Substrate specificity of acetylxylan esterase from Schizophyllum commune: mode of action on acetylated carbohydrates.

Substrate specificity of a purified acetylxylan esterase from Schizophyllum commune was investigated on a variety of methyl per-O-acetyl glycopyranosides, methyl di-O-acetyl-beta-D-xylopyranosides and acetylated polysaccharides. The enzyme preferentially deacetylated the 3-position of methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside and 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside. Removal of the 3-acetyl group from the xylopyranoside was accompanied by a slower deacetylation at positions 2 and 4. A similarly slower, accompanying deacetylation occurred primarily at position 2 with the glucopyranoside. Such specificity corresponds well to the expected function of the esterase in acetylxylan degradation. Of the three possible diacetates of methyl beta-D-xylopyranoside, the 3,4-diacetate was found to be the most rapidly deacetylated. Unexpectedly, products of its deacetylation were a mixture of 2- and 4-monoacetate. The formation of the methyl 2-O-acetyl-beta-D-xylopyranoside involved an enzyme-mediated acetyl group transfer because the rate of the enzyme-catalyzed reaction exceeded the rate of spontaneous migration of acetyl groups. This is the likely mechanism for acetyl removal from position 2 in the native substrate. The enzyme exhibited the highest regioselectivity with methyl 2,3,4,6-tetra-O-acetyl-beta-D-mannopyranoside. An 80% conversion of this substrate to methyl 4,6-di-O-acetyl-beta-D-mannopyranoside, a new mannose derivative, was achieved. In contrast to the majority of lipases and esterases exploited for regioselective deacetylation, the S. commune acetylxylan esterase did not attack the C-6 acetyl linkages in methyl hexopyranosides when other acetyl groups were available.

A new paradigm for graduate research and training in the biomedical sciences and engineering.

98Emphasis on the individual investigator has fostered discovery for centuries, yet it is now recognized that the complexity of problems in the biomedical sciences and engineering requires collaborative efforts from individuals having diverse training and expertise. Various approaches can facilitate interdisciplinary interactions, but we submit that there is a critical need for a new educational paradigm for the way that we train biomedical engineers, life scientists, and mathematicians. We cannot continue to train graduate students in isolation within single disciplines, nor can we ask any one individual to learn all the essentials of biology, engineering, and mathematics. We must transform how students are trained and incorporate how real-world research and development are done-in diverse, interdisciplinary teams. Our fundamental vision is to create an innovative paradigm for graduate research and training that yields a new generation of biomedical engineers, life scientists, and mathematicians that is more diverse and that embraces and actively pursues a truly interdisciplinary, team-based approach to research based on a known benefit and mutual respect. In this paper, we describe our attempt to accomplish this via focused training in biomechanics, biomedical optics, mathematics, mechanobiology, and physiology. The overall approach is applicable, however, to most areas of biomedical research.

Action of acetylxylan esterase from Trichoderma reesei on acetylated methyl glycosides.

Substrate specificity of purified acetylxylan esterase (AcXE) from Trichoderma reesei was investigated on partially and fully acetylated methyl glycopyranosides. Methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside was deacetylated at positions 2 and 3, yielding methyl 4-O-acetyl-beta-D-xylopyranoside in almost 90% yield. Methyl 2,3-di-O-acetyl beta-D-xylopyranoside was deacetylated at a rate similar to the fully acetylated derivative. The other two diacetates (2,4- and 3,4-), which have a free hydroxyl group at either position 3 or 2, were deacetylated one order of magnitude more rapidly. Thus the second acetyl group is rapidly released from position 3 or 2 after the first acetyl group is removed from position 2 or 3. The results strongly imply that in degradation of partially acetylated beta-1,4-linked xylans, the enzyme deacetylates monoacetylated xylopyranosyl residues more readily than di-O-acetylated residues. The T. reesei AcXE attacked acetylated methyl beta-D-glucopyranosides and beta-D-mannopyranosides in a manner similar to the xylopyranosides.

Substrate specificity and mode of action of acetylxylan esterase from Streptomyces lividans.

The substrate specificity of purified acetylxylan esterase (AcXE) from Streptomyces lividans was investigated on partially and fully acetylated methyl glycopyranosides. The enzyme exhibited deacetylation regioselectivity on model compounds which provided insights pertaining to its function in acetylxylan degradation. The enzyme catalyzed double deacetylation of methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside and methyl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside at positions 2 and 3. Two methyl xylopyranoside diacetates, which had a free hydroxyl group at position 2 or 3, i.e. the derivatives that most closely mimic monoacetylated xylopyranosyl residues in acetylxylan, were deacetylated 1 to 2 orders of magnitude faster than methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside and methyl 2,3-di-O-acetyl-beta-D-xylopyranoside. These observations explain the double deacetylation. The second acetyl group is released immediately after the first one is removed from the fully acetylated methyl beta-D-xylo- and -glucopyranoside. The results suggest that in acetylxylan degradation the enzyme rapidly deacetylates monoacetylated xylopyranosyl residues, but attacks doubly acetylated residues much more slowly. Evidence is also presented that the St. lividans enzyme could be the first real substrate-specific AcXE.

Optical glucose sensing in biological fluids: an overview.

Recent technological advancements in the photonics industry have led to a resurgence of interest in optical glucose sensing and to realistic progress toward the development of an optical glucose sensor. Such a sensor has the potential to significantly improve the quality of life for the estimated 16 million diabetics in this country by making routine glucose measurements more convenient. Currently over 100 small companies and universities are working to develop noninvasive or minimally invasive glucose sensing technologies, and optical methods play a large role in these efforts. This article reviews many of the recent advances in optical glucose sensing including optical absorption spectroscopy, polarimetry, Raman spectroscopy, and fluorescent glucose sensing. In addition a review of calibration and data processing methods useful for optical techniques is presented.

The use of polarized laser light through the eye for noninvasive glucose monitoring.

BACKGROUND: In order to optimally manage diabetes mellitus, it is recommended blood glucose levels be monitored several times daily so an appropriate action can be taken to maintain tight control of these levels within a normal physiological range. All commercially available devices to measure blood glucose concentrations require the extraction of a drop of blood, normally obtained via the lancing of a finger. The main drawback to this method is the pain, often leading to low patient compliance. Therefore, a noninvasive glucose sensing method would greatly facilitate the management of diabetes. METHODS: In this article, we describe in vitro and in vivo results from a laser-based optical polarimetry system using the anterior chamber of the eye as a potential method to noninvasively monitor glucose levels in the body. RESULTS: It is shown, in vitro, that glucose can be predicted in the presence of albumin at physiological levels and, through the use of a novel light coupling mechanism, it is demonstrated that a polarimetric signal can be detected, in vivo, through a rabbit eye. CONCLUSIONS: Although the commercial production of a feasible noninvasive glucose monitoring method is still years away, laser-based polarimetry remains a viable alternative due to its potential to extract concentration information using the eye as a unique optical window into the body.

Measurement of the glucose transport time delay between the blood and aqueous humor of the eye for the eventual development of a noninvasive glucose sensor.

In the recent past, several noninvasive optically based methods have been proposed for physiologic glucose sensing. One proposed optical sensing site has been the eye, which, due to its unique optical properties, can be considered as a transparent optical window into the body. In particular, the aqueous humor within the anterior chamber of the eye has been shown to contain glucose levels correlated to those of blood. Concern, however, has been expressed that using the aqueous humor solution as a measure of blood glucose may be problematic due to the potential transport time delay between the blood and the aqueous humor glucose concentrations. This investigation was performed to measure the transport time delay in a rabbit model. The time delay between the blood and aqueous humor glucose concentrations was measured invasively in five New Zealand White rabbits over a series of weeks. An anesthesia protocol containing the drug Xylazine was used to elevate the blood glucose levels to a level commonly seen in diabetic patients. The difference between the glucose peak location times occurring in the blood and aqueous humor glucose response was measured and defined as the transport time delay. The average transport time lag was measured to be under 5 min. This measured time delay indicates that, indeed, the eye could potentially be used as a sensing site for indirect blood glucose measurements and may eventually aid the development of a noninvasive glucose sensor due to its unique optical properties compared to other biological tissues.

Noninvasive glucose sensing utilizing a digital closed-loop polarimetric approach.

A polarimetric glucose sensor utilizing a digital closed-loop controller was designed and implemented during this study. Its potential as a noninvasive glucose sensor was evaluated in vitro for both glucose-doped water and bovine aqueous humor mediums. A physiological hyperglycemic concentration range was used in both calibration and validation of each set of experiments. Ideally, the end application of this system could estimate blood glucose concentrations indirectly by measuring the amount of rotation of a light beam's polarization state after it propagates through the aqueous humor contained within the anterior chamber of the eye. The polarimeter designed in this study differs from similar investigated systems in that it utilizes a digital closed-loop control system. This type of controller was implemented in order to further improve system repeatability and stability without sacrificing accuracy. Unique to this investigation, independent validation sets other than those used to create each respective calibration model were obtained. The results of the glucose-doped water experiments yielded mean standard errors of prediction for calibration and validation of 6.91 and 8.84 mg/dl, respectively. The mean standard errors of prediction during calibration and validation of the glucose-doped aqueous humor experiments were higher at 27.20 and 27.47 mg/dl, respectively, due to medium degradation over time while exposed to air.

Monte Carlo modeling for implantable fluorescent analyte sensors.

A Monte Carlo simulation of photon propagation through human skin and interaction with a subcutaneous fluorescent sensing layer is presented. The algorithm will facilitate design of an optical probe for an implantable fluorescent sensor, which holds potential for monitoring many parameters of biomedical interest. Results are analyzed with respect to output light intensity as a function of radial distance from source, angle of exit for escaping photons, and sensor fluorescence (SF) relative to tissue autofluorescence (AF). A sensitivity study was performed to elucidate the effects on the output due to changes in optical properties, thickness of tissue layers, thickness of the sensor layer, and both tissue and sensor quantum yields. The optical properties as well as the thickness of the stratum corneum, epidermis, (tissue layers through which photons must pass to reach the sensor) and the papillary dermis (tissue distal to sensor) are highly influential. The spatial emission profile of the SF is broad compared that of the tissue fluorescence and the ratio of sensor to tissue fluorescence increases with distance from the source. The angular distribution of escaping photons is more concentrated around the normal for SF than for tissue AF. The information gained from these simulations will be helpful in designing appropriate optics for collection of the signal of interest.

Noninvasive optical polarimetric glucose sensing using a true phase measurement technique.

The focus of this paper was to describe the development and testing of a noninvasive true phase optical polarimetry sensing system to monitor in vivo glucose concentrations. To demonstrate the applicability of this optical sensor for glucose measurement, we first calibrated the system and then tested it in vitro using both a glass test cell filled with glucose solution in the physiologic range, with a path length of 0.9 cm to approximate one centimeter path length present in the anterior chamber of the eye, and then on an excised human eye. Our technique used helium neon laser light which was coupled through a rotating linear polarizer along with two stationary linear polarizers and two detectors to produce reference and signal outputs whose amplitudes varied sinusoidally with a frequency of twice the angular velocity of the rotating polarizer, and whose phase was proportional to the rotation of the linear polarization vector passing through the glucose solution.

Application of a multivariate technique to Raman spectra for quantification of body chemicals.

Raman spectroscopy is a highly specific technique for the identification of molecules by way of the associated characteristic spectra. The aim of this feasibility study is to assess the combination of the multivariate calibration technique of Partial Least-Squares with Raman spectroscopy for the estimation of glucose, lactic acid, and urea concentrations in the presence of each other in a water substrate. The instrument is a CCD-based Raman spectrometer utilizing the 514.5 nm argon laser line. The estimates for the analyte concentrations yielded a standard deviation of concentration residuals of 20.71 mg/dL for glucose, 12.92 mg/dL for lactic acid, and 19.07 mg/dL for urea.

Simultaneous optical and nuclear magnetic resonance spectroscopy for monitoring cardiac energetics in vivo.

There are a number of applications in which it is useful to simultaneously collect data from what are traditionally separate instrumentation modalities. In particular, in vivo physiological investigations in which data from parallel experiments must be correlated would benefit from simultaneous data collection through 1) elimination of subject variability, 2) elimination of treatment variability, and 3) a reduction in the number of animal preparations required. Here we describe the simultaneous collection of fluo-3 optical fluorescence and 31P nuclear magnetic resonance (NMR) spectra to measure intracellular calcium levels and high-energy phosphate metabolism, respectively, in vivo. This work is part of ongoing research into the profound anoxia tolerance exhibited by the hearts of certain turtle species. An NMR compatible optical fluorescence spectrometer was constructed and tested. In the 31-cm bore of a 2 T superconducting magnet, NMR and optical spectra were collected every 10-15 min from the in situ, in vivo hearts of anesthetized turtle subjects prior to and during one to three hours of anoxia. It was found that while PCr stores became significantly depleted during anoxia, beta-adenosine triphosphate (ATP) levels remained within 20% of control values, and intracellular diastolic calcium levels did not vary by more than 10%. The ability to make simultaneous phosphorus and calcium measurements on a single subject is important to understanding the exact relationship between phosphorus energy state and maintenance of calcium homeostasis.

Isolation and partial characterization of an extracellular glucansucrase from Leuconostoc mesenteroides NRRL B-1355 that synthesizes an alternating (1 goes to 6), (1 goes to 3)-alpha-D-glucan.

Leuconostoc mesenteroides NRRL B-1355 grows on sucrose to produce two extracellular alpha-D-glucans. Although both are termed dextrans, they are chemically and physically distinct, and can be separated by fractional ethanol precipitation into fractions designated L and S. Fraction L is similar to B-512F dextran, having 95% alpha-(1 goes to 6) linkages and 5% alpha-(1 goes to 3) branch linkages, but fraction S has an alternating sequence of alpha-(1 goes to 6) and alpha-(1 goes to 3) linkages. Because of its structural differences from dextran, its different physical characteristics, and its resistance to hydrolysis by endodextranase, we have named glucan S, alternan, and the enzyme that synthesizes it from sucrose, alternansucrase. Alternansucrase has been isolated by two different methods. The first involves removal of the fraction L glucan from the culture fluid via hydrolysis by an endodextranase, followed by chromatography on Bio-Gel A5m. The void-volume fraction synthesizes only alternan, whereas the slower-migrating, second fraction synthesizes mainly dextran, together with some alternan. The second method utilized hydrophobic chromatography on O-(phenoxyacetyl) cellulose; a portion of the alternansucrase did not bind, whereas the bound portion, removed by eluting with detergent, contained both alternansucrase and dextransucrase. The glucans were identified by physical appearance, the concentration of ethanol required for precipitation, periodate-oxidation behavior, and susceptibility to hydrolysis by endodextranase. Also studied was the inhibition of the enzymes by 3-deoxy-3-fluoro-alpha-D-glucopyranosyl fluoride, tris(hydroxymethyl)aminomethane, 2-aminoethanol, and octyl beta-D-glucopyranoside.

The hydrolytic and transferase action of alternanase on oligosaccharides.

Alternanase is an enzyme which endo-hydrolytically cleaves the alpha-(1-->3), alpha-(1-->6)-linked D-glucan, alternan. The main products are isomaltose, alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-D-Glc and the cyclic tetrasaccharide cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. It is also capable of acting on oligosaccharide substrates. The cyclic tetrasaccharide is slowly hydrolyzed to isomaltose. Panose and the trisaccharide alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-D-Glc both undergo transglycosylation reactions to give rise to the cyclic tetrasaccharide plus D-glucose, with panose being converted at a much faster rate. The tetrasaccharide alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc is hydrolyzed to D-glucose plus the trisaccharide alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-D-Glc. Alternanase does not act on isomaltotriose, theanderose (6(Glc)-O-alpha-D-Glcp sucrose), or alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glc. The enzyme releases 4-nitrophenol from 4-nitrophenyl alpha-isomaltoside, but not from 4-nitrophenyl alpha-D-glucopyranoside, 4-nitrophenyl alpha-isomaltotrioside, or 4-nitrophenyl alpha-isomaltotetraoside.

The formation of alpha-D-(1----3) branch linkages by a D-glucansucrase from Streptococcus mutans 6715 producing a soluble D-glucan.

An exocellular D- glucansucrase that synthesizes a water-soluble, alpha-D-(1----6)-linked D-glucan having a high proportion of alpha-D-(1----3) branches was purified from the culture broth of Streptococcus mutans 6715. The rate of incorporation of D-[14C]glucose from [14C]sucrose into D-glucan of high molecular weight by this enzyme was increased (stimulated) by the presence of exogenous Leuconostoc mesenteroides B- 512F dextran, and it was found that this dextran could act as an acceptor. A highly branched dextran, containing 45-50% of alpha-D-(1----3) branch linkages, did not stimulate the enzyme nearly so much as B- 512F dextran, which has a low degree (5%) of alpha-D-(1----3) branches. We interpret this as evidence that the stimulating effects of dextran are not due to priming. If they were, the more highly branched dextran should have produced the greatest stimulation per unit weight, because a much greater number of nonreducing-end, priming sites would be available. We show that the D- glucansucrase was capable of transferring D-glucosyl groups from sucrose to B- 512F dextran to form alpha-D-(1----3) branches, thereby rendering the dextran more resistant to hydrolysis by endodextranase . The presence of 1.6M ammonium sulfate caused the enzyme to synthesize a D-glucan having a much higher percentage of alpha-D-(1----3) linkages.

Enzymic alpha-galactosylation of a cyclic glucotetrasaccharide derived from alternan.

Alternanase catalyzes the hydrolysis of alternan, an alpha-(1-->3)-alpha-(1-->6)-D-glucan produced by Leuconostoc mesenteroides, resulting in the formation of a cyclic tetramer cyclo -->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->(2) (cGlc(4)). Two alpha-galactosidases, one from coffee bean and the other produced by a fungus, currently described as Thermomyces lanuginosus, were found to catalyze an efficient 6-O-alpha-D-galactopyranosylation of cGlc(4). The attachment of a nonreducing alpha-D-galactopyranosyl residue to the cGlc(4) molecule opens new possibilities for future applications of the cyclic tetramer, since the D-galactopyranosyl residue can be easily modified by D-galactose oxidase to introduce a reactive aldehyde group. The results also extend our knowledge about the synthetic potential of T. lanuginosus alpha-galactosidase.

Disproportionation reactions catalyzed by Leuconostoc and Streptococcus glucansucrases.

Glucansucrases from Leuconostoc mesenteroides NRRL B-512F and Streptococcus mutans 6715 were found to utilize a number of D-gluco-oligosaccharides as D-glucosyl donors and as acceptors. These donors included isomaltotriose and its homologs, panose, maltotriose, and dextran. In each case, D-glucosyl groups were transferred from the donor to an acceptor sugar. When the donor sugar also acted as an acceptor, disproportionation reactions occurred. Isomaltotriose, for example, gave rise to isomaltose and isomaltotetraose initially, and to a series of isomalto-oligosaccharides eventually. In addition to forming alpha-D-(1----6) linkages in the reactions, dextransucrase from S. mutans 6715 was capable of forming alpha-D-(1----3)-linked products.

X-ray structure determination and modeling of the cyclic tetrasaccharide cyclo.

The cyclic tetrasaccharide cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->] is the major compound obtained by the action of endo-alternases on the alternan polysaccharide. Crystals of this cyclo-tetra-glucose belong to the orthorhombic space group P2(1)2(1)2(1) with a = 7.620(5), b = 12.450(5) and c = 34.800(5) A. The asymmetric unit contains one tetrasaccharide together with five water molecules. The tetrasaccharide adopts a plate-like overall shape with a very shallow depression on one side. The shape is not fully symmetrical and this is clearly apparent on comparing the (phi, psi) torsion angles of the two alpha-(1-->6) linkages. There is almost 10 degrees differences in phi and more than 20 degrees differences in psi. The hydrogen bond network is asymmetric, with a single intramolecular hydrogen bond: O-2 of glucose ring 1 being the donor to O-2 of glucose ring 3. These two hydroxyl groups are located below the ring and their orientation, dictated by this hydrogen bond, makes the floor of the plate. Among the five water molecules, one located above the center of the plate occupies perfectly the shallow depression in the plate shape formed by the tetrasaccharide. Molecular dynamics simulation of the tetrasaccharide in explicit water allows rationalization of the discrepancies observed between the X-ray structures and data obtained previously by NMR.

Design of a self-cleaning thermoresponsive nanocomposite hydrogel membrane for implantable biosensors.

Following implantation of a biosensor, adhesion of proteins and cells and eventual fibrous encapsulation will limit analyte diffusion and impair sensor performance. A thermoresponsive nanocomposite hydrogel was developed as a self-cleaning biosensor membrane to minimize the effect of the host response and its utility for an optical glucose sensor, demonstrated here. It was previously reported that thermoresponsive nanocomposite hydrogels prepared from photopolymerization of an aqueous solution of N-isopropylacrylamide (NIPAAm) and polysiloxane colloidal nanoparticles released adhered cells with thermal cycling. However, poly(N-isopropylacrylamide) hydrogels exhibit a volume phase transition temperature (VPTT) of approximately 33-34 degrees C, which is below body temperature. Thus, the hydrogel would be in a collapsed state in vivo, which would ultimately limit diffusion of the target analyte (e.g., glucose) to the encapsulated sensor. In this study, the VPTT of the nanocomposite hydrogel was increased by introducing N-vinylpyrrolidone (NVP) as a comonomer, so that the hydrogel was in the swollen state in vivo. This thermoresponsive nanocomposite hydrogel was prepared by the photopolymerization of an aqueous solution of NIPAAm, NVP, and polysiloxane colloidal nanoparticles. In addition to a VPTT a few degrees above body temperature, the hydrogel also exhibited good mechanical strength, glucose diffusion, and in vitro cell release upon thermal cycling. Thus, this nanocomposite hydrogel may be useful as a biosensor membrane to minimize biofouling and extend the lifetime and efficiency of implantable glucose sensors and other biosensors.