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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.

Culture filtrates of 22 mushrooms were screened for extracellular prolyl oligopeptidase activity. Four strains with relatively high enzyme activity were all from inky cap mushrooms. The production of Coprinopsis clastophylla prolyl oligopeptidase was associated with the growth of the fungus and the enzyme was not released by cell lysis. The enzyme was purified 285-fold to a specific activity of 52.05 U/mg. It was purified to a single band on a native polyacrylamide gel. However, the enzyme separated into three bands on a sodium dodecyl sulfate-polyacrylamide gel with mobility corresponding to molecular weights of approximately 84, 60, and 26 kDa. The results of tandem mass spectrometric analysis revealed that the 60 kDa protein was likely a degradation product of the 84 kDa protein. The isoelectric point of the purified enzyme was 5.2. The purified enzyme had an optimal pH and temperature of 8.0 and 37°C, respectively. Diisopropyl fluorophosphate (DFP), p-chloromercuribenzoaic acid (PCMB), Hg(2+), and Cu(2+) strongly inhibited C. clastophylla prolyl oligopeptidase. This enzyme is a serine peptidase and one or more cysteine residues of the enzyme are close to the active site.

Soybean is an excellent source of plant-derived protein, and soy products are one of the important protein sources for vegetarians in Taiwan. Soybean production in Taiwan is not self-sufficient. Taiwan imports 2.5 million tons of soybeans annually. More than 90% of the imported soybeans are genetically modified. To provide an objective assessment on safety of genetically modified soybean and for post-market monitoring, we conducted a comparative assessment on key component compositions between imported genetically modified and organic soybeans. All the soybean samples were purchased from the local market to simulate the status of Taiwanese consumers purchasing soybeans. The genetically modified soybean samples were herbicide-tolerant soybeans. The contents of the proximate, the amino acid composition, the fatty acid composition, vitamins, minerals, isoflavones, and anti-nutritional factors of soybean samples were analyzed. Most contents of the key components of the genetically modified soybean had no significant difference with those of the organic soybean in this study. However, the contents of cysteine and α-linoleic acid were significantly lower in the genetically modified soybean samples, and the contents of crude fat, palmitic acid, stearic acid, oleic acid, linoleic acid, vitamin K1, and calcium were significantly higher in the genetically modified soybean samples. But they were all within the range of reference values. A total of 314 pesticide residues in each of the samples were analyzed. Only glyphosate residue was detected in the genetically modified soybean samples, but it is well below the threshold prescribed by the government. In summary, the imported genetically modified and organic soybeans purchased from Taiwan market were shown to be substantially equivalent in composition.