来源于耐碱真菌Pseudallescheria sp.JSM-2的碱性木聚糖酶基因的克隆、表达及其性质研究

Cloning,Expression and Characterization of an Alkaline Xylanase from Alkali-tolerant Fungus Pseudallescheria sp. JSM-2

从造纸废水中分离得到的耐碱真菌Pseudallescheria sp.JSM-2的DNA为模板,利用同源克隆和TAIL-PCR的方法,获得了一个碱性木聚糖酶基因xyl11-1。该基因DNA和cDNA分别为797bp和678bp。该基因的推测蛋白N-端有一个18个氨基酸的信号肽序列和一个含207个氨基酸的成熟蛋白。编码成熟蛋白的cDNA序列在毕赤酵母GS115中重组表达后,进一步纯化并进行酶学性质测定。重组XYL11-1的最适pH为6.5,在pH4.5～9.0范围有50%以上的酶活;在pH4.5～12.0范围具有良好的pH稳定性;最适温度为50℃;以燕麦木聚糖为底物,比活为2618U/mg;且对中性和碱性蛋白酶具有极好的抗性。该酶作用底物范围广,包括各种木聚糖、纤维素和葡聚糖,易于工业化发酵生产,具有在纸浆脱墨、动物饲料、鱼类饵料中的应用潜力。

An alkali-tolerant fungal strain, Pseudallescheria sp. JSM-2, was isolated from the wastewater of a paper mill. By using degenerate PCR and TAIL-PCR techniques, a full-length xylanase gene, xy111-1, was cloned from the genomic DNA of strain JSM-2. The complete genomic and chromosomal DNA sequences of xy111-1 consist of 797 bp and 678 bp, respectively. Deduced XYL11-1 contains a putative 18-residue signal peptide at N-terminus and a C-terminal 207-residue polypeptide. Recombinant XYL11-1 produced in Pichia pastoris GS115 was purified and characterized. The optimal pH of XYL11-1 was 6.5, and the enzyme exhibited more than 50% of the maximal activity at pH 4.5 -9.0. XYLll-1 was stable at pH 4.5 - 12.0. The optimal temperature was found to be 50 ℃. XYL11-1 had a specific activity of 2 618 U/mg towards oat spelt xylan, and was strongly resistant to various neutral and alkaline proteases. In addition to hydrolysis of different xylans, XYL11-1 had broad substrate specificity, including various xylans, cellulose and glucan. Moreover, fermentation of XYL11-1 was easily handled. These superior properties of XYL11-1 make it advantageous for applying in the animal and fish feed and kraft pulp industries.