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Changing the substrate/cofactor specificity of an enzyme requires multiple mutations at spatially adjacent positions around the substrate pocket. However, this is challenging when solely based on crystal structure information because enzymes undergo dynamic conformational changes during the reaction process. Herein, we proposed a method for estimating the contribution of each amino acid residue to substrate specificity by deploying a phylogenetic analysis with logistic regression. Since this method can estimate the candidate amino acids for mutation by ranking, it is readable and can be used in protein engineering. We demonstrated our concept using redox cofactor conversion of the

Co-culture is a promising way to alleviate metabolic burden by dividing the metabolic pathways into several modules and sharing the conversion processes with multiple strains. Since an intermediate is passed from the donor to the recipient via the extracellular environment, it is inevitably diluted. Therefore, enhancing the intermediate consumption rate is important for increasing target productivity. In the present study, we demonstrated the enhancement of mevalonate consumption in Escherichia coli by adaptive laboratory evolution and applied the evolved strain to isoprenol production in an E. coli (upstream: glucose to mevalonate)-E. coli (downstream: mevalonate to isoprenol) co-culture. An engineered mevalonate auxotroph strain was repeatedly sub-cultured in a synthetic medium supplemented with mevalonate, where the mevalonate concentration was decreased stepwise from 100 to 20 µM. In five parallel evolution experiments, all growth rates gradually increased, resulting in five evolved strains. Whole-genome re-sequencing and reverse engineering identified three mutations involved in enhancing mevalonate consumption. After introducing nudF gene for producing isoprenol, the isoprenol-producing parental and evolved strains were respectively co-cultured with a mevalonate-producing strain. At an inoculation ratio of 1:3 (upstream:downstream), isoprenol production using the evolved strain was 3.3 times higher than that using the parental strain.

Due to its easy availability, preparation, handling and non-toxic nature, Equisetum arvense horsetail extract was chosen as a reducing, stabilizing and functionalizing agent for Au and bi-phasic Au/ZrO₂ nanoparticle phytosynthesis-inorganic nanoparticle synthesis mediated by plant extract. We studied Au and bi-phasic Au/ZrO₂ nanoparticles in colloids by various physical-chemical and analytical methods over 5 weeks. Dynamic Light Scattering and Scanning Transmission Electron Microscopy compared core and hydrodynamic diameters of nanoparticles. ζ-potential measurement indirectly determined nanoparticles stability in liquid medium. Ultraviolet-Visible Spectroscopy characterized basic absorbance maxima for both Au and the bi-phasic Au/ZrO₂ system. Finally, total metal concentration was determined using Inductively Coupled Plasma Mass Spectrometry. ζ-potential measurements proved satisfactory stability of both Au (-13.4 to -17 mV) and Au/ZrO₂ nanoparticles (-14.1 to -17.5 mV) over the experimental period. Scanning Transmission Electron Microscopy with Selected Area Diffraction analysis confirmed nanoparticles crystalline nature, and we determined 24 nm and 40 nm core nanogold diameters in Au and Au/ZrO₂ nanoparticle colloids. Dynamic light scattering analysis confirmed the dichotomy between particle sizes in liquid medium in the hundreds of nanometers measured, and long-term measurements confirmed reasonable colloid stability-a paramount parameter for potential nanoparticles applications; especially in heterogeneous catalysis.

Our hypothesis introduced (i) Tilia sp. leachate as the basic platform for Ag-AgCl nanoparticle phytosynthesis as a new bionanotechnological protocol, (ii) determination of Ag-AgCl colloidal properties during periodic temperature changes and (iii) confirmation of formed colloid as an active and fundamental catalytic tool for degradation of organic pollutants. Easy-to-prepare Tilia sp. leachate was mixed with silver precursor to form the Ag-AgCl nanoparticle system. We used SEM and FTIR to determine Tilia matrix organic/inorganic compounds and then performed STEM, ICP-MS, UV/VIS and XRD analysis to phytosynthesize Ag-AgCl nanoparticles. We confirmed that Tilia sp. leachate contained specific biomolecules with nanoparticle synthesis potential. Colloidal Ag-AgCl nanoparticles revealed dominant spherical morphology with uniform mean diameter from 14 to 16 nm. There were no significant differences observed in ζ-potential, ionic strength, hydrodynamic dimension or pH value during 5 weeks with periodic temperature changes, thus confirming stable colloidal properties. In addition, this specialized application of Ag-AgCl nanoparticles was performed by effective 4-nitrophenol catalysis at low Ag-AgCl NP concentration and very rapid reaction kinetics.

Recent advances in biotechnology have enabled the generation of antibodies with high affinity for the surfaces of specific inorganic materials. Herein, we report the synthesis of functional materials from multiple nanomaterials by using a small bispecific antibody recombinantly constructed from gold-binding and ZnO-binding antibody fragments. The bispecific antibody-mediated spontaneous linkage of gold and ZnO nanoparticles forms a binary gold-ZnO nanoparticle composite membrane. The relatively low melting point of the gold nanoparticles and the solubility of ZnO in dilute acidic solution then allowed for the bottom-up synthesis of a nanoporous gold membrane by means of a low-energy, low-environmental-load protocol. The nanoporous gold membrane showed high catalytic activity for the reduction of p-nitrophenol to p-aminophenol by sodium borohydride. Here, we show the potential utility of nanoparticle pairing mediated by bispecific antibodies for the bottom-up construction of nanostructured materials from multiple nanomaterials.

Due to its easy availability, preparation, handling and non-toxic nature

Diabody 31, a bispecific therapeutic antibody, is a heterodimer comprised of two types of chimeric single-chain variable fragments (scFv), that has been identified as a clone with high cytotoxicity against cancer cells. Diabody 31 is inactive as a homodimer and monomer, yet active as a heterodimer in solution. Herein, we examined the association between two kinds of diabodies, LH- and HL type, based on the behavior of four kinds of their constituent chimera scFvs, by size analysis. More than half of the LH diabody fraction, including two types of chimeric scFV that were equally expressed, and nearly all HL diabody fraction components, including one chimeric HL-L scFv, were in inactive form and changed components dynamically as time passed. The association of these diabodies corresponded to that of the scFv chimera, indicating that analysis of these chimeras could predict diabody quality. Furthermore, addition of the deficient HL-R chimera scFv, comprised of an unbalanced chimeric scFv ratio, to the HL diabody fraction increased cancer cell cytotoxicity, with maximal effects obtained upon repeating the process five times. These findings provide insights into the efficient construction of a functional diabody by noting the characteristics of associated diabodies and the molar ratio of expressed chimera scFvs.

Molecular evolution based on mutagenesis is widely used in protein engineering. However, optimal proteins are often difficult to obtain due to a large sequence space. Here, we propose a novel approach that combines molecular evolution with machine learning. In this approach, we conduct two rounds of mutagenesis where an initial library of protein variants is used to train a machine-learning model to guide mutagenesis for the second-round library. This enables us to prepare a small library suited for screening experiments with high enrichment of functional proteins. We demonstrated a proof-of-concept of our approach by altering the reference green fluorescent protein (GFP) so that its fluorescence is changed into yellow. We successfully obtained a number of proteins showing yellow fluorescence, 12 of which had longer wavelengths than the reference yellow fluorescent protein (YFP). These results show the potential of our approach as a powerful method for directed evolution of fluorescent proteins.

Molecular evolution based on mutagenesis is widely used in protein engineering. However, optimal proteins are often difficult to obtain due to a large sequence space that requires high costs for screening experiments. Here, we propose a novel approach that combines molecular evolution with machine learning. In this approach, we conduct two rounds of mutagenesis where an initial library of protein variants is used to train a machine-learning model to guide mutagenesis for the second-round library. This enables to prepare a small library suited for screening experiments with high enrichment of functional proteins. We demonstrated a proof-of-concept of our approach by altering the reference green fluorescent protein (GFP) so that its fluorescence is changed to yellow while improving its fluorescence intensity. Using 155 and 78 variants for the initial and the second-round libraries, respectively, we successfully obtained a number of proteins showing yellow fluorescence, 12 of which had better fluorescence performance than the reference yellow fluorescent protein (YFP). These results show the potential of our approach as a powerful platform for accelerated discovery of functional proteins.

The cytotoxicity of T cell-recruiting antibodies with their potential to damage late-stage tumor masses is critically dependent on their structural and functional properties. Recently, we reported a semi-high-throughput process for screening highly cytotoxic small bispecific antibodies (i.e., diabodies). In the present study, we improved the high-throughput performance of this screening process by removing the protein purification stage and adding a stage for determining the concentrations of the diabodies in culture supernatant. The diabodies were constructed by using an Escherichia coli expression system, and each diabody contained tandemly arranged peptide tags at the C-terminus, which allowed the concentration of diabodies in the culture supernatant to be quantified by using a tag-sandwich enzyme-linked immunosorbent assay. When estimated diabody concentrations were used to determine the cytotoxicity of unpurified antibodies, results comparable to those of purified antibodies were obtained. In a surface plasmon resonance spectroscopy-based target-binding assay, contaminants in the culture supernatant prevented us from conducting a quantitative binding analysis; however, this approach did allow relative binding affinity to be determined, and the relative binding affinities of the unpurified diabodies were comparable to those of the purified antibodies. Thus, we present here an improved high-throughput process for the simultaneous screening and determination of the binding parameters of highly cytotoxic bispecific antibodies.

Small bispecific antibodies that induce T-cell–mediated cytotoxicity have the potential to damage late-stage tumor masses to a clinically relevant degree, but their cytotoxicity is critically dependent on their structural and functional properties. Here, we constructed an optimized procedure for identifying highly cytotoxic antibodies from a variety of the T-cell–recruiting antibodies engineered from a series of antibodies against cancer antigens of epidermal growth factor receptor family and T-cell receptors. By developing and applying a set of rapid operations for expression vector construction and protein preparation, we screened the cytotoxicity of 104 small antibodies with diabody format and identified some with 103-times higher cytotoxicity than that of previously reported active diabody. The results demonstrate that cytotoxicity is enhanced by synergistic effects between the target, epitope, binding affinity, and the order of heavy-chain and light-chain variable domains. We demonstrate the importance of screening to determine the critical rules for highly cytotoxic antibodies.

A novel strategy for the preparation of protein-decorated gold nanoparticles (Au NPs) was developed inside Escherichia coli cells, where an artificial oxidoreductase, composed of antibody-binding protein (pG), Bacillus stearothermophilus glycerol dehydrogenase (BsGLD) and a peptide tag with gold-binding affinity (H6C), was overexpressed in the cytoplasm. In situ formation of Au NPs was promoted by a natural electron-donating cofactor, nicotinamide adenine dinucleotide (NAD), which was regenerated to the reduced form of NADH by the catalytic activity of the fusion protein (pG-BsGLD-H6C) overexpressed in the cytoplasm of E. coli, with the concomitant addition of exogenous glycerol to the reaction system. The fusion protein was self-immobilized on Au NPs inside the E. coli cells, which was confirmed by SDS-PAGE and western blotting analyses of the resultant Au NPs. Finally, the IgG binding ability of the pG moiety displayed on Au NPs was evaluated by an enzyme-linked immunosorbent assay.

養殖ブリから採血された血液に対してタンパク質変性，酵素分解，限外ろ過の順に処理を施してヘモグロビンから酵素処理ヘム鉄（HIP）を調製し，濃縮させる各行程について検討した．プロテアーゼ分解の後，限外ろ過して得られた透過液と残渣について乾燥質量と鉄イオンの分布を分析するとともに，各試料に含まれるペプチド成分の分子量測定を行った．その結果，プロテアーゼによってヘモグロビンが目的通りに分解されていること，限外ろ過膜によってHIPはろ過残渣として定量的に回収されていることが示された．ろ過残渣として得られたHIPは分子量2,400–7,300程度の成分を含んでいた．分画分子量の異なる限外ろ過膜での透過実験の結果，分子構造が剛直なヘム鉄を主成分とするHIPは，分画分子量20,000の膜を用いた場合でも95%以上がろ過残渣として回収されることが示された．

廃棄物系バイオマスである卵殻膜および羽毛を吸着剤として用いた貴金属の分離について検討した．タンパク質を多く含むこれらのバイオマスは，Au(III)，Pd(II)およびPt(IV)の貴金属イオンに対して選択的な吸着能を有し，貴金属イオンの分離回収に利用可能であることが明らかになった．タンパク質高含有の卵殻膜が羽毛に比べて高い吸着能力を示し，卵殻膜についてはpH 5以下の領域でAu(III)イオンに対して高い吸着能を示すことがわかった．その吸着挙動は卵殻膜に含まれるタンパク質とAu(III)イオンの錯形成が大きく関わっており，水溶液中のAu(III)イオン種に影響されることが示唆された．

Bacterial cellulose (BC) has attracted attention for preparing advanced materials due to its microfibrous structure. In this study, quaternary ammonium bacterial cellulose (QABC) as well as quaternary ammonium plant cellulose (QAPC) as adsorbents for proteins have been prepared. Introduction of the quaternary ammonium group was conducted under different conditions to determine the best conditions for modification. The fibrous structures of QABC and QAPC were quite different and determined by the starting material. The adsorption capacities of hemoglobin and an anionic dye, thymol blue, on the adsorbents QABC and QAPC which were prepared under different conditions have been investigated. QABC showed a higher adsorption capacity for hemoglobin compared with QAPC, but a lower adsorption capacity for thymol blue.

Crystalline assemblies of fluorescent molecules have different functional properties than the constituent monomers, as well as unique optical characteristics that depend on the structure, size, and morphological homogeneity of the crystal particles. In this study, we selected peptides with affinity for the surface of perylene crystal particles by exposing a peptide-displaying phage library in aqueous solution to perylene crystals, eluting the surface-bound phages by means of acidic desorption or liquid–liquid extraction, and amplifying the obtained phages in Escherichia coli. One of the perylene-binding peptides, PeryBPb1: VQHNTKYSVVIR, selected by this biopanning procedure induced perylene molecules to form homogenous planar crystal nanoparticles by means of a poor solvent method, and fusion of the peptide to a fluorescent protein enabled one-pot formation of protein-immobilized crystalline nanoparticles. The nanoparticles were well-dispersed in aqueous solution, and Forster resonance energy transfer from the perylene crystals to the fluorescent protein was observed. Our results show that the crystal-binding peptide could be used for simultaneous control of perylene crystal morphology and dispersion and protein immobilization on the crystals.

Here, we report a new approach for the biofabrication of protein-immobilized gold nanoparticles (Au NPs), using oxidoreductase with gold-binding peptide-tagged recombinant proteins. The reduction of Au ions to Au(0) was achieved using a natural electron-donating cofactor, nicotinamide adenine dinucleotide, which was regenerated by the glycerol dehydrogenase (GLD) enzyme. First, we selected the A3 peptide (AYSSGAPPMPPF) as a gold binding moiety. The A3 peptide was introduced to the C-terminus of fusion proteins of immunoglobulin G (IgG)-binding domains of protein G and protein A. In the presence of the recombinant protein, the GLD-catalyzed cofactor reduction resulted in the efficient in situ fabrication of Au NPs immobilized with the fusion protein. Moreover, the protein-immobilized Au NPs were shown to have IgG binding activity. Although the A3 peptide had the ability to stabilize Au NPs, the results suggested that its binding affinity for Au NPs was unexpectedly weaker than that of His-tag. A cysteine residue was thus introduced to a recombinant protein adjacent to the A3 peptide. Finally, an artificial peptide, comprising A3 sequence with the C-terminal single cysteine residue, enabled the stable display of a fusion protein while maintaining its IgG binding activity through the Au-S bond. This enzyme-assisted one-pot methodology for protein-Au NPs conjugation offers one potent route for the facile fabrication of biomolecule-decorated metal NPs.

Here we report the enzymatic synthesis of gold nanoparticles (Au NPs) by an engineered Escherichia coli harboring an NADH cofactor regeneration system coupled with glycerol dehydrogenase, which can be triggered by the addition of exogenous glycerol.

A new enzymatic approach for the fabrication of gold nanoparticles (Au NPs) decorated with a functional protein was developed using a recombinant glycerol dehydrogenase from Bacillus stearothermophilus (BsGLD) that was tagged with an N-terminal protein G domain and a C-terminal (His)6Cys tag. The recombinant BsGLD promoted the synthesis of the Au NPs via the catalytic reduction of nicotinamide adenine dinucleotide using glycerol as a substrate, and the recombinant BsGLD was self-immobilized on the Au NPs as a result of the protein G's binding capability for IgG antibody.

We report a one-pot biological approach to fabricate gold nanoparticle (AuNP)-ZZ domain conjugates using peptide-functionalized proteins that can simultaneously direct both biomineralization and surface modification of AuNPs. In addition, immuno-AuNPs are readily prepared through the specific binding of antibodies to the ZZ domain on the AuNPs.