Influence of heat treatment in the presence of certain metal ions on the spectral-fluorescent properties of an amylolytic enzyme preparation

Тепловая обработка ферментных препаратов приводит к заметному изменению их активности в результате определенных конформационных изменений в молекуле фермента. На основе изучения ультрафиолетовых спектров авторы судят об изменении полярности окружения остатков триптофана при добавлении ионов цинка, алюминия и кальция. В работе приведены результаты изучения спектров флуоресценции ферментного препарата – ультраконцентрата глюкоамилазы Asp. awamori после тепловой обработки и добавления ионов некоторых металлов для обнаружения структурных изменений в молекуле фермента при этих воздействиях. Установлено, что совместное влияние ионов металлов и теплового воздействия на водный раствор ферментного препарата ультраконцентрата глюкоамилазы Asp. awamori приводит к повышению интенсивности свечения и смещению λmax в спектрах флуоресценции ультраконцентрата глюкоамилазы Asp. awamori в область более коротких длин волн. Это указывает на увеличение гидрофобности окружения триптофана и подтверждает конформационные изменения в молекуле фермента, происходящие под действием ионов некоторых металлов и при тепловом воздействии. The heat treatment of enzyme preparations leads to a noticeable change in their activity as a result of certain conformational changes in the enzyme molecule. On mastering the study of ultraviolet spectra, the authors judge a change in the polarity of the environment of tryptophan residues with the addition of zinc, aluminum and calcium ions. The paper presents the results of studying the fluorescence spectra of an enzyme preparation: glucoamylase ultraconcentrate Asp. awamori after heat treatment and the addition of certain ions of some metals to detect structural changes in the enzyme molecule under these influences. It was found that the combined effect of metal ions and thermal effects on the aqueous solution of the enzyme preparation of glucoamylase ultraconcentrate Asp. awamori, leads to an increase in luminescence intensity and a shift of λmax in the fluorescence spectra of Asp. awamori glucoamylase ultraconcentrate to the region of shorter wavelengths. This indicates an increase in the hydrophobicity of the tryptophan environment and provides confirmation of conformational changes in the enzyme molecule under the action of ions of certain metals and upon thermal exposure.

COVID-19 and Diabetes Mellitus: The Link and Clinical Implications

<b><i>Background:</i></b> Coronavirus disease 2019 (COVID-19) is a pandemic viral infection that has ravaged the world in recent times, and the associated morbidity and mortality have been much more pronounced in those with noncommunicable disease. Diabetes mellitus is one of commonest noncommunicable diseases associated with worsening clinical status in COVID-19 patients. <b><i>Summary:</i></b> The aim of this review was to evaluate the receptors and pathogenetic link between diabetes and COVID-19. Both disease conditions involve inflammation with the release of inflammatory markers. The roles of angiotensin-converting enzyme molecule and dipeptidyl peptidase were explored to show their involvement in COVID-19 and diabetes. Pathogenetic mechanisms such as impaired immunity, microangiopathy, and glycemic variability may explain the effect of diabetes on recovery of COVID-19 patients. The effect of glucocorticoids and catecholamines, invasion of the pancreatic islet cells, drugs used in the treatment of COVID-19, and the lockdown policy may impact negatively on glycemic control of diabetic patients. The outcome studies between diabetic and nondiabetic patients with COVID-19 were also reviewed. Some drug trials are still ongoing to determine the suitability or otherwise of some drugs used in diabetic patients with COVID-19, such as dapagliflozin trial and linagliptin trial.

Chemical Synthesis of an Enzyme Containing an Artificial Catalytic Apparatus

With the goal of investigating electronic aspects of the catalysis of peptide bond hydrolysis, an analogue of HIV-1 protease was designed in which a non-peptide hydroxy-isoquinolinone artificial catalytic apparatus replaced the conserved Asp25–Thr26–Gly27 sequence in each 99-residue polypeptide chain of the homodimeric enzyme molecule. The enzyme analogue was prepared by total chemical synthesis and had detectable catalytic activity on known HIV-1 protease peptide substrates. Compared with uncatalyzed hydrolysis, the analogue enzyme increased the rate of peptide bond hydrolysis by ∼108-fold. Extensions of this unique approach to the study of enzyme catalysis in HIV-1 protease are discussed.

The effects of macromolecular crowding and surface charge on the properties of an immobilized enzyme: activity, thermal stability, catalytic efficiency and reusability

The microenvironment around an immobilized enzyme molecule significantly influences the properties of the immobilized enzyme.

Ultrahigh-throughput–directed enzyme evolution by absorbance-activated droplet sorting (AADS)

Ultrahigh-throughput screening, in which members of enzyme libraries compartmentalized in water-in-oil emulsion droplets are assayed, has emerged as a powerful format for directed evolution and functional metagenomics but is currently limited to fluorescence readouts. Here we describe a highly efficient microfluidic absorbance-activated droplet sorter (AADS) that extends the range of assays amenable to this approach. Using this module, microdroplets can be sorted based on absorbance readout at rates of up to 300 droplets per second (i.e., >1 million droplets per hour). To validate this device, we implemented a miniaturized coupled assay for NAD+-dependent amino acid dehydrogenases. The detection limit (10 μM in a coupled assay producing a formazan dye) enables accurate kinetic readouts sensitive enough to detect a minimum of 1,300 turnovers per enzyme molecule, expressed in a single cell, and released by lysis within a droplet. Sorting experiments showed that the AADS successfully enriched active variants up to 2,800-fold from an overwhelming majority of inactive ones at ∼100 Hz. To demonstrate the utility of this module for protein engineering, two rounds of directed evolution were performed to improve the activity of phenylalanine dehydrogenase toward its native substrate. Fourteen hits showed increased activity (improved >4.5-fold in lysate; kcat increased >2.7-fold), soluble protein expression levels (up 60%), and thermostability (Tm, 12 °C higher). The AADS module makes the most widely used optical detection format amenable to screens of unprecedented size, paving the way for the implementation of chromogenic assays in droplet microfluidics workflows.