Discovery of long-chain polyamines embedded in the biosilica on the Bacillus cereus spore coat.

Biosilicification is the process by which organisms incorporate soluble, monomeric silicic acid, Si(OH)4, in the form of polymerized insoluble silica, SiO2. Although the mechanisms underlying eukaryotic biosilicification have been intensively investigated, prokaryotic biosilicification has only recently begun to be studied. We previously reported that biosilicification occurs in the gram-positive, spore-forming bacterium Bacillus cereus, and that silica is intracellularly deposited on the spore coat as a protective coating against acids, although the underlying mechanism is not yet fully understood. In eukaryotic biosilicifying organisms, such as diatoms and siliceous sponges, several relevant biomolecules are embedded in biogenic silica (biosilica). These biomolecules include peptides, proteins, and long-chain polyamines. In this study, we isolated organic compounds embedded in B. cereus biosilica to investigate the biomolecules involved in the prokaryotic biosilicification process and identified long-chain polyamines with a chemical structure of H2N-(CH2)4-[NH-(CH2)3]n-NH2 (n: up to 55). Our results demonstrate the common presence of long-chain polyamines in different evolutionary lineages of biosilicifying organisms, i.e., diatoms, siliceous sponges, and B. cereus, suggesting a common mechanism underlying eukaryotic and prokaryotic biosilicification.

Chromatographic purification of small extracellular vesicles using an affinity column for phospholipid membranes.

OBJECTIVES: This study aimed to investigate whether chromatography using an ExoPUA column, an affinity column for phospholipid membranes, could potentially serve as an efficient, rapid, scalable, and reproducible method for purifying small extracellular vesicles (sEVs). RESULTS: We used the ExoPUA column connected to a fast-performance liquid chromatography system. One-step chromatographic purification of sEVs from culture supernatant using the ExoPUA protocol resulted in an 82 ± 16-fold increase in purity with a yield of 38 ± 5% of sEVs. The purified sEVs contained CD9, CD63, TSG101, and miRNA (miR-21), but not the endoplasmic reticulum protein Calnexin. Transmission electron microscopy indicated that the purified sEVs were intact. The purification performance of the ExoPUA protocol showed superior results in terms of yield compared to that of the differential ultracentrifugation method, the most commonly used method for purifying sEVs in laboratories, and purity compared to that of the DEAE chromatography protocol. CONCLUSION: The sEVs were effectively purified in the bind-elute mode and the ExoPUA column can be refreshed and sterilized with sodium hydroxide (NaOH), having high potential for multiple sEV purification in a scalable and industrial manner.

A novel salt- and organic solvent-tolerant phosphite dehydrogenase from Cyanothece sp. ATCC 51142.

Phosphite dehydrogenase (PtxD) is a promising enzyme for NAD(P)H regeneration. To expand the usability of PtxD, we cloned, expressed, and analyzed PtxD from the marine cyanobacterium Cyanothece sp. ATCC 51142 (Ct-PtxD). Ct-PtxD exhibited maximum activity at pH 9.0°C and 50°C and high stability over a wide pH range of 6.0-10.0. Compared to previously reported PtxDs, Ct-PtxD showed increased resistance to salt ions such as Na+, K+, and NH4 +. It also exhibited high tolerance to organic solvents such as ethanol, dimethylformamide, and methanol when bound to its preferred cofactor, NAD+. Remarkably, these organic solvents enhanced the Ct-PtxD activity while inhibiting the PtxD activity of Ralstonia sp. 4506 (Rs-PtxD) at concentrations ranging from 10% to 30%. Molecular electrostatic potential analysis showed that the NAD+-binding site of Ct-PtxD was rich in positively charged residues, which may attract the negatively charged pyrophosphate group of NAD+ under high-salt conditions. Amino acid composition analysis revealed that Ct-PtxD contained fewer hydrophobic amino acids than other PtxD enzymes, which reduced the hydrophobicity and increased the hydration of protein surface under low water activity. We also demonstrated that the NADH regeneration system using Ct-PtxD is useful for the coupled chiral conversion of trimethylpyruvic acid into L-tert-leucine using leucine dehydrogenase under high ammonium conditions, which is less supported by the Rs-PtxD enzyme. These results imply that Ct-PtxD might be a potential candidate for NAD(P)H regeneration in industrial applications under the reaction conditions containing salt and organic solvent.

Gatekeeper Residue Replacement in a Phosphite Transporter Enhances Mutational Robustness of the Biocontainment Strategy.

Biocontainment is a key methodology to reduce environmental risk through the deliberate release of genetically modified microorganisms. Previously, we developed a phosphite (HPO32-)-dependent biocontainment strategy, by expressing a phosphite-specific transporter HtxBCDE and phosphite dehydrogenase in bacteria devoid of their indigenous phosphate (HPO42-) transporters. This strategy did not allow Escherichia coli to generate escape mutants (EMs) in growth media containing phosphate as a phosphorus source using an assay with a detection limit of 1.9 × 10-13. In this study, we found that the coexistence of a high dose of phosphate (>0.5 mM) with phosphite in the growth medium allows the phosphite-dependent E. coli strain to generate EMs at a frequency of approximately 5.4 × 10-10. In all EMs, the mutation was a single amino acid substitution of phenylalanine to cysteine or serine at position 210 of HtxC, the transmembrane domain protein of the phosphorus compound transporter HtxBCDE. Replacement of the HtxC F210 residue with the other 17 amino acids revealed that HtxC F210 is crucial in determining substrate specificity of HtxBCDE. Based on the finding of the role of HtxC F210 as a "gatekeeper" residue for this transporter, we demonstrate that the replacement of HtxC F210 with amino acids resulting from codons that require two simultaneous point mutations to generate phosphate permissive HtxC mutants can reduce the rate of EM generation to an undetectable level. These findings also provide novel insights into the functional classification of HtxBCDE as a noncanonical ATP-binding cassette transporter in which the transmembrane domain protein participates in substrate recognition.

Engineering Cofactor Specificity of a Thermostable Phosphite Dehydrogenase for a Highly Efficient and Robust NADPH Regeneration System.

Nicotinamide adenine dinucleotide phosphate (NADP)-dependent dehydrogenases catalyze a range of chemical reactions useful for practical applications. However, their dependence on the costly cofactor, NAD(P)H remains a challenge which must be addressed. Here, we engineered a thermotolerant phosphite dehydrogenase from Ralstonia sp. 4506 (RsPtxD) by relaxing the cofactor specificity for a highly efficient and robust NADPH regeneration system. The five amino acid residues, Cys174-Pro178, located at the C-terminus of ß7-strand region in the Rossmann-fold domain of RsPtxD, were changed by site-directed mutagenesis, resulting in four mutants with a significantly increased preference for NADP. The catalytic efficiency of mutant RsPtxDHARRA for NADP (K cat/K M)NADP was 44.1 µM-1 min-1, which was the highest among the previously reported phosphite dehydrogenases. Moreover, the RsPtxDHARRA mutant exhibited high thermostability at 45°C for up to 6 h and high tolerance to organic solvents, when bound with NADP. We also demonstrated the applicability of RsPtxDHARRA as an NADPH regeneration system in the coupled reaction of chiral conversion of 3-dehydroshikimate to shikimic acid by the thermophilic shikimate dehydrogenase of Thermus thermophilus HB8 at 45°C, which could not be supported by the parent RsPtxD enzyme. Therefore, the RsPtxDHARRA mutant might be a promising alternative NADPH regeneration system for practical applications.

Photo-triggered cargo release from liposome chlorin e6-bearing pullulan hybrid nanoparticles via membrane permeabilization.

A liposome chlorin e6-bearing pullulan nanogel hybrid was prepared as a light-triggered payload release platform. The current system enabled manipulation of the release profile of model drugs encapsulated by liposomes. Gelatin hydrogels that comprised hybrid nanoparticles could successfully control the delivery of cargo molecules to human mesenchymal stem cells with light stimuli without injury to the cells.

Application of peptides with an affinity for phospholipid membranes during the automated purification of extracellular vesicles.

Extracellular vesicles (EVs), such as exosomes, have garnered increasing interest because of their potential clinical applications that range from diagnostics to therapeutics. The development of an automated and reproducible EV purification platform would therefore aid the introduction of EV biomarkers and therapies into the clinic. Here, we demonstrate that K8- as well as K-16 peptides (containing 8 and 16 lysine residues with dissociation constants of 102 nM and 11.6 nM for phosphatidylserine, respectively) immobilized on magnetic beads can capture small EVs (< 0.2 µm) from culture supernatants of MCF7 human breast cancer cells. Importantly, the bound EVs could be dissociated from the beads under mild conditions (e.g. 0.5 M NaCl), and the isolated EVs had the typical shapes of EVs under SEM and TEM with a mean particle size of 99 nm. Using the peptide-immobilized beads, we adapted a pre-existing bench top instrument for magnetic separation to perform automated EV purification with higher purity and yield than that obtained using the standard ultracentrifugation method.

Arginine-mediated dissociation of single cells and cell sheets from a polystyrene culture dish.

Here, we report a novel non-enzymatic cell dissociation method, based on our finding that adherent cells dissociate rapidly from the polystyrene culture dish when incubated in an l- or d-arginine-containing solution. We also demonstrate the successful detachment of confluent NIH/3T3 cell monolayers from the culture dish as a cell sheet by the addition of an arginine solution.

Live-cell imaging of macrophage phagocytosis of asbestos fibers under fluorescence microscopy.

BACKGROUND: Frustrated phagocytosis occurs when an asbestos fiber > 10 µm in length is engulfed imperfectly by a macrophage, and it is believed to be associated with chromosomal instability. Few studies have focused on dynamic cellular imaging to assess the toxicity of hazardous inorganic materials such as asbestos. One reason for this is the relative lack of fluorescent probes available to facilitate experimental visualization of inorganic materials. We recently developed asbestos-specific fluorescent probes based on asbestos-binding proteins, and achieved efficient fluorescent labeling of asbestos. RESULTS: Live-cell imaging with fluorescent asbestos probes was successfully utilized to dynamically analyze asbestos phagocytosis. The fluorescently labeled asbestos fibers were phagocytosed by RAW 264.7 macrophages. Internalized fibers of < 5 µm in length were visualized clearly via overlaid phase contrast and fluorescence microscopy images, but they were not clearly depicted using phase contrast images alone. Approximately 60% of the cells had phagocytosed asbestos fibers after 2 h, but over 96% of cells remained alive even 24 h after the addition of asbestos fibers. Immediate cell death was only observed when an asbestos fiber was physically pulled from a cell by an external force. Notably, at 24 h after the addition of asbestos fibers an approximately 4-fold increase in the number of binucleated cells was observed. Monitoring of individual cell divisions of cells that had phagocytosed asbestos suggested that binucleated cells were formed via the inhibition of cell separation, by asbestos fibers of > 10 µm in length that were localized in the proximity of the intercellular bridge. CONCLUSIONS: Fluorescently labeled asbestos facilitated visualization of the dynamic biological processes that occur during and after the internalization of asbestos fibers, and indicated that (i) frustrated phagocytosis itself does not lead to immediate cell death unless the asbestos fiber is physically pulled from the cell by an external force, and (ii) macrophages that have phagocytosed asbestos can divide but sometimes the resulting daughter cells fuse, leading to the formation of a binucleated cell. This fusion only seemed to occur when a comparatively long asbestos fiber (> 10 µm) was shared by two daughter cells.

Insulin sensor cells for the analysis of insulin secretion responses in single living pancreatic ß cells.

Investigation of the functions of insulin-secreting cells in response to glucose in single-living cells is essential for improving our knowledge on the pathogenesis of diabetes. Therefore, it is desired to develop a new convenient method that enables the direct detection of insulin secreted from single-living cells. Here, insulin-sensor-cells expressing a protein-based insulin-detecting probe immobilized on the extracellular membrane were developed to evaluate the insulin-secretion response in single-living pancreatic ß cells. The protein-based insulin-detecting probe (NαLY) was composed of a bioluminescent protein (nano-luc), the αCT segment of the insulin receptor, L1 and CR domains of the insulin receptor, and a fluorescent protein (YPet). NαLY exhibited a bioluminescence resonance energy transfer (BRET) signal in response to insulin; thus, cells of Hepa1-6 line were genetically engineered to express NαLY on the extracellular membrane. The cells were found to act as insulin-sensor-cells, exhibiting a BRET signal in response to insulin. When the insulin-sensor-cells and pancreatic ß cells (MIN6 cell line) were cocultured and stimulated with glucose, insulin-sensor-cells nearby pancreatic ß cells showed the spike-shaped BRET signal response, whereas the insulin-sensor-cells close to one pancreatic ß cell did not exhibit such signal response. However, all the insulin-sensor-cells showed a gradual increase in BRET signals, which were presumably attributed to the increase in insulin concentrations in the culture dish, confirming the function of these insulin-sensor-cells. Therefore, we demonstrated that heterogenetic insulin secretion in single-living pancreatic ß cells could be measured directly using the insulin sensor cells.

Synthetic Phosphorus Metabolic Pathway for Biosafety and Contamination Management of Cyanobacterial Cultivation.

Recent progress in genetic engineering and synthetic biology have greatly expanded the production capabilities of cyanobacteria, but concerns regarding biosafety issues and the risk of contamination of cultures in outdoor culture conditions remain to be resolved. With this dual goal in mind, we applied the recently established biological containment strategy based on phosphite (H3PO3, Pt) dependency to the model cyanobacterium Synechococcus elongatus PCC 7942 ( Syn 7942). Pt assimilation capability was conferred on Syn 7942 by the introduction of Pt dehydrogenase (PtxD) and hypophosphite transporter (HtxBCDE) genes that allow the uptake of Pt, but not phosphate (H3PO4, Pi). We then identified and disrupted the two indigenous Pi transporters, pst (Synpcc7942_2441 to 2445) and pit (Synpcc7942_0184). The resultant strain failed to grow on any media containing various types of P compounds other than Pt. The strain did not yield any escape mutants for at least 28 days with a detection limit of 3.6 × 10-11 per colony forming unit, and rapidly lost viability in the absence of Pt. Moreover, growth competition of the Pt-dependent strain with wild-type cyanobacteria revealed that the Pt-dependent strain could dominate in cultures containing Pt as the sole P source. Because Pt is rarely available in aquatic environments this strategy can contribute to both biosafety and contamination management of genetically engineered cyanobacteria.

A Novel Biocontainment Strategy Makes Bacterial Growth and Survival Dependent on Phosphite.

There is a growing demand to develop biocontainment strategies that prevent unintended proliferation of genetically modified organisms in the open environment. We found that the hypophosphite (H3PO2, HPt) transporter HtxBCDE from Pseudomonas stutzeri WM88 was also capable of transporting phosphite (H3PO3, Pt) but not phosphate (H3PO4, Pi), suggesting the potential for engineering a Pt/HPt-dependent bacterial strain as a biocontainment strategy. We disrupted all Pi and organic Pi transporters in an Escherichia coli strain expressing HtxABCDE and a Pt dehydrogenase, leaving Pt/HPt uptake and oxidation as the only means to obtain Pi. Challenge on non-permissive growth medium revealed that no escape mutants appeared for at least 21 days with a detection limit of 1.94 × 10-13 per colony forming unit. This represents, to the best of our knowledge, the lowest escape frequency among reported strategies. Since Pt/HPt are ecologically rare and not available in amounts sufficient for the growth of the Pt/HPt-dependent bacteria, this strategy offers a reliable and practical method for biocontainment.

Differential Counting of Asbestos Using Phase Contrast and Fluorescence Microscopy.

Considering the increasing use of various asbestos substitutes, asbestos risk management in many industries may require accurate techniques for detecting and distinguishing asbestos from non-asbestos fibers. Using fluorescently labeled asbestos-binding proteins, we previously developed a novel method for detection and counting of asbestos fibers under fluorescence microscopy (FM). This method can provide speedy, on-site detection and identification of the asbestos fibers and has higher sensitivity than phase contrast microscopy (PCM). However, current asbestos exposure limits are derived from risk assessments based on epidemiological studies that were conducted using PCM fiber counts. Therefore, the sensitivity of asbestos testing should be maintained at PCM level to properly assess compliance with these limit values. Here, we developed and tested a novel application of FM as a differential counting method that complements PCM analysis and is fully compatible with the PCM-based epidemiological data. In the combined PCM-FM method, the fluorescent asbestos-binding probe is applied prior to filter clearing. The method makes it possible to easily switch between two microscopic techniques while analyzing the same fields of view: PCM is used for counting fibers, and FM for differentiating asbestos from non-asbestos fibers. Using airborne dust samples from demolition sites in Japan, we compared PCM-FM with scanning electron microscopy (SEM)-based differential counting method. Statistical analysis indicated a slight conservative bias of PCM-FM method, combined with relatively high variability across the full range of fiber concentrations in our sample set. Using correlative microscopy, we also evaluated the specificity of FM staining, which is a potential cause of variability between the two methods. The energy-dispersive X-ray analysis indicated that ~95% of fluorescently stained fibers in the demolition site samples were correctly identified as asbestos. While further research is needed to fully clarify the causes of variability between FM- and SEM-based differential counting, PCM-FM could be used for rapid and selective detection of asbestos fibers in field samples.

Application of volcanic ash particles for protein affinity purification with a minimized silica-binding tag.

We recently reported that the spore coat protein, CotB1 (171 amino acids), from Bacillus cereus mediates silica biomineralization and that the polycationic C-terminal sequence of CotB1 (14 amino acids), designated CotB1p, serves as a silica-binding tag when fused to other proteins. Here, we reduced the length of this silica-binding tag to only seven amino acids (SB7 tag: RQSSRGR) while retaining its affinity for silica. Alanine scanning mutagenesis indicated that the three arginine residues in the SB7 tag play important roles in binding to a silica surface. Monomeric l-arginine, at concentrations of 0.3-0.5 M, was found to serve as a competitive eluent to release bound SB7-tagged proteins from silica surfaces. To develop a low-cost, silica-based affinity purification procedure, we used natural volcanic ash particles with a silica content of ∼70%, rather than pure synthetic silica particles, as an adsorbent for SB7-tagged proteins. Using green fluorescent protein, mCherry, and mKate2 as model proteins, our purification method achieved 75-90% recovery with ∼90% purity. These values are comparable to or even higher than that of the commonly used His-tag affinity purification. In addition to low cost, another advantage of our method is the use of l-arginine as the eluent because its protein-stabilizing effect would help minimize alteration of the intrinsic properties of the purified proteins. Our approach paves the way for the use of naturally occurring materials as adsorbents for simple, low-cost affinity purification.

Rapid on-site detection of airborne asbestos fibers and potentially hazardous nanomaterials using fluorescence microscopy-based biosensing.

A large number of peptides with binding affinity to various inorganic materials have been identified and used as linkers, catalysts, and building blocks in nanotechnology and nanobiotechnology. However, there have been few applications of material-binding peptides in the fluorescence microscopy-based biosensing (FM method) of environmental pollutants. A notable exception is the application of the FM method for the detection of asbestos, a dangerous industrial toxin that is still widely used in many developing countries. This review details the selection and isolation of asbestos-binding proteins and peptides with sufficient specificity to distinguish asbestos from a large variety of safer fibrous materials used as asbestos substitutes. High sensitivity to nanoscale asbestos fibers (30-35 nm in diameter) invisible under conventional phase contrast microscopy can be achieved. The FM method is the basis for developing an automated system for asbestos biosensing that can be used for on-site testing with a portable fluorescence microscope. In the future, the FM method could also become a useful tool for detecting other potentially hazardous nanomaterials in the environment.

The C-Terminal Zwitterionic Sequence of CotB1 Is Essential for Biosilicification of the Bacillus cereus Spore Coat.

UNLABELLED: Silica is deposited in and around the spore coat layer of Bacillus cereus, and enhances the spore's acid resistance. Several peptides and proteins, including diatom silaffin and silacidin peptides, are involved in eukaryotic silica biomineralization (biosilicification). Homologous sequence search revealed a silacidin-like sequence in the C-terminal region of CotB1, a spore coat protein of B. cereus. The negatively charged silacidin-like sequence is followed by a positively charged arginine-rich sequence of 14 amino acids, which is remarkably similar to the silaffins. These sequences impart a zwitterionic character to the C terminus of CotB1. Interestingly, the cotB1 gene appears to form a bicistronic operon with its paralog, cotB2, the product of which, however, lacks the C-terminal zwitterionic sequence. A ΔcotB1B2 mutant strain grew as fast and formed spores at the same rate as wild-type bacteria but did not show biosilicification. Complementation analysis showed that CotB1, but neither CotB2 nor C-terminally truncated mutants of CotB1, could restore the biosilicification activity in the ΔcotB1B2 mutant, suggesting that the C-terminal zwitterionic sequence of CotB1 is essential for the process. We found that the kinetics of CotB1 expression, as well as its localization, correlated well with the time course of biosilicification and the location of the deposited silica. To our knowledge, this is the first report of a protein directly involved in prokaryotic biosilicification. IMPORTANCE: Biosilicification is the process by which organisms incorporate soluble silicate in the form of insoluble silica. Although the mechanisms underlying eukaryotic biosilicification have been intensively investigated, prokaryotic biosilicification was not studied until recently. We previously demonstrated that biosilicification occurs in Bacillus cereus and its close relatives, and that silica is deposited in and around a spore coat layer as a protective coating against acid. The present study reveals that a B. cereus spore coat protein, CotB1, which carried a C-terminal zwitterionic sequence, is essential for biosilicification. Our results provide the first insight into mechanisms required for biosilicification in prokaryotes.

Development of an automated asbestos counting software based on fluorescence microscopy.

An emerging alternative to the commonly used analytical methods for asbestos analysis is fluorescence microscopy (FM), which relies on highly specific asbestos-binding probes to distinguish asbestos from interfering non-asbestos fibers. However, all types of microscopic asbestos analysis require laborious examination of large number of fields of view and are prone to subjective errors and large variability between asbestos counts by different analysts and laboratories. A possible solution to these problems is automated counting of asbestos fibers by image analysis software, which would lower the cost and increase the reliability of asbestos testing. This study seeks to develop a fiber recognition and counting software for FM-based asbestos analysis. We discuss the main features of the developed software and the results of its testing. Software testing showed good correlation between automated and manual counts for the samples with medium and high fiber concentrations. At low fiber concentrations, the automated counts were less accurate, leading us to implement correction mode for automated counts. While the full automation of asbestos analysis would require further improvements in accuracy of fiber identification, the developed software could already assist professional asbestos analysts and record detailed fiber dimensions for the use in epidemiological research.

Application of a phosphite dehydrogenase gene as a novel dominant selection marker for yeasts.

The use of antibiotic resistance markers in the commercial application of genetically modified microorganisms is limited due to restrictions on the release of antibiotics and their resistance genes to the environment. To avoid contamination by other microorganisms, the development of a dominant selection marker with low environmental risks is still needed. Here we demonstrated a new selection system for Schizosaccharomyces pombe and Saccharomyces cerevisiae using a bacterial phosphite dehydrogenase gene (ptxD). A Sz. pombe transformant carrying ptxD under a strong promoter or on a multicopy plasmid grew on a minimal medium containing phosphite (Pt) as a sole source of phosphorus. To adapt this system to S. cerevisiae strains, codon optimization of ptxD was necessary. The codon-optimized ptxD system appeared effective in not only laboratorial but also industrial S. cerevisiae strains that are diploid or polyploid. Since Pt is a safe and inexpensive chemical, ptxD could be used as a novel dominant selection marker applicable on an industrial scale.

Affinity purification of recombinant proteins using a novel silica-binding peptide as a fusion tag.

We recently reported that silica is deposited on the coat of Bacillus cereus spores as a layer of nanometer-sized particles (Hirota et al. 2010 J Bacteriol 192: 111-116). Gene disruption analysis revealed that the spore coat protein CotB1 mediates the accumulation of silica (our unpublished results). Here, we report that B. cereus CotB1 (171 amino acids [aa]) and its C-terminal 14-aa region (corresponding to residues 158-171, designated CotB1p) show strong affinity for silica particles, with dissociation constants at pH 8.0 of 2.09 and 1.24 nM, respectively. Using CotB1 and CotB1p as silica-binding tags, we developed a silica-based affinity purification method in which silica particles are used as an adsorbent for CotB1/CotB1p fusion proteins. Small ubiquitin-like modifier (SUMO) technology was employed to release the target proteins from the adsorbed fusion proteins. SUMO-protease-mediated site-specific cleavage at the C-terminus of the fused SUMO sequence released the tagless target proteins into the liquid phase while leaving the tag region still bound to the solid phase. Using the fluorescent protein mCherry as a model, our purification method achieved 85 % recovery, with a purity of 95 % and yields of 0.60 ± 0.06 and 1.13 ± 0.13 mg per 10-mL bacterial culture for the CotB1-SUMO-mCherry and CotB1p-SUMO-mCherry fusions, respectively. CotB1p, a short 14-aa peptide, which demonstrates high affinity for silica, could be a promising fusion tag for both affinity purification and enzyme immobilization on silica supports.