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M S Mel'nik

Publications and source records attributed to M S Mel'nik.

6 recordsLinked to original sources
PubMed1991

[Cellobiohydrolase from Clostridium thermocellum, synthesized by a recombinant E. coli strain].

Clostridium thermocellum cellobiohydrolase was isolated in preparative amounts from the recombinant strain of E. coli K12 C600 carrying plasmid pCU 304 with a C. thermocellum chromosomal DNA insertion. The isolation procedure included chromatography on Ultrogel AcA 44, ion-exchange chromatography on DEAE-Sepharose CL-6B, rechromatography on Ultrogel and FPLC on Mono Q resulting in a 17.6% yield and 1530-fold purification. According to data from sodium dodecylsulfate polyacrylamide gel electrophoresis performed under nondenaturing conditions and analytical gel isoelectrofocusing, the enzyme preparation contains only one active protein band with Mr 56.2 +/- 1.0 kDa and pI 4.15. The enzyme does not reduce the viscosity of the CM-cellulose solution but forms reducing sugars from this soluble substrate. Cellobiose (93-97%) is the major component produced by the enzyme from crystalline and amorphous cellulose (specific activity 2.3 x 10(-3) and 2.8 x 10(-2) U/mg, respectively). The activity optimum of the enzyme is at pH 5.6, 60 degrees C. The half-inactivation time at 60 degrees C and 65 degrees C is 450 and 15.5 min, respectively. The action pattern of the enzyme on the low molecular fluorogenic cellooligosaccharides suggests that the enzyme pertains to typical cellobiohydrolases.

Binding Sites↗
PubMed1989

[A new type of Clostridium thermocellum endoglucanase produced by the recombinant strain of E. coli. Some properties and identification in donor cells].

The properties of endoglucanase produced by the recombinant strain of E. coli carrying plasmid pCU 104 with a 2.9 kb insert of chromosomal DNA of C. thermocellum encoding the multiple forms of the 35.5 kD polypeptide (pI 4.3-4.7) were studied. The enzyme has a broad pH optimum of activity (6.0-7.5). The half-inactivation time for different forms of the enzyme at 65 degrees C is similar and is equal to 25-30 minutes. The enzyme is related to endoglucanases weakly adsorbed on cellulose (Kp = 0.065 1/g). Hydrolysis of microcrystalline cellulose is completed within 7 days (7-9%) and is accompanied by the formation of cellobiose and cellotriose. The enzyme splits dyed lichenan (mixed 1,3-1,4-beta-glucane) at a higher rate than the dyed CM-cellulose. A guinea pig antiserum to enzyme isoforms with a pI of 4.46-4.54 was obtained. Using direct solid phase immunoenzymatic analysis, it was demonstrated that all the enzyme isoforms under study (pI 4.3-4.7) are immunologically related (serum titers for different enzyme isoforms vary from 1:20,000 to 1:50,000). In the original culture fluid of C. thermocellum, the antigen related to the enzyme isolated from the recombinant strain was unobserved. However, SDS-PAAG electrophoresis of SDS- and mercaptoethanol-treated culture fluids revealed among 11 protein bands at least 4 antigens interacting with antibodies (Mr = 107, 76, 67 and 37 kD), although their antibody titers were far lower and did not exceed 1:300-1:500. The cumulative data suggest that the endoglucanase under study is not identical to the earlier described enzymes encoded by the cel A- and ceI B-genes of C. thermocellum.

Animals↗
PubMed1982

[Methylpyrophosphate, the simplext organic substrate of yeast inorganic phyrophosphatase].

The interaction between yeast inorganic pyrophosphatase and the simplest organic substrate, methylpyrophosphate, was studied. Methylpyrophosphate hydrolysis occurred most intensively in the presence of Zn2+ less intensively in the presence of CO2+ and Mn2+ and did not occur at all in the presence of Mg2+. The complex convertible into reaction products contains two Zn2+ and one molecule of zinc methylpyrophosphate per one active center. The activator metal ions are linked with pyrophosphatase consecutively; each of the two enzyme forms produced can bind to the substrate. The values of the maximal velocity and the constants of the substrate binding to the pyrophosphatase were calculated.

Binding Sites↗
PubMed1982

[Some features of methylpyrophosphate hydrolysis by yeast inorganic pyrophosphatase].

The interaction of yeast inorganic pyrophosphatase with methylpyrophosphate was studied. In the presence of Mg2+ the rate of hydrolysis of the methylpyrophosphate-Zn2+ complex by the enzyme was shown to decrease. This was accompanied by competition of Zn2+ and Mg2+ for one site of Me2+ binding on the enzyme. The kinetics of combined hydrolysis of zinc methylpyrophosphate and zinc pyrophosphate were studied. It was found that both substrates are hydrolyzed at the same active site of the enzyme. Free methylpyrophosphate when bound to a specific phosphorylation site on the enzyme surface accelerated magnesium pyrophosphate hydrolysis. Some kinetic parameters of this hydrolysis were determined.

Binding Sites↗
PubMedDate not supplied

[Cellulases from microorganisms].

Compositions of cellulase-hemicellulase systems of aerobic fungi (hyphomycetes, ascomycetes, and basidiomycetes), aerobic bacteria, actinomycetes, as well as anaerobic fungi and bacteria, are considered in the context of modern structural classification of glycosyl hydrolases. A new nomenclature of cellulases and relative enzymes based on their structural classification is reviewed. Some opportunities of cellulase improvement by means of protein engineering are discussed.

Bacteria, Aerobic↗