Special enzymes are necessary for producing gluten-free foods, and specific proteolytic enzymes with gluten-degrading activity may be used as oral treatments for celiac disease. Enzymes of the kind were sought, identified, and preliminarily characterized in two strains of the alkaliphilic microscopic fungi Sodiomyces alkalinus and S. magadiensis. Post-glutamine cleaving activity was for the first time observed in the strains along with proline-cleaving activities of dipeptidyl peptidase 4 (DPP4) and proline aminopeptidase (PAP), allowing efficient hydrolysis of both proline/glutamine-rich gluten peptides and whole gluten. The optimal pH and pH-dependent stability were determined for the peptidases in question. All of the enzymes shown to cleave the prolyne/glutamine-containing bonds were assigned to the serine peptidase group and were found to be stable in moderately acidic and alkaline conditions. Owing to their activities, the peptidases are promising as tools to produce gluten-free foods and to design diets for gluten-intolerant patients.

The review is dedicated to search for peptidase groups responsible for fungal growth and for inhibitors of these peptidases. Available data on diversity, significance, distribution and peculiarities of proteolytic enzymes of fungi of the genera Aspergillus, Penicillium, Trichoderma and Alternaria are discussed. According to analysis of the data we concluded that serine, metalloand glutamic peptidases are necessary for fungal growth. Thus, these enzymes are considered as perspective targets for inhibitors that may serve as the reason to search for such inhibitors and development of novel biocides on their basis that protect works of art against biodestruction.

Extracellular serine proteases from the culture liquids of two strains of an entomopathogenic fungus Cordyceps militaris were isolated and characterized. Their activity was demonstrated to depend on the composition of the culture medium. Electrophoretically homogeneous preparations of the secreted enzymes were obtained by affinity chromatography on bacitracin-silochrome and gel filtration on Superdex G-75. Molecular weight of the proteins was 24 kDa.

Electrophoretically pure preparation of a secreted subtilisin-like protease was isolated from the culture liquid of an entomopathogenic fungus C. militaris strain KP-97-2. The enzyme had an optimum activity at 45°C and pH 7–9. Data were collected on the substrate specificity of the enzyme and its stability at different values of pH and temperature. Adsorption and desorption of the extracellular protease on the cuticle of Nauphoeta cinerea was studied in function of pH.

Proteolytic enzymes of the fungi attract attention of investigators due to many reasons among which their large diversity, wide substrate specificity, stability under extreme conditions (pH, temperature) are most important. Their functional significance, including various processes, from hydrolysis of macromolecular substrates under deficiency of nitrogen compounds to initiation and maintenance of pathogenesis, is also very interesting. The present review deals with classification and biochemical properies of extracellular fungal proteinases, their physiological role as well as fields of practical application. Much attention is given to pecularities of extracellular proteinases of xylotrophic basidiomycetes-- an exceedingly important group of the fungi from the point of functioning of biological communities, and their participation in biodestruction of plant waste which are also used as source of food and medicinal preparations. Special attention is focused on regulation of synthesis and secretion of extracellular proteinases of xylotrophic basidiomycetes.

Протеолитические ферменты грибов привлекают внимание исследователей по ряду причин, среди которых наиболее значимы их большое разнообразие, широкая субстратная специфичность, устойчивость в экстремальных условиях. Не менее интересна их функциональная роль, включающая целый ряд процессов – от гидролиза макромолекулярных субстратов в условиях крайне ограниченного содержания азотистых веществ до инициирования и поддержания процесса патогенеза. В представленном обзоре обсуждаются особенности внеклеточных протеаз ксилотрофных базидиомицетов, исключительно важной группы грибов с точки зрения функционирования биологических сообществ и участия этих грибов в процессах биодеструкции растительных отходов, к тому же интенсивно используемых человеком в качестве источника питания и лекарственных средств. Особое внимание в обзоре уделено вопросам, касающимся классификации грибных протеаз, их биохимических свойств, а также физиологической роли и регуляции активности в процессе роста грибов.

he presence of proteins in the culture liquid of filamentous fungi under study was found to induce the secretion of proteinases. The inhibitory analysis of the major extracellular proteinases of the saprotrophic fungus Trichoderma harzianum and the phytopathogenic fungus Alternaria alternata showed that they both belong to the group of serine proteinases. The substrate specificity of these proteinases and their sensitivity to inhibitors suggest that the enzyme of T. harzianum is a subtilisin-like proteinase and the enzyme of A. alternata is a trypsin-like proteinase. This difference between the proteinases may reflect the physiological difference between their producers (saprotroph and phytopathogen).

Physicochemical and functional characteristics of plant protein proteinase inhibitors as antistress biopolymers were studied to determine the mechanisms for plant resistance to phytopathogens and to obtain disease-resistant cereal and leguminous cultures. The activity of trypsin, chymotrypsin, and subtilisin inhibitors varied in monocotyledonous and dicotyledonous cultures. Study varieties of leguminous and cereal cultures were shown to contain endogenous inhibitors specific to proteinases of phytopathogenic fungi Fusarium, Colletotrichum, Helminthosporium, and Botrytis. These inhibitors were characterized by species specificity and variety specificity. Protease inhibitors from buckwheat seeds inhibited proteases of fungal pathogens and suppressed germination of spores and growth of the fungal mycelium. Our results suggest that proteinaceous inhibitors of proteinases are involved in the protective reaction of plants under stress conditions.

Complete amino acid sequence of IT1 protease inhibitor and partial amino acid sequences of IT2 and IT4 protease inhibitors from buckwheat Fagopyrum esculentum Moench seeds were determined by automatic Edman degradation and mass spectrometry. IT1 inhibitor comprises 69 amino acid residues and its molecular mass is 7743.8 D. N-terminal 48 amino acid residues of IT2 inhibitor are identical to similar sequence of IT1 inhibitor. The sequence of 10 amino acid residues of IT4 inhibitor is completely identical to a part of the sequence of IT1 inhibitor C-terminally adjacent to its active site. Analysis of amino acid sequences of IT1, IT2 and IT4 inhibitors suggests that the proteins are the members of the potato proteinase inhibitor 1 family and include Arg-Asp residues in their active site.

The SH-proteinase which performs the second stage of hydrolysis of the main storage protein, 13S globulin, is localized in protein bodies and vacuoles of 4-day-old buckwheat seedlings. In the presence of 1.0 mM dithiothreitol (DTT) under conditions of microdialysis at pH 5.3, modified 13S globulin is hydrolyzed by the protein body extracts by 85% and by the vacuole extracts by 98%. Cysteine proteinase activity accounts for 90% of the proteolytic activity of protein bodies and for 59% of that in vacuoles. Protein body vacuolization is accompanied by a sharp increase in aspartyl proteinase activity which is also capable of hydrolysing modified 13S globulin from 2% in protein bodies to 34% in vacuoles. No hydrolyzing activity of modified 13S globulin was found in the complex protein bodies (PB I fraction) of dry buckwheat seeds, indicating that this activity appears during germination.