Detection, purification and partial characterization of beta-amylase from trophozoites of Entamoeba invadens.
Trophozoites of Entamoeba invadens were able to ingest glucopolysaccharides and metabolize them. An activity capable of degrading these substrates was purified to apparent homogeneity. The enzyme was identified as beta-amylase (alpha-1,4-D-glucan maltohydrolase EC 3.2.1.2): It was active against glycogen, amylose and amylopectin in a ratio of 100:198:133 and was also able to attack linear alpha-1,4-glucooligosaccharides with more than three glucose moieties. All degradation experiments yielded maltose as reaction product, and no free glucose could be detected. While amylose was completely degraded, amylolysis of glycogen and amylopectin yielded limit dextrins besides maltose. The enzyme was completely inactive against cyclohexaamylose, cycloheptaamylose and pullulan, indicating its lack of endo-glucosidase specificity. Hydrolysis of 4-nitrophenyl-maltoheptaoside resulted in the successive removal of maltose units from the non-reducing end yielding 4-nitrophenyl-maltopentaoside, -trioside and -glucoside. No 4-nitrophenyl-glycosides with even numbered glucose moieties were formed from this substrate. The enzyme exhibited a relative molecular mass of M(r) = 45,000 +/- 5% by gel filtration and sodium dodecyl sulfate (SDS) gel electrophoresis and the N-terminal sequence 1 VEVNVMLPL 9. Optimal hydrolysis was observed at pH 5.5 and a temperature of 42 degrees C. On the basis of inhibition by mercury ions and p-chloro-mercurybenzoate and abrogation of this effect by thiol reagents beta-amylase was identified as sulfhydryl-enzyme.
Serafimova
F
F
Fachbereich Biologie/Chemie, Abteilung Biochemie, Universität Osnabrück, Germany.
Franz
A
A
Werries
E
E
D000818:Animals; D050260:Carbohydrate Metabolism; D004747:Entamoeba / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D017421:Sequence Analysis; D013379:Substrate Specificity; D013696:Temperature; D001614:beta-Amylase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D001614:beta-Amylase