4.3 Degradation and modification of cellulose acetates by biological systems

A survey is given on recent findings in the enzymology of cellulose acetate degradation. Acetyl esterases have been identified as the principal enzymes, initiating cellulose acetate degradation as a prerequisite for endoglucanase-catalyzed cellulose acetate depolymerisation. Acetyl esterases are provided by nature to deacetylate naturally occurring partly acetylated polysaccharides, i.e. xylan and chitin. Accordingly they are not designed to attack high DS cellulose acetate. Under these circumstances acetyl esterases require a pretreatment of cellulose acetate, leading to some reduction in DS, in case highly substituted material should be degraded. One of these treatments is composting under the conditions of which a partial deacetylation may occur under the action of heat and high pH, facilitating the accessibility for acetyl esterases. However from the present knowledge it cannot be excluded that certain microbial specialists exist, being capable to degrade high DS cellulose acetate.     

应用激光解吸质谱和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析蛇毒精氨酸酯酶的纯度和分子量

应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)和SDS-聚丙烯酰胺凝胶电泳对吉林省两地市的同种白眉蝮蛇蛇毒中具有抗栓塞药效的精氨酸酯酶进行了分析和比较。MALDI-TOF-MS法具有快速、准确度高、灵敏度高的优点,两种方法结合,互为补充,取得了令人满意的结果,MALDI-TOF-MS完全可以直接用作蛇毒成分分离过程中重要的研究手段。     

An eleven amino acid residue deletion expands the substrate specificity of acetyl xylan esterase II (AXE II) from <Emphasis Type="Italic">Penicillium purpurogenum</Emphasis>

The soft-rot fungus Penicillium purpurogenum secretes to the culture medium a variety of enzymes related to xylan biodegradation, among them three acetyl xylan esterases (AXE I, II and III). AXE II has 207 amino acids; it belongs to family 5 of the carbohydrate esterases and its structure has been determined by X-ray crystallography at 0.9 A resolution (PDB 1G66). The enzyme possesses the alpha/beta hydrolase fold and the catalytic triad typical of serine esterases (Ser90, His187 and Asp175). AXE II can hydrolyze esters of a large variety of alcohols, but it is restricted to short chain fatty acids. An analysis of its three-dimensional structure shows that a loop that covers the active site may be responsible for this strict specificity. Cutinase, an enzyme that hydrolyzes esters of long chain fatty acids and shows a structure similar to AXE II, lacks this loop. In order to generate an AXE II with this broader specificity, the preparation of a mutant lacking residues involving this loop (Gly104 to Ala114) was proposed. A set of molecular simulation experiments based on a comparative model of the mutant enzyme predicted a stable structure. Using site-directed mutagenesis, the loop's residues have been eliminated from the AXE II cDNA. The mutant protein has been expressed in Aspergillus nidulans A722 and Pichia pastoris, and it is active towards a range of fatty acid esters of up to at least 14 carbons. The availability of an esterase with broader specificity may have biotechnological applications for the synthesis of sugar esters.     

Improvement of electrophoretic detection of antitrypsin activity in fish blood and seminal plasma

The original method of Uriel and Berges for detection of trypsin inhibitors lacks specificity due to masking effects of nonspecific esterases. We report a modification of this method based on inhibition of esterases in samples by phenylmethylsulfonyl fluoride (PMSF). This method can be particularly useful for characterization profiles of antitrypsin activity in seminal plasma of salmonid fish where esterases and inhibitors migrate at the same mobility.     

Insight into Substrate Preference of Two Chimeric Esterases by Combining Experiment and Molecular Simulation

Better understanding of the relationship between the substrate preference and structural module of esterases is helpful to novel enzyme development. For this purpose, two chimeric esterases AAM7 and PAR, constructed via domain swapping between two ancient thermophilic esterases, were investigated on their molecular simulation(including homology modeling, substrates docking and substrate binding affinity validation) and enzymatic assay(specific activities and activation energies calculating). Our results indicate that the factors contributing to the substrate preference of many enzymes especially the broad-specificity enzymes like esterases are multiple and complicated, the substrate binding domains or binding pockets are important but not the only factor for substrate preference.     

Trimethyl lock: a stable chromogenic substrate for esterases

p-Nitrophenyl acetate is the most commonly used substrate for detecting the catalytic activity of esterases, including those that activate prodrugs in human cells. This substrate is unstable in aqueous solution, limiting its utility. Here, a stable chromogenic substrate for esterases is produced by the structural isolation of an acetyl ester and p-nitroaniline group using a trimethyl lock moiety. Upon ester hydrolysis, unfavorable steric interactions between the three methyl groups of this o-hydroxycinnamic acid derivative encourage rapid lactonization to form a hydrocoumarin and release p-nitroaniline. This "prochromophore" could find use in a variety of assays.     

Micro two-dimensional gel electrophoresis of serum and testis esterases from different strains of mice

Two-dimensional electrophoresis with time-dependent polyacrylamide gradient gel electrophoresis (PAGGE) in the second dimension was applied to the separation of native molecular forms of esterases from serum and testis of four strains of mice (C57BL/6J, Swiss OF1, F1 hybrid derived from these two populations and Tfm). In Phast System, a modified pH 3-9 gradient, a linear 8-25% gel gradient and a migration time corresponding to 300 Vh, were found to provide the best conditions for esterase analysis. About 70 esterase-active fractions could be separated with good reproducibility. The variants were characterized by their pI (3.9-7.35), their relative mobility and the visual estimation of their susceptibility towards neuraminidase and different esterase inhibitors. In the two tissues, the distribution of the esterase variants corresponded to a 50-500 kDa molecular mass range of calibration proteins, but most of the serum and testis-specific isoforms were confined to the 59-72 kDa range. All serum variants contained a terminal N-acetylneuraminic acid residue, whereas only the testicular esterases in common with those in serum appeared sensitive to neuraminidase. Cholinesterases with a low relative mobility and carboxylesterases with a high relative mobility were detected in serum, while carboxylesterases accounted for the greatest part in the testis which also contained cholinesterases and acetylesterases. Minor interspecies differences were found between C57BL/6J and Swiss OF1 esterases. The expression of two variants which differed between these two species seemed intermediate for the hybrid originating from these two populations. Two new spots were detected in the two-dimensional map of esterases from the strain bearing the Tfm mutation.     

Mapping the substrate selectivity of new hydrolases using colorimetric screening: lipases from Bacillus thermocatenulatus and Ophiostoma piliferum,esterases from Pseudomonas fluorescens and Streptomyces diastatochromogenes

Recent advances in biochemistry and molecular biology have simplified the discovery and preparation of new hydrolases. Although these hydrolases might solve problems in organic synthesis, measuring their selectivity, especially enantioselectivity, remains tedious and time consuming. Recently, we developed a colorimetric screening method to measure the enantioselectivity of hydrolases. Here we apply this rapid screening method to map the substrate selectivity of four new hydrolases: lipases from the thermophilic Bacillus thermocatenulatus (DSM 730, BTL2) and a filamentous fungus Ophiostoma piliferum (NRRL 18917, OPL) and esterases from two bacteria, Pseudomonas fluorescens (SIK-W1, esterase I, PFE) and Streptomyces diastatochromogenes (Tü 20, SDE). We screened a general library of 29 substrates and a chiral library of 23 pairs of enantiomers. All four hydrolases catalysed the hydrolysis of unnatural substrates, but the two lipases accepted a broader range of substrates than the two esterases. As expected, the two lipases favoured more hydrophobic substrates, while the two esterases showed a preference for smaller substrates. Several moderately enantioselective reactions were identified for the solketal esters: BTL2, butyrate, E=7.9 (R); octanoate, E=4.9 (R) and 3-bromo-2-methyl propionate methyl esters, PFE, E=12 (S); SDE, E=5.6 (S). OPL showed low enantioselectivity toward all substrates tested. The current colorimetric screen could not measure the selectivity for several slow-reacting substrates. Traditional screening identified high enantioselectivity of BTL2 and PFE toward one of these slow substrates, 1-phenylethyl acetate (E>50).     

Probing lipase/esterase libraries for lipid A hydrolases--discovery of biocatalysts for the detoxification of bacterially-expressed recombinant protein

In our ongoing efforts to develop new methods for lipopolysaccharide (LPS) detoxification, we have screened lipase/esterase libraries for the ability to deacylate the 2'- and 3'-fatty acid chains from lipid A: the most active esterases were successfully employed to inactivate LPSs in a crude concentrated cell supernatant of E. Coli containing a recombinant single chain antibody (scFv).     

用于测量农药残留的小麦酯酶的选择

为研制探测农药残留的生物传感器，研究了农药乐果[O,O-二甲基S－（N－甲基胺基甲酰甲戎）二硫代磷酸酯]对各种小麦植物酯酶的抑制，从小麦中提取植物酶，以农药乐果为抑制剂，采用分光光度法研究了乐果对各种小麦酯酶活性的影响；研究显示，不同品种小麦酯酶对农药乐果的敏感度不同，在所研究的品种中，豫麦39和小麦周9对乐果较敏感，研究结果说明了选择小麦酶源的必要性。     

Diazo compounds for the bioreversible esterification of proteins

A diazo compound is shown to convert carboxylic acids to esters efficiently in an aqueous environment. The basicity of the diazo compound is critical: low basicity does not lead to a reaction but high basicity leads to hydrolysis. This reactivity extends to carboxylic acid groups in a protein. The ensuing esters are hydrolyzed by human cellular esterases to regenerate protein carboxyl groups. This new mode of chemical modification could enable the key advantages of prodrugs to be translated from small-molecules to proteins.     

The synthesis of antibiotic sugars

The term antibiotic sugar denotes the carbohydrate component of an antibiotic. Two alternative approaches are adopted for the synthesis of these neutral and amino sugars, many of which have unusual structures: One route starts from fairly simple non-carbohydrate precursors, the other depending upon the modification of a carbohydrate precursor.     

Physangulidines A, B, and C: three new antiproliferative withanolides from Physalis angulata L

Bioassay-directed fractionation of the whole plant of Physalis angulata L. afforded three new antiproliferative withanolides with an unusual carbon framework: physangulidines A (1), B (2), and C (3). Structures of the three isomeric withanolides were determined by a combination of HRMS, NMR spectroscopic, and X-ray crystallographic methods. Each has shown significant antiproliferative activity against DU145 prostate cancer cells. Physangulidine A (1) was further tested against a wide range of additional cancer cell lines and found to exhibit significant antiproliferative activity.     

A One-Pot Approach to Multi-Substituted Fused Aziridines: Formal Intermolecular [2+1] Cycloaddition Reaction between Saccharin-Derived Cyclic Ketimines and Sulfur Ylides

Herein a concise and efficient method for the synthesis of aziridines has been developed. The reaction proceeds through a formal intermolecular [2+1] cycloaddition reaction of saccharin-derived cyclic ketimines with sulfur ylides. This methodology features high atom-economy, broad substrate scope and an operationally simple procedure, affording the aziridines with excellent yields and diastereoselectivity (>20 : 1 dr). In the presence of base, the aziridine products could be readily converted to benzothiazines by an unusual rearrangement reaction.     

Macrocyclic sulfamides: synthesis, hybridization, and metal binding properties

Four cyclophanes incorporating the cyclosulfamide sub-unit have been synthesised in high yield. The X-ray crystal structures of three of them, and of cyclosulfamide itself, provide useful insight into the hybridisation of such compounds. The ionophoric properties of the macrocycles are also reported, with the sulfamides showing unusual selectivity for rubidium (benzyl trimer), barium (pyridyl dimer) and silver (pyridyl dimer and trimer).     

Anticancer Aminoferrocene Derivatives Inducing Production of Mitochondrial Reactive Oxygen Species

Elevated levels of reactive oxygen species (ROS) and deficient mitochondria are two weak points of cancer cells. Their simultaneous targeting is a valid therapeutic strategy to design highly potent anticancer drugs. The remaining challenge is to limit the drug effects to cancer cells without affecting normal ones. We have previously developed three aminoferrocene (AF)-based derivatives, which are activated in the presence of elevated levels of ROS present in cancer cells with formation of electron-rich compounds able to generate ROS and reduce mitochondrial membrane potential (MMP). All of them exhibit important drawbacks including either low efficacy or high unspecific toxicity that prevents their application in vivo up to date. Herein we describe unusual AF-derivatives lacking these drawbacks. These compounds act via an alternative mechanism: they are chemically stable in the presence of ROS, generate mitochondrial ROS in cancer cells, but not normal cells and exhibit anticancer effect in vivo.     

掺铥硫氧化钇的特殊余辉性质

迄今为止,稀土长余辉磷光体已见文献或专利公开报道的激活离子主要有适于紫外光激发的三价铈离子(Ce³⁺)和三价镨离子(Pr³⁺)、适于可见光激发的铕离子(Eu³⁺和Eu²⁺)及钐离子(Sm³⁺,Sm²⁺),尚未涉及到铥离子Tm³⁺或Tm²⁺.我们在Tm³⁺离子激活的硫氧化钇体系Y₂O₂S:Tm³⁺中发现了长余辉荧光特性.特别是在该磷光体中还发现了一种非常特殊的余辉现象.     

Novel etching phenomena in poly(3‐hydroxy butyrate) and poly(oxymethylene) spherulites

Poly‐3‐hydroxy butyrate has been etched and studied under scanning and transmission electron microscopes. It displays three of the following unusual features: (1) spherulites develop in a loose spiral rather than radial structure, which appears to reflect the chiral nature of the polymer; (2) in the banded spherulitic structure, lamellae oriented flat‐on to the surface are etched more deeply in relation to edge‐on lamellae; and (3) material crystallized at high temperature is less resistant to etching than that crystallized at low temperature, whereas the most rapid rate of etching appears to be where growth occurred at an intermediate temperature where the growth rate was at its maximum. The second and third phenomena are contrary to what is found in polymers such as polyethylene and polyethylene terephthalate and are attributed to excess free volume in the material located between the main lamellar bundles. Polyoxymethylene also displays the same unusual relationship of etching rate with crystallization temperature. © 2001 John Wiley & Sons, Inc. J Polym Sci Part B: Polym Phys 40: 124–133, 2002     

Isoelectric focusing in immobilized pH gradients of phosphoglucomutase and esterases from the spiny lobster

A method is described for detecting polymorphisms of cephalothorax and tail homogenates of 25 puerulus staged Panulirus argus in phosphoglucomutase (PGM) and esterases. Isoelectric focusing in immobilized pH gradients was used. In the pH 6.0-8.0 interval for phosphoglucomutase and in the pH 3.5-5.0 and 4.2-4.9 ranges for esterases, both enzymes appeared as polymorphic band patterns. These could be explained by one locus with 2 alleles for phosphoglucomutase and 3 loci with 2, 3 and 4 alleles for esterases. Esterases exhibit a more extensive polymorphism in immobilized pH gradients than in polyacrylamide gel electrophoresis.     

Lewis acid-catalyzed rearrangement of 2,2-dichloronorbornane to 1-chloronorbornane

The mechanism for the unusual AlCl(3)-catalyzed rearrangement of 2,2-dichloronorbornane to 1-chloronorbornane in pentane has been elucidated; the reaction, which also yields four isomeric dichloronorbornanes, occurs in three steps: (1). ionization to form the 2-chloro-2-norbornyl cation, which was fully characterized by two-dimensional (1)H and (13)C NMR in SbF(5)/SO(2)ClF; (2). Wagner-Meerwein shift to yield the 1-chloro-2-norbornyl cation, which was partially characterized by (1)H NMR; and (3). hydride abstraction.