Molecular Cloning and Heterologous Expression of the Isopullulanase Gene from Aspergillus Niger A.T.C.C

Biochem. J. (1997) 323, 757–764 (Printed in Great Britain) 757 Molecular cloning and heterologous expression of the isopullulanase gene from Aspergillus niger A.T.C.C. 9642 Hiroyoshi AOKI, Yopi and Yoshiyuki SAKANO* Department of Applied Biological Science, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwaicho, Fuchu, Tokyo 183, Japan Isopullulanase (IPU) from Aspergillus niger A.T.C.C. (American Penicillium minioluteum (61%) and Arthrobacter sp. (56%). Type Culture Collection) 9642 hydrolyses pullulan to isopanose. When the ipuA gene was expressed in Aspergillus oryzae, the IPU is important for the production of isopanose and is used in expressed protein (recombinant IPU) had IPU activity and was the structural analysis of oligosaccharides with α-1,4 and α-1,6 immunologically reactive with antibodies raised against native glucosidic linkages. We have isolated the ipuA gene encoding IPU. The substrate specificity, thermostability and pH profile of IPU from the filamentous fungi A. niger A.T.C.C. 9642. The recombinant IPU were identical with those of the native enzyme, ipuA gene encodes an open reading frame of 1695 bp (564 amino but recombinant IPU (90 kDa) was larger than the native enzyme acids). IPU contained a signal sequence of 19 amino acids, and (69–71 kDa). After deglycosylation with peptide-N-glycosidase the molecular mass of the mature form was calculated to be F, the deglycosylated recombinant IPU had the same molecular 59 kDa. IPU has no amino-acid-sequence similarity with the mass as deglycosylated native enzyme (59 kDa). This result other pullulan-hydrolysing enzymes, which are pullulanase, suggests that the carbohydrate chain of recombinant IPU differed neopullulanase and glucoamylase. However, IPU showed from that of the native enzyme. a high amino-acid-sequence similarity with dextranases from INTRODUCTION gene (ipuA) and show that the deduced amino acid sequence of IPU is similar to those of the Penicillium and Arthrobacter Pullulan, an α-glucan produced by Aureobasidium pullulans,is dextranases. We also characterized the ipuA gene expressed in used in various fields, including the food, pharmaceutical and Aspergillus oryzae M-2-3 and the enzymic properties of the chemical industries [1]. Pullulan is a linear polymer which is expressed protein (recombinant IPU). composed of maltotriose units with α-1,6 glucosidic linkages (Figure 1). Enzymes that hydrolyse pullulan have been classified into four groups on the basis of their cleavage sites on pullulan [2]. They are (i) pullulanase (EC 3.2.1.41) [1], (ii) Thermo- MATERIALS AND METHODS actinomyces ulgaris α-amylase (TVA; EC 3.2.1.1) [3] and Determination of internal amino acid sequence neopullulanase (EC 3.2.1.135) [4], (iii) isopullulanase (IPU) [5] IPU F2 was previously purified from mycelia of A. niger A.T.C.C. and (iv) glucoamylase (EC 3.2.1.3) [1] (Figure 1). As pullulan- 9642 [7]. IPU F2 was partially digested with Staphylococcus V8 hydrolysing enzymes can precisely recognize the structural protease (Boehringer Mannheim) by the method of Cleveland et differences between α-1,4 or α-1,6 glucosidic linkages, they are al. [8]. After the peptide fragments of IPU F2 were separated by applied in the structural analysis of oligosaccharides and poly- SDS}15%-PAGE, they were electrotransferred to a PVDF saccharides. membrane (Immobilon-P; Millipore Co.) and stained with IPU (pullulan 4-glucanohydrolase (isopanose-forming), EC Coomassie Brilliant Blue by the method of Matsudaira [9]. 3.2.1.57) was purified from Aspergillus niger A.T.C.C. (American Peptide fragments on the membrane were cut out and analysed Type Culture Collection) 9642 [5]. IPU hydrolyses pullulan and by automated Edman degradation on an ABI 477A protein panose (Glcα1-6Glcα1-4Glc) to produce isopanose (Glcα1- sequencer (Applied Biosystems). 4Glcα1-6Glc), and glucose and isomaltose, respectively. How- ever, IPU does not attack starch or dextran. IPU was purified as an extracellular enzyme in a solid culture on wheat bran (extracellular IPU) and as a cell-bound enzyme Cloning strategy of IPU cDNA and genomic DNA from the cell wall of mycelia in a submerged culture (cell-bound (I) cDNA cloning from an A. niger A.T.C.C. 9642 cDNA library IPU) [6]. Furthermore, cell-bound IPU was separated into two different glycosylated forms, IPU F1 (pI 5.0) and IPU F2 (pI 4.9) Total RNA was isolated from mycelia of a submerged culture of by Mono-P HR 5}20 chromatofocusing [7]. In the present paper A. niger A.T.C.C. 9642 by the method of Okayama et al. [10]. we describe the cloning and nucleotide sequencing of the IPU Poly(A)-rich RNA was purified from the total RNA using a Abbreviations used: IPU, isopullulanase; PNGase F, peptide-N-glycosidase F; TVA, Thermoactinomyces vulgaris α-amylase; DIG, digoxigenin; GCG, Genetics Computer Group; ORF, open reading frame; A.T.C.C., American Type Culture Collection. * To whom correspondence should be addressed. The nucleotide sequence data reported in this paper have been submitted to the DDBJ/EMBL/Genbank nucleotide sequence database under the accession number D85240. 758 H. Aoki, Yopi and Y. Sakano Figure 2 Nnucleotide sequences of primers used for PCR The annealing regions and restriction enzyme sites of the primers are shown by broken and double underlining respectively. The start and stop codons of the isopullulanase gene are indicated. digestion and Southern hybridization using the cDNA of λIP101 as a probe. The HindIII--EcoRI region of the genomic DNA insert of pIPE was sequenced. (III) Cloning of the full-length cDNA by PCR A. niger total RNA was purified from mycelia by the acid guanidine thiocyanate}phenol}chloroform (AGPC) method [13]. Single-strand cDNA (25 µl) was synthesized from 10 µg of the total RNA using an AMV (avian myeloblastosis virus) Reverse Transcriptase First-strand cDNA Synthesis Kit (Life Science Inc., St. Petersburg, FL, U.S.A.) and an oligo(dT) primer according to the manufacturer’s instructions. PCR was performed using the first-strand cDNA as a template. The PCR reaction mixture (100 µl) contained 1 µl of the first- strand cDNA solution, 2.5 units of Taq DNA polymerase (TaKaRa), 2.5 mM dNTP, 10 µg}ml RNase A, and 0.5 µM IPUNT and RTR1 primers (Figure 2) in a standard reaction Figure 1 Action patterns of pullulan-hydrolysing enzymes buffer. The PCR reaction mixture was incubated at 37 mC for 1 h Arrows indicate the attack points of pullulan-hydrolysing enzymes on pullulan. to increase the efficiency of PCR prior to the PCR cycle. The PCR cycle program was 95 mC for 2 min (denaturation), 55 mC for 1 min (annealing), and 72 mC for 2 min (extension) for two cycles, followed by 33 cycles at an elevated annealing temperature mRNA purification kit (Pharmacia Biotech). Double-strand (60 mC). The amplified cDNA of IPU (1.2 kb) was digested with cDNA was synthesized from poly(A)-rich RNA using an EcoRI and XbaI, and subcloned into pUC119. This constructed oligo(dT) primer and a cDNA synthesis kit (Pharmacia Biotech). plasmid was named pRT1, and the nucleotide sequence of the A cDNA library was constructed in a λgt11 vector (Stratagene). amplified cDNA was determined. & The cDNA library (1.5¬10 plaques) was immunoscreened with a polyclonal rabbit antibody raised against purified native IPU Southern-blot analysis F2 [7]. Three positive plaques (λIP101, 201 and 401) were obtained and subcloned into pUC119 [11]. The nucleotide DNA was digested with restriction endonucleases, electro- sequences of these clones were determined. phoresed on an 0.8%-agarose gels, and alkaline-transferred to nylon membranes (Hybond N; Amersham International). The (II) Cloning of the IPU-encoding gene from an A. niger A.T.C.C. 9642 cDNA probe subcloned from λIP101 was labelled by DIG- genomic DNA library dUTP using a DIG DNA labelling and detection kit. Hybridization and detection with the DIG labelled cDNA probe The genomic DNA of A. niger A.T.C.C. 9642 was purified by the were carried out according to the manufacturer’s instructions. method of Hynes et al. [12]. The genomic DNA was completely digested with EcoRI. The EcoRI fragments were separated by Nucleotide sequencing and analysis 0.8%-agarose-gel electrophoresis and 6.6–2.3 kb fragments were extracted from the gel and ligated into the EcoRI site of pUC119. DNA sequence analysis was carried out by the dideoxy-chain- % The genomic DNA library (1.0¬10 colonies) was screened by termination method [14] using an AutoRead Sequencing Kit and colony hybridization [11] using the cDNA of λIP101 as a probe. an A.L.F. DNA Sequencer II (Pharmacia Biotech). Both strands The cDNA probe was labelled with digoxigenin (DIG)-dUTP were independently and completely sequenced. Nucleotide using a DIG DNA labelling and detection kit (Boehringer sequences were analysed with the Genetics Computer Group Mannheim). Hybridization and detection of DIG-labelled probe (GCG, Madison, WI, U.S.A.) sequence-analysis software pack- was performed according to the manufacturer’s instructions. A age version 8.0.1. The hydrophobicity and hydrophilicity of the positive clone, pIPE, was analysed by restriction-endonuclease amino acid sequence of IPU was analysed by the PepPlot Cloning and expression of the isopullulanase gene from Aspergillus niger 759 Figure 3 Restriction-endonuclease map of the cloned genomic DNA and cDNA of isopullulanase-encoding gene from A. niger A.T.C.C. 9642 (A) genomic DNA; (B) cDNA. cDNAs in λIP101-401 were obtained by immunoscreening the cDNA library; the cDNA in pRT1 was cloned by PCR using first-strand cDNA transcribed from A. niger A.T.C.C. 9642 mRNA. The white arrow represents the ORF of isopullulanase.

Load more