Cloning of exoinulinase gene from Penicillium janthinellum strain B01 and its high-level expression in Pichia pastoris

Abstract

Aims: The aim of this study is to improve exoinulinase production by expression of a cloned exoinulinase gene inuA1 (GenBank accession no. ) from Penicillium janthinellum strain B01 in Pichia pastoris. Methods and Results: 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 succesfully 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. Conclusions: An exoinulinase gene of P. janthinellum strain B01 was cloned, sequenced and over‐expressed successfully in P. pastoris. Significance and Impact of the Study: 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.

Direct production of bioethanol from Jerusalem artichoke inulin by gene-engineeringSaccharomyces cerevisiae6525 with exoinulinase gene

“…a-factors) as well as more detailed studies on the cellular localization of the inulinase protein in yeast. Surprisingly, although the expression level of inulinase in S. cerevisiae was less than that in Picha pastoris reported earlier (Wang et al 2011), it greatly enhanced the production of ethanol from inulin, as described further in the text.…”

“…Previously, it was reported that the inulinase activity of a yeast transformant with an inulinase gene integrated into its genome remained very stable during five sequential batch cultivations in a nonselective medium. In contrast, the inulinase activity of a transformant carrying the same gene in a plasmid (Ycplacc33) decreased gradually Wang et al 2011). The integration strategy used in the present study not only facilitates stable recombination of the heterologous gene, but also increases the possibility of integrating multiple copies (Figure 1) contained the constitutive promoter PGK1 (782 bp) and the effective terminator CYC1 (249 bp).…”

“…The inuA1 gene encoding exo-inulinase was amplified from Penicillium janthinellum strain B01 (Wang et al 2011;Sandionigi et al 2012). We used Saccharomyces cerevisiae 6525 (kindly supplied by Dr. Bai, Dalian, China), a strain with good fermentation ability, as the host for harboring the inulinase gene.…”

“…The recombinant inulinase activity in the supernatants of the transformants obtained earlier was determined according to Wang et al (2011). We determined the concentration of reducing sugars in the mixture using the 3,5-dinitrosalicylic (DNS) acid assay with fructose as the standard (Miller 1959).…”

Characterization of an exo-inulinase fromArthrobacter: A novel NaCl-tolerant exo-inulinase with high molecular mass

“…For example, the molecular masses of 9 characterized inulinases are over the range of 49. 8 24,26 and the exo-inulinase from P. janthinellum B01 is also inhibited by 1 mM Na C . 24 Compared with the above exo-inulinases, InuAHJ7 is different: the exo-inulinase has a molecular mass of 95.1 kDa and shows tolerance to 3.0%-20.0% (w/v) Na C and 1.0 and 10.0 mM Zn 2C and Pb 2C .…”