Effect of polygalacturonase and feruloyl esterase from Aspergillus tubingensis on demucilage and quality of coffee beans

To explore the potential for the application of Aspergillus tubingensis enzyme extract in coffee processing, the effects of crude enzymes on coffee beans were studied. Yields of polygalacturonase and feruloyl esterases obtained from solid-state fermentation using A. tubingensis, with pectin and de-starched wheat bran as carbon sources, were 15.9 U/mL and 2.44 U/mL, respectively. Crude enzyme extracts removed the mucilage of coffee cherries within 3 h, which is substantially more efficient than traditional fermentation. Additionally, the viscosity of coffee mucilage was reduced to 80% by a 3-h treatment with the crude enzyme extract at 50 °C. The titratable acidity and organic acids in coffee beverages were also decreased to half the amounts of those in the traditionally fermented group. The total chlorogenic acid in the green beans decreased to 67.3%; however, a decline of only 14.3% was observed in the traditionally fermented group. On the other hand, chlorogenic acid lactones in the roasted beans were reduced to 63.9%, and a 37.2% decline in the chlorogenic acid content was detected.     

阿魏酸酯酶A的基因克隆与表达及其水解产物的纯化

为在毕赤酵母中表达来源于米曲霉Aspergillus oryzae的A型阿魏酸酯酶并研究其水解功能,探讨大孔树脂对其水解产物阿魏酸的纯化条件及纯化效果,以米曲霉A.oryzae CICC 40186总RNA为模板,通过RT-PCR技术克隆出了米曲霉阿魏酸酯酶A(AorFaeA)成熟肽的编码基因AorfaeA,并借助pPIC9K质粒实现了其在毕赤酵母GS115中的异源表达。SDS-PAGE分析结果显示纯化后的重组阿魏酸酯酶(reAorFaeA)为单一条带,其表观分子质量约39.0 kDa。以阿魏酸甲酯为底物,经高效液相色谱法测得该酶的最高酶活为58.35 U/mg。利用reAorFaeA和木聚糖酶复合酶水解去淀粉麦麸制备阿魏酸,用大孔树脂纯化小麦麸皮阿魏酸粗提液,所测树脂中HPD-300型大孔树脂的吸附量和解吸率较高,以50%的乙醇为洗脱液,当流速为1.0 mL/min时洗脱效果较好。在该纯化条件下,阿魏酸的回收率为92%,质量分数由原材料中的0.13%富集提高到10.55%。这些研究为阿魏酸的酶法"绿色生产"及应用奠定了坚实的理论基础。     

Molecular aspects of hereditary spastic paraplegia

Hereditary spastic paraplegia (HSP) is a clinically and genetically heterogeneous group of neurodegenerative disorders characterized by progressive lower limbs spasticity and weakness. What was first thought to be a small group of rare Mendelian disorder has now become a large group that includes many complex syndromes. While large families with defined modes of inheritance were used for the initial HSP gene discovery, new sequencing technologies have recently allowed the study of small families, with the identification of many new disease causative genes. These discoveries are slowly leading to a better understanding of the molecular mechanisms underlying HSP with the identification of precise disease pathways. These insights may lead to new therapeutic strategies for what is a group of largely untreatable diseases. This review looks at the key players involved in HSP and where they act in their specific pathways.     

Synthesis of 4-nitrophenyl caffeate and its use in assays of caffeoyl esterases

We have prepared 4-nitrophenyl caffeate by a combination of standard procedures of organic synthesis and enzymatic deacetylation. Based on hydrolysis of 4-nitrophenyl caffeate, a convenient spectrophotometric assay was developed for specific monitoring of caffeoyl esterase. The method is fast and easy to perform, and it requires no expensive equipment. Its reliability was tested on eight enzyme preparations comprising various combinations of caffeoyl, feruloyl, and acetyl esterase as well as protease activities.     

Non-lipolytic and lipolytic sequence-related carboxylesterases: A comparative study of the structure–function relationships of rabbit liver esterase 1 and bovine pancreatic bile-salt-activated lipase

To differentiate esterases from lipases at the structure–function level, we have compared the kinetic properties and structural features of sequence-related esterase 1 from rabbit liver (rLE) and bile-salt-activated lipase from bovine pancreas (bBAL). In contrast to rLE, bBAL hydrolyses water-insoluble medium and long chain esters as vinyl laurate, trioctanoin and olive oil. Conversely, rLE and bBAL are both active on water-soluble short chain esters as vinyl acetate, vinyl propionate, vinyl butyrate, tripropionin, tributyrin and p-nitrophenyl butyrate. However, the enzymes show distinctive kinetic behaviours. rLE displays maximal activity at low substrate concentration, below the critical micelle concentration, whereas bBAL acts preferencially on emulsified esters, at concentration exceeding the solubility limit. Comparison of the 3D structures of rLE and bBAL shows, in particular, that the peptide loop at positions 116–123 in bBAL is deleted in rLE. This peptide segment interacts with a bile salt molecule thus inducing a conformational transition which gives access to the active site. Inhibition studies and manual docking of a bulky ester molecule as vinyl laurate in the catalytic pocket of rLE and bBAL show that the inability of the esterase to hydrolyse large water-insoluble esters is not due to steric hindrance. It is hypothesized that esterases lack specific hydrophobic structures involved both in the stabilization of the lipase–lipid adsorption complex at interfaces and in the spontaneous transfer of a single substrate molecule from interface to the catalytic site.     

Cell Lysis of Cyanobacteria and Its Implications for Nutrient Dynamics

The dynamics of nutrients, such as phosphorus, nitrogen, and carbohydrates, during cyanobacteria cell lysis was investigated under darkness incubation in the laboratory. The cell lysis rate of cyanobacteria sampled from Lake Taihu was measured using an esterase assay. Based on particulate esterase activity, the calculated cyanobacteria lysis rate was 0.094 d^–1. During 30 days of darkness incubation, Chlorophyll a concentration decreased from 56 μg L^–1 to 2.0 μg L^–1. Parallel to this, total particulate carbohydrate concentration decreased rapidly. The fluctuation of dissolved organic carbon concentration was a function of the production of non‐carbohydrate by cyanobacteria and the decomposition of carbohydrate by bacteria. Total dissolved carbohydrates and dissolved polysaccharides concentrations showed a similar pattern, declining at the beginning of the experiment and keeping relatively stable, thereafter. In contrast, the concentration of dissolved monosaccharides remained constant during the entire process. The concentrations of NH₄⁺ and PO₄^3– increased at the early stage, and then decreased afterwards. A gradual decrease in NO₃^– concentration after day 8 indicated that anaerobic conditions might be produced during the cell lysis process. The present results demonstrated cyanobacteria cell lysis has a big influence on the nutrient status of the surrounding water. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Further Study on the Esterase Patterns of Sibling Species in the Drosophila saltans Subgroup (saltans Group): Intraspecific and Interspecific Variations in the Development

Twenty of the 32 esterase bands previously detected in the adults of D. prosaltans, D. saltans and D.␣austrosaltans were found in larvae and pupae studied in this work. The results showed that, in addition to expressing the highest number of esterase bands, the adult stage of the three species exhibited the highest degree of expression (amount of synthesis) for most of the bands. Differences between larval and pupal stages were detected in the degree of expression (amount of synthesis) of the bands and in the frequency of samples expressing them. The frequencies of expression of the bands corresponding to genes in loci 1–3 were greater in pupae than in larvae while the frequencies of expression of the bands corresponding to genes in loci 4–9 were predominantly expressed in larvae or were equal in both developmental stages. Like the adults, larvae, pupae and empty pupal cases (which were also studied in this work) showed specific esterases. Taken together, the observations showed that, in the species studied, every developmental stage is characterized by specific bands and by specific frequency and degree of expression of the bands shared with other stages.     

猪肝酯酶研究进展

猪肝酯酶是手性合成中重要的水解酶,对它的生化特性研究较早。近几年猪肝酯酶的基因克隆研究也取得了较大进展。重组酶具有选择特异性更高的特点,与生化提取方法相比成本较低廉。基因工程技术的开展使猪肝酯酶在化学工业、生物制药及相关领域中有了更加广泛的应用前景．     

Preparation of enantiomerically pure (S)-flurbiprofen by an esterase from Pseudomonas sp. KCTC 10122BP

Esterase PF1-K from Pseudomonas sp. KTCC 10122BP was overproduced by the fed-batch culture of Escherichia coli. The soluble expression of esterase PF1-K was achieved by shifting the culture temperature from 37 to 25 °C at the time of IPTG induction. The enzyme was partially purified to about 75% purity by a single-step hydrophobic interaction column chromatography. The purified enzyme exhibited a fairly high enantioselectivity towards the hydrolysis of rac-flurbiprofen ethyl ester. The enzymatic chiral resolution was further improved by optimizing the reaction conditions in terms of reaction rate and enantioselectivity. The optimal reaction conditions were found to be 40 °C, pH 10.5 and 600 mM of initial rac-flurbiprofen ethyl ester. After 90 min of batch reaction under the optimal conditions, 50% of the initial rac-flurbiprofen was hydrolyzed with an enantiomeric excess of 99%.     

苜蓿组织培养中球形胚发生时特异蛋白质和同工酶分析

试验在苜蓿组织培养中,对球形胚形成过程中特异蛋白质表达的模式、过氧化物酶及酯酶同工酶酶谱变化进行研究,结果表明：苜蓿组织培养中从胚性愈伤组织到球形胚发育的进程中,顺序消失和出现了11种中小分子量多肽;过氧化物酶同工酶酶谱发生了显著的变化;酯酶同工酶酶谱变化不大,但其总活力对于维持体细胞胚胎发生是必须的。