Effect of different incubation conditions and stress factors on microbial transglutaminase production, partial purification and characterisation of the enzyme

In this study, microbial production of transglutaminase (TG) from different strains, development of fermentation conditions, determination of stress factors affecting enzyme production, partial purification by different methods and characterization of enzyme have been carried out. Microbial transglutaminase (mTG) production (0.036 Unit/ml) was achieved in starch-glucose based medium (GNB) at pH 6.0 and 30°C using Streptomyces mobaraensis (NRRL B-3729). At the same conditions, an increase of ~3.75 fold in enzyme activity was achieved with orbital incubation and the production period was shortened. In addition, an activity of 0.423 Unit/ml was obtained with a controlled fermentation (500 rpm, 10 cm3 air/min) where more efficient mixing and aeration was provided, resulting in an increase of 3.13 and 11.75 fold in enzyme activity compared to orbital and static incubation, respectively. The effect of stress factors on enzyme production has been examined, and the salts and trypsin provided a significant increase in enzyme activity. In the partial purification step, the yield was 93% and the purification fold was 1.18 for the precipitation with 80% ammonium sulfate salt saturation, and the yield was 98% and the purification fold was 1.00 for the 80% ethanol concentration. Kinetic parameters of the partially purified enzyme obtained after ammonium sulfate precipitation and dialysis were determined and the Michaelis constant (Km) was calculated as 6.35 mM and the maximum velocity (Vmax) as 0.35 Unit/ml. While the optimum temperature of the enzyme was 37°C and pH 5.8, the stability was observed to be high (~ 93%) at 55°C and pH 6.0. There was a loss of mTG enzyme activity at the presence of certain metal ions (Fe+2, Co+2, Zn+2), and an increase was determined in EDTA. These results demonstrate that different fermentation strategies can be used to increase mTG production and characterization of purified enzymes is important in terms of their utility in industrial applications.