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Aspergillus fumigatus Produces Two Arabinofuranosidases From Glycosyl Hydrolase Family 62: Comparative Properties of the Recombinant Enzymes

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Abstract

The genes of two α-l-arabinofuranosidases (AbfI and II) from family GH 62 have been identified in the genome of Aspergillus fumigatus wmo. Both genes have been expressed in Pichia pastoris and the enzymes have been purified and characterized. AbfI is composed of 999 bp, does not contain introns and codes for a protein (ABFI) of 332 amino acid residues. abfII has 1246 bp, including an intron of 51 bp; the protein ABFII has 396 amino acid residues; it includes a family 1 carbohydrate-binding module (CBM) in the N-terminal region, followed by a catalytic module. The sequence of ABFI and the catalytic module of ABFII show a 79 % identity. Both enzymes are active on p-nitrophenyl α-l-arabinofuranoside (pNPAra) with KM of 94.2 and 3.9 mM for ABFI and II, respectively. Optimal temperature for ABFI is 37 °C and for ABFII 42 °C, while the pH optimum is about 4.5 to 5 for both enzymes. ABFII shows a higher thermostability. When assayed using natural substrates, both show higher activity over rye arabinoxylan as compared to wheat arabinoxylan. ABFII only is active on sugar beet pulp arabinan and both are inactive towards debranched arabinan. The higher thermostability, higher affinity for pNPAra and wider activity over natural substrates shown by ABFII may be related to the presence of a CBM. The availability of the recombinant enzymes may be useful in biotechnological applications for the production of arabinose.

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Acknowledgments

This work has been supported by grants from the Fondo Nacional de Ciencia y Tecnología (FONDECYT) (1130180), and Universidad Andrés Bello (DI-478-14/R and DI-31-12/R).

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Authors and Affiliations

  1. Departamento de Ciencias Biológicas, Universidad Andrés Bello, Santiago, Chile

    Rodrigo Pérez & Jaime Eyzaguirre

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  1. Rodrigo Pérez
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  2. Jaime Eyzaguirre
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Correspondence to Jaime Eyzaguirre.

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The authors declare that they have no competing interests.

Additional information

Highlights

The gene coding for two GH 62 arabinofuranosidases (abfI and II) have been sequenced.

Both genes have been heterologously expressed in Pichia pastoris.

The enzymes (ABFI and II) have been purified and characterized.

Both enzymes belong to sub-family GH 62-1, but ABFII possesses a carbohydrate-binding module.

ABFII shows higher stability and broader substrate specificity.

Electronic Supplementary Material

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Fig. S1

Nucleotide sequence of abfI and amino acid sequence of ABFI. The translation start and termination codons are in bold. (DOCX 99 kb)

Fig. S2

Nucleotide sequence of abfII and amino acid sequence of ABFII. The translation start and termination codons are in bold. The intron sequence is in small letters. (DOCX 19 kb)

Fig. S3

CLUSTAL W alignment of the amino acid sequences of ABFI and of ABFII. The sequence of the CBM of ABFII is underlined. The catalytic residues are boxed. Vertical lines show the end of the signal peptides as predicted by SignalP. (DOCX 43 kb)

Fig. S4

Promoter sequences of the abfI (A) and abfII (B) genes. The TATA box of abfI is highlighted. (DOCX 146 kb)

Fig. S5

Multiple sequence alignment of a set of fungal GH 62 ABFs. The alignment was performed with CLUSTAL Omega. The catalytic amino acids are boxed. The sequences are from: Aspergillus nidulans (EAA59562); Aspergillus niger (CAK38069); Aspergillus sojae (BAA 85252); Aspergillus tubingensis (CAB01408); Bipolaris zeicola (AAG42252); Chrysosporium lucknowense (AED84983); Penicillium capsulatum (CAM07245); Penicillium chrysogenum (BAG71682); Podospora anserina (CAP62366); Trichoderma reesei (AAP57750); Ustilago maydis (XP_011391032); Coprinopsis cinerea (AB557888); Scytalidium thermophilum (AbfA, AHZ56658); Scytalidium thermophilum (AbfC, AHZ56660); Penicillium funiculosum (abf62a, b and c; De La Mare et al. 2013); ABFI (this work; KU160789); ABFII (this work; KU160799) (DOCX 30 kb)

Table S1

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Table S2

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Pérez, R., Eyzaguirre, J. Aspergillus fumigatus Produces Two Arabinofuranosidases From Glycosyl Hydrolase Family 62: Comparative Properties of the Recombinant Enzymes. Appl Biochem Biotechnol 179, 143–154 (2016). https://doi.org/10.1007/s12010-016-1984-0

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