Semi-rational evolution of ω-transaminase from Aspergillus terreus for enhancing the thermostability / 生物工程学报

ω-transaminase (ω-TA) is a natural biocatalyst that has good application potential in the synthesis of chiral amines. However, the poor stability and low activity of ω-TA in the process of catalyzing unnatural substrates greatly hampers its application. To overcome these shortcomings, the thermostability of (R)-ω-TA (AtTA) from Aspergillus terreus was engineered by combining molecular dynamics simulation assisted computer-aided design with random and combinatorial mutation. An optimal mutant AtTA-E104D/A246V/R266Q (M3) with synchronously enhanced thermostability and activity was obtained. Compared with the wild- type (WT) enzyme, the half-life t1/2 (35 ℃) of M3 was prolonged by 4.8-time (from 17.8 min to 102.7 min), and the half deactivation temperature (T1050) was increased from 38.1 ℃ to 40.3 ℃. The catalytic efficiencies toward pyruvate and 1-(R)-phenylethylamine of M3 were 1.59- and 1.56-fold that of WT. Molecular dynamics simulation and molecular docking showed that the reinforced stability of α-helix caused by the increase of hydrogen bond and hydrophobic interaction in molecules was the main reason for the improvement of enzyme thermostability. The enhanced hydrogen bond of substrate with surrounding amino acid residues and the enlarged substrate binding pocket contributed to the increased catalytic efficiency of M3. Substrate spectrum analysis revealed that the catalytic performance of M3 on 11 aromatic ketones were higher than that of WT, which further showed the application potential of M3 in the synthesis of chiral amines.

Enhanced pyruvic acid yield in an osmotic stress-resistant mutant of Yarrowia lipolytica

BACKGROUND: Pyruvic acid (PA), a vital α-oxocarboxylic acid, plays an important role in energy and carbon metabolism. The oleaginous yeast Yarrowia lipolytica (Y. lipolytica) has considerable potential for the production of PA. An increased NaCl concentration reportedly increases the biomass and PA yield of Y. lipolytica. RESULTS: To increase the yield of PA, the NaCl-tolerant Y. lipolytica A4 mutant was produced using the atmospheric and room temperature plasma method of mutation. The A4 mutant showed growth on medium containing 160 g/L NaCl. The PA yield of the A4 mutant reached 97.2 g/L at 120 h (0.795 g/g glycerol) in a 20-L fermenter with glycerol as the sole carbon source, which was 28.9% higher than that of the parental strain. CONCLUSION: The PA yield from Y. lipolytica can be improved by increasing its NaCl tolerance.

Presence of bound substrate in lactate dehydrogenase from carp liver.

While attempting to purify UDP-galactose 4-epimerase from carp liver extract at pH 8.0, it was observed that the preparation even after dialysis could reduce NAD to NADH, interfering epimerase assay. The NAD reduction activity and the epimerase were co-eluted in a series of chromatographic steps. Mass spectrometric analysis of semi-purified fraction revealed that carp liver lactate dehydrogenase (LDH) contained bound lactate which was converted to pyruvate in the presence of NAD. The enzyme-bound lactate and the association with epimerase stabilized LDH from trypsin digestion and thermal inactivation at 45°C by factors of 2.7 and 4.2 respectively, as compared to substrate-free LDH. LDH and epimerase do not belong to any one pathway, but are the rate-limiting enzymes of two different pathways of carbohydrate metabolism. Typically, strongly associated enzymes work in combination, such as two enzymes of the same metabolic pathway. In that background, co-purification of LDH and epimerase as reloaded in this study was an unusual phenomenon.

Toxicological effect and biochemical alterations induced by different fractions of Euphorbia royleana latex in freshwater harmful vector snail Lymnaea acuminata.

Laboratory evaluation was made to assess the molluscicidal activity of different fractions of Euphorbia royleana (Family- Euphorbiaceae) latex obtained through sephadex gel column against freshwater snail Lymnaea (Radix) acuminata Lamarack. This snail is the vector of liver fluke, Fasciola hepatica Linnaeus and Fasciola gigantica Cobbold, which causes endemic fascioliasis in cattle and livestock. The toxic effect of the different fractions was time dependent and fifth fraction obtained through benzene: ethyl acetate (5:5) had maximum molluscicidal activity against Lymnaea acuminata. There was a significant negative correlation between LC values and exposure periods thus increase in exposure time, the LC50 value of V fraction of Euphorbia royleana latex was decreased from 14.28 mg/l (24 hr) to 9.28 mg/l (96 hr) against Lymnaea acuminata. After exposure to sub-lethal concentrations of this fraction there were significant time and dose dependent alterations observed in pyruvate, lactate levels, ALAT, AAT, AChE and cytochrome oxidase enzyme activities in different body tissues of Lymnaea acuminata. It is proposed that the fifth fraction of E. royleana latex can be used as a molluscicide for controlling the harmful snail population from aquatic ecosystem without any harm due to their reversible toxic action.

Cell volume changes affect gluconeogenesis in the perfused liver of the catfish Clarias batrachus.

In addition to lactate and pyruvate, some amino acids were found to serve as potential gluconeogenic substrates in the perfused liver of Clarias batrachus. Glutamate was found to be the most effective substrate, followed by lactate, pyruvate, serine, ornithine, proline, glutamine, glycine, and aspartate. Four gluconeogenic enzymes, namely phosphoenolpyruvate carboxykinase (PEPCK), pyruvate carboxylase (PC), fructose 1,6-bisphosphatase (FBPase) and glucose 6-phosphatase (G6Pase) could be detected mainly in liver and kidney, suggesting that the latter are the two major organs responsible for gluconeogenic activity in this fish. Hypo-osmotically induced cell swelling caused a significant decrease of gluconeogenic efflux accompanied with significant decrease of activities of PEPCK, FBPase and G6Pase enzymes in the perfused liver. Opposing effects were seen in response to hyperosmotically induced cell shrinkage. These changes were partly blocked in the presence of cycloheximide, suggesting that the aniso-osmotic regulations of gluconeogenesis possibly occurs through an inverse regulation of enzyme proteins and/or a regulatory protein synthesis in this catfish. In conclusion, gluconeogenesis appears to play a vital role in C. batrachus in maintaining glucose homeostasis, which is influenced by cell volume changes possibly for proper energy supply under osmotic stress.

Toxic and sub-lethal effects of oleandrin on biochemical parameters of fresh water air breathing murrel, Channa punctatus (Bloch.).

Active compound oleandrin extracted from Nerium indicum (Lal Kaner) leaf has potent piscicidal activity. The piscicidal activity of oleandrin on freshwater fish C. punctatus was both time and dose dependent. Exposure to sub-lethal doses of oleandrin for 24hr and 96hr to fish caused significant alteration in the level of total protein, total free amino acid, nucleic acid, glycogen, pyruvate, lactate and enzyme protease, phosphatases, alanine aminotransferase, aspartate aminotransferase and acetylcholinesterase activity in liver and muscle tissues. The alterations in all the above biochemical parameters were also significantly time and dose dependent. The results show a significant recovery in all the above biochemical parameters, in both liver and muscle tissues of fish after the 7th day of the withdrawal of treatment. Toxicity persistence test of oleandrin on juvenile Labeo rohita shows that fish seed of common culturing carp can be released into rearing ponds after three days of oleandrin treatment. It supports the view that the oleandrin is safer and may be useful substitute of other piscicides for removing the unwanted freshwater fishes from aquaculture ponds.

Effect of whole body gamma radiation on hepatic LDH activity, lactate, pyruvate concentration and rate of oxygen consumption in Bufo melanostictus.

Whole body Co60 gamma radiation induced changes in lactic dehydrogenase (LDH) activity, pyruvate, lactate content and rate of oxygen (O2) consumption in a tropical hibernating anuran (Bufo melanostictus). In 3.5 and 7 Gy treated groups, a significant increase in LDH activity and lactate/pyruvate ratio was observed, whereas a significant decrease in O2 consumption rate was observed in treated animals on post-irradiation day (PID) 1, 5 and 10. Increase in LDH activity was observed on PID-1 in both the treated groups, reached to a peak on PID-5 in 7 Gy treated group and then declined on PID-10.

Inmunorreactividad de enolasa en el epitelio seminífero senil humano / Immunoreactivity to enolase in the human senil seminiferous epithelium

El metabolismo energético intracelular compartimentalizado, activa la enzimo enolasa para formar ácido pirúvico. Este, substrato energético, debe ingresar a la mitocondria para continuar hacia el ciclo de losácidos tricarboxílicos. Sin embargo, durante la proliferación del epitelio seminífero ocurre una distribución y pérdida citoplasmática progresiva y disposición de las mitocondrias a nivel del flagelo inicial. en el presente estudio se describe la inmunoreactividad de la enzimo enolasa en las diferentes poblaciones celulares del epitelio seminífero, en testiculo humano senil. Se trabajo con un paciente de 70 años, sometido a orquiectomía subcapsular terapéutica. El tejido testicular fue fijado inmediatamente en formalina taponada al 10 por ciento y mantenido por 12 horas. Luego se procesó por técnicas histológicas corrientes e incluyo en parafina para obtener secciones de 5 um. Posteriormente se procedió a la reacción inmunohistoquímica para enolasa y revelación con complejo avidina-biotina. Finalmente se cuantificaron las distintas poblaciones celulares del epitelio seminífero con reacción positivo o negativa. Los resultados preliminares se expresan en porcentajes de células positivas respecto del total de células contadas (40x. se observó que la totalidad de las células de Sértoli presentaron reacción negativa a la enolasa. En el epitelio seminífero se encontró que el 76 por ciento de las gonias (gonias tipo A y B) mostraron reacción negativa a la enolasa, mientras que en citos 1 se redujo al 10 por ciento y ausencia total en espermátidas y espermatozoides. Por lo tanto, las célula somáticas (de origen mesodérmico), del epitelio seminífero presentarían isoenzimas enolasas de reactividad diferente en la relación con la línea germinal (espermatogonias). Adicionalmente, en la línea germinal la inmunoreactividad a enolasa disminuye mientras progresa la espermatogénesis

Rat liver responsiveness to gluconeogenic substrates during insulin-induced hypoglycemia

Hepatic responsiveness to gluconeogenic substrates during insulin-induced hypoglycemia was investigated. For this purpose, livers were perfused with a saturating concentration of 2 mM glycerol, 5 mM L-alanine or 5 mM L-glutamine as gluconeogenic substrates. All experiments were performed 1 h after an ip injection of saline (CN group) or 1 IU/kg of insulin (IN group). The IN group showed higher (P<0.05) hepatic glucose production from glycerol, L-alanine and L-glutamine and higher (P<0.05) production of L-lactate, pyruvate and urea from L-alanine and L-glutamine. In addition, ip injection of 100 mg/kg glycerol, L-alanine and L-glutamine promoted glucose recovery. The results indicate that the hepatic capacity to produce glucose from gluconeogenic precursors was increased during insulin-induced hypoglycemia.

Time sequence of changes in the responsiveness of glycogen breakdown to adrenergic agonists in perfused liver of rats with insulin-induced hypoglycemia

The time-course changes of the responsiveness of glycogen breakdown to a- and Beta-adrenergic agonists during insulin-induced hypoglycemia (IIH) were investigated. Blood glucose levels were decreased prior to the alteration in the hepatic responsiveness to adrenergic agonists. The activation of hepatic glucose production and glycogenolysis by phenylephrine (2 µM) and isoproterenol (20 µM) was decreased in IIH. The changes in the responsiveness of glycogen catabolism were first observed for isoproterenol and later for phenylephrine. Hepatic ß-adrenergic receptors showed a higher degree of adrenergic desensitization than did a-receptors. Liver glycogen synthase activity, glycogen content and the catabolic effect of dibutyryl cyclic AMP (the Beta-receptor second messenger) were not affected by IIH.

Transient improvement of pyruvate metabolism after coenzyme Q therapy in Kearns-Sayre syndrome: MRS study

Coenzyme Q therapy has been used to support metabolic derangements in patients with mitochondrial encephalomyopathies. Biochemical analysis of the living human brain can be performed by magnetic resonance spectroscopy (MRS). We report upon a KSS patient who was serially imaged with localized proton MRS to monitor the efficacy of CoQ treatment. A 17-year-old girl with KSS was serially imaged with localized proton MRS performed on a GE 1.5 T SIGNA MRI/MRS system. The elevated lactate contents of lesions decreased after one month of CoQ therapy but were re-elevated 10 months after treatment. We conclude that MRS presents us with a powerful tool for monitoring the effects of therapeutic trials in mitochondrial encephalomyopathies.

The metabolic effects of estriol in female rat liver

The effects of estriol on oxygen uptake, glucose release, lactate and pyruvate production, beta-hydroxybutyrate and acetoacetate production in perfused rat liver as well as, carbon uptake in rat liver and intracellular calcium in isolated Kupffer cells were investigated. Basal oxygen consumption of perfused liver increased significantly in estriol or ethanol-treated rats. But these increased effects were blocked by gadolinium chloride pretreatment. In a metabolic study, pretreatment with estriol resulted in a decrease in glucose production and in glycolysis while an increase in ketogenesis. A more oxidized redox state of the mitochondria was indicated by increased ratios of perfusate [lactate]/[pyruvate] and decreased ratios of perfusate [beta-hydroxybutyrate]/[acetoacetate]. Carbon uptake of Kupffer-cell increased significantly in estriol-treated rats. But these increased uptake were not shown in rats pre-treated by gadolinium chloride blocking phagocytosis. In isolated Kupffer cells from estriol-treated rats, intracellular calcium was more significantly increased after addition of lipopolysaccharide (LPS) than in controls. These findings suggest that the metabolic effects of estriol (two mg per 100 mg body wt) can be summarized to be highly toxic in rat liver, and these findings suggest that oral administration of estrogens may induce hepatic dysfunctions and play a role in the development of liver disease.

Channeling of TCA cycle intermediates in Saccharomyces cerevisiae.

Metabolism of 13C labeled substrates viz. glucose and pyruvate in S. cerevisiae has been studied by 13C Nuclear Magnetic Resonance Spectroscopy. C3-Pyruvate, alanine and lactate, and C2-acetate are produced from [1-13C]glucose. The pyruvate, entering TCA cycle, leads to preferential labeling of C2-glutamate. [2-13C]Glucose results in labeling of C2-pyruvate, alanine and lactate. Some C3-pyruvate is also produced, indicating the routing of the label from glucose through pentose phosphate pathway (PPP). In TCA cycle the C2-pyruvate preferentially labels the C3-glutamate. The NMR spectra, obtained with [2-13C]pyruvate as substrate, confirm the above observations. These results suggest that the intermediates of TCA cycle are transferred from one enzyme active site to another in a manner that allows only restricted rotation of the intermediates. That is, the intermediates are partially channeled.

Status of ammonia, glutamate, lactate and pyruvate during Plasmodium yoelii infection and pyrimethamine treatment in mice.

Ammonia, lactate, glutamate and pyruvate levels in blood, liver, brain, spleen and kidney were determined during Plasmodium yoelii infection and pyrimethamine treatment in mice. Ammonia and lactate levels showed significant increase with rise in parasitaemia except in spleen where decrease in the lactate levels was observed. The glutamate level displayed a marked decrease in blood, liver and splenic tissues, whereas, significant increase in glutamate level in kidney was observed, although its level in cerebral tissue remained unaltered. The pyruvate level in blood and liver showed a noticeable decrease but brain, spleen and kidney registered an elevation of the same due to the parasitic infection. Pyrimethamine (oral) treatment (10 mg/kg body weight) to infected mice (5-10%) for four days brought back the altered levels of the above cellular constituents in different tissues to normal, a week after cessation of drug treatment.