produce, and strong in anti-interference resistance, but suffering from insatisfactory specificity. In this study, a strategy of Adjacent Site Saturation Mutation (ASSM) was designed to improve the specificity of transcription factor CadR, which acted as the recognition element and determined the specificity of whole cell Cd2+ biosensors. A specific saturated library was constructed using the strategy of adjacent mutation. After two rounds of high-throughput visual screening, a whole-cell biosensor with good response to Cd2+, and with significant weakened Hg2+ interference was obtained. The optimized whole-cell biosensor showed a linear dynamic concentration range from 500 nM to 100 μM, a detection limit of 0.079 μM, and has satisfactory specificity and anti-interference. The ASSM strategy proposed in this study can provide a new method for the application of synthetic biology in food safety detection, indicating the importance of whole-cell biosensors for the detection of heavy metals.

Engineering a human P2X2 receptor with altered ligand selectivity in yeast

P2X receptors are a family of ligand gated ion channels found in a range of eukaryotic species including humans but are not naturally present in the yeast Saccharomyces cerevisiae. We demonstrate the first recombinant expression and functional gating of the P2X2 receptor in baker’s yeast. We leverage the yeast host for facile genetic screens of mutant P2X2 by performing site saturation mutagenesis at residues of interest, including SNPs implicated in deafness and at residues involved in native binding. Deep mutational analysis and rounds of genetic engineering yield mutant P2X2 F303Y A304W, which has altered ligand selectivity towards the ATP analog AMP-PNP. The F303Y A304 variant shows over 100-fold increased intracellular calcium amplitudes with AMP-PNP compared to the WT receptor and has a much lower desensitization rate. Since AMP-PNP does not naturally activate P2X receptors, the F303Y A304 P2X2 may be a starting point for downstream applications in chemogenetic cellular control. Interestingly, the A304W mutation selectively destabilizes the desensitized state, which may provide a mechanistic basis for receptor opening with suboptimal agonists. The yeast system represents an inexpensive, scalable platform for ion channel characterization and engineering by circumventing the more expensive and time-consuming methodologies involving mammalian hosts.

Engineering the activity and thermostability of a carboxylic acid reductase in the conversion of vanillic acid to vanillin

Vanillin is a valuable natural product that can be used as a fragrance and additive. Recent research in the biosynthesis of vanillin has brought attention to a key enzyme, carboxylic acid reductase (CAR), which catalyzes the reduction of vanillic acid to vanillin. Nevertheless, the biosynthesis of vanillin is hampered by the low activity and stability of CAR. As such, a rational design campaign was conducted on a well-documented carboxylic acid reductase from Segniliparus rugosus (SrCAR), using vanillic acid as the model substrate. After combined active site saturation and iterative site-specific mutagenesis, the best quadruple mutant N292H/K524S/A627L/E1121W (M3) was successfully obtained. In comparison to the wildtype SrCAR, M3 demonstrated a 4.2-fold increase in catalytic efficiency (kcat/Km), and its half-life (t1/2) was enhanced by 3.8 times up to 385.08 minutes at 40 °C. In silico docking and molecular dynamics simulation provided insights into the improved activity and stability. In the subsequent preparative-scale reaction with 100 mM (16.8 g L−1) vanillic acid, the whole cell catalysis utilizing M3 produced 10.15 g·L−1 of vanillin and 1.11 g·L−1 of vanillyl alcohol, respectively. This work demonstrates a dual improvement in the activity and thermal stability of SrCAR, thereby potentially facilitating the application of carboxylic acid reductase in the biosynthesis of vanillin.

Increase in the solubility of uvsY using a site saturation mutagenesis library for application in a lyophilized reagent for recombinase polymerase amplification.

Recombinase uvsY from bacteriophage T4, along with uvsX, is a key enzyme for recombinase polymerase amplification (RPA), which is used to amplify a target DNA sequence at a constant temperature. uvsY, though essential, poses solubility challenges, complicating the lyophilization of RPA reagents. This study aimed to enhance uvsY solubility. Our hypothesis centered on the C-terminal region of uvsY influencing solubility. To test this, we generated a site-saturation mutagenesis library for amino acid residues Lys91-Glu134 of the N-terminal (His)6-tagged uvsY. Screening 480 clones identified A116H as the variant with superior solubility. Lyophilized RPA reagents featuring the uvsY variant A116H demonstrated enhanced performance compared to those with wild-type uvsY. The uvsY variant A116H emerges as an appealing choice for RPA applications, offering improved solubility and heightened lyophilization feasibility.

Engineering A-type Dye-Decolorizing Peroxidases by Modification of a Conserved Glutamate Residue.

Dye-decolorizing peroxidases (DyPs) are recently identified microbial enzymes that have been used in several Biotechnology applications from wastewater treatment to lignin valorization. However, their properties and mechanism of action still have many open questions. Their heme-containing active site is buried by three conserved flexible loops with a putative role in modulating substrate access and enzyme catalysis. Here, we investigated the role of a conserved glutamate residue in stabilizing interactions in loop 2 of A-type DyPs. First, we did site saturation mutagenesis of this residue, replacing it with all possible amino acids in bacterial DyPs from Bacillus subtilis (BsDyP) and from Kitasatospora aureofaciens (KaDyP1), the latter being characterized here for the first time. We screened the resulting libraries of variants for activity towards ABTS and identified variants with increased catalytic efficiency. The selected variants were purified and characterized for activity and stability. We furthermore used Molecular Dynamics simulations to rationalize the increased catalytic efficiency and found that the main reason is the electron channeling becoming easier from surface-exposed tryptophans. Based on our findings, we also propose that this glutamate could work as a pH switch in the wild-type enzyme, preventing intracellular damage.

Structure-guided directed evolution of deacetylase leads to increased specificity of chloroacetamides and effective production of chloroamines

O-3-chloro-2-propenyl hydroxylamine (chloroamine) is the primary material for the production of cyclohexenone herbicides, such as clethodim, which are widely used in agricultural production due to their high efficiency, low toxicity, and good environmental safety. In this study, a bioenzymatic strategy is proposed for the synthesis of chloroamines in order to address the polluting nature of traditional chemical synthesis. Using easily synthesized chloroacetamides as the model substrate, histone deacetylase 10 (zHDAC10) from zebrafish was used as the biocatalyst for the deacetylation process, resulting in the production of chloroamides. Through a structure-directed mutagenesis and site saturation mutagenesis strategy, a zHDAC10-M2 (A24RE274K) was created, leading to approximately an 8-fold enhancement of activity in comparison to the zHDAC10-WT, with a high catalytic efficiency of 40 mM and a 24-hour conversion of over 94% on a 100-mL preparative scale. Structural analysis and molecular dynamics showed that the mutation of 24 and 274 sites to positively charged amino acids enabled them to form hydrogen bonds with the terminal Cl of chloroamines, thereby stabilizing the state of the substrate in the active pocket and thus improving the catalytic efficiency. The biosynthetic route established in this study provides a new promising way for the production of chloroamines.

Experimental modification in thermal stability of oligomers by alanine substitution and site saturation mutagenesis of interfacial residues

For certain industrial applications, the stability of protein oligomers is important. In this study, we demonstrated an efficient method to improve the thermal stability of oligomers using the trimeric protein chloramphenicol acetyltransferase (CAT) as the model. We substituted all interfacial residues of CAT with alanine to detect residues critical for oligomer stability. Mutation of six of the forty-nine interfacial residues enhanced oligomer thermal stability. Site saturation mutagenesis was performed on these six residues to optimize the side chains. About 15% of mutations enhanced thermal stability by more than 0.5 °C and most did not disrupt activity of CAT. Certain combinations of mutations further improved thermal stability and resistance against heat treatment. The quadruple mutant, H17V/N34S/F134A/D157C, retained the same activity as the wild-type after heat treatment at 9 °C higher temperature than the wild-type CAT. Furthermore, combinations with only alanine substitutions also improved thermal stability, suggesting the method we developed can be used for rapid modification of industrially important proteins.

Antibody-directed evolution reveals a mechanism for enhanced neutralization at the HIV-1 fusion peptide site

The HIV-1 fusion peptide (FP) represents a promising vaccine target, but global FP sequence diversity among circulating strains has limited anti-FP antibodies to ~60% neutralization breadth. Here we evolve the FP-targeting antibody VRC34.01 in vitro to enhance FP-neutralization using site saturation mutagenesis and yeast display. Successive rounds of directed evolution by iterative selection of antibodies for binding to resistant HIV-1 strains establish a variant, VRC34.01_mm28, as a best-in-class antibody with 10-fold enhanced potency compared to the template antibody and ~80% breadth on a cross-clade 208-strain neutralization panel. Structural analyses demonstrate that the improved paratope expands the FP binding groove to accommodate diverse FP sequences of different lengths while also recognizing the HIV-1 Env backbone. These data reveal critical antibody features for enhanced neutralization breadth and potency against the FP site of vulnerability and accelerate clinical development of broad HIV-1 FP-targeting vaccines and therapeutics.

Effect of solute concentration on microstructure evolution during static recrystallization in Mg-0.2%Ce alloy using cellular automata

A new cellular automata model has been developed to evaluate the effect of solute drag on the microstructure and texture evolution during static recrystallization of Mg and its alloys in the temperature range of 200 – 400 °C. In the present model, the site saturation nucleation was assumed and all the nuclei are present at the grain boundaries. The growth of each nucleus depends on the solute concentration and dislocation density difference between neighboring grains. The microstructural analysis of the newly developed cellular automata model showed the evolution of grain size at different temperatures compared to the equilibrium microstructure containing the equilibrium concentration of solute atoms at the grain boundaries. Further, the texture evolution showed that there is an increase in the texture strength with an increase in the annealing temperature when the solute concentration was lower than the equilibrium concentration at the boundaries, whereas the texture strength decreased with increasing the annealing temperature when the solute concentration was higher than the equilibrium solute concentration at the boundaries. The present model shows that solute drag pressure increased continuously with increasing the annealing temperature when the solute concentration was lower than the equilibrium concentration at the boundaries. However, the solute drag pressure decreased with an increase in the annealing temperature when the solute concentration was higher than the equilibrium solute concentration at the boundaries.

Surface engineering of cyclodextrin glycosyltransferase reveals structural compactness and rigidity responsible for enhanced organic solvents resistance

Cyclodextrin glycosyltransferase (CGTase) is a preferable biocatalyst for production of cyclodextrin and phenols glycosylated derivatives due to its cost-effective donors and broad range of acceptors. Organic solvents (OSs) are often beneficial for solubilizing hydrophobic substrates and product specificity, but often harmful towards CGTases. Herein, using Paenibacillus macerans CGTase (PmCGTase) as a template and DMSO as a screening solvent, semi-rational engineering was performed at 18 surface residues by a developed high throughput screening method. Using site saturation mutagenesis and iterative saturation mutagenesis, 5 OSs-resistant variants (G539I, G539V, G539I/R146F, G539V/R146A, G539I/R146F/D147N) were obtained. Compared with WT, the best variant G539I/R146F/D147N showed a 79.3 % enhanced residual activity at 30 % DMSO and a 60 % increased t1/2 value at both 40 and 50 °C. In addition, a decreased Km value and an increased kcat value led to 41.9 % higher catalytic efficiency of G539I/R146F/D147N than WT in 15 % DMSO. This variant was also applied to glycosylation of sophoricoside in 20 % DMSO, leading to 26.2 % increased conversion. Molecular dynamics demonstrates that its enhanced OSs resistance may be attributed to the strengthened structural compactness and rigidity. Our results provide guidance for engineering OSs resistance of PmCGTase, which would further improve its application potential.