College of Life Science and Technology, Guangxi University, Nanning, Guangxi, P.R. China.
Prep Biochem Biotechnol. 2011;41(2):201-17. doi: 10.1080/10826068.2011.547384.
An intracellular α-glucosidase with high transglycosylation activity was purified from a mutant strain of Aspergillus niger M-1 by sequential chromatography using a DEAE-cellulose 52 column, a DEAE-Sepharose CL-6B column, and a Sephadex G-100 column. The molecular mass of the purified enzyme was determined to be 116 kD with no subunits and a pI of 5.23. Maximal α-glucosidase activity occurred at pH 6.0 and 50°C. The N-terminal amino acid sequences were identified as N-SVPGTEYVV-. The presence of Ca(2+) enhanced the enzyme activity by 20%, while the α-glucosidase activity was strongly inhibited by p-chloromercuribenzoate, N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride, monochloroacetic acid, and 2-mercaptoethanol. In addition, Ag(+), n-bromosuccinimide, and acetylacetone inhibited enzyme activity by 70%, 50%, and 22%, respectively. K(m) values of 4.32 m mol L(-1) and V(max) of 3.10 × 10(-2) mol L(-1) min(-1) were found for methyl-α-D-glucopyranoside (α-MG). Maltose was identified as the preferred substrate. The high-performance liquid chromatography (HPLC) analysis indicated that the oligosaccharide products contained 10.54% of isomaltose, 8.08% of panose, and 9.29% of isomaltotriose, and the amount of glucose, maltose, maltotriose, and maltotetrose was dropped from 22.21% to 15.80% using the purified enzyme in the solution of 25% maltose and 3% glucose. This intracellular α-glucosidase has potential applications in the synthesis of sugar derivatives and the investigation of associated mechanisms.
一种具有高转糖苷活性的细胞内α-葡萄糖苷酶从黑曲霉 M-1 的突变株中通过顺序色谱法用 DEAE-纤维素 52 柱、DEAE-Sepharose CL-6B 柱和 Sephadex G-100 柱进行纯化。该酶的分子量为 116kD，没有亚基，等电点为 5.23。最大的α-葡萄糖苷酶活性出现在 pH 6.0 和 50°C。N-末端氨基酸序列被鉴定为 N-SVPGTEYVV-。Ca(2+)的存在使酶活性提高了 20%，而对氯汞苯甲酸、N-(3-二甲氨基丙基)-N-乙基碳二亚胺盐酸盐、一氯乙酸和 2-巯基乙醇强烈抑制α-葡萄糖苷酶的活性。此外，Ag(+)、N-溴代丁二酰亚胺和乙酰丙酮分别抑制酶活性 70%、50%和 22%。发现甲基-α-D-吡喃葡萄糖苷（α-MG）的 K(m)值为 4.32mmol/L，V(max)值为 3.10×10(-2)mol/L min(-1)。麦芽糖被鉴定为首选底物。高效液相色谱（HPLC）分析表明，低聚糖产物中含有 10.54%异麦芽糖、8.08%潘糖和 9.29%异麦芽三糖，而在 25%麦芽糖和 3%葡萄糖溶液中使用纯化酶后，葡萄糖、麦芽糖、麦芽三糖和麦芽四糖的量从 22.21%下降到 15.80%。这种细胞内α-葡萄糖苷酶在糖衍生物的合成和相关机制的研究中有潜在的应用。
