Lee Hee Kyoung, Jang Won Young, Kim Young Ho
Laboratory of Immunobiology, School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu, Republic of Korea.
AMB Express. 2023 May 5;13(1):42. doi: 10.1186/s13568-023-01551-w.
Four GH16 family β-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 °C and 7.0, respectively. The enzyme was stable up to 50 °C and over a pH range of 5.0-8.0. The kinetic parameters, including Km, Vmax, kcat, and kcat/Km, of GH16B β-agarases for agarose were 14.40 mg/mL, 542.0 U/mg, 576.3 s, and 4.80 × 10 s M, respectively. The addition of 1 mM MnCl 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 µg/mL) under continuous magnetic stirring at 50 °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 ~ 650 mg NA4/~ 900 mg NA6 (i.e., ~ 85.3% of the theoretical maximum yield). These findings suggest that the recombinant thermostable GH16B β-agarase is useful for agarose liquefaction to produce NA4 and NA6.
从琼脂分解菌纤维弧菌属菌株KY-GH-1中分离出4种GH16家族β-琼脂酶（GH16A、GH16B、GH16C和GH16D），并在大肠杆菌系统中表达，比较了它们的活性。只有GH16B（597个氨基酸，63.8 kDa）带有N端22个氨基酸的信号序列，分泌到培养上清液中，并表现出强大的内切琼脂糖水解活性，最终产物为新琼脂四糖（NA4）和新琼脂六糖（NA6）。该酶活性的最适温度和pH分别为50℃和7.0。该酶在50℃以下以及pH值为5.0-8.0的范围内稳定。GH16Bβ-琼脂酶对琼脂糖的动力学参数，包括Km、Vmax、kcat和kcat/Km，分别为14.40 mg/mL、542.0 U/mg、576.3 s和4.80×10 s M。添加1 mM MnCl和15 mM三（2-羧乙基）膦可增强酶活性。当以琼脂糖或新琼脂寡糖为底物时，酶催化的终产物为NA4和NA6，而以琼脂寡糖为底物时，除NA4和NA6外还产生琼脂五糖。在50℃连续磁力搅拌下，用该酶（1.6 μg/mL）处理9%[w/v]的融化琼脂糖14小时，可使琼脂糖有效液化成NA4和NA6。通过Sephadex G-15柱色谱从酶解产物（9%[w/v]琼脂糖，20 mL）中纯化NA4和NA6，得到约650 mg NA4/约900 mg NA6（即理论最大产量的约85.3%）。这些发现表明，重组热稳定GH16Bβ-琼脂酶可用于琼脂糖液化以生产NA4和NA6。
