摘要:

To clone the malolactic enzyme gene fromLactobacillussp. 89, construction of a shuttle vector able to express itself inLactobacillussp. 89 andEscherichia coliwas undertaken. The shuttle plasmid pLE16 resulted from the union of pBR328 and of the pLB10 plasmid extracted fromLactobacillus bulgaricus10. The bacterial transformation inLactobacillussp. 89 was performed by electroporation, and the clones were selected on MRS medium with 30 μg∙mL−1chloramphenicol added. Fifty percent of the clones fromLactobacillussp. 89 lost their resistance to chloramphenicol following 28 generations when the selection pressure was not maintained. The restriction map of pLE16 (7600 bp) was established using several restriction enzymes.Key words: malolactic enzyme, shuttle plasmid,Escherichia coli,Lactobacillus, electroporatio

ISSN:0008-4166    

DOI:10.1139/m92-011

出版商:NRC Research Press    

年代:1992

数据来源: NRC

摘要:

Two lipolytic proteins (61 and 57 kDa) present in a Sephadex G-100 fraction of extracellular lipase fromGeotrichum candidumATCC 66592 were separated using high-performance liquid chromatography. Crossed electrofocusing immunoelectrophoresis was used to demonstrate that the 61-kDa lipase fraction contained two forms of lipase with pI 4.5 and 4.7. However, when deglycosylated with endoglycosidase H, the two forms gained an identical pI, 4.6. The 57-kDa lipase fraction contained one form of lipase with pI close to 4.5. Although the 61- and 57-kDa lipases were immunologically identical, the substrate specificity differed. Thus, the 61-kDa lipase hydrolysed palmitic acid methyl ester at an initial velocity of hydrolysis that was 60% of the initial velocity of hydrolysis of oleic acid methyl ester, whereas the 57-kDa lipase hydrolysed palmitic acid methyl ester at an initial velocity of hydrolysis that was only7% of the initial velocity of hydrolysis of oleic acid methyl ester.Key words:Geotrichum candidum, lipases, multiple forms, deglycosylation, substrate specificity.

ISSN:0008-4166    

DOI:10.1139/m92-012

出版商:NRC Research Press    

年代:1992

数据来源: NRC

摘要:

A multiplex polymerase chain reaction was developed to simultaneously detect the presence of toxin A and toxin B genes ofClostridium difficile. A 1050-bp fragment of the toxin B gene and a 1217-bp fragment of the toxin A gene were amplified from 42 toxic strains ofC.difficile; however, from 10 nontoxic strains the toxin gene fragments were not amplified; these data demonstrate that this multiplex polymerase chain reaction procedure can be used to differentiate between toxic and nontoxic strains. This sensitive and specific multiplex polymerase chain reaction forC.difficiletoxins may prove to be a valuable diagnostic procedure.Key words:Clostridium difficile, polymerase chain reaction, bacterial toxins.

ISSN:0008-4166    

DOI:10.1139/m92-013

出版商:NRC Research Press    

年代:1992

数据来源: NRC