Bernard Y. Tao

Oligosaccharide synthesis by enzymatic transglycosylation

Agricultural Research Service, United States Department of Agriculture, Northern Regional Research Center, 1815 N. University St., Peoria, IL 61604, U.S.A. 2 Biochemical and Food Process Engineering, Agricultural Engineering Department, Purdue University, West Lafayette, IN 47907, U.S.A.

Polymerase chain reaction (PCR) techniques for site-directed mutagenesis

Polymerase chain reaction (PCR) technology has revolutionized the process of isolating and amplifying segments of DNA. One powerful application of PCR is its use in precise site-directed mutagenesis (SDM). SDM provides an elegant tool for scientists and engineers to explore biocatalytic mechanisms and processes to understand the structural-functional relationships of enzymes and other proteins. This article reviews techniques and methodology used in site-directed mutagenesis of genes by PCR.

Synthesis of Cellulose-Fatty Acid Esters for Use as Biodegradable Plastics*

Long-chain fatty acid carbohydrate esters (FACE) were synthesized by the acid chloride-pyridine reaction to different degrees of substitution (DS). The hydrolyzed soybean oil was used as the source of unsaturated fatty acids. High molecular weight FACE polymers are insoluble in common solvents, such as benzene, toluene, THF, etc., and are highly water resistant. However, FACE polymers of hydrolyzed cellulose (MW 180 kD) are soluble/swellable in toluene and can be cast into tough, flexible films. FACE polymer properties of tensile strength and clasticity vary with degree of substitution and polymer size.

Can Soy Methyl Esters Reduce Fluid Transport and Improve Durability of Concrete

Many durability problems in concrete are caused by the transport of water or fluid containing aggressive ions. As a result, many seek to reduce the absorption and flow of fluids in concrete through the use of admixtures or topical sealants that refine or block pores. Results are presented of a pilot study that evaluated the potential use of soy methyl ester–polystyrene blends (SME–PS) as either an admixture or a topical treatment or both. Results are presented for tests on the fresh properties, hardened properties, and transport properties of concrete containing SME–PS. Dramatic reductions in fluid absorption can be obtained with SME–PS, with little noticeable influence on set time, strength, or shrinkage. Eventually the application of SME–PS to concrete may have the potential to be used as a method to penetrate into the pore structure at saw cuts and reduce long-term damage.

Solubilization conditions for hydrophobic membrane protein, oleosin, in soybeans

Oleosins are amphipathic proteins found in soybeans that give stability to oil-bodies by surrounding them. Furthermore, oleosins have broad biotechnological applications in protein purification and in immobilization as a tagging parther. In this study we provide the proper combinations of detergents and chaotropes for solubilization of oleosins. The solubilization effects of each detergent were compared by the results of SDS-PAGE analysis, from the density measurement of each band using an image analysis program. Among the twelve chemicals tested, 3-(decyldimethylammonio) propanesulfonale inner salt (SB 3–10), nonaethylene glycol octylphenyl ether (NP-40), and n-dodecyl-β-d-maltoside (DM) were determined to be effective for oleosin solubilization. In addition, urea, thiourea, and reducing agents played roles in enhancing the effects of the detergents. The best results were obtained with 2.5% of each detergent in combination with 4 M urea and 2 M thiourea at room temperature. The zwitterionic detergent, SB 3–10, is recommended as the best non-denaturant for the solubilization of soybean oleosins with the advantage of its easy removal by dialysis due to its low molecular weight and a high critical micelle concentration.

Can Soy Methyl Esters Improve Concrete Pavement Joint Durability

Although many concrete pavements provide excellent long-term performance, some pavements (primarily in the Midwest) have shown premature deterioration at the joints. This premature deterioration is a concern because such deterioration can shorten the life of pavements that are otherwise functioning well. Previous work has hypothesized that these joints may be susceptible to preferential fluid saturation, which can lead to freeze–thaw damage or chemical degradation. This work examines the use of soy methyl ester–polystyrene (SME-PS) blends as a method to reduce the rate of fluid ingress into the pore system of the concrete and thereby make the concrete more resistant to deterioration. SME-PS is derived from soybeans and has demonstrated an ability to reduce fluid absorption in concrete when used as a topical treatment. A series of experiments was developed to evaluate the effectiveness of various dosage rates of SME-PS for increasing concrete durability at pavement joints. The experiments show that SME-PS reduces fluid ingress, salt ingress, and the potential for freeze–thaw damage. As a result of the positive experimental results, the Indiana Department of Transportation is conducting field trials that use SME-PS on concrete pavements that are beginning to show signs of premature deterioration with the expectation that SME-PS will extend the life of the joints and thereby reduce maintenance cost and extend the life of concrete pavements.

Detection of native and recombinant Bacillus macerans cyclodextrin glycosyltransferase using microtiter elisa techniques

Abstract Direct, sandwich, and competitive ELISA methods were developed to quantify Bacillus macerans cyclodextrin glycosyltransferase (CGTase) and its thioredoxin fusion molecule using rabbit polyclonal antibodies. The direct ELISA method was used to demonstrate equivalent molar response of both native CGTase and the thioredoxin-CGTase fusion protein to antibody binding. Sandwich ELISA showed the most sensitive detection range (0.2 ∼ 50 μg ml−1) against CGTase. We improved a competitive ELISA method by coating the microtiter plate with denatured CGTase in 6 m guanidine-HCI solution, resulting in enhanced, rapid response. This competitive ELISA method was specific, precise, and rapid when the method was used to quantify CGTase and monitor production of CGTase and its thioredoxin fusion protein in the recombinant E. coli. These methods are specifically useful when detecting and quantifying recombinant CGTase proteins in which mutations may have reduced or eliminated enzymatic activity.

A simple method to express soluble, highly active cyclodextrin glycosyltransferase in recombinant E. coli

The activity of soluble cyclodextrin glycosyltransferase was increased by 40% without formation of inclusion bodies by induction at low temperature with the addition of 10 mM CaCl2 into the medium, when the corresponding gene was expressed in two recombinant E. coli. Furthermore, the protein expressed at 25 °C had approximately 30% higher specific activity than that expressed at 30 °C. The two different hosts, promoters, and media showed the same result, indicating that this simple method can be used in expressing other proteins.

A robust fuel flexible combustion control strategy for biodiesel with variable fatty acid composition during mixing-controlled combustion:

Biodiesel is a diesel fuel alternative which is produced from renewable and domestically available sources. The use of biodiesel generally lowers carbon dioxide, carbon monoxide and particulate matter emissions. However, there are certain challenges associated with the use of biodiesel, mainly (1) lower fuel energy density, (2) increased nitrogen oxide (NO x ) emissions and (3) fuel variability due to feedstock and processing differences. In prior efforts, the authors have demonstrated that the first two of these challenges can be overcome for different blend fractions of soy-based biodiesel by using a control algorithm incorporating energy-based fueling for torque control and combustible oxygen mass fraction control for NO x regulation. However, in addition to overcoming these combustion-related challenges, in this work, the authors consider the extension of these techniques to biodiesel generated from oils/fats of varying composition. The type of oil/fat from which the biodiesel is derived will impact the fuel properties via variation in the fuel’s fatty acid composition. The fuel’s fatty acid composition can also be altered by an additional processing done in order to change certain fuel properties. For example, the saturation level of biodiesel can be reduced in order to lower the fuel cloud point, making it suitable for colder climates. The effect of variation in the fuel fatty acid structure on the previously developed control algorithm is studied in this work. It is shown both theoretically and experimentally that the proposed control algorithms are robust to variation in the fatty acid composition of biodiesel due to the fact that biodiesels with very different fatty acid compositions exhibit minor changes in heating values and fuel oxygen mass fraction. As such, the control technique is suitable for use with variable blend fractions of biodiesel produced from different feedstocks as well as fuel processed to improve cold weather operation.

Assessment of efficacy and reactive gas species generation for orange juice decontamination using high voltage atmospheric cold plasma

Summary form only given. While multiple studies have demonstrated atmospheric cold plasma as an effective non-thermal technology for eliminating bacteria, spores, and biological contaminants from food and non-food surfaces1, few report the application of this technique to liquid food within a package2,3. In this study, we use plasma spectroscopy to characterize the high voltage atmospheric cold plasma (HVACP)2 generated by a dielectric barrier discharge (DBD) in orange juice (OJ) within a package containing either air or modified atmosphere (MA65). We also evaluate the effectiveness of HVACP for decontaminating Salmonella enterica serovar Typhi (S. Typhi) in OJ. We exposed 50 ml of OJ both directly and indirectly to 90 kV for up to 120 s and used optical emission spectroscopy (OES) and optical absorption spectroscopy (OAS) to characterize the reactive gas species (RGS). The sample was stored in a refrigerator at 4°C for 24 h following treatment. Conductivity, pH and hydrogen peroxide content was measured before and after the treatments. Treating 50 ml of OJ containing S. Typhi for 120 s with direct treatment resulted in a 2.9 log10 reduction in air and a 4.2 log10 reduction in MA65 24 h after treatment. Using indirect treatment resulted in a 3.8 log10 reduction in MA65. OES and OAS indicated that the levels of OH, N2, N2+, and O- generated in OJ varied with package gas and whether the application was direct or indirect. These differences in RGS between fill gases and contact methods may impact the decontamination efficiency of S. Typhi in OJ. The implications of RGS on microorganism decontamination and potential plasmas systems will be discussed.