Binding of carbohydrates to solid supports Part 3: Reaction of sugar hydrazones with polystyrene substituted with aromatic aldehydes

The hydrazones of glucose and N-acetylglucosamine, as models for the residues at the reducing termini of glycans, were covalently and reversibly bound in good yield to hydroxybenzaldehydo ligands attached to a polymer support. The binding, by a sugar azine linkage, occurred within two hours at room temperature at neutral pH, and efficient recoveries of sugars from the beads were achieved by displacement with aqueous hydrazine hydrate, ethanolic benzaldehyde, or aqueous acetone. Enzyme modification of glycans was demonstrated by separation of the products of hydrolysis of lactose hydrazone with -galactosidase, using hydroxybenzaldehyde-derivatized polystyrene beads. Addition of a spacer arm to aminopolystyrene beads, for binding of reducing sugars as Amadori compounds to the aromatic amine function, was also investigated.     

Binding of carbohydrates to solid supports: evaluation of a prototype system

Mono- and disaccharides were covalently and irreversibly bound to aminopolystyrene beads in good yield by heating in dilute aqueous solution. The degree and stability of sugar binding were determined by chemical and radiochemical methods and the accessibility of the bound sugars was demonstrated by exoglycosidase hydrolysis and by an enzyme-linked lectin-binding assay using Concanavalin A.     

Bismuth(III) complexes with 2-acetylpyridine- and 2-benzoylpyridine-derived hydrazones: Antimicrobial and cytotoxic activities and effects on the clonogenic survival of human solid tumor cells

Complexes [Bi(2AcPh)Cl₂]·0.5H₂O (1), [Bi(2AcpClPh)Cl₂] (2), [Bi(2AcpNO₂Ph)Cl₂] (3), [Bi(2AcpOHPh)Cl₂]·2H₂O (4), [Bi(H2BzPh)Cl₃]·2H₂O (5), [Bi(H2BzpClPh)Cl₃] (6), [Bi(2BzpNO₂Ph)Cl₂]·2H₂O (7) and [Bi(H2BzpOHPh)Cl₃]·2H₂O (8) were obtained with 2-acetylpyridine phenylhydrazone (H2AcPh), its -para-chloro-phenyl- (H2AcpClPh), -para-nitro-phenyl (H2AcpNO₂Ph) and -para-hydroxy-phenyl (H2AcpOHPh) derivatives, as well as with the 2-benzoylpyridine phenylhydrazone analogues (H2BzPh, H2BzpClPh, H2BzpNO₂Ph, H2BzpOHPh).Upon coordination to bismuth(III) antibacterial activity against Gram-positive and Gram-negative bacterial strains significantly improved except for complex (4).The cytotoxic effects of the compounds under study were evaluated on HL-60, Jurkat and THP-1 leukemia, and on MCF-7 and HCT-116 solid tumor cells, as well as on non-malignant Vero cells. In general, 2-acetylpyridine-derived hydrazones proved to be more potent and more selective as cytotoxic agents than the corresponding 2-benzoylpyridine-derived counterparts.Exposure of HCT-116 cells to H2AcpClPh, H2AcpNO₂Ph and complex (3) led to 99% decrease of the clonogenic survival. The IC₅₀ values of these compounds were three-fold smaller when cells were cultured in soft-agar (3D) than when cells were cultured in monolayer (2D), suggesting that they constitute interesting scaffolds, which should be considered in further studies aiming to develop new drug candidates for the treatment of colon cancer.     

Application of a reaction model to improve calculation of the sugar recovery standard for sugar analysis

A kinetic model was applied to improve determination of the sugar recovery standard (SRS) for biomass analysis. Three sets of xylose (0.10-1.00 g/L and 0.999-19.995 g/L) and glucose (0.206-1.602 g/L) concentrations were measured by HPLC following reaction of each for 1 h. Then, parameters in a kinetic model were fit to the resulting sugar concentration data, and the model was applied to predict the initial sugar concentrations and the best SRS value (SRS(p)). The initial sugar concentrations predicted by the model agreed with the actual initial sugar concentrations. Although the SRS(e) calculated directly from experimental data oscillated considerably with sugar concentration, the SRS(p) trend was smooth. Statistical analysis of errors and application of the F-test confirmed that application of the model reduced experimental errors in SRS(e). Reference SRS(e) values are reported for the three series of concentrations.     

Improved activity retention of enzymes deposited on solid supports

Enzymes deposited on solid support usually show good stability when operated in organic solvents. Decreased stability of the enzyme preparations was noticed when low enzyme loadings were used (e.g., with Celite as support; less than 1 mg enzyme/g). It was possible to avoid the activity loss by the addition of an additive which protects the enzyme during the immobilization. Proteins (such as albumin, gelatin, and casein) and poly(ethylene glycol) were effective additives whereas amino acids, monomeric carbohydrates, and polysaccharides had no effect. The amount of additive needed for stabilization was shown to depend on the structure of the support, more additive being required for a support with high porosity. The stabilizing effect was investigated in a series of glyceryl-controlled-pore glass (CPG) with varying specific surface areas (9.5-180 m(2)/g). The minimum addition of albumin, giving full stabilization, on the different supports correlated to a monolayer coverage of the surface, approximately 2-3 mg protein/m(2). The effect of the additive was less pronounced when increasing amounts of enzyme were immobilized (5-40 mg enzyme/g Celite). The effect of the additives was studied using mandelonitrile lyase, but alpha-chymotrypsin and lipase P were also shown to be stabilized. (c) 1993 John Wiley & Sons, Inc.     

双酶偶联转化果糖制备含有稀少糖的混合糖液

酶转化法是功能性稀少糖生产的重要途径,但单一稀少糖转化酶的转化率普遍较低。文中提出构建双酶偶联转化系统提高转化效率的思路,即利用D-阿洛酮糖3-差向异构酶(D-psicose 3-epimerase,DPE)和L-鼠李糖异构酶(L-rhamnose isomerase,L-RhI)双酶偶联反应,催化D-果糖生成D-阿洛酮糖和D-阿洛糖等功能性稀少糖。DPE和L-RhI加酶量的比例为1∶10,其中DPE的浓度为0.05 mg/mL;转化反应的最佳温度为60℃,最适pH为9.0。当D-果糖浓度为2%时,反应10 h达到平衡,此时D-阿洛酮糖和D-阿洛糖的产量分别为5.12和2.04 g/L。利用文中提出的双酶偶联系统可以将果葡糖浆等富含果糖的低附加值原料转化为含有功能性稀少糖的高附加值混合糖液。     

The Arabidopsis SUCROSE UNCOUPLED-6 gene is identical to ABSCISIC ACID INSENSITIVE-4: involvement of abscisic acid in sugar responses

In plants, sugars act as signalling molecules that control many aspects of metabolism and development. Arabidopsis plants homozygous for the recessive sucrose uncoupled-6 (sun6) mutation show a reduced sensitivity to sugars for processes such as photosynthesis, gene expression and germination. The sun6 mutant is insensitive to sugars that are substrates for hexokinase, suggesting that SUN6 might play a role in hexokinase-dependent sugar responses. The SUN6 gene was cloned by transposon tagging and analysis showed it to be identical to the previously described ABSCISIC ACID INSENSITIVE-4 (ABI4) gene. Our analysis suggests the involvement of abscisic acid and components of the abscisic acid signal transduction cascade in a hexokinase-dependent sugar response pathway. During the plant life cycle, SUN6/ABI4 may be involved in controlling metabolite availability in an abscisic acid- and sugar-dependent way.     

Extra-weak chemiluminescence of drugs. XII. Effect of the molar ratio of amino acid to sugar on extra-weak chemiluminescence in the Maillard reaction

The extra-weak chemiluminescence in the Maillard reaction caused by the reaction between L -lysine and D -arabinose was measured, and a linear relationship was found between the chemiluminescence and the amount of L -lysine added. After a 1-hour reaction equimolar amounts of D -arabinose and L -lysine were consumed regardless of the initial concentration of D -arabinose. The chemiluminescence of the Maillard reaction originates from Maillard reaction products formed by the equimolar reaction between sugar and amino acid and depends on the concentration of amino acid.     

Antagonistic potential of certain legumes to Meloidgyne incognita on sugar beet

Under greenhouse conditions, a pot experiment was conducted to clarify the potential of using some legumes as intercropped plants for reducing the root-knot nematode Meloidogyne incognita infecting sugar beet (Beta vulgaris L.) cv. DS-9004 compared to non-legume plant, garlic and non-intercropped plants. The obtained results revealed that all legumes including chickpea, Egyptian clover, faba bean, fenugreek, lentil and lupin significantly (p ≤ 0.05) reduced nematode criteria on the roots of sugar beet at different degrees. Chickpea and Egyptian clover reduced the number of galls on the roots of sugar beet as the percentage of reductions were 54 and 50%, respectively, followed by lupin and fenugreek, while garlic achieved 72% reduction compared to non-intercropped plants. Lupin reduced the number of egg masses by 59% followed by Egyptian clover and fenugreek (32%), three months after the treatment. On the other hand, six months after the treatment, chickpea reduced the number of galls by 55.7% followed by lupin (53.4%) and Egyptian clover (52.3%) and the percentage of reduction of egg masses behaved the same trend. Also, the treatments improved plant growth criteria of sugar beet, weight of roots (tubers) and the percentage of total soluble solids (TSS).     

Reversible immobilization of cephalosporin C acylase on epoxy supports coated with polyethyleneimine

In this study, a recombinant cephalosporin C acylase (CCA) was covalently or physically immobilized on an epoxy-activated support LX1000-EPC4 (EP) or its derivatives, EP-polyethyleneimine (EP-PEI) and EP-ethylenediamine (EP-EDA) with cationic groups on the surface. Zeta potential was used as a tool for activated carrier analysis and immobilization analysis. The EP-PEI (the cationic polymer PEI grafted support) showed higher zeta potential than EP-EDA (the small diamine EDA modified support) and EP support. Among these three supports, immobilization of CCA on EP-PEI had the highest specific activity according to the range of enzyme loadings. Michaelis constant K_m values of EP-PEI-CCA and EP-EDA-CCA were 22?mM and 30?mM, respectively, which were lower than that of the free enzyme (43?mM), suggesting that the support’s zeta potential is related to the affinity of the enzyme for the substrate. The enzyme immobilized on EP-PEI showed a much higher thermal stability (stabilization factor of 32-fold compared with the free enzyme) than that on the EP-EDA (stabilization factor of 5.5-fold) and EP supports (stabilization factor of 1.7-fold). The adsorption of CCA on EP-PEI support was very strong and reversible. The CCA could be thoroughly desorbed using a high concentration of NaCl (e.g., 2 M) at low pH value (pH 3.0). The regenerated EP-PEI support could then be reused for enzyme immobilization.     

Protein immobilization to alumina supports: I. characterization of alumina-organophosphate ligand interactions and use in the attachment of papain

The chemical adsorption of organic phosphate compounds to alumina has been used to create surface linkers for protein immobilization. A number of particulate alumina supports were screened for their physical properties and ability to bind organic phosphate compounds. Two aluminas, termed C1 and CPC, were selected based on their suitability for subsequent testing as protein immobilization supports. Papain was successfully immobilized to these supports when derivatized with phosphate compounds containing free terminal carboxyl groups. Protein binding was enhanced when support carboxyl groups were activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The level of papain immobilization was dependent upon the length of the linker used and the mass of protein exposed to the support. (c) 1992 John Wiley & Sons, Inc.     

Bruising of sugar beet roots and consequential sugar loss: current understanding and research needs

Bruising of sugar beet roots and the consequential sugar loss do not receive the attention they deserve within the sugar beet industry. Recent harvester tests have indicated that current levels of bruising damage could be decreased with existing technology. There is, however, little understanding of biological factors affecting susceptibility to bruising of sugar beet roots. This paper examines the available information on causes of bruising, the significance of some tissue characteristics, the processes of sugar loss following bruising and agronomic, physiological and biochemical considerations relevant to bruising and the sugar loss that follows. Some research needs are identified in conclusion.     

Analysis of root images from auger sampling with a fast procedure: a case of application to sugar beet

Manual line-intersect methods for estimating root length are being progressively replaced by faster and more accurate image analysis procedures. These methods even allow the estimation of some more root parameters (e.g., diameter), but still require preliminary labour-intensive operations. Through a task-specific macro function written in a general-purpose image analysis programme (KS 300 – Zeiss), the processing time of root images was greatly reduced with respect to skeletonisation methods by using a high-precision algorithm (Fibrelength). This has been previously proposed by other authors, and estimates length as a function of perimeter and area of the digital image of roots. One-bit binary images were acquired, aiming at large savings in computer memory, and automatic discrimination of roots against extraneous objects based on their elongation index (perimeter²/area), was performed successfully. Of four tested spatial resolutions (2.9, 5.9, 8.8, 11.8 pixel mm^–1), in clean samples good accuracy in root length estimation was achieved at 11.8 pixel mm^–1, up to a root density of 5 cm cm^–2 on the scanner bed. This resolution is theoretically suitable for representing roots at least 85 m wide. When dealing with uncleaned samples, a thick layer of water was useful in speeding up spreading of roots on the scanner bed and avoiding underestimation of their length due to overlaps with organic debris. A set of fibrous root samples of sugar beet (Beta vulgaris var. saccharifera L.) collected at harvest over two years at Legnaro (NE Italy) was analysed by applying the above procedure. Fertilisation with 100 kg ha^–1 of nitrogen led to higher RLD (root length density in soil) in shallow layers with respect to unfertilised controls, whereas thicker roots were found deeper than 80 cm of soil without nitrogen.     

动态血糖监测在老年2型糖尿病患者降糖治疗中的应用价值

目的：通过对老年2型糖尿病患者进行动态血糖监测了解降糖治疗的疗效,评价动态血糖监测系统的应用价值,确定其在治疗老年2型糖尿病患者中的地位。方法：选取2008年8月至2013年8月住院的老年2型糖尿病患者95例,随机分为对照组48例和观察组47例,对照组行予常规的指尖血糖监测,观察组行动态血糖监测,比较两组患者血糖的控制情况。结果：观察组患者治疗后平均血糖、高血糖持续时间、低血糖持续时间、血糖最大波波动幅度、平均血糖波动幅度、血清糖化白蛋白及餐后2h血糖等与治疗前相比较比较,差异均有统计学意义,P〈0.05。对照组患者治疗后平均血糖、高血糖持续时间、低血糖持续时间、血糖最大波波动幅度、平均血糖波动幅度、血清糖化白蛋白及餐后2h血糖等与治疗前相比较比较,差异均无统计学意义,P〉0.05。两组患者空腹血糖治疗前、后差异均无统计学意义,P〉0.05。结论：动态血糖监测系统用于监测老年2型糖尿病患者的降糖治疗疗效优于常规血糖检测。     

动态血糖监测在老年2 型糖尿病患者降糖治疗中的应用价值

目的：通过对老年2 型糖尿病患者进行动态血糖监测了解降糖治疗的疗效,评价动态血糖监测系统的应用价值,确定其在 治疗老年2 型糖尿病患者中的地位。方法：选取2008 年8 月至2013 年8 月住院的老年2 型糖尿病患者95 例,随机分为对照组 48 例和观察组47 例,对照组行予常规的指尖血糖监测,观察组行动态血糖监测,比较两组患者血糖的控制情况。结果：观察组患 者治疗后平均血糖、高血糖持续时间、低血糖持续时间、血糖最大波波动幅度、平均血糖波动幅度、血清糖化白蛋白及餐后2h 血 糖等与治疗前相比较比较,差异均有统计学意义,P＜0.05。对照组患者治疗后平均血糖、高血糖持续时间、低血糖持续时间、血糖 最大波波动幅度、平均血糖波动幅度、血清糖化白蛋白及餐后2h血糖等与治疗前相比较比较,差异均无统计学意义,P＞0.05。两 组患者空腹血糖治疗前、后差异均无统计学意义,P＞0.05。结论：动态血糖监测系统用于监测老年2 型糖尿病患者的降糖治疗疗 效优于常规血糖检测。     

Binding of oxidized and reduced cytochrome c2 to photosynthetic reaction centers: plasmon-waveguide resonance spectroscopy

The dissociation constants for the binding of oxidized and reduced wild-type cytochrome c(2) from Rhodobacter capsulatus and the lysine 93 to proline mutant of cytochrome c(2) to photosynthetic reaction centers (Rhodobacter sphaeroides) has been measured to high precision using plasmon-waveguide resonance spectroscopy. For the studies reported, detergent-solubilized photosynthetic reaction center was exchanged into a phosphatidylcholine lipid bilayer to approximate the physiological environment. At physiologically relevant ionic strengths ( approximately 100 mM), we found two binding sites for the reduced wild-type cytochrome (K(D) = 10 and 150 nM), with affinities that decrease with decreasing ionic strength (2-5-fold). These results implicate nonpolar interactions as an important factor in determining the dissociation constants. Taking advantage of the ability of plasmon-waveguide resonance spectroscopy to reslove the contribution of changes in mass and of structural anisotropy to cytochrome binding, we can demonstrate very different properties for the two binding sites. In contrast, the oxidized wild-type cytochrome only binds to a single site with a K(D) of 10 nM at high ionic strength, and this site has properties similar to the low-affinity site for binding the reduced cytochrome. The binding of oxidized cytochrome c(2) has a strong ionic strength response, with the affinity decreasing approximately 30-fold in going from high to low ionic strength. The K93P mutant binds to a single site in both redox states, which is similar, in terms of mass and structural anisotropy, to the oxidized wild-type site, with the affinity of the mutant oxidized state being approximately 30-fold weaker than that of the oxidized wild-type cytochrome at high ionic strength. Thus, reduced wild-type cytochrome can bind to both the high- and low-affinity sites, while the oxidized wild-type cytochrome and both redox states of the mutant cytochrome can only bind to the low-affinity site, possibly the consequence of the more stable structure of reduced wild-type cytochrome. In aggregate, the results are consistent with a model in which a transient conformational change in the region 88-102 in the cytochrome three-dimensional structure, the so-called hinge region, drives the dissociation of the oxidized cytochrome from the reaction center-cytochrome complex, facilitating turnover.     

The amino‐terminal tail of Hxt11 confers membrane stability to the Hxt2 sugar transporter and improves xylose fermentation in the presence of acetic acid

Hxt2 is a glucose repressed, high affinity glucose transporter of the yeast Saccharomyces cerevisiae and is subjected to high glucose induced degradation. Hxt11 is a sugar transporter that is stably expressed at the membrane irrespective the sugar concentration. To transfer this property to Hxt2, the N‐terminal tail of Hxt2 was replaced by the corresponding region of Hxt11 yielding a chimeric Hxt11/2 transporter. This resulted in the stable expression of Hxt2 at the membrane and improved the growth on 8% d ‐glucose and 4% d ‐xylose. Mutation of N361 of Hxt11/2 into threonine reversed the specificity for d ‐xylose over d ‐glucose with high d ‐xylose transport rates. This mutant supported efficient sugar fermentation of both d ‐glucose and d ‐xylose at industrially relevant sugar concentrations even in the presence of the inhibitor acetic acid which is normally present in lignocellulosic hydrolysates. Biotechnol. Bioeng. 2017;114: 1937–1945. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Use of water to evaluate hydrophobicity of organically-modified xerogel enzyme supports

Silica xerogels are a new class of materials suitable for the immobilization of enzymes for various applications including biotransformations and biosensors. The physicochemical properties of xerogels, such as hydrophobicity, can be manipulated by the introduction of organically-modified silicates. This allows the immobilization matrix to be engineered to suit the enzyme and its application. Interfacial activation of lipase is a phenomenon in which the enzyme displays increased activity when it is bound to a hydrophobic interface. Lipase was entrapped in organically-modified xerogels in which the hydrophobicity of the enzyme support was modulated by the selection of different alkyltrimethoxysilane co-precursors and the ratio in which they were combined with tetramethyl orthosilicate. Interaction between the enzyme support and water was investigated with two methods to quantitatively assess the hydrophobicity of the entrapment matrix. The contact angle formed between the xerogel and water was used to determine hydrophobicity on a macroscopic level. Temperature-controlled water desorption was used to determine hydrophobicity on a microscopic level. Both methods were suitable for quantitatively discriminating between hydrophobic and hydrophilic materials. Further, the hydrophobicity of the enzyme support influenced the hydrolytic activity of the entrapped lipase under non-aqueous conditions. The specific activity of lipase increased only when entrapped in xerogels which could be classified as hydrophobic materials, that is with contact angles greater than 90 degrees or hydrophobicity values as determined by water desorption greater than 0.65.     

Studies on sugar isomerases of Lactobacillus sp.: Whole cell immobilization of d-glucose isomerase

A new soil isolate of Lactobacillus sp. grown in Yamanaka medium under submerged conditions showed the presence of d-glucose, d-xylose and d-ribose isomerases in washed cell suspension and cell free extracts. d-Xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) and d-ribose isomerase (d-ribose ketol-isomerase, EC 5.3.1.20) activities reached a maximum in 48 h of growth and then declined. d-Glucose isomerase (d-glucose 6-phosphate isomerase, d-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) activity was maximum after 72 h and remained constant for ～120 h of growth. d-Glucose isomerase activity increased with the increase in number of generations of culture and reached a maximum in 5–6 generations, whereas d-xylose and d-ribose isomerase activities decreased. The washed and starved whole cells could be heat treated and immobilized on the rough surface of glass rods or glass slides using acetone treatment. The heat treated immobilized cells showed only the presence of d-glucose isomerase activity and showed no d-xylose and d-ribose isomerase activities. d-Glucose isomerase activity of heat treated immobilized cells was inhibited less by sorbitol, mannitol, sodium arsenate, cysteine and calcium ions than the free d-glucose isomerase activity in fresh untreated washed whole cells and cell free extracts. EDTA inhibition had the same effect for both forms. Ca²⁺inhibition could be reversed by adding Mg²⁺ions.     

Metabolic regulation of leaf senescence: interactions of sugar signalling with biotic and abiotic stress responses

Sugars are important signals in the regulation of plant metabolism and development. During stress and in senescing leaves, sugars often accumulate. In addition, both sugar accumulation and stress can induce leaf senescence. Infection by bacterial and fungal pathogens and attack by herbivores and gall-forming insects may influence leaf senescence via modulation of the sugar status, either by directly affecting primary carbon metabolism or by regulating steady state levels of plant hormones. Many types of biotic interactions involve the induction of extracellular invertase as the key enzyme of an apoplasmic phloem unloading pathway, resulting in a sourcesink transition and an increased hexose/sucrose ratio. Induction of the levels of the phytohormones ethylene and jasmonate in biotic interactions results in accelerated senescence, whereas an increase in plant- or pathogen-derived cytokinins delays senescence and results in the formation of green islands within senescing leaves. Interactions between sugar and hormone signalling also play a role in response to abiotic stress. For example, interactions between sugar and abscisic acid (ABA) signalling may be responsible for the induction of senescence during drought stress. Cold treatment, on the other hand, can result in delayed senescence, despite sugar and ABA accumulation. Moreover, natural variation can be found in senescence regulation by sugars and in response to stress: in response to drought stress, both drought escape and dehydration avoidance strategies have been described in different Arabidopsis accessions. The regulation of senescence by sugars may be key to these different strategies in response to stress.