Purification and biochemical characterization of feruloyl esterases from Aspergillus terreus MTCC 11096

Aspergillus terreus MTCC 11096 isolated from the soils of agricultural fields cultivating sweet sorghum was previously identified to produce feruloyl esterases (FAEs). The enzymes responsible for feruloyl esterase activity were purified to homogeneity and named as AtFAE‐1, AtFAE‐2, and AtFAE‐3. The enzymes were monomeric having molecular masses of 74, 23 and 36 kDa, respectively. Active protein bands were identified by a developed pH‐dependent zymogram on native PAGE. The three enzymes exhibited variation in pH tolerance ranging between pH 5–8 and thermostability of up to 55°C. Inhibition studies revealed that the serine residue was essential for feruloyl esterase activity; moreover aspartyl and glutamyl residues are not totally involved at the active site. Metal ions such as Ca²⁺, K⁺, and Mg²⁺ stabilized the enzyme activity for all three FAEs. Kinetic data indicated that all three enzymes showed catalytic efficiencies (k_cat/K_m) against different synthesized alkyl and aryl esters indicating their broad substrate specificity. The peptide mass fingerprinting by MALDI/TOF‐MS analysis and enzyme affinity toward methoxy and hydroxy substituents on the benzene ring revealed that the AtFAE‐1 belonged to type A while AtFAE‐2 and AtFAE‐3 were type C FAE. The FAEs could release 65 to 90% of ferulic acid from agrowaste substrates in the presence of xylanase. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:924–932, 2013     

Improved enantioselectivity of thermostable esterase ST0071 from archaeon Sulfolobus tokodaii by site-saturation mutagenesis

An archaeon GGG(A)X-type esterase (ST0071) can catalyze the hydrolysis of various acetates of secondary alcohols, but shows low enantioselectivity. Using structure-guided site-saturation mutagenesis, we successfully identified a G274W variant that has excellent selectivity compared with that of wild-type ST0071.     

Heterochiral tetrapeptide self-assembly into hydrogel biomaterials for hydrolase mimicry

Self-assembling short peptides have attracted great interest as enzyme mimics, especially if the catalytic activity resides solely in the supramolecular structure so that it can be switched on/off as needed by controlling assembly/disassembly. Among the various enzyme classes, hydrolases find wide application in biomaterials, and their mimetics often contain His residues, in addition to either divalent cations or other amino acids to mimic the catalytic site. This work reports two self-assembling tetrapeptides based on the Ser-His motif for catalysis and the Phe-Phe motif to drive amyloid structure formation. Both peptides form thermoreversible hydrogels in phosphate buffer at neutral pH that display a mild esterase-like activity, as demonstrated on the hydrolysis of 4-nitrophenyl acetate as a model substrate, although presence of Ser did not enhance catalytic activity. The systems are characterised by circular dichroism, transmission electron microscopy, oscillatory rheology and Thioflavin T fluorescence as an amyloid stain, to provide further insights that may assist the future design of improved supramolecular catalysts.     

寄主植物对斜纹夜蛾酯酶和乙酰胆碱酯酶活性的影响

利用生物测定与生物化学的方法,就取食不同寄主植物的2个斜纹夜蛾[Spodoptera litura(Fab.)]品系对丙溴磷和灭多威的敏感性及其体内的酯酶与乙酰胆碱酯酶的活性变化进行了研究。结果表明,取食不同寄主植物的斜纹夜蛾对丙溴磷和灭多威的敏感性不同。在敏感品系中,取食不同食料的斜纹夜蛾后代对灭多威的敏感性顺序为:烟草<棉花<大豆<人工饲料;对丙溴磷而言,敏感性顺序则为:棉花<烟草<大豆<人工饲料。而在田间抗性品系中,对丙溴磷的敏感性顺序为:棉花<烟草<人工饲料<大豆;对灭多威的敏感性顺序为:棉花<烟草<大豆<人工饲料。此外,在田间抗性和敏感品系中,取食不同食料的斜纹夜蛾体内的酯酶活性之间均存在显著差异,对其乙酰胆碱酯酶的活性也有程度不同的诱导效应,但并不会引起乙酰胆碱酯酶的质变。     

Esterases as a marker for growth of BY-2 tobacco cells and early somatic embryos of the Norway spruce

Growth is one of the basic properties of biological systems. The methods which are commonly used for the determination of growth are usually difficult and not very accurate. In the present work we decided to use esterase activity as a growth marker in tobacco suspension culture (BY-2 line) and in early somatic embryos of Norway spruce (clone 2/32) grown on a semi-solid medium. Esterase activity correlates well with the classical growth characteristics of BY-2 and spruce early somatic embryos. Determination of esterase activity is based on spectrophotometric and spectrofluorimetric detection of reaction products, which arise from the enzymatic hydrolysis of two substrates (p -nitrophenyl acetate and fluorescein diacetate) by esterase. The spectrophotometric method enabled us to detect approximately 10⁴ BY-2 cells and 25 spruce embryos whereas the more sensitive spectrofluorimetric method allowed us to detect approximately 800 BY-2 cells and 5 early somatic embryos of Norway spruce.     

Ultrastructural and ultracytochemical study of the middle ear epithelium in the chicken,Gallus gallus domesticus

The fine structure of the epithelium lining the tympanic cavity of the chicken was studied by TEM and SEM. In addition, the distribution of nonspecific esterase activity in the epithelium was investigated by TEM. Ultrastructural study revealed the presence of disk-like apical protrusions of the epithelial cells, previously not observed in other cell types. The protrusions contained some cytoplasmic organelles and were characterized by a ring-shaped thickening around their periphery. The ring was made up of a granulo-filamentous material. Our observations clearly indicate the existence of an apocrine secretory mechanism, consisting of a progressive detachment of disk-like protrusions from the apex of the epithelial cells. The ultracytochemical study demonstrated nonspecific esterase activity on the epithelial surface and in the secretory vesicles. We propose that nonspecific esterase is a marker for middle ear surfactant in birds.     

Exogenous melatonin affects lipids and enzyme activities in mink (Mustela vison) liver

Exogenous melatonin as subcutaneous 2.7-mg implants was given to eight female and male minks in late July with an equal number of animals in the control groups. The liver enzyme activities and major lipids of liver and plasma were measured in October-November. Melatonin had very pronounced effects on the lipid and carbohydrate metabolism of the minks and there was also a clear sexual dimorphism. In the males, melatonin decreased the lipase esterase activity of the liver. In the liver of the females, however, melatonin increased the glucose-6-phosphatase activity. Due to melatonin treatment the liver triacylglycerol contents diminished in both sexes. At the same time, in the females the liver cholesterol levels were decreased. In the plasma lipids, the only change was a fall in the polar lipids of the melatonin-treated females. Melatonin seems to be responsible for the metabolic changes associated with the onset of wintering, especially for the acceleration of the deposition of subcutaneous fat reserves. The smaller females experience the effects of exogenous melatonin more rapidly than the males. Perhaps the smaller body size requires an earlier onset of metabolic preparation for the winter.