Nuttawee Niamsiri

Co-culturing of Pichia guilliermondii enhanced volatile flavor compound formation by Zygosaccharomyces rouxii in the model system of Thai soy sauce fermentation.

The roles of salt-tolerant yeasts such as Zygosaccharomyces rouxii, Candida versatilis, and Candida etchellsii in the production of volatile flavor compounds (VFCs) in soy sauce fermentation have been well documented. However, the knowledge of VFC production by other salt-tolerant yeasts is still limited. In this work, the roles of Z. rouxii and Pichia guilliermondii strains in VFC production were investigated in moromi medium as a model system for soy sauce fermentation. Inoculation of a single culture of either Z. rouxii or P. guilliermondii as well as co-cultures of these two yeasts into moromi medium showed increased numbers of viable yeast at around 0.7 to 1.9 log CFU/mL after 7days of cultivation at 30°C. During cultivation, both single and co-cultures displayed survival over a 7-day time period, compared with the controls (no culture added). Overall, yeast inoculation enhanced the production of VFCs in the moromi media with higher amounts of ethanol, alcohols, furanones, esters, aldehyde, acid, pyrone and phenols, known as important characteristic flavor compounds in soy sauce. Moreover, the co-culture produced more alcohols, furanones, esters, maltol and benzoic acid than the single culture of Z. rouxii.

Engineering of Chimeric Class II Polyhydroxyalkanoate Synthases

ABSTRACT PHA synthase is a key enzyme involved in the biosynthesis of polyhydroxyalkanoates (PHAs). Using a combinatorial genetic strategy to create unique chimeric class II PHA synthases, we have obtained a number of novel chimeras which display improved catalytic properties. To engineer the chimeric PHA synthases, we constructed a synthetic phaC gene from Pseudomonas oleovorans (phaC1Po) that was devoid of an internal 540-bp fragment. Randomly amplified PCR products (created with primers based on conserved phaC sequences flanking the deleted internal fragment) were generated using genomic DNA isolated from soil and were substituted for the 540-bp internal region. The chimeric genes were expressed in a PHA-negative strain of Ralstonia eutropha, PHB−4 (DSM 541). Out of 1,478 recombinant clones screened for PHA production, we obtained five different chimeric phaC1Po genes that produced more PHA than the native phaC1Po. Chimeras S1-71, S4-8, S5-58, S3-69, and S3-44 exhibited 1.3-, 1.4-, 2.0-, 2.1-, and 3.0-fold-increased levels of in vivo activity, respectively. All of the mutants mediated the synthesis of PHAs with a slightly increased molar fraction of 3-hydroxyoctanoate; however, the weight-average molecular weights (Mw) of the PHAs in all cases remained almost the same. Based upon DNA sequence analyses, the various phaC fragments appear to have originated from Pseudomonas fluorescens and Pseudomonas aureofaciens. The amino acid sequence analyses showed that the chimeric proteins had 17 to 20 amino acid differences from the wild-type phaC1Po, and these differences were clustered in the same positions in the five chimeric clones. A threading model of PhaC1Po, developed based on homology of the enzyme to the Burkholderia glumae lipase, suggested that the amino acid substitutions found in the active chimeras were located mostly on the protein model surface. Thus, our combinatorial genetic engineering strategy proved to be broadly useful for improving the catalytic activities of PHA synthase enzymes.

In vitro self-assembly of gold nanoparticle-coated poly(3-hydroxybutyrate) granules exhibiting plasmon-induced thermo-optical enhancements.

Polyhydroxyalkanoate (PHA) synthase attached to gold nanoparticles (AuNP) produce poly(3-hydroxybutyrate) (PHB) upon the addition of 3-hydroxybutyrate-CoA, and then coalesce to form micrometer-sized AuNP-coated PHB granules. These AuNP-coated PHB granules are potential theranostic agents that have enhanced imaging capabilities and are capable of heating upon near-infrared laser irradiation. The AuNP-coated PHB exhibited 11-fold enhancement in surface-enhanced Raman scattering over particles prior polymerization. Stained AuNP-coated PHB exhibited a 6-fold enhancement in fluorescence intensity as well as a 1.3-fold decrease in photobleaching rate compared to PHB granules alone. The granules were also shown to emit heat when illuminated at 808 nm with a 3.9-fold increase in heating rate compared to particles alone.

Glutaminase-producing Meyerozyma (Pichia) guilliermondii isolated from Thai soy sauce fermentation

In this study, 34 yeast isolates were obtained from koji and moromi samples of Thai soy sauce fermentation. However, the most interesting yeast strain was isolated from the enriched 2 month-old (M2) moromi sample and identified as Meyerozyma (Pichia) guilliermondii EM2Y61. This strain is a salt-tolerant yeast that could tolerate up to 20% (w/v) NaCl and produce extracellular and cell-bound glutaminases. Interestingly, its glutaminases were more active in 18% (w/v) NaCl which is a salt concentration in moromi. The extracellular glutaminases activity was found to be much higher than that of cell-bound glutaminase. The highest specific activity and stability of the extracellular glutaminase were found in 18% (w/v) NaCl at pH4.5 and 37°C. A challenge test by adding partially-purified extracellular glutaminase from M. guilliermondii EM2Y61 into 1 month-old (M1) moromi sample showed an increased conversion of L-glutamine to L-glutamic acid. This is the first report of glutaminase producing M. guilliermondii isolated from the moromi of Thai soy sauce fermentation. The results suggested the potential application of M. guilliermondii EM2Y61 as starter yeast culture to increase l-glutamic acid during soy sauce fermentation.

Polymer-lipid-PEG hybrid nanoparticles as photosensitizer carrier for photodynamic therapy

Polymer-lipid-PEG hybrid nanoparticles were investigated as carriers for the photosensitizer (PS), 5,10,15,20-Tetrakis(4-hydroxy-phenyl)-21H,23H-porphine (pTHPP) for use in photodynamic therapy (PDT). A self-assembled nanoprecipitation technique was used for preparing two types of core polymers poly(d,l-lactide-co-glycolide) (PLGA) and poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) with lipid-PEG as stabilizer. The resulting nanoparticles had an average particle size of 88.5±3.4nm for PLGA and 215.0±6.3nm for PHBV. Both nanoparticles exhibited a core-shell structure under TEM with high zeta potential and loading efficiency. X-ray powder diffraction analysis showed that the encapsulated pTHPP molecules in polymeric nanoparticles no longer had peaks of free pTHPP in the crystalline state. The pTHPP molecules encapsulated inside the polymeric core demonstrated improved photophysical properties in terms of singlet oxygen generation and cellular uptake rate in a FTC-133 human thyroid carcinoma cell line, compared to non-encapsulated pTHPP. The pTHPP-loaded polymer-lipid-PEG nanoparticles showed better in vitro phototoxicity compared to free pTHPP, in both time- and concentration-dependent manners. Overall, this study provides detailed analysis of the photophysical properties of pTHPP molecules when entrapped within either PLGA or PHBV nanoparticle cores, and demonstrates the effectiveness of these systems for delivery of photosensitizers. The two polymeric systems may have different potential benefits, when used with cancer cells. For instance, the pTHPP-loaded PLGA system requires only a short time to show a PDT effect and may be suitable for topical PDT, while the delayed photo-induced cytotoxic effect of the pTHPP-loaded PHBV system may be more suitable for cancer solid tumors. Hence, both pTHPP-encapsulated polymer-lipid-PEG nanoparticles can be considered promising delivery systems for PDT cancer treatment.

Bionanofabrication polyhydroxyalkanoates (PHAS) micro-/nanostructures on solid surfaces and its applications in nanobiotechnology

Bionanofabrication is a novel fabrication process that takes advantage of the specificity and catalytic efficiency of biological systems to create novel nanoscale structures. Polyhydroxyalkanoates (PHAs) are a family of aliphatic polyesters produced by a variety of microorganisms as a reserve of carbon and energy. PHAs can be combined from more than 100 different monomers to give materials with widely different physical properties. PHAs are biocompatible, biodegradable and demonstrate piezo electric and non-linear optical properties making them potential useful for tissue engineering, drug delivery, degradable packaging and smart materials. The enzymes involved in the synthesis of PHAs have been harnessed in our laboratory to produce novel polymers in vitro both in bulk and on solid surfaces. Site-specific attachment of the key catalytic enzyme, PHA synthase, on nanofabricated surfaces and subsequent addition of 3-(R)-hydroxybutyryl-CoA substrates (HB-CoA), allows us to create spatially ordered polyhydroxybutyrate (PHB) polymeric structures via in situ enzymatic surface-initiated polymerization (ESIP). By varying the reaction conditions we have optimized the PHB polymer growth at the interface and the resulting material characterized by fluorescence microscopy and atomic force microscopy. In the absence of additives such as bovine serum albumin, the PHB polymer synthesized on the surfaces formed very distinct and uniform granular structures on Au patterned surfaces. The average size of PHB granules was measured to be approximately 0.5 to 1 μm in diameter and 100 nm in height from the Au surfaces. In the presence of bovine serum albumin, the average size of PHB granules and PHB film thickness sinificantly increased to be approximately 1 to 5 μm in diameter and 500 nm to 1 μm in height, respectively, uniformly covering patterned surfaces. We believe that the use of this novel enzymatic approach offers many practical applications in different areas. For example, it can be employed to generate biocompatible PHAs coated solid surfaces for tissue engineering, promoting cell attachment and growth. As a result, one of our goals is to employ ESIP for in situ solid-phase synthesis of novel functionalized PHAs micro-/nanostructures with a wide range of mechanical, thermal, and biocompatible properties. In addition to biocompatible surface coatings, we envision that the novel polymeric micro-/nano-structures can be built in spaces that cannot be accessed by convention lithographic tools or other fabrication process. For example, PHB structures can be formed in situ inside microfluidic channels to produce rapid microfluidic mixing. Currently, we are investigating the use of in situ synthesized PHB polymer on specific Au patterned surfaces such as straight ridges and staggered herringbone patterns to act as passive micromixers inside microfluidic channels.

Improved Hardness of Nanocomposite Films on PMMA Sheet Using Beadmilled-SiO2 Nanoparticle in Dowanol PM

The main objective of this study was to prepare bead milled-silica nanoparticles (SiO2) as reinforcing materials for transparent hard coating films. SiO2 dispersed in Dowanol PM without any stabilizer was used as a main component in the nanocomposite hard coating films to improve hardness of Poly methyl methacrylate (PMMA) sheets. However, the major challenge in hard coating formulation is the dispersion of nanoscale SiO2 particles. Bead milling machine (MiniCer, NETZSCH, Germany) equipped with different sizes of zirconia (ZrO2) beads (0.1, 0.5, and 1.0 mm) was used for dispersing 40wt% SiO2 in Dowanol PM to achieve target sizes of 200, 500, and 800 nm. The dispersed nanoparticles were characterized by UV-visible spectroscopy for their optical transmission, transmission electron microscopy (TEM) for particle morphologies, and dynamic light scattering technique (DLS) for the particles sizes. The milled-SiO2 nanoparticles were stable in Dowanol PM as suspensions with their particles sizes closed to the target sizes. The 200-nm suspension showed the longest storage time without any aggregate formation. Whereas, the dispersed nanoparticles suspensions with the particles size >500 nm formed agglomerates during storage. The SiO2/MTMS nanocomposite coating film was then prepared coated milled-SiO2 suspension in Dowanol PM on PMMA sheets. The film with 200 nm SiO2 showed the highest transparency (92% at 550 nm) which was like the uncoated PMMA sheets. At thickness of 3-microns, SiO2/MTMS nanocomposite films could improve pencil hardness of PMMA sheets from

A sacrificial process for fabrication of biodegradable polymer membranes with submicron thickness.

A new sacrificial molding process using a single mask has been developed to fabricate ultrathin 2-dimensional membranes from several biocompatible polymeric materials. The fabrication process is similar to a sacrificial microelectromechanical systems (MEMS) process flow, where a mold is created from a material that can be coated with a biodegradable polymer and subsequently etched away, leaving behind a very thin polymer membrane. In this work, two different sacrificial mold materials, silicon dioxide (SiO2 ) and Liftoff Resist (LOR) were used. Three different biodegradable materials; polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and polyglycidyl methacrylate (PGMA), were chosen as model polymers. We demonstrate that this process is capable of fabricating 200-500 nm thin, through-hole polymer membranes with various geometries, pore-sizes and spatial features approaching 2.5 µm using a mold fabricated via a single contact photolithography exposure. In addition, the membranes can be mounted to support rings made from either SU8 or PCL for easy handling after release. Cell culture compatibility of the fabricated membranes was evaluated with human dermal microvascular endothelial cells (HDMECs) seeded onto the ultrathin porous membranes, where the cells grew and formed confluent layers with well-established cell-cell contacts. Furthermore, human trabecular meshwork cells (HTMCs) cultured on these scaffolds showed similar proliferation as on flat PCL substrates, further validating its compatibility. All together, these results demonstrated the feasibility of our sacrificial fabrication process to produce biocompatible, ultra-thin membranes with defined microstructures (i.e., pores) with the potential to be used as substrates for tissue engineering applications.

Viscoelastic Property and Cell Adhesion Process of Cultured Fibroblasts on Different Self-assembled Monolayers Monitored by Acoustic Wave Biosensor

A quartz crystal microbalance (QCM) technique was used to reveal the effect of alkanethiol self-assembled monolayers (SAMs) on the viscoelastic property and cell adhesion process of cultured fibroblasts (L929), by measuring the change in frequency (Δf ) and resistance (ΔR). Four types of SAMs having different functional groups including hydroxyl (OH), carboxylic acid (COOH), amine (NH2) and methyl (CH3) were used in this study. The QCM measurements during cell adhesion showed that floating cells were rapidly adsorbed on all functionalized surfaces as a soft cell monolayer which their stiffness was increased by cell spreading process. Initially, the Δf and ΔR were both positive shifts for COOH, OH and NH2 surfaces indicated fluid-like behavior of adherent cells. A multi-step decreasing of both Δf and ΔR were observed in case of COOH and OH surfaces represented solid-like behavior of cell spreading. The CH3 surface was used as a control because no cell adhesion occurs. QCM responses and morphological changes during cell adhesion process were resulted from viscoelastic nature of mammalian cells. The changes of cell morphology and viscoelastic property during cell adhesion process strongly depend on the surface functionality which can be observed by QCM technique.

Bionanofabrication polyhydroxyalkanoates (PHAS) micro-/nano-structures on solid surfaces and its applications in nanobiotechnology

Summary form only given. Bionanofabrication is a novel fabrication process that takes advantage of the specificity and catalytic efficiency of biological systems to create novel nanoscale structures. Polyhydroxyalkanoates (PHAs) are a family of aliphatic polyesters produced by a variety of microorganisms as a reserve of carbon and energy. PHAs can be combined from more than 100 different monomers to give materials with widely different physical properties. PHAs are biocompatible, biodegradable and demonstrate piezo electric and non-linear optical properties making them potential useful for tissue engineering, drug delivery, degradable packaging and smart materials. The enzymes involved in the synthesis of PHAs have been harnessed in our laboratory to produce novel polymers in vitro both in bulk and on solid surfaces. Site-specific attachment of the key catalytic enzyme, PHA synthase, on nanofabricated surfaces and subsequent addition of 3-(R)-hydroxybutyryl-CoA substrates (HB-CoA), allows us to create spatially ordered polyhydroxybutyrate (PHB) polymeric structures via in situ enzymatic surface-initiated polymerization (ESIP). By varying the reaction conditions we have optimized the PHB polymer growth at the interface and the resulting material characterized by fluorescence microscopy and atomic force microscopy. In the absence of additives such as bovine serum albumin, the PHB polymer synthesized on the surfaces formed very distinct and uniform granular structures on Au patterned surfaces. The average size of PHB granules was measured to be approximately 0.5 to 1 mum in diameter and 100 nm in height from the Au surfaces. In the presence of bovine serum albumin, the average size of PHB granules and PHB film thickness significantly increased to be approximately 1 to 5 mum in diameter and 500 nm to 1 mum in height, respectively, uniformly covering patterned surfaces. We believe that the use of this novel enzymatic approach offers many practical applications in different areas. For example, it can be employed to generate biocompatible PHAs coated solid surfaces for tissue engineering, promoting cell attachment and growth. As a result, one of our goals is to employ ESIP for in situ solid-phase synthesis of novel functionalized PHAs micro-/nano- structures with a wide range of mechanical, thermal, and biocompatible properties. In addition to biocompatible surface coatings, we envision that the novel polymeric micro-/nano-structures can be built in spaces that cannot be accessed by convention lithographic tools or other fabrication process. For example, PHB structures can be formed in situ inside microfluidic channels to produce rapid microfluidic mixing. Currently, we are investigating the use of in situ synthesized PHB polymer on specific Au patterned surfaces such as straight ridges and staggered herringbone patterns to act as passive micromixers inside microfluidic channels