Hydroxamate-based colorimetric method for direct screening of transglutaminase-producing bacteria.
Microbial transglutaminase (MTGase) is a commercial enzyme that has been applied to many protein containing foods to improve their textural property. The screening of MTGase-producing microorganisms from various sources might lead to the discovery of a new MTGase with different characteristics. This report demonstrates the use of a direct detection method for MTGase-producing bacteria grown on an agar plate by filter paper disc (FPD) assay. The principle of the assay is the formation of a red burgundy color by the hydroxamate-ferric complex. The color developed intensity was linearly correlated by the concentration of hydroxamic acid in the range of 0.1-0.8 μM and was visually scored at 4 levels: 0, 1, 2 and 3. Streptoverticillium mobaraense DSM 40847, a positive MTGase-producer, was chosen for the verification and improving of the proposed method. The colonies grown on the nutrient agar plate at 37°C for 24 h were covered with FPDs and 30 μl of substrates (CBZ-Gln-Gly and hydroxylamine). After incubation, 10 μl of the ferric-TCA-HCl solution was placed on the FPD. The optimal time taken to catalyze the formation of CBZ-Gln-Gly-hydroxamic acid by the MTGase and the time taken for the hydroxamate-ferric complex to form color were 180 and 60 min, respectively. Using this assay, 30 of 189 colonies isolated from wastewater and floating-floc samples showed MTGase-positive colonies which were well correlated to the quantitative screening of MTGase activity (R(2) = 0.9758). The results revealed that the FPD assay could be used for the qualitative screening of MTGase-producing bacteria.
Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai 90112, Thailand.
Chaiwut
C
Bourneow
Soottawat
S
Benjakul
Aran
A
H-Kittikun
D000362:Agar; D001419:Bacteria / Q000201:enzymology*; D003124:Colorimetry / Q000379:methods*; D003470:Culture Media / Q000737:chemistry; D006877:Hydroxamic Acids / Q000378:metabolism*; D008403:Mass Screening / Q000379:methods*; D011503:Transglutaminases / Q000032:analysis*
D003470:Culture Media; D006877:Hydroxamic Acids; D000362:Agar; D011503:Transglutaminases