Site Saturation Research Articles

Improving heterologous production of phenylpropanoids in Saccharomyces cerevisiae by tackling an unwanted side reaction of Tsc13, an endogenous double-bond reductase.

Phenylpropanoids, such as flavonoids and stilbenoids, are of great commercial interest, and their production in Saccharomyces cerevisiae is a very promising strategy. However, to achieve commercially viable production, each step of the process must be optimised. We looked at carbon loss, known to occur in the heterologous flavonoid pathway in yeast, and identified an endogenous enzyme, the enoyl reductase Tsc13, which turned out to be responsible for the accumulation of phloretic acid via reduction of p-coumaroyl-CoA. Tsc13 is an essential enzyme involved in fatty acid synthesis and cannot be deleted. Hence, two approaches were adopted in an attempt to reduce the side activity without disrupting the natural function: site saturation mutagenesis identified a number of amino acid changes which slightly increased flavonoid production but without reducing the formation of the side product. Conversely, the complementation of TSC13 by a plant gene homologue essentially eliminated the unwanted side reaction, while retaining the productivity of phenylpropanoids in a simulated fed batch fermentation.

Sigma Phase in Superduplex Stainless Steel: Formation, Kinetics and Microstructural Path

The superduplex stainless steels (SDSS) are widely used in chemical, oil and gas industries, to pipelines and storage material facilities. In welding process or working in temperature elevated, secondary phases may appear in the form of precipitates, as the sigma phase (σ) which is an intermetallic compound. This compound is harmful to the properties of steel, deteriorating its mechanical properties, such as decreasing corrosion resistance and toughness. In this paper it is analyzed the formation, kinetics and microstructural evolution of sigma phase in SDSS UNS S32750 after isothermal aging at 700oC, 750oC and 800oC. In this work sigma phase kinetics is studied by JMAK theory and by two microstructural path descriptors, SV, interfacial area per unit of volume between sigma phase and austenite, and , mean chord length of sigma, both in function of the VV, volumetric fraction of sigma, known in the literature as microstructural partial path (MP). The MP formulation is common in recrystallization studies, but so far has not been used in the sigma phase precipitation studies, being applied here for the first time. The results indicated that the sigma phase nucleates by site saturation with anisotropic linear impingement. This means that sigma phase nucleates on edges.

Amino acid substitutions in random mutagenesis libraries: lessons from analyzing 3000 mutations.

The quality of amino acid substitution patterns in random mutagenesis libraries is decisive for the success in directed evolution campaigns. In this manuscript, we provide a detailed analysis of the amino acid substitutions by analyzing 3000 mutations of three random mutagenesis libraries (1000 mutations each; epPCR with a low-mutation and a high-mutation frequency and SeSaM-Tv P/P) employing lipase A from Bacillus subtilis (bsla). A comparison of the obtained numbers of beneficial variants in the mentioned three random mutagenesis libraries with a site saturation mutagenesis (SSM) (covering the natural diversity at each amino acid position of BSLA) concludes the diversity analysis. Seventy-six percent of the SeSaM-Tv P/P-generated substitutions yield chemically different amino acid substitutions compared to 64% (epPCR-low) and 69% (epPCR-high). Unique substitutions from one amino acid to others are termed distinct amino acid substitutions. In the SeSaM-Tv P/P library, 35% of all theoretical distinct amino acid substitutions were found in the 1000 mutation library compared to 25% (epPCR-low) and 26% (epPCR-high). Thirty-six percent of distinct amino acid substitutions found in SeSaM-Tv P/P were unobtainable by epPCR-low. Comparison with the SSM library showed that epPCR-low covers 15%, epPCR-high 18%, and SeSaM-Tv P/P 21% of obtainable beneficial amino acid positions. In essence, this study provides first insights on the quality of epPCR and SeSaM-Tv P/P libraries in terms of amino acid substitutions, their chemical differences, and the number of obtainable beneficial amino acid positions.

Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering.

Improvements in DNA synthesis and sequencing have underpinned comprehensive assessment of gene function in bacteria and eukaryotes. Genome-wide analyses require high-throughput methods to generate mutations and analyze their phenotypes, but approaches to date have been unable to efficiently link the effects of mutations in coding regions or promoter elements in a highly parallel fashion. We report that CRISPR-Cas9 gene editing in combination with massively parallel oligomer synthesis can enable trackable editing on a genome-wide scale. Our method, CRISPR-enabled trackable genome engineering (CREATE), links each guide RNA to homologous repair cassettes that both edit loci and function as barcodes to track genotype-phenotype relationships. We apply CREATE to site saturation mutagenesis for protein engineering, reconstruction of adaptive laboratory evolution experiments, and identification of stress tolerance and antibiotic resistance genes in bacteria. We provide preliminary evidence that CREATE will work in yeast. We also provide a webtool to design multiplex CREATE libraries.

Inversion of Extender Unit Selectivity in the Erythromycin Polyketide Synthase by Acyltransferase Domain Engineering.

Acyltransferase (AT) domains of polyketide synthases (PKSs) select extender units for incorporation into polyketides and dictate large portions of the structures of clinically relevant natural products. Accordingly, there is significant interest in engineering the substrate specificity of PKS ATs in order to site-selectively manipulate polyketide structure. However, previous attempts to engineer ATs have yielded mutant PKSs with relaxed extender unit specificity, rather than an inversion of selectivity from one substrate to another. Here, by directly screening the extender unit selectivity of mutants from active site saturation libraries of an AT from the prototypical PKS, 6-deoxyerythronolide B synthase, a set of single amino acid substitutions was discovered that dramatically impact the selectivity of the PKS with only modest reductions of product yields. One particular substitution (Tyr189Arg) inverted the selectivity of the wild-type PKS from its natural substrate toward a non-natural alkynyl-modified extender unit while maintaining more than twice the activity of the wild-type PKS with its natural substrate. The strategy and mutations described herein form a platform for combinatorial biosynthesis of site-selectively modified polyketide analogues that are modified with non-natural and non-native chemical functionality.

Increasing the Thermostable Sugar-1-Phosphate Nucleotidylyltransferase Activities of the Archaeal ST0452 Protein through Site Saturation Mutagenesis of the 97th Amino Acid Position.

It is typically difficult to increase enzymatic activity by introducing substitutions into a natural enzyme. However, it was previously found that the ST0452 protein, a thermostable enzyme from the thermophilic archaeon Sulfolobus tokodaii, exhibited increased activity following single amino acid substitutions of Ala. In this study, ST0452 proteins exhibiting a further increase in activity were created using a site saturation mutagenesis strategy at the 97th position. Kinetic analyses showed that the increased activities of the mutant proteins were principally due to increased apparent kcat values. These mutant proteins might suggest clues regarding the mechanism underlying the reaction process and provide very important information for the design of synthetic improved enzymes, and they can be used as powerful biocatalysts for the production of sugar nucleotide molecules. Moreover, this work generated useful proteins for three-dimensional structural analysis clarifying the processes underlying the regulation and mechanism of enzymatic activity.

Improvement of alkalophilicity of an alkaline xylanase Xyn11A-LC from Bacillus sp. SN5 by random mutation and Glu135 saturation mutagenesis.

BackgroundFamily 11 alkaline xylanases have great potential economic applications in the pulp and paper industry. In this study, we would improve the alkalophilicity of family 11 alkaline xylanase Xyn11A-LC from Bacillus sp. SN5, for the better application in this field.ResultsA random mutation library of Xyn11A-LC with about 10,000 clones was constructed by error-prone PCR. One mutant, M52-C10 (V116A and E135V), with improved alkalophilicity was obtained from the library. Site-directed mutation showed that the mutation E135V was responsible for the alkalophilicity of the mutant. The variant E135V shifted the optimum pH of the wild-type enzyme from 7.5 to 8.0. Compared to the relative activities of the wild type enzyme, those of the mutant E135V increased by 17.5, 18.9, 14.3 and 9.5 % at pH 8.5, 9.0, 9.5 and 10.0, respectively. Furthermore, Glu135 saturation mutagenesis showed that the only mutant to have better alkalophilicity than E135V was E135R. The optimal pH of the mutant E135R was 8.5, 1.0 pH units higher than that of the wild-type. In addition, compared to the wild-type enzyme, the mutations E135V and E135R increased the catalytic efficiency (k cat/K m) by 57 and 37 %, respectively. Structural analysis showed that the residue at position 135, located in the eight-residue loop on the protein surface, might improve the alkalophilicity and catalytic activity by the elimination of the negative charge and the formation of salt-bridge.ConclusionsMutants E135V and E135R with improved alkalophilicity were obtained by directed evolution and site saturation mutagenesis. The residue at position 135 in the eight-residue loop on the protein surface was found to play an important role in the pH activity profile of family 11 xylanases. This study provided alkalophilic mutants for application in bleaching process, and it was also helpful to understand the alkaline adaptation mechanism of family 11 xylanases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0310-9) contains supplementary material, which is available to authorized users.

Effects of Surface Epitope Coverage on the Sensitivity of Displacement Assays that Employ Modified Nanoparticles: Using Bisphenol A as a Model Analyte.

With the ever-increasing use of nanoparticles in immunosensors, a fundamental study on the effect of epitope density is presented herein, with a small molecule epitope, on the performance of the displacement assay format in an enzyme-linked immunosorbent assay (ELISA). Thiolated bisphenol A (BPA) functionalized gold nanoparticles (cysBPAv-AuNPs) and specific anti-BPA antibodies are employed for this purpose. It is shown that the displacement of cysBPAv-AuNPs bound to the immobilized antibodies was influenced by both the avidity of bound cysBPAv-AuNPs and the concentration of free BPA to displace it. The importance of surface epitope density was that it changed the number of epitopes in close proximity to the antibody-binding site. This then influenced the avidity of cysBPAv-AuNPs bound to the immobilized antibody. Furthermore, the molar epitope concentration in an assay appears to affect the degree of antibody binding site saturation. Controlling surface epitope density of the functionalized nanoparticles and molar epitope concentration in an assay leads to a decrease of the concentration of free BPA required to displace the bound cysBPAv-AuNP, and hence better assay performance with regards to the D50 value and dynamic range in the displacement assay.

Abstract 2071: SW43-DOX - a potential new targeted drug for the treatment of liver malignancies - An in vitro evaluation of receptor affinity and tumor internalization

Abstract Purpose: The σ2-receptor is known to be overexpressed on many rapidly growing cancer cells, thus represents a tumor biomarker and is potentially attractive for targeted tumor therapy. We have synthesized the conjugate SW43-DOX consisting of the σ2-receptor agonist SW43 and the anticancer agent Doxorubicin (DOX). The purpose of this study was to compare the half maximal effective concentration (EC50) of SW43-DOX and DOX and to evaluate the receptor affinity and internalization of SW43-DOX in hepatocellular carcinoma and other cancer cell lines in vitro. Material & Methods: SW43 was synthesized and conjugated with Doxorubicin (A Chemtek Inc.) For saturation binding & internalization assays, the chelator L-NETA was attached & loaded with Lu177. Hep G2, Hep 3B, Panc-1 & HT-29 cell lines were evaluated. EC50 was assessed by treatment with various concentrations ranging from 0.5-700 μM of SW43-DOX/Doxorubicin (CellTiter-Glo®; Promega). Saturation binding assay: σ2-receptor blocking was achieved with 10-100 μM SW43 for 15 min. at RT prior to adding 1-300 μM SW43-DOX-Lu177 in HBSS buffer (+0.1% BSA) incubating for 3.5h on ice. Internalization assay: After receptor blocking (7.5 μM SW43; 15 min.), cells were incubated with 75 nM SW43-DOX-Lu177 for .5, 1 & 2h at 37°C/5% CO2. The cell surface bound fraction from the internalization assay was scavenged by incubation with HBSS (+20 mM NaOAc; pH4.0; 10 min; 37°C/5% CO2). Cells were lysed with 0.5% SDS, radioactivity measured (Cobra -counter, Packard Bell) & normalized to whole cell protein. Statistics: EC50: Dose response non-linear fitting model; Internalization: ANOVA + Bonferroni post-hoc test, Saturation binding: One site saturation non-linear regression fit model. Prism V6.0. Results: EC50 showed lower needed concentration (μM) for SW43-DOX compared to Doxorubicin: Hep G2 (12.0 (95%CI: 10.3-13.9) vs. 81.5 (95%CI: 66.7-99.5); p<0.001), Hep 3B (8.0 (95%CI: 7.2-9) vs. 17.6 (95%CI: 14.1-22); p<0.001), Panc-1 (21.59 (95%CI: 15.44-30.2) vs. 64.5 (95%CI: 50.3-82.6); p = 0.31), and HT-29 (20.7 (95%CI: 18.3-23.3) vs. 179.8 (95%CI: 65.9-593.5); p<0.001). Maximal specific cell surface binding capacity (Bmax; pmol/mg): Hep G2 (21.5; 95%CI: 14.3-28.6), Hep 3B (35.2; 95%CI: 27.9-42.5), Panc-1 (66.5; 95%CI: 51.6-78.2) & HT-29 (33.4; 95%CI: 8.43-58.5). Specific binding affinity (KD) in nM: Hep G2 (49.2; 95%CI: 2.2-96.3), Hep 3B (40.8; 95%CI: 13.4-68.1), Panc-1 (96.4; 95%CI: 51.4-141.5), HT-29 (31.3; 95%CI: -40.5-84.4). Specific internalization was evident at all time points for all cell lines (p<0.05). Specific internalization (pmol/mg ±SD) after 2h was 29.5 ±6.6 (Panc-1), 10.3 ±1.6 (HT-29), 18.2 ±2.36 (Hep G2) & 30.7 ±8.2 (Hep 3B). Conclusion: SW43-DOX exerts a higher antitumoral effect than Doxorubicin & binds specifically onto the cell surface with high affinity & subsequently specific uptake via the σ2-receptor. Citation Format: Johannes M. Ludwig, Yongkang Gai, Sun Lingyi, Dexing Zeng, Hyun S. Kim. SW43-DOX - a potential new targeted drug for the treatment of liver malignancies - An in vitro evaluation of receptor affinity and tumor internalization. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2071.

Directed evolution of adenylosuccinate synthetase from Bacillus subtilis and its application in metabolic engineering

Adenylosuccinate synthetase (EC. 6.3.4.4) encoded by purA in Bacillus subtilis, catalyzing the first step of the conversion of IMP to AMP, plays an important role in flux distribution in the purine biosynthetic pathway. In this study, we described the use of site saturation mutagenesis to obtain a desired enzyme activity of adenylosuccinate synthetase and its application in flux regulation. Based on sequence alignment and structural modeling, a library of enzyme variants was created by a semi-rational evolution strategy in position Thr238 and Pro242. Other than purA deletion, the leaky mutation purAP242N partially reduced the flux towards AMP derived from IMP and increased the riboflavin synthesis precursor GTP, while also kept the requirement of ATP synthesis for cell growth. PurAP242N was introduced into an inosine-producing strain and resulted in an approximately 4.66-fold increase in inosine production, from 0.088±0.009g/L to 0.41±0.051g/L, in minimal medium without hypoxanthine accumulation. These results underline that the directed evolution of adenylosuccinate synthetase could tailor its activities and adjust metabolic flux. This mutation may provide a promising application in purine-based product accumulation, like inosine, guanosine and folate which are directly stemming from purine pathway in B. subtilis.

Engineering of Methylation State Specific 3xMBT Domain Using ELISA Screening.

The ε-amino group of lysine residues may be mono-, di- or tri-methylated by protein lysine methyltransferases. In the past few years it has been highly considered that methylation of both histone and non-histone proteins has fundamental role in development and progression of various human diseases. Thus, the establishment of tools to study lysine methylation that will distinguish between the different states of methylation is required to elucidate their cellular functions. The 3X malignant brain tumor domain (3XMBT) repeats of the Lethal(3)malignant brain tumor-like protein 1 (L3MBTL1) have been utilized in the past as an affinity reagent for the identification of mono- and di-methylated lysine residues on individual proteins and on a proteomic scale. Here, we have utilized the 3XMBT domain to develop an enzyme-linked immunosorbent assay (ELISA) that allows the high-throughput detection of 3XMBT binding to methylated lysines. We demonstrated that this system allows the detection of methylated peptides, methylated proteins and PKMT activity on both peptides and proteins. We also optimized the assay to detect 3XMBT binding in crude E. coli lysates which facilitated the high throughput screening of 3XMBT mutant libraries. We have utilized protein engineering tools and generated a double site saturation 3XMBT library of residues 361 and 411 that were shown before to be important for binding mono and di-methylated substrates and identified variants that can exclusively recognize only di-methylated peptides. Together, our results demonstrate a powerful new approach that will contribute to deeper understanding of lysine methylation biology and that can be utilized for the engineering of domains for specific binders of other post-translational modifications.

A comparative study on the isochronal and isothermal crystallization kinetics of Co46.45Fe25.55Ta8B20 soft magnetic metallic glass with high thermal stability

The crystallization kinetics of Co46.45Fe25.55Ta8B20 soft magnetic metallic glass with high thermal stability has been systematically investigated during isochronal and isothermal annealing treatments. The kinetics parameters have been calculated using different approaches, such as Kissinger, Ozawa, Augis–Bennett, Gao and Johnson-Mehl-Avrami (JMA) for isochronal crystallization and JMA for the isothermal regime. According to the isoconversional approach, it has been shown that the nucleation of the (Co,Fe)21Ta2B6 phase during continuous heating needs a higher energy barrier compared to its growth, where the average activation energy value is smaller than the apparent one. The Avrami exponent values for isochronal crystallization at all heating rates, calculated according to the Gao model, shows a good agreement with those determined by the JMA model at the beginning of crystallization. The JMA model demonstrates that the average Avrami exponent is around 2 for isochronal crystallization, revealing a three-dimensional diffusion-controlled crystal growth with a decreasing nucleation rate. It has been shown that based on the JMA model, the site saturation occurs for crystallized fraction (α) beyond 0.3 in the isochronal condition, so that the normal grain growth (NGG) model with an exponent of 0.32 can be applied for α ≥ 0.5. The results manifest that the average Avrami exponent and the overall nucleation rate of (Co,Fe)21Ta2B6 crystals in the isothermal and isochronal conditions are comparable and the nucleation process occurs over a notably larger crystallized fraction in the isothermal condition.

Structural and Mechanistic Insights Derived from Saturation Mutagenesis of CcdB

CcdB is a 101-residue homodimeric protein, which is part of the toxin-antitoxin (CcdB-CcdA) module important in F-plasmid maintenance in E. coli. We generated a site saturation mutagenesis library of CcdB. This mutant library was subjected to phenotypic screening in a strain with and without the F-plasmid and sequenced using Illumina sequencing. From a comparison of mutational tolerance at each residue in these two strains, we were able to identify the active site residues of CcdB involved in Gyrase binding, solely from mutant phenotypes. Average mutational tolerance at each individual residue further correlated well with residue depth.It is important to understand the mechanism by which binding of antitoxin to toxin in complex with its cellular results in toxin release and target rejuvenation. While an allosteric mechanism has been proposed previously, a transient ternary complex of Gyrase:CcdB:CcdA, which is an essential component for allostery, could not be experimentally detected (De Jonge et al, 2009). We identified CcdB residues important in CcdA binding from the deep sequencing data. This information was used to design SPR experiments to probe for ternary complex formation. We were able to detect a transient ternary complex between the C-terminal residues (61-72) of CcdA and the CcdB-Gyrase complex. The present study led to further insights into the binding mechanism of CcdA to CcdB and the rejuvenation mechanism whereby CcdA extracts CcdB from its complex with Gyrase.Thus saturation mutagenesis combined with deep sequencing is a powerful tool to elucidate the structural and functional determinants of a protein.

Site Saturation Mutagenesis Applications on Candida methylica Formate Dehydrogenase.

In NADH regeneration, Candida methylica formate dehydrogenase (cmFDH) is a highly significant enzyme in pharmaceutical industry. In this work, site saturation mutagenesis (SSM) which is a combination of both rational design and directed evolution approaches is applied to alter the coenzyme specificity of NAD+-dependent cmFDH from NAD+ to NADP+ and increase its thermostability. For this aim, two separate libraries are constructed for screening a change in coenzyme specificity and an increase in thermostability. To alter the coenzyme specificity, in the coenzyme binding domain, positions at 195, 196, and 197 are subjected to two rounds of SSM and screening which enabled the identification of two double mutants D195S/Q197T and D195S/Y196L. These mutants increase the overall catalytic efficiency of NAD+ to 5.6 × 104-fold and 5 × 104-fold value, respectively. To increase the thermostability of cmFDH, the conserved residue at position 1 in the catalytic domain of cmFDH is subjected to SSM. The thermodynamic and kinetic results suggest that 8 mutations on the first residue can be tolerated. Among all mutants, M1L has the best residual activity after incubation at 60°C with 17%. These studies emphasize that SSM is an efficient method for creating “smarter libraries” for improving the properties of cmFDH.

A Structure-Based Design Protocol for Optimizing Combinatorial Protein Libraries.

Protein variant libraries created via site-directed mutagenesis are a powerful approach to engineer improved proteins for numerous applications such as altering enzyme substrate specificity. Conventional libraries commonly use a brute force approach: saturation mutagenesis via degenerate codons that encode all 20 natural amino acids. In contrast, this chapter describes a protocol for designing "smarter" degenerate codon libraries via direct combinatorial optimization in "library space." Several case studies illustrate how it is possible to design degenerate codon libraries that are highly enriched for favorable, low-energy sequences as assessed using a standard all-atom scoring function. There is much to gain for experimental protein engineering laboratories willing to think beyond site saturation mutagenesis. In the common case that the exact experimental screening budget is not fixed, it is particularly helpful to perform a Pareto analysis to inspect favorable libraries at a range of possible library sizes.

Combined and Iterative Use of Computational Design and Directed Evolution for Protein-Ligand Binding Design.

The advantages of computational design and directed evolution are complementary, and only through combined and iterative use of both approaches, a daunting task such as protein-ligand interaction design, can be achieved efficiently. Here, we describe a systematic strategy to combine structure-guided computational design, iterative site saturation mutagenesis, and yeast two-hybrid system (Y2H)-based phenotypic screening to engineer novel and orthogonal interactions between synthetic ligands and human estrogen receptor α (hERα) for the development of novel gene switches.

Exposing a β-Lactamase "Twist": the Mechanistic Basis for the High Level of Ceftazidime Resistance in the C69F Variant of the Burkholderia pseudomallei PenI β-Lactamase.

Around the world, Burkholderia spp. are emerging as pathogens highly resistant to β-lactam antibiotics, especially ceftazidime. Clinical variants of Burkholderia pseudomallei possessing the class A β-lactamase PenI with substitutions at positions C69 and P167 are known to demonstrate ceftazidime resistance. However, the biochemical basis for ceftazidime resistance in class A β-lactamases in B. pseudomallei is largely undefined. Here, we performed site saturation mutagenesis of the C69 position and investigated the kinetic properties of the C69F variant of PenI from B. pseudomallei that results in a high level of ceftazidime resistance (2 to 64 mg/liter) when expressed in Escherichia coli. Surprisingly, quantitative immunoblotting showed that the steady-state protein levels of the C69F variant β-lactamase were ∼4-fold lower than those of wild-type PenI (0.76 fg of protein/cell versus 4.1 fg of protein/cell, respectively). However, growth in the presence of ceftazidime increases the relative amount of the C69F variant to greater than wild-type PenI levels. The C69F variant exhibits a branched kinetic mechanism for ceftazidime hydrolysis, suggesting there are two different conformations of the enzyme. When incubated with an anti-PenI antibody, one conformation of the C69F variant rapidly hydrolyzes ceftazidime and most likely contributes to the higher levels of ceftazidime resistance observed in cell-based assays. Molecular dynamics simulations suggest that the electrostatic characteristics of the oxyanion hole are altered in the C69F variant. When ceftazidime was positioned in the active site, the C69F variant is predicted to form a greater number of hydrogen-bonding interactions than PenI with ceftazidime. In conclusion, we propose "a new twist" for enhanced ceftazidime resistance mediated by the C69F variant of the PenI β-lactamase based on conformational changes in the C69F variant. Our findings explain the biochemical basis of ceftazidime resistance in B. pseudomallei, a pathogen of considerable importance, and suggest that the full repertoire of conformational states of a β-lactamase profoundly affects β-lactam resistance.

Phase transformation mechanism of low-carbon high strength low alloy steel upon continuous cooling

The transformation characteristics of low-carbon high strength low alloy steel for various cooling rates were systematically investigated by means of dilatometric measurements and microstructure observations. According to the results, it is recognised that the increase of the cooling rate could lead to microstructure evolution from a mixture of polygonal ferrite, acicular ferrite and bainite ferrite to the dual phase of acicular ferrite and bainite ferrite. The kinetics mechanism of the phase transformation was further studied by a modified analytical phase transformation model, which involves site saturation, diffusion/interface-controlled growth, impingement correction for randomly distributed growing particles. It is demonstrated that diffusion-controlled polygonal ferrite and acicular ferrite phase transformation precedes the interface-controlled bainite ferrite phase transformation. For the diffusion-controlled growth, the transformation is slowed down with the increase of the cooling rate, which prevents the diffusion process to some degree and increases the diffusion activation energy QD. For the interface-controlled growth, the interface migration activation energy shows a declining trend with the increase of cooling rate, thus promoting the transformation.

Modeling particulate matter concentrations measured through mobile monitoring in a deletion/substitution/addition approach

Abstract Land use regression modeling (LUR) through local scale circular modeling domains has been used to predict traffic-related air pollution such as nitrogen oxides (NOX). LUR modeling for fine particulate matters (PM), which generally have smaller spatial gradients than NOX, has been typically applied for studies involving multiple study regions. To increase the spatial coverage for fine PM and key constituent concentrations, we designed a mobile monitoring network in Monroe County, New York to measure pollutant concentrations of black carbon (BC, wavelength at 880 nm), ultraviolet black carbon (UVBC, wavelength at 3700 nm) and Delta-C (the difference between the UVBC and BC concentrations) using the Clarkson University Mobile Air Pollution Monitoring Laboratory (MAPL). A Deletion/Substitution/Addition (D/S/A) algorithm was conducted, which used circular buffers as a basis for statistics. The algorithm maximizes the prediction accuracy for locations without measurements using the V-fold cross-validation technique, and it reduces overfitting compared to other approaches. We found that the D/S/A LUR modeling approach could achieve good results, with prediction powers of 60%, 63%, and 61%, respectively, for BC, UVBC, and Delta-C. The advantage of mobile monitoring is that it can monitor pollutant concentrations at hundreds of spatial points in a region, rather than the typical less than 100 points from a fixed site saturation monitoring network. This research indicates that a mobile saturation sampling network, when combined with proper modeling techniques, can uncover small area variations (e.g., 10 m) in particulate matter concentrations.

Protein engineering of a nitrilase from Burkholderia cenocepacia J2315 for efficient and enantioselective production of (R)-o-chloromandelic acid.

The nitrilase-mediated pathway has significant advantages in the production of optically pure aromatic α-hydroxy carboxylic acids. However, low enantioselectivity and activity are observed on hydrolyzing o-chloromandelonitrile to produce optically pure (R)-o-chloromandelic acid. In the present study, a protein engineering approach was successfully used to enhance the performance of nitrilase obtained from Burkholderia cenocepacia strain J2315 (BCJ2315) in hydrolyzing o-chloromandelonitrile. Four hot spots (T49, I113, Y199, and T310) responsible for the enantioselectivity and activity of BCJ2315 were identified by random mutagenesis. An effective double mutant (I113M/Y199G [encoding the replacement of I with M at position 113 and Y with G at position 199]), which demonstrated remarkably enhanced enantioselectivity (99.1% enantiomeric excess [ee] compared to 89.2% ee for the wild type) and relative activity (360% of the wild type), was created by two rounds of site saturation mutagenesis, first at each of the four hot spots and subsequently at position 199 for combination with the selected beneficial mutation I113M. Notably, this mutant also demonstrated dramatically enhanced enantioselectivity and activity toward other mandelonitrile derivatives and, thus, broadened the substrate scope of this nitrilase. Using an ethyl acetate-water (1:9) biphasic system, o-chloromandelonitrile (500 mM) was completely hydrolyzed in 3 h by this mutant with a small amount of biocatalyst (10 g/liter wet cells), resulting in a high concentration of (R)-o-chloromandelic acid with 98.7% ee, to our knowledge the highest ever reported. This result highlights a promising method for industrial production of optically pure (R)-o-chloromandelic acid. Insight into the source of enantioselectivity and activity was gained by homology modeling and molecular docking experiments.