[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.
Mel'nik
M S
MS
Kapkov
D V
DV
Mogutov
M A
MA
Rabinovich
M L
ML
Klesov
A A
AA
D000818:Animals; D002480:Cellulase / Q000032:analysis* / Q000037:antagonists & inhibitors; D003013:Clostridium / Q000201:enzymology*; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006168:Guinea Pigs; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007124:Immunoenzyme Techniques; D007700:Kinetics; D008970:Molecular Weight
D004274:DNA, Recombinant; C056374:endoglucanase 1; D002480:Cellulase
Novyĭ tip éndogliukanazy Clostridium thermocellum, obrazuemoĭ rekombinantnym shtammom E. coli. Svoĭstva i identifikatsiia v organizme donora.