Characterization and large-scale production of recombinant Streptoverticillium platensis transglutaminase.
Recombinant Streptomyces platensis transglutaminase (MtgA) produced by the Streptomyces lividans transformant 25-2 was purified by ammonium sulfate fractionation, followed by CM-Sepharose CL-6B fast flow, and blue-Sepharose fast flow chromatography. The purification factor was approximately 33.2-fold, and the yield was 65%. The molecular weight of the purified recombinant MtgA was 40.0 KDa as estimated by SDS-PAGE. The optimal pH and the temperature for the enzyme activity were 6.0 and 55 degrees C, respectively, and the enzyme was stable at pH 5.0-6.0 and at temperature 45-55 degrees C. Enzyme activity was not affected by Ca(2+), Li(+), Mn(2+), Na(+), Fe(3+), K(+), Mg(2+), Al(3+), Ba(2+), Co(2+), EDTA, or IAA but was inhibited by Fe(2+), Pb(2+), Zn(2+), Cu(2+), Hg(2+), PCMB, NEM, and PMSF. Optimization of the fermentation medium resulted in a twofold increase of recombinant MtgA activity in both flasks (5.78 U/ml) and 5-l fermenters (5.39 U/ml). Large-scale productions of the recombinant MtgA in a 30-l air-lift fermenter and a 250-l stirred-tank fermenter were fulfilled with maximal activities of 5.36 and 2.54 U/ml, respectively.
Lin
Shie-Jea
SJ
Bioresource Collection and Research Center, Food Industry Research and Development Institute, 331 Shih-Pin Road, 30062, Hsinchu, Taiwan.
Hsieh
Yi-Fang
YF
Lai
Li-An
LA
Chao
Mei-Li
ML
Chu
Wen-Shen
WS
D002412:Cations / Q000494:pharmacology; D003470:Culture Media; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D048170:Streptomyces lividans / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D013306:Streptomycetaceae / Q000201:enzymology* / Q000235:genetics; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D011503:Transglutaminases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D002412:Cations; D003470:Culture Media; D011994:Recombinant Proteins; D011503:Transglutaminases