ABSTRACT
We previously isolated two α-L-arabinofuranosidases (ABFs), termed AFQ1 and AFS1, from the culture filtrate of Penicillium chrysogenum 31B. afq1 and afs1 complementary DNAs encoding AFQ1 and AFS1 were isolated by in vitro cloning. The deduced amino acid sequences of AFQ1 and AFS1 are highly similar to those of Penicillium purpurogenum ABF 2 and ABF 1, respectively, which belong to glycoside hydrolase (GH) families 51 and 54, respectively. Pfam analysis revealed an "Alpha-L-AF_C" domain in AFQ1 and "ArabFuran-catal" and "AbfB" domains in AFS1. Semi-quantitative RT-PCR analysis indicated that the afq1 gene was constitutively expressed in P. chrysogenum 31B at a low level, although the expression was slightly induced with arabinose, arabinitol, arabinan, and arabinoxylan. In contrast, expression of the afs1 gene was strongly expressed by the above four carbohydrates and less strongly induced by galactan. Recombinant enzymes (rAFQ1 and rAFS1) expressed in Escherichia coli were active against both p-nitrophenyl α-L-arabinofuranoside and polysaccharides with different specificities. (1)H-NMR analysis revealed that rAFS1 degraded arabinofuranosyl side chains that were both singly and doubly linked to the backbones of arabinoxylan and L-arabinan. On the other hand, rAFQ1 preferentially released arabinose linked to C-3 of single-substituted xylose or arabinose residues in the two polysaccharides.
Subject(s)
Gene Expression , Glycoside Hydrolases/genetics , Glycoside Hydrolases/metabolism , Penicillium chrysogenum/enzymology , Carbohydrate Metabolism , DNA, Fungal/chemistry , DNA, Fungal/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Gene Expression Profiling , Gene Expression Regulation, Enzymologic , Glycoside Hydrolases/chemistry , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Penicillium chrysogenum/genetics , Protein Structure, Tertiary , Real-Time Polymerase Chain Reaction , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
We previously described five arabinanolytic enzymes secreted by Penicillium chrysogenum 31B into the culture medium. Here, we describe a sixth such enzyme, termed AbnS1. Analysis of the reaction products of debranched arabinan revealed that AbnS1 cleaved the substrate in an endo manner. The optimum temperature of AbnS1 was 60°C, which was much higher than that of a cold-adapted endo-arabinanase (Abnc) produced by this strain. The abns1 cDNA gene encoding AbnS1 was isolated by in vitro cloning. The deduced amino acid sequence of AbnS1 had 70% identity with that of Abnc. Pfam analysis revealed a Glyco_hydro_43 domain at positions 28 to 318 of AbnS1. Semi-quantitative reverse transcription-polymerase chain reaction analysis indicated that the abns1 gene was constitutively expressed in P. chrysogenum 31B at a low level, although the expression was only slightly induced with arabinose and arabinan. In contrast, expression of the abnc gene encoding Abnc was strongly induced by arabinose, arabinitol, and arabinan. Using debranched arabinan as substrate, recombinant AbnS1 (rAbnS1) accumulated arabinobiose and arabinotriose as the major products. Recombinant Abnc (rAbnc) released mainly arabinotriose and lesser amounts of arabinose and arabinobiose than did rAbnS1. Branched arabinan was completely degraded to arabinose by the action of rAbnS1 or rAbnc in combination with α-L: -arabinofuranosidase.
