Enzymatic characterization of the product of an aminopeptidase-like gene from Aspergillus oryzae

Transcription

1 Enzymatic characterization of the product of an aminopeptidase-like gene from Aspergillus oryzae Ken-Ichi Kusumoto 1, Mayumi Matsushita 1, Ikuyo Furukawa 1, Satoshi Suzuki 1, Yoshinao Koide 2, Hiroki Ishida 3, Youhei Yamagata 4, Michio Takeuchi 5, Yutaka Kashiwagi 1 ( 1 Natl. Food Res. Inst., 2 Amano Enzyme, 3 Gekkeikan, 4 Grad. Sch. Agric. Sci.,Tohoku Univ., 5 Tokyo Univ. of Agric. Tech.)

2 Abstract: Aspartyl aminopeptidase from Aspergillus oryzae has high substrate specificity, degrading only amino-terminal acidic amino acids from peptides, and may be suitable for processing physiological peptides. However, little is known about the biochemical properties of this enzyme and its efficient production in A. oryzae. The gene encoding aspartyl aminopeptidase was overexpressed under a takaamylase gene promoter, with His-tag linker in A. oryzae, during cultivation in a cobalt-containing medium. The enzyme was extracted from mycelia and purified with immobilized nickel ion absorption chromatography using a buffer containing cobalt ion and imidazole. The active fraction was further purified with gel filtration chromatography. The resultant, electrophoretically pure enzyme displayed a molecular mass of 520 kda. This enzyme displayed high affinity to peptide substrate rather than synthetic substrates. Thus, recombinant A. oryzae aspartyl aminopeptidase was purified to homogeneity with an increased specific activity, when cultivated in a cobalt ion-rich medium. Moreover, the use of suitable metal ions in microbial cultivation and purification processes may help increase specific activity of other metalloproteases and their functional analysis.

3 3Aodap1 (AO ) 1. aminopeptidase I zinc metalloprotease (M18) 2. Its deduced amino acid sequence is 52 % identical to yhr113w of Saccharomyces cerevisiae, whose enzymatic activity was reported. 3. The gene yhr113w encodes aspartyl aminopeptidase (DAP, acidic amino acid-specific aminopeptidase) 4. The existence of DAP was reported for yeast and mammals. 5. This gene is expressed in A. oryzae (EST data).

4 Construction of a vector plasmid dapa PCR product attb1 attb2 dapa attp4 ccdb attp2 Donor vector BP reaction attl1 attl2 dapa pkka17 (Entry clone) AmyBPro HA-His6 attl4 attr1 attr2 attl3 Pg5 Pa Pg3 HH attr4 ccdb attr3 Fig. Schematic view of construction of vector plasmid pkka17l1. For detail explanation of MultiSite Gateway system, see Invitrogen Website ( AmyBPro, promoter region of AmyB; AmyBTer, terminator region of AmyB; HA-His6, HAtag linked to His-tag; ccdb, a gene for control of cell death. A DNA or plasmid containing ccdb will be obtained as a biproduct of both BP and LR reaction, which is not shown in this figure. pgdp (Destination vector) ptra LR reaction attb4 attb1 attb2 attb3 AmyBPro dapa HA-His6 ptra pkka17l1

5 Isolation of Aodap1-overexpressing strain of A. oryzae ProAmyB Aodap1 His-tag pucori pkka17l1 (7.1kb) Ampr ptra Plasmid for aminopeptidase overexpression Transformation of A. oryzae RIB40 Selection of the strain carrying the plasmid DNA Western blot analysis of cell-free extract with anti-his-tag antibody M Lanes 1-5: Transformants 1-5 kd ~52kDa Dap1 protein (54kDa from genome information) (57kDa with His-tag and linker) Confirmation of the target protein production

6 Enzymatic activity of cell-free extract of Aodap1- overexpressing strain Control strain, repressive condition Control strain, inductive condition Specific activity (mu/mg protein) Overexpressing strain, repressive condition Overexpressing strain, inductive condition Asp-pNA* Asp + pna Glu-pNA* Glu + pna 0 Asp-pNA hydrolyzing activity Glu-pNA hydrolyzing activity *pna; para-nitrophenyl anilide Control strain: Aspergillus oryzae RIB40(host strain) Overexpressing strain: Plasmid 1-introdusing strain Repressive condition: Liquid culture with glucose as carbon source Inductive condition: Liquid culture with starch as carbon source DAP from mammals and yeast does not hydrolyze synthetic substrate

7 Purification of DAP from its overexpressing strain Purification of recombinant DAP Cultivated mycelia (cultivated with CDS containing starch and 1 mm CoCl 2 ) Frozen and powdered to prepare crude extract (CE) (20 mm Tris-HCl, ph7.5, 10% glycerol, 20 mm imidazole, 250 mm KCl) SDS-PAGE(CBB stain) CE FT A G kda 15,000 rpm (22,400g) x 20 min Supernatant Loaded to Ni-IMAC(BIO-RAD) column Elution with buffer containing imidazole Buffer A (FT*) : 20 mm imidazole Buffer G :100 mm imidazole Measurement of Asp-pNA hydrolyzing activity *FT; frow through Specific activity CE G2 frc mu/mg 1405 mu/mg

8 Gel filtration analysis of DAP kda Total protein (absorbance at 595 nm) Total protein (X 10-3 absorbance at 280 nm) Activity (nmol/min/ml) Fraction Number (0.6 ml per tube) Fig. Separation of recombinant A. oryzae aspartyl aminopeptidase by Superose 6 gel filtration chromatography. Dotted line with, aspartyl aminopeptidase activity; solid line with, absorbance at 595 nm assayed with protein assay kit; solid line with no symbol, absorbance at 280 nm.

9 SDS-PAGE analysis of purified DAP M kda Fig. Purification of recombinant aspartyl aminopeptidase from A. oryzae. Coomassie Brilliant blue-stained SDS polyacrylamide gel (5-15%) of 0.6 ml of each fraction (23-34) eluted from the Superose 6 column. Fraction numbers are indicated above the lanes. M, protein molecular mass marker.

10 Reactivity against the synthetic substrates 120% 100% 80% 60% 40% 20% 0% Asp-pNA Leu-pNA Pro-pNA Glu-pNA After reaction with 1 mm of each substrates for 20 min at 30, released pna was measured with spectrophotometer. Relative activity against each substrate is shown. (pna amount released from Asp-pNA as 100 %) Besides the described substrates above, no activity was detected with pna substrates of Gly, Met, Lys, Arg, Ala,and Val.

11 Reactivity of DAP against natural peptide 0 min A 5 min A 30 min 20 h B B A B A Time (min) Time (min) Time (min) Time (min) Fig. Hydrolysis of angiotensin II by purified aspartyl aminopeptidase from A. oryzae as analyzed by HPLC. Conditions of enzyme reaction and HPLC are described in MATERIALS AND METHODS. Reaction time is shown in each chromatographic profile. Peak A, angiotensin II; peak B, angiotensin III. Angiotensin III was identified by comparison with the retention time of the authentic standard. Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu (Substrate: Angiotensin II) Asp + Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu (Product: Angiotensin III) No more reaction

12 Summary kb coding region of Aodap1 was obtained by PCR with RIB40 genomic DNA. 2. Isolation of Aspergillus oryzae transformant overexpressing Aodap1, with AmyB promoter linked at N-terminus and His-tag at its carboxy-terminus. 3. Purification of the gene product (DAP) with guidance of hydrolyzing activity for Asp-p-nitroanilide (Asp-pNA), with using Ni-IMAC column chromatography. The purified DAP appeared single band at ca. 54 kda by SDS-PAGE. DAP hydrolyzed Asp-pNA and Glu-pNA, but have no activity against pna substrates of Gly, Met, Leu, Lys, Arg, Ala, Val, and Pro. 4. Angiotensin II was hydrolyzed by DAP to produce only angiotensin III which was des-asp angiotensin II. 5. Aodap1 is proved to encode acidic amino acid-specific aminopeptidase. DAP of A. oryzae hydrolyzes synthetic substrates (pna substrates) in addition to natural peptides. This character is different from DAP of mammals or yeast. 6. Gel filtration profile suggested DAP of A. oryzae might be nonamer (520 kda). Acknowledgements We greatly appreciate the help of Prof. Dr. K. Kitamoto and Dr. J. Maruyama of the Department of Biotechnology, University of Tokyo, for providing plasmids pg5'pa, and pg3'hh, to construct the plasmid pkka17l1. K.-I. Kusumoto and M. Matsushita- Morita equally contributed to this work. This study was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).