Heterologous expression and characterization of a novel xanthan lyase
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摘要: 黄原胶裂解酶是一种黄原胶修饰酶,对黄原胶的改性及新型黄原胶寡糖的制备具有十分重要的意义。本实验从一株性能优良的黄原胶降解菌Microbacterium sp.XT11中首次克隆出黄原胶降解酶编码基因xly。该基因编码理论分子量为122027 u的蛋白质,其N端含有一条35 aa的分泌信号肽,C端含有一段392 aa的碳水化合物结合域CBM。随后,在去除分泌信号肽及CBM的截短酶的N端融合了谷胱甘肽巯基转移酶亲和纯化标签（GST）,所形成的融合蛋白XLY-GST能够在大肠杆菌BL21（DE3）中高水平可溶表达。纯化的XLY-GST表现出与天然黄原胶裂解酶一致的酶学性质,其最适作用温度为40℃,最适p H为6.0,对碱性环境具有一定的耐受力（最高耐受p H10.5）。Ca2+和Mn2+对该裂解酶有显著地激活作用,而Cu2+则对该酶有一定的抑制作用。XLY-GST对黄原胶侧链的乙酰基和丙酮酸基团修饰程度表现出较高的特异性。本实验所取得的研究结果对黄原胶裂解酶的制备及黄原胶侧链修饰的研究奠定了基础。Abstract: Xanthan lyase is a kind of xanthan- modifying enzymes which are powerful tools for xanthan modification and xantho- oligosaccharide preparation.In this paper,a novel xanthan lyase- encoding gene xly was cloned and sequenced for the first time,from an excellent xanthan- degrading strain Microbacterium sp.XT11.The xly gene encoded a 122027 u protein,including a 35- amino acid signal peptide at N terminus and a 392- amino acid carbohydrate binding module( CBM) at C terminus. A mature enzyme without signal peptide and CBM was fused with a GST tag and the resulted XLY- GST could be expressed in Escherichia coli BL21( DE3) in a high level.The purified XLY- GST showed similar features with that of the wild type xanthan lyase.It was optimally active at p H6.0 and 40 ℃ and alkali- tolerant at a high p H value of 10.5.The metal ions including Ca2 +and Mn2 +strongly stimulated xanthan lyase activity but ion Cu2 +was its inhibitor.XLY- GST was specific on the pyruvated and mannosyl residue in the intact xanthan molecule.Our work should be valuable for preparing the xanthan lyase and studying the modification of xanthan side chain.
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Keywords:
- xanthan lyase /
- xanthan modification /
- enzyme characterization
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