Extracellular production of a thermostable Cellvibrio endolytic β-agarase in Escherichia coli for agarose liquefaction

초록

Four GH16 family beta-agarases (GH16A, GH16B, GH16C, and GH16D), originated from an agarolytic bacterium Cellvibrio sp. KY-GH-1, were expressed in an Escherichia coli system and their activities were compared. Only GH16B (597 amino acids, 63.8 kDa), with N-terminal 22-amino acid signal sequence, was secreted into the culture supernatant and demonstrated a robust endolytic agarose hydrolyzing activity for producing neoagarotetraose (NA4) and neoagarohexaose (NA6) as end products. The optimal temperature and pH for the enzyme activity were 50 degrees C and 7.0, respectively. The enzyme was stable up to 50 degrees C and over a pH range of 5.0-8.0. The kinetic parameters, including Km, Vmax, kcat, and kcat/Km, of GH16B beta-agarases for agarose were 14.40 mg/mL, 542.0 U/mg, 576.3-s(-1), and 4.80 x-10(6) s(-1) M-1, respectively. The addition of 1 mM -MnCl2 and 15 mM tris(2- carboxyethyl)phosphine enhanced the enzymatic activity. When agarose or neoagaro- oligosaccharides were used as substrates, the end products of enzymatic catalysis were NA4 and NA6, whereas agaropentaose was produced along with NA4 and NA6 when agaro-oligosaccharides were used as substrates. Treatment of 9%[w/v] melted agarose with the enzyme (1.6 mu g/mL) under continuous magnetic stirring at 50 degrees C for 14 h resulted in efficient agarose liquefaction into NA4 and NA6. Purification of NA4 and NA6 from the enzymatic hydrolysate (9%[w/v] agarose, 20 mL) via Sephadex G-15 column chromatography yielded similar to 650 mg NA4/similar to 900 mg NA6 (i.e., similar to 85.3% of the theoretical maximum yield). These findings suggest that the recombinant thermostable GH16B ss-agarase is useful for agarose liquefaction to produce NA4 and NA6.

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