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Title 

Specific detection of pullulanase type I in polyacrylamide gels

Authors 

Chul Ho Kim

Publisher 

Wiley-Blackwell

Issue Date 

1994

Citation 

FEMS Microbiology Letters, vol. 116, no. 3, pp. 327-331

Keywords 

activity stainingdebranching enzymepullulanpullulanase type Istarchzymogrampullulanasearticlepolyacrylamide gel electrophoresispriority journal

Abstract 

A specific detection of pullulanase type I which hydrolyzes the α-1,6- glycosidic linkages on pullulan and starch was developed using impregnation of gels with soluble starch and staining for amylose with iodine. It was a simple and highly sensitive zymogram method capable of detecting as little as 0.001 unit of pullulanase type I activity in polyacrylamide gels after electrophoresis. After fractionation of crude enzyme using DEAE ion exchange chromatography to avoid possible co-migration of amylolytic enzymes which disturb the interaction between amylose and iodine, the high and critical sensitivity of the detection was achieved. The specific detection is based on the fact that when pullulanase type I hydrolyzes the α-1,6-glycosidic bonds in soluble starch increased amounts of α-1,4-linked amylose is formed which yields more intensely blue colored conjugate with iodine. Thus, blue bands on the lighter background signal the presence of pullulanase type I. In contrast, amylolytic enzymes give 'white' bands on the lightly stained background because they remove amylose. This procedure is effective in enzyme screening to distinguish debranching enzyme (pullulanase type I) from other pullulan-degrading enzymes.

ISSN 

0378-1097

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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