Purification and characterization of paralytic shellfish toxin-transforming enzyme, sulfocarbamoylase I, from the Japanese bivalve Peronidia venulosa.

The Japanese bivalve Peronidia venulosa contains paralytic shellfish toxin (PST)-transforming enzymes that convert the weakly toxic C-toxins to the more potent decarbamoyl toxins. The enzyme was purified 154-fold with a yield of 0.26% and was named sulfocarbamoylase I. It was found to be a protein with an estimated molecular weight of 300 kDa by gel filtration column chromatography. Observation of a single band equivalent to 150 kDa on SDS-PAGE with or without reducing agents suggested it to be a homodimer with ionically bound subunits. The enzyme catalyzes the hydrolysis of the carboxyl bond in the N-sulfocarbamoyl moiety of PSP-toxins. The sulfonyl moiety in the carbamoyl side chain of substrates is essential for enzyme recognition. The N-terminal amino acid sequences of nine tryptic peptides were determined by the Edman degradation method. In a database search using the BLAST program, no protein that shows remarkable homology was retrieved. Several characteristics of the enzyme were also compared with those of another PST-transforming enzyme, carbamoylase I, which was previously isolated from the Japanese clam Mactra chinensis.

Assessment of Microbiological and Chemical Quality of Bubble Tea Beverages Vended in Taiwan.

Bubble tea beverages (n = 105) purchased from vendors in Taiwan were tested to determine their microbiological and chemical quality. Nearly half of the tested samples (48.6%, 51 of 105) had aerobic plate counts (APCs) higher than the Taiwan Food and Drug Administration guideline of 4.0 log CFU/mL, and 55 (52.4%) had coliform counts (most probable number [MPN]) higher than the 10 MPN/mL guideline. Escherichia coli, Salmonella, Staphylococcus aureus, sweeteners, preservatives, maleic acid, and coumarin were not detected in any sample. However, catechins were not detected to 188 mg/mL, and caffeine was 10.1 to 457.6 mg/mL. Bubble tea samples obtained from vendors in southern Taiwan had a mean APC of 2.6 log CFU/mL and a mean coliform count of 61.7 MPN/mL; these values were significantly lower (P < 0.05) than those from samples collected from vendors in northern, eastern, or central Taiwan. Samples obtained from southern Taiwan had the highest mean catechin concentrations of 21.3 mg/mL (P < 0.05). About 60% (63 of 105) of the bubble tea samples were not labeled with the origin of the tea leaves, which is in violation of Taiwanese food labeling regulations. In general, the bubble tea beverages tested had satisfactory microbial and chemical qualities.

Purification and characterization of paralytic shellfish toxin transforming enzyme from Mactra chinensis.

A carbamoylase, which catalyzes hydrolysis of the carbamoyl (or N-sulfocarbamoyl) moiety of paralytic shellfish toxins, was purified from the digestive glands of the Japanese clam Mactra chinensis. Using five steps of column chromatography, 290 microg of Carbamoylase I showing homogeneity on SDS-PAGE was obtained. Carbamoylase I was revealed to be a glycoprotein, having estimated molecular weight of 190 kDa. Observation of single band equivalent to 94 kDa on SDS-PAGE under reducing conditions suggested it to be a homodimer. The optimal temperature and pH were 20 degrees C and 7.0. Carbamoylase I did not require a divalent cation and its activity was inhibited by the serine proteinase inhibitors, benzenesulfonyl fluoride and 4-(2-aminoethyl)-benzenesulfonyl fluoride. Carbamoylase I hydrolyzed both carbamate and N-sulfocarbamate toxins. The presence or absence of a hydroxyl moiety at the N-1 position of the substrate toxins did not significantly alter the reaction rate, but the stereochemistry of sulfate esters at C-11 greatly affected it. The K(m) was 3.02 microM for saxitoxin as a substrate. Nineteen amino acids of the N-terminal sequence were identified by the Edman method. MALDI-TOF-MS/MS spectra of (18)O-labeled tryptic peptides indicated the possible internal amino acid sequences of five peptides.