Получены новые биокатализаторы – препараты субтилизина Карлсберг, иммобилизованного на хитозане (дезацетилированном производном хитина). Препараты охарактеризованы по содержанию фермента, гидролитической активности и способности катализировать образование пептидной связи в среде органических растворителей. Исследовано влияние формы и состава биокомпозита (содержания фермента и сшивающего реагента – глутарового альдегида), а также рН буфера на биокаталитические свойства иммобилизованного фермента в реакциях гидролиза пептидной связи. Изучена синтазная активность полученных образцов в средах диметилформамид-ацетонитрил (6 : 4) в реакции образования Z-Ala-Ala-Leu-Phe-pNA в зависимости от времени реакции. Выход продукта достигал количественного уже через 40 мин реакции.

Commercial preparations of trypsin, varying in activity, were immobilized in a cryogel of polyvinyl alcohol, activated by dialdehydes (terephthalic, succinic, or glutaric) or divinyl sulfone. All preparations of the immobilized enzyme exhibited hydrolytic activity and retained stability for 8 months. In an organic solvent environment, specimens of immobilized trypsin catalyzed the synthesis of N-carbobenzoxy-L-phenylalanyl-L-arginyl-L-leucine p-nitroanilide from N-carbobenzoxy-L-phenylalanyl-L-argininine methyl ester (or N-carbobenzoxy-L-phenylalanyl-L-arginine) and L-leucine p-nitroanilide, as well as the formation of N-carbobenzoxy-L-alanyl-L-alanyl-L-arginyl-L-phenylalanine p-nitroanilide from N-carbobenzoxy-L-alanyl-L-alanyl-L-arginine and L-phenylalanine p-nitroanilide. The presence of small amounts of water in organic solvents was prerequisite to the biocatalysts manifesting synthase activity in reactions of peptide bond formation.

Разработан химико-энзиматический синтез новых высокоспецифичных флуорогенных субстратов цистеиновых протеиназ семейства папаина – Abz-Phe-Ala-pNA (I) и Glp-Phe-Ala-Amc (II) (Abz, pNA, Glp и Amc – o-аминобензоил, n-нитроанилид, пироглутамил и 4-амино-7-метилкумарид соответственно). Субстрат (I) был получен в водно-органической среде с использованием нативного химотрипсина. Субстрат (II) – в смеси DMF–MeCN под действием химотрипсина и субтилизина Карлсберг, иммобилизованных на криогеле поливинилового спирта. Гидролиз субстрата (I) папаином, фицином и бромелаином сопровождался повышением интенсивности флуоресценции в 15 раз, субстрата (II) – изменением спектра флуоресценции. Показана однозначность ферментативного гидролиза субстратов после остатка Ala. Определена удельная активность гидролиза субстратов папаином, бромелаином и фицином. Показано, что наибольшую активность по обоим субстратам проявлял папаин. Активность всех исследованных протеиназ была существенно выше по субстрату (II), чем по субстрату (I). Минимально определяемые концентрации папаина составили по субстрату (I) – 2.4 ? 10-10 М, по (II) – 1.2 ? 10-11 М. Установлена высокая селективность цистеиновых протеиназ по отношению к Glp-Phe-Ala-Amc.

Исследованы активность и стабильность нативных субтилизинов Карлсберг и 72 и их комплексов с додецилсульфатом натрия (SDS) в средах, содержащих органические растворители. Определены кинетические константы гидролиза специфических хромогенных пептидных субстратов Z-Ala-Ala-Leu-pNA и Glp-Ala-Ala-Leu-pNA нативными субтилизинами Карлсберг и 72. Установлено, что субтилизин Карлсберг в комплексе с SDS в средах безводных органических растворителей является эффективным катализатором синтеза пептидов с полифункциональными аминокислотами в Р1 и Р'1 -положениях (Glu, Arg, Asp), содержащими незащищенные боковые ионогенные группы.

Предложена серия флуорогенных тетра-, пента- и гексапептидных субстратов с внутримолекулярным тушением флуоресценции общей формулы Abz-X-Phe-Phe-Y-Ded или (-pNA), где X = Ala, Ala-Ala, Val-Ala; Υ = -, Ala, Ala-Ala. Определены кинетические параметры гидролиза субстратов пепсином, катепсином D, гастриксином человека, пепсином свиньи, химозином теленка, аспергиллопепсином А. Показано, что синтезированные соединения - эффективные субстраты аспартильных протеиназ различного происхождения.A series of fluorogenic tetra-, penta-, and hexapeptide substrates of the general structure Abz-X-Phe-Phe-Y-Ded (or -pNa in place of -Ded), where X = Ala, Ala-Ala, or Val-Ala and Υ = -, Ala, or Ala-Ala, were proposed. Kinetic parameters of hydrolysis of these substrates by pepsin, cathepsin D, human gastricsin, pig pepsin, calf chymosin, and aspergillopepsin A were determined. The compounds synthesized proved to be effective substrates for aspartyl proteases of diverse origins.

The subtilisin-sodium dodecyl sulfate complex was shown to catalyze the coupling of peptide segments on a solid phase in organic medium. By a two-stage enzymic condensation of peptide fragments on aminosilochrom (A) containing Met-Ala-Gly as a spacer, Dnp(or Boc)-Ala-Ala-Leu-Ala-Ala-Glu(OMe)-Met-Ala-Gly-A and Z-Ala-Ala-Glu(OMe)-Ala-Ala-Leu-Met-Ala-Gly-A were obtained. It was shown that the condensation products can be split off from the support using the Met residue cleavage by BrCN.

Проведено сравнительное изучение растворимости, стабильности и активности нативного субтилизина-72 и его комплекса с SDS в ряде полярных органических растворителей. Показана возможность катализа образования пептидной связи субтилизином, суспендированным в ацетонитриле, и комплексом SDS-субтилизин, растворенным в этаноле и изопропаноле. С помощью SDS-субтилизина получены: трипептид Z-Ala-Ala-Leu-pNA, тетрапептиды общей формулы A-Ala-Ala-PpP,-В, где А = Z или Abz; Р, = Leu, Phe, Met, Trp, Ile, Tyr, Phe(N0 2), Glu(OCH 3), P', = Leu, Phe, Glu, Ala, Ile, Val, Arg; В = NH 2, pNA, Ded; пентапептиды Z-Ala-Ala-Leu-Ala-Ala-pNA и Z-Ala-Ala-Leu-Ala-Phe-pNA, гексапептид Abz-Val-Ala-Phe-Phe-Ala-Ala-Ded. Под действием SDS-субтилизина проведена олигомеризация трипептида H-Phe-Ala-Leu-OCH 3 в этаноле с получением 63% три- и 37% тетраолигомеров соответственно. Показано, что SDS-субтилизин является более эффективным катализатором, чем нативный фермент в виде суспензии.

Stepwise application of affinity chromatography on bacitracin-silochrome, gel filtration on Acrylex P-10, rechromatography on bacitracin-Sepharose 4B and gel filtration on Sephadex G-15, a homogeneous metalloproteinase (M(r) = 35,000 Da) has been isolated from the cultural filtrate of B. megaterium strain 599. The amino acid composition and N-terminal sequence (20 amino acids) of the enzyme have been determined. The proteinase is not inhibited by diisopropyl-fluorophosphate, is inhibited by o-phenanthroline, EDTA, and Zn2+, and is activated by Co2+. The enzyme has a peak activity at 60-65 degrees C. The maximum of the enzymatic activity after hydrolysis of synthetic substrates is at pH 6.5-7.0. The enzyme is stable at pH 7.0-9.0 and retains its stability at 45-60 C for several hours. In acid media the enzyme undergoes irreversible inactivation. The dependence of kcat/Km on pH points to the involvement of an ionogenic group with pKa 7.5 in the catalytic act, most probably of the imidazole group of histidine. The metalloproteinase hydrolyzes synthetic peptide substrates at the bonds formed by the amino groups of hydrophobic amino acids-Phe, Leu, Ile and Val.

Porcine pepsin behaviour during the synthesis of peptide p-nitroanilides and esters has been studied. In many cases, especially when long-chain peptides, such as Z-Ala-Ala-Phe-Leu-Ala-Ala-OMe, were synthesized, pepsin disappeared from the solution, being entrapped by the product precipitate rather than inactivated. Sorption of the enzyme on the product might be partially responsible for this effect. The active enzyme could be eluted from the precipitate by NaCl and isopropanol. Non-proteolytic proteins (lysozyme, bovine albumin, carbonic anhydrase) could also co-precipitate with pepsin.

Pepsin was shown to catalyze synthesis of esters or p-nitroanilides tri-, tetra-, penta- and hexapeptides of general formula Z-X-Y-B, where X = Ala-Phe, Phe-Met, Ala-Ala-Glu, Ala-Ala-Phe, Ala-Ala-Leu, Ala-Ala-Trp, Ala-Ala-Met. Y = Ala, Leu, Val, Phe, Arg, Ala-Ala, Gly-Gly, Leu-Ala-Ala, Phe-Ala-Ala. B = OMe, pNA. The reactions were carried out in dimethylformamide-water solutions at pH 4.6 by equimolar ratio of amino- and carboxyl components (with the exception of Arg-pNA taken in 2-fold excess). The amount of pepsin in the reaction approached 1:1700 enzyme: substrate molar ratio although it might be improved--up to 1:3.10(5) for relatively long peptides.

A homogeneous serine proteinase was isolated from cultural filtrates of the extreme halophilic bacteria Halobacterium mediterranei 1538 using affinity chromatography on bacitracin-Sepharose, ultrafiltration and gel filtration on Sephadex G-75, with a 48% yield and 260-fold purification. The enzyme was completely inactivated by specific inhibitors of serine proteinases, PMSF and DFP, as well as by Hg2+ and PCMB. The enzyme activity was strongly dependent of NaCl concentration, the enzyme being inactivated below 0.75 M NaCl. Inactivation of the enzyme was also seen in the presence of 2-7% organic solvents. The pH optimum for Glp-Ala-Ala-Leu-pNA hydrolysis is 8.0-8.5; Km is 0.14 mM, kcat is 36.9 s-1. The stability optimum lies at pH 5.5-8.0, temperature optimum is at 55 degrees C. The enzyme molecular weight is 41,000 Da; pI is 7.5. The substrate specificity of the enzyme is comparable to that of secretory subtilisins; the extent of protein substrate hydrolysis is similar to that of proteinase K. The N-terminal sequence of Halobacterium mediterranei serine proteinase, Asp-Thr-Ala-Asn-Asp-Pro-Lys-Tyr-Gly-Ser-Gln-Tyr-Ala-Pro-Gln-Lys-Val-Asn- Ala- Asp-, reveals a 50% homology with the aminoterminal sequence of Thermoactinomyces vulgaris serine proteinase. Hence, the serine proteinase secreted by halophilic bacteria may be considered as a structural and functional analog of eubacterial enzymes.

p-Nitroanilides of antranyloyltripeptides of the general structure Abz-Ala-Ala-P'1-pNA (P'1 = Phe, Leu, Ile, Val) containing intramolecularly quenched fluorescent groups (Abz is a fluorogenic group and pNA is a quencher of fluorescence) were prepared by combination of chemical and enzymatic methods. Thermolysin and metalloproteinases from Legionella pneumophila and Thermoactinomyces species were shown to hydrolyse Ala-P'1 bond of the peptides with simultaneous 4-7 fold increase in fluorescence. Kinetic parameters for enzymatic hydrolysis of the substrates were determined. Metalloendopeptidases can be assayed in the presence of serine proteinases (of the subtilisin type) using Abz-Ala-Ala-Ile-pNA or Abz-Ala-Ala-Val-pNA.

Two methods of isolation of pure aspergillopepsin A have been developed. The first method is based on sequential chromatography of a crude preparation on gramicidin C-sepharose 4B and ECTEOLA-cellulose (35% yield, 100-fold purification). The second method consists in sequential chromatography of evaporated extract of Asp. awamori surface culture on Acrylex P-10, aminosilochrome and ECTEOLA-cellulose (40% yield, 430-fold purification). The methods discussed may be used to isolate pure aspergillopepsin A preparations in order to establish the primary structure of the enzyme.