ABSTRACT
Endpolygalacturonase I from Stereum purpureum has been identified as a causative substance for the silver-leaf disease in apples. It possesses a unique pro-sequence in the C-terminal region that lacks endpolygalacturonases from any other origin. In this study, we analyzed and compared enzymatic characteristics between pro-form (pro-endoPG I) and mature form processed by V8 protease (endoPG I) and described the suppression activity of the pro-sequence. Of note, the optimal pH for pro-endoPG I activity shifted to pH 4.0 from pH 4.5-5.0 of endoPG I. The kinetic parameters indicated that the activity inhibition resulted from a pH-independent decrease of substrate affinity and pH-dependent deterioration of velocity by the pro-sequence. Analysis of site-directed mutations within pro-endoPG I showed that its α-helical structure includes two glutamates (E364 and E366) and alanine (A365), and its orientation by prolines (especially P348) in the pro-sequence played a significant role in its suppression activity. As for mutations in the mature domain, a marked reduction of suppression was observed for enzymes with mutations in H150, R220 and K253, indicating that the pro-sequence interacts with the active cleft by a few ionic bonds.
Subject(s)
Agaricales/enzymology , Enzyme Precursors/chemistry , Enzyme Precursors/metabolism , Polygalacturonase/chemistry , Polygalacturonase/metabolism , Agaricales/genetics , Amino Acid Sequence , Hydrogen-Ion Concentration , Kinetics , Malus/microbiology , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Pectins/metabolism , Polygalacturonase/genetics , Sequence Homology, Amino Acid , Serine Endopeptidases/metabolismABSTRACT
Continuous monitoring of ammonia removal by microbial complexes and observation of their morphology were carried out using a microdevice. Consumption of NH(4)(+) ions by active sludge could clearly be recorded over 48 h. Aggregation of the sludge could be observed in parallel by using confocal reflection microscopy.
Subject(s)
Ammonia/metabolism , Microbiological Techniques/methods , Sewage/microbiology , Microscopy, Confocal/methodsABSTRACT
The feasibility of a method to monitor biofilm development non-destructively in a microfluidic device was addressed. Here, we report that biofilm growth could be non-destructively monitored by an image analysis technique based on modification of confocal reflection microscopy.
Subject(s)
Bacterial Load/methods , Biofilms/growth & development , Image Interpretation, Computer-Assisted/methods , Microfluidic Analytical Techniques/methods , Microscopy, Confocal/methods , Streptococcus mutans/cytology , Streptococcus mutans/growth & development , Bacterial Load/instrumentation , Bioreactors , Cell Culture Techniques , Microfluidic Analytical Techniques/instrumentationABSTRACT
The feasibility of a method to nondestructively measure planktonic bacterial growth in a microfluidic device was addressed. Here, we report that the growth of Pseudomonas aeruginosa in a microfluidic device could be measured by a three-dimensional image analysis technique based on confocal reflection microscopy in a time-course.
