Colloid chitin azure is a dispersible, low-cost substrate for chitinase measurements in a sensitive, fast, reproducible assay.
Chitin and its derivatives are widely used as biomedical materials because of their versatility and biocompatibility. Chitinases are enzymes that produce chito-oligosaccharides from chitin. The assay of chitinase activity is difficult because few appropriate substrates are available. In this study, the authors developed an efficient and low-cost chitinase assay using colloidal chitin azure. The assay feasibility is evaluated and compared with traditional assays employing colloidal chitin and chitin azure. The authors found that the optimum pH for determining chitinase activity using colloid chitin azure was pH 5 or 8. The method was sensitive, and the assay was complete within 30 min. When the assay was used to measure chitinase activities produced by 2 strains of chitinolytic bacteria, BCTS (an Escherichia coli BL21 [DE3] expressing a secretory recombinant chitinase) and AS1 (a chitinolytic bacterium with low levels of chitinase), it was shown that cultivation in Bushnell-Haas selection medium caused AS1 to secrete a higher level of chitinase than was secreted when the bacterium grew in other media. In summary, colloid chitin azure is a sensitive, feasible, reproducible, and low-cost substrate for the assay of chitinase activity.
Shen
Chia-Rui
CR
Graduate Institute and Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Kweishan, Taoyuan, Taiwan.
Chen
Yu-Sheng
YS
Yang
Ching-Jen
CJ
Chen
Jeen-Kuan
JK
Liu
Chao-Lin
CL
D001399:Azure Stains / Q000737:chemistry / Q000191:economics; D001672:Biocompatible Materials / Q000378:metabolism*; D001681:Biological Assay; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D003102:Colloids; D004396:Coloring Agents / Q000737:chemistry / Q000191:economics; D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005240:Feasibility Studies; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013997:Time Factors
D001399:Azure Stains; D001672:Biocompatible Materials; D003102:Colloids; D004396:Coloring Agents; D003470:Culture Media; D011994:Recombinant Proteins; D002686:Chitin; D002688:Chitinases