Tag Archives: substrate specificity
Isolation and characterization of Bacillus sp. IMV B-7883 proteases
O. V. Gudzenko*, L. D. Varbanets
Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine, Kyiv, Ukraine;
*e-mail: alena.gudzenko81@gmail.com
Received: 20 July 2023; Revised: 12 September 2023;
Accepted: 27 October 2023; Available on-line: 06 November 2023
The representatives of Bacillus are some of the best protease producers studied so far since they exhibit broad substrate specificity, significant activity, stability, simple downstream purification, short period of fermentation and low cost. Earlier, we showed that Bacillus sp. IMV B-7883 strain synthesizes an extracellular proteases, which exhibit elastolytic and fibrinogenolytic activity. The aim of the work was to isolate and purify these enzymes from the culture liquid of the Bacillus sp. IMV B-7883 strain, as well as to study their properties. Isolation and purification of proteases was carried out by precipitation of the culture liquid with ammonium sulfate, gel permeation and ion exchange chromatography and rechromatography on Sepharose 6B. As a result, proteases with elastolytic and fibrinogenolytic activity with a molecular weight of 23 and 20 kDa respectively were isolated with elastase activity increased by 63.6 and fibrinogenolytic activity by 44.1 times. The enzyme with elastase activity had a pH-optimum of 7.0 and hydrolyzed only elastin, while the enzyme with fibrinogenolytic activity was an alkaline protease with a pH-optimum of 8.0 and in addition to fibrinogen, showed specificity for fibrin and, in trace amounts, for collagen.
Substrate specificity of Cryptococcus albidus and Eupenicillium erubescens α-L-rhamnosidases
Е. V. Gudzenko, L. D. Varbanets
Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine, Kyiv;
е-mail: varbanets@serv.imv.kiev.ua
The substrate specificity of Cryptococcus albidus and Eupenicillium erubescens α-L-rhamnosidases has been investigated. It is shown that the enzymes are able to act on synthetic and natural substrates, such as naringin, neohesperidin. α-L-Rhamnosidases hydrolysed the latter ones very efficiently, in this case E. erubescens enzyme was characterized by higher values of Vmax in comparison with the enzyme of C. albidus. However the C. albidus α-L-rhamnosidase showed greater affinity for naringin and neohesperidin than the enzyme of E. erubescens (Km 0.77 and 3.3 mM and 5.0 and 3.0 mM, respectively). As regards the synthetic derivatives of monosaccharides, both enzymes exhibited narrow specificity for glycon: E. erubescens α-L-rhamnosidase – only to the p-nitrophenyl-α-L-rhamnopiranoside (Km 1.0 mM, Vmax 120 µmol/min/mg protein), and C. albidus – to p-nitrophenyl-α-D-glucopyranoside (Km 10 mM, Vmax 5 µmol/min/mg protein). Thus, it was found that the enzyme preparations of E. erubescens and C. albidus are differed by their substrate specificity. The ability of E. erubescens and C. albidus α-L-rhamnosidases to hydrolyse natural substrates: naringin and neohesperidin, evidences for their specificity for α-1,2-linked L-rhamnose. Based on these data, we can predict the use of E. erubescens and C. albidus α-L-rhamnosidases in various industries, food industry in particular. This is also confirmed by the fact that the investigated α-L-rhamnosidases were stable at 20% concentration of ethanol and 500 mM glucose in the reaction mixture.
Microbial α-amylases: physico-chemical properties, substrate specificity and domain structure
K. V. Avdiyuk, L. D. Vаrbanets
Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine;
e-mail: varbanets@serv.imv.kiev.ua
The current literature data on producers, physico-chemical properties and substrate specificity of α-amylases produced by microbes from different taxonomic groups such as bacteria, fungi and yeasts are discussed in the survey. Synthesis of α-amylase majority is an inducible process which is stimulated in the presence of starch or products of its hydrolysis. It is possible to increase enzymes activity level by optimization of cultivation conditions of strains-producers. α-Аmylases, isolated from different sources are distinguished in their physico-chemical properties, particularly in their molecular weights, рН- and thermooptimums, inhibitors and activators. The enzymes hydrolyse soluble starch, аmylose, аmylopectin, glycogen, maltodextrins, α- and β-cyclodextrins and other carbohydrate substrates. It is well known that α-amylases belong to GH-13 family of glycosyl-hydrolases, which contain the catalytic domain A as (β/α)8-barrel. In addition to domain А, α-аmylases contain two other domains: В and С, which are localized approximately on opposite sides of (β/α)8-barrel. Most of the known α-amylases contain calcium ion, which is located on the surface between domains А and В and plays an important role in stability and activity of the enzyme.
Proteolytic activity of IgGs from blood serum of wistar rats at experimental rheumatoid arthritis
Yu. Ya. Kit1, S. L. Myronovsky3, I. I. Kril’2,
A. M. Havrylyuk2, V. V. Chop’yak2, R. S. Stoika1
1Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv;
2Danylo Halytsky Lviv National Medical University, Ukraine;
3Ivan Franko National University of Lviv, Ukraine;
e-mail: kit@cellbiol.lviv.ua
The aim of this work was to study the proteolytic activity of IgGs purified from blood serum of Wistar rats at experimental rheumatoid arthritis (ERA) induced by an injection of bovine collagen of type II. Twenty rats were immunized with a preparation of bovine collagen II (Sigma-Aldrich, USA) in the presence of complete Freund’s adjuvant. ERA development was determined by inflammation in limbs of treated animals. IgG preparations were isolated from blood serum of immunized and non-immunized animals by precipitation of antibodies with 33% ammonium sulfate followed by chromatography on the Protein G-Sepharose column. Human histone H1, bovine collagen II, calf thymus histones, myelin basic protein (MBP), bovine serum albumin (BSA), and bovine casein were used as substrates of the proteolytic activity of IgGs. It was found that IgG preparations from blood serum of rats with ERA were capable of cleaving histone H1 and MBP, however, they were catalytically inactive towards collagen II, casein, BSA, and core histones. IgGs from blood serum of non-immunized rats were proteolytically inactive towards all used protein substrates. Thus, we demonstrated that immunization of rats with bovine collagen II induced IgG-antibodies possessing the proteolytic activity towards histone H1 and MBP. This activity might be associated with the development of inflammatory processes in the immunized rats.