Hydroxamate-based colorimetric method for direct screening of transglutaminase-producing bacteria

Abstract

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.

Microbial transglutaminase and its application in the food industry. A review

“…The literature reports on the potential use of plant materials such as soy, rice, maize and wheat flour, corn steep liquor, wheat middlings or malt extract as a source of nitrogen (Zhu et al 1995 ). Bourneow et al ( 2012 ) demonstrated that peptone was the best source of nitrogen for transglutaminase extraction from the Streptomyces sp. P20 and S .…”

“…C2361 0.77 Bourneow et al ( 2001 ) Providencia sp. C1112 0.92 Bourneow et al ( 2001 ) Streptoverticillium mobaraense 0.9–3.4 Bourneow et al ( 2012 ); Zheng et al ( 2002 ); Zhu et al ( 1996 ); Gerber et al ( 1994 ); Ando et al ( 1989 ) Streptomyces platensis M5218 0.66 Lin et al ( 2006 ) Streptomyces hygroscopicus n/a Cui et al ( 2007 ) Streptomyces lividans n/a Lin et al ( 2004 , 2007 ) Streptomyces lividans JT46/pAE053 2.2 Lin et al ( 2006 ) Streptomyces lydicus 1.3 Bourneow et al ( 2001 ) Streptomyces platensis 1.4 Lin et al ( 2006 ); Bourneow et al ( 2001 ) Streptomyces sioyansis 3.3 Bourneow et al ( 2001 ) Streptoverticillium griseocarneum 1.46 Gerber et al ( 1994 ); Lin et al ( 2003 ) Streptoverticillium ladakanum NRRL–3191 0.28–1.55 Téllez-Luis et al ( 2004a , b ) Streptoverticillium sp. s–8112 1.46 Kanaji et al ( 1993 ) …”

MICROBIAL TRANSGLUTAMINASE OF INDONESIAN Streptomyces sp. : Screening and Production in several media

“…Samples from Gadjah Mada University were firstly screened qualitatively using filter paper disc (FPD) method developed by Bourneow et al (2012) with slight modification. The positive colonies were then quantitatively assayed for their enzyme activity (Grossowicz et al, 1950) with slight modification.…”