Formation of covalent beta-linked carbohydrate-enzyme intermediates during the reactions catalyzed by alpha-amylases

Porcine pancreatic and Bacillus amyloliquefaciens alpha-amylases were examined for the formation of covalent carbohydrate intermediates during reaction. The enzymes were precipitated and denatured by adding 10 volumes of acetone. When these denatured enzymes were mixed with methyl alpha-6-[(3)H]-maltooligosaccharide glycosides and chromatographed on BioGel P-2, no carbohydrate was found in the protein void volume peak. When the enzymes were added to the methyl alpha-6-[(3)H]-maltooligosaccharide glycosides and allowed to react for 15s at 1 degrees C and then precipitated and denatured with 10 volumes of acetone, (3)H-labeled carbohydrates were found in the BioGel P-2 protein void volume peak, indicating the formation of enzyme-carbohydrate covalent intermediates. (1)H NMR analysis of the denatured enzyme from the reaction with methyl alpha-maltooligosaccharide glycosides confirmed that carbohydrate was attached to the denatured enzyme. (1)H NMR saturation-transfer analysis further showed that the carbohydrate was attached to the denatured enzyme by a beta-configuration. This configuration is what would be expected for an enzyme that catalyzes the hydrolysis of alpha-(1-->4) glycosidic linkages by a two-step, S(N)2 double-displacement reaction to give retention of the alpha-configuration of the substrates at the reducing-end of the products.     

Hexose monophosphate shunt,the role of its metabolites and associated disorders: A review

The hexose monophosphate (HMP) shunt acts as an essential component of cellular metabolism in maintaining carbon homeostasis. The HMP shunt comprises two phases viz. oxidative and nonoxidative, which provide different intermediates for the synthesis of biomolecules like nucleotides, DNA, RNA, amino acids, and so forth; reducing molecules for anabolism and detoxifying the reactive oxygen species during oxidative stress. The HMP shunt is significantly important in the liver, adipose tissue, erythrocytes, adrenal glands, lactating mammary glands and testes. We have researched the articles related to the HMP pathway, its metabolites and disorders related to its metabolic abnormalities. The literature for this paper was taken typically from a personal database, the Cochrane database of systemic reviews, PubMed publications, biochemistry textbooks, and electronic journals uptil date on the hexose monophosphate shunt. The HMP shunt is a tightly controlled metabolic pathway, which is also interconnected with other metabolic pathways in the body like glycolysis, gluconeogenesis, and glucuronic acid depending upon the metabolic needs of the body and depending upon the biochemical demand. The HMP shunt plays a significant role in NADPH₂ formation and in pentose sugars that are biosynthetic precursors of nucleic acids and amino acids. Cells can be protected from highly reactive oxygen species by NADPH ₂. Deficiency in the hexose monophosphate pathway is linked to numerous disorders. Furthermore, it was also reported that this metabolic pathway could act as a therapeutic target to treat different types of cancers, so treatments at the molecular level could be planned by limiting the synthesis of biomolecules required for proliferating cells provided by the HMP shunt, hence, more experiments still could be carried out to find additional discoveries.     

不同酿酒酵母菌株来源异麦芽糖酶IMA1的克隆、表达及表征

[目的]异麦芽糖酶IMA1在充分利用含有α-1,6-O-糖苷键的低聚糖中起着关键作用。[方法]在本研究中,对来自4株酿酒酵母菌株（包括3株嗜酸性菌株）来源的异麦芽糖酶IMA1进行克隆、表达、纯化和表征。[结果]研究发现,4种异麦芽糖酶IMA1表现出类似的pH和温度依赖性,但表现出不同的动力学参数和热稳定性。IMA1-A对α-MG（α-甲基葡糖苷）表现出最高的结合亲和力、转换数、催化效率和热稳定性。结构和序列分析表明,2个远离活性位点和底物结合位点的氨基酸的差异对异麦芽糖酶IMA1的动力学参数和热稳定性有重要影响。[结论]本研究结果对进一步研究异麦芽糖酶IMA1的结构-功能关系奠定了基础。     

Hybrid physics-based and data-driven modeling for bioprocess online simulation and optimization

Model-based online optimization has not been widely applied to bioprocesses due to the challenges of modeling complex biological behaviors, low-quality industrial measurements, and lack of visualization techniques for ongoing processes. This study proposes an innovative hybrid modeling framework which takes advantages of both physics-based and data-driven modeling for bioprocess online monitoring, prediction, and optimization. The framework initially generates high-quality data by correcting raw process measurements via a physics-based noise filter (a generally available simple kinetic model with high fitting but low predictive performance); then constructs a predictive data-driven model to identify optimal control actions and predict discrete future bioprocess behaviors. Continuous future process trajectories are subsequently visualized by re-fitting the simple kinetic model (soft sensor) using the data-driven model predicted discrete future data points, enabling the accurate monitoring of ongoing processes at any operating time. This framework was tested to maximize fed-batch microalgal lutein production by combining with different online optimization schemes and compared against the conventional open-loop optimization technique. The optimal results using the proposed framework were found to be comparable to the theoretically best production, demonstrating its high predictive and flexible capabilities as well as its potential for industrial application.     

Chromatographic approach to study the configurational stability of Ni(II) complexes of amino‐acid Schiff bases possessing stereogenic nitrogen

Herein, we disclose the design of a model Ni(II) complex of glycine Schiff base possessing single‐nitrogen stereogenic center, which was successfully used for high‐performance liquid chromatography (HPLC)‐assisted assessment of its configurational stability. The major finding is that the configurational stability of the Ni(II)‐coordinated nitrogen is profoundly dependent on the reaction conditions used, in particular the solvent, and can range from inconsequential (t_½ less than 5 min) to virtually completely stable (t_½ 90 y). The discovery reported in this study most likely to be of certain theoretical and synthetic value.     

Absolute configuration of phomactatriene diterpenoids obtained by Wagner‐Meerwein rearrangement of epimeric verticillols

The epimeric diterpenes (+)‐(1S,3E,7E,11S,12S)‐verticilla‐3,7‐dien‐12‐ol ( 1 ), isolated from Bursera suntui, and (+)‐(1S,3E,7E,11S,12R)‐verticilla‐3,7‐dien‐12‐ol ( 2 ), isolated from Bursera kerberi, gave the same Wagner‐Meerwein rearrangement product (?)‐(1E,4Z,8Z,11S,12R)‐phomacta‐1,(15)4,8‐triene ( 3 ). The Et₂O:BF₃‐induced transformations evidence that verticillenes and phomactanes, both containing the bicyclo[9.3.1]pentadecane skeleton, are biogenetically related through the verticillen‐12‐yl cation ( A ⁺ ), which also is a key intermediate in the biosynthetic pathways to generate antitumor taxanes. Molecular modeling using the Monte Carlo protocol, followed by density functional theory (DFT) geometry optimization employing the hybrid functionals B3LYP and B3PW91, both with the DGDZVP basis set, secured the configuration of 3 as followed from the good agreement between the calculated and experimental vibrational circular dichroism spectra. Similar DFT calculations allowed determining the absolute configuration of (+)‐(1R,4R,5R,8S,9S,11S,12R,15R)‐1,15:4,5:8,9‐triepoxyphomactane ( 9 ), which surprisingly derives from epoxidation of the second minimum energy conformer of 3 .     

亚热带河口养殖塘沉积物有机碳矿化及其影响因子

亚热带河口的养殖塘是温室气体二氧化碳(CO2)的重要排放源,但其沉积物有机碳矿化动力学特征目前尚未厘清.以我国东南沿海亚热带闽江、木兰溪以及九龙江河口区的6个凡纳滨对虾(Penaeus vannamei)养殖塘为研究对象,对沉积物取样,并进行为期60 d的室内厌氧培养,对养殖塘沉积物CO2累积产量曲线利用一阶动力学模型...     

Conformational Priming of RepA-WH1 for Functional Amyloid Conversion Detected by NMR Spectroscopy

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Novel kinetic analysis of enzymatic dipeptide synthesis: effect of pH and substrates on thermolysin catalysis.

The point of maximum activity is specific to a particular substrate-enzyme system but may vary with different substrates and the same enzyme. The specificity of enzymes has, however, been generally reported only at their "optimal" pH. In this article, we introduce the Michaelis-Menten equation taking pH into account, and apply it to the pH-activity profile of the thermolysin-catalyzed dipeptide synthesis. It has been reported to date that the pH-activity profile of thermolysin follows a bell-shaped curve with a maximal activity at or near pH 7.0. The profiles obtained in this study, however, indicated that the optimal pH varied from 5.8 (for F-AspPheOMe) to 7.3 (for Z-ArgPheOMe), and the order of thermolysin activity was greatly dependent on the pH of reaction media. We have succeeded in evaluating the substrates-induced change of the dissociation states of the active site of thermolysin using the hydrophobicity of substrates. We have obtained apparent kinetic parameters which are independent of the pH of reaction media. The apparent specificity of thermolysin which were independent of pH of the reaction media was in order L-Leu > L-Asp > L-Arg > L-Ala > L-Gly > L-Val and Z > Boc = F at P1 and P2 positions, respectively.