Key Laboratory of Marine Biology, Nanjing Agricultural University, Nanjing, 210095 Jiangsu, China.
J Appl Microbiol. 2011 Dec;111(6):1371-80. doi: 10.1111/j.1365-2672.2011.05145.x. Epub 2011 Sep 23.
The aim of this study is to improve exoinulinase production by expression of a cloned exoinulinase gene inuA1 (GenBank accession no. JF961344) from Penicillium janthinellum strain B01 in Pichia pastoris.
A full-length cDNA of exoinulinase gene (inuA1) was cloned from P. janthinellum strain B01 using RACE PCR. An open reading frame (ORF) of 2115 bp is interrupted by a single intron of 67 bp. The fragment encodes a signal peptide with 20 amino acids and a mature protein with 684 amino acids. The inuA1 was subcloned to the pPICZαC expression vector and successfully over-expressed in Pichia pastoris X-33. The highest activity of exoinlinase reached 272.8 U ml(-1) in the fermentation liquid. It was c. 11-fold of that produced by wild-strain B01. A large amount of fructose was identified after the hydrolysis of inulin with the crude recombinant exoinulinase. The recombinant exoinulinase was purified and characterized. The molecular weight of the purified recombinant exoinulianse was 100 kDa. The mass spectrometry result indicated that the purified protein was indeed recombinant exoinulinase. The optimal pH and temperature of the purified recombinant exoinulianse were 4.5 and 50°C, respectively.
An exoinulinase gene of P. janthinellum strain B01 was cloned, sequenced and over-expressed successfully in P. pastoris.
Only a few genes have been cloned from P. janthinellum because its molecular biology is poorly understood. In this study, we cloned and over-expressed inuA1 gene of P. janthinellum in P. pastoris. This recombinant exoinulinase can be used to hydrolyse inulin to produce fructose and facilitate the biofuel production from inulin resources.
本研究旨在通过在毕赤酵母中表达从青霉（Penicillium janthinellum）菌株 B01 克隆的内切菊粉酶基因 inuA1（GenBank 登录号 JF961344）来提高外切菊粉酶的产量。
使用 RACE-PCR 从青霉（Penicillium janthinellum）菌株 B01 中克隆了全长内切菊粉酶基因（inuA1）。一个 2115bp 的开放阅读框（ORF）被一个 67bp 的单内含子打断。该片段编码一个 20 个氨基酸的信号肽和一个 684 个氨基酸的成熟蛋白。inuA1 被亚克隆到 pPICZαC 表达载体中，并在毕赤酵母 X-33 中成功过表达。发酵液中内切菊粉酶的最高活性达到 272.8 U/ml，比野生菌株 B01 产生的活性高 11 倍。用粗制重组内切菊粉酶水解菊粉后，鉴定出大量的果糖。纯化和表征了重组内切菊粉酶。纯化的重组内切菊粉酶的分子量为 100 kDa。质谱结果表明，纯化的蛋白确实是重组内切菊粉酶。纯化的重组内切菊粉酶的最适 pH 和温度分别为 4.5 和 50°C。
成功地从青霉（Penicillium janthinellum）菌株 B01 克隆、测序并在毕赤酵母中过表达了内切菊粉酶基因。
由于青霉（Penicillium janthinellum）的分子生物学研究较少，因此仅从该菌中克隆了少数几个基因。在本研究中，我们在毕赤酵母中克隆并过表达了青霉（Penicillium janthinellum）的 inuA1 基因。这种重组内切菊粉酶可用于水解菊粉生产果糖，并促进从菊粉资源生产生物燃料。
