Atan1p—an extracellular tannase from the dimorphic yeast Arxula adeninivorans: molecular cloning of the ATAN1 gene and characterization of the recombinant enzyme

Abstract

The tannase-encoding Arxula adeninivorans gene ATAN1 was isolated from genomic DNA by PCR, using as primers oligonucleotide sequences derived from peptides obtained after tryptic digestion of the purified tannase protein. The gene harbours an ORF of 1764 bp, encoding a 587-amino acid protein, preceded by an N-terminal secretion sequence comprising 28 residues. The deduced amino acid sequence was similar to those of tannases from Aspergillus oryzae (50% identity), A. niger (48%) and putative tannases from A. fumigatus (52%) and A. nidulans (50%). The sequence contains the consensus pentapeptide motif (-Gly-X-Ser-X-Gly-) which forms part of the catalytic centre of serine hydrolases. Expression of ATAN1 is regulated by the carbon source. Supplementation with tannic acid or gallic acid leads to induction of ATAN1, and accumulation of the native tannase enzyme in the medium. The enzymes recovered from both wild-type and recombinant strains were essentially indistinguishable. A molecular mass of ∼320 kDa was determined, indicating that the native, glycosylated tannase consists of four identical subunits. The enzyme has a temperature optimum at 35-40• C and a pH optimum at ∼6.0. The enzyme is able to remove gallic acid from both condensed and hydrolysable tannins. The wildtype strain LS3 secreted amounts of tannase equivalent to 100 U/l under inducing conditions, while the transformant strain, which overexpresses the ATAN1 gene from the strong, constitutively active A. adeninivorans TEF1 promoter, produced levels of up to 400 U/l when grown in glucose medium in shake flasks.

Cloning, Production, and Functional Expression of the Bacteriocin Enterocin A, Produced by Enterococcus faecium T136, by the Yeasts Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, and Arxula adeninivorans

“…However, no EntA production by A. adeninivorans G1212EA was detectable ( Table 3). The nitrite reductase promoter (P AYNI1 ) used for the expression of entA in A. adeninivorans G1212EA is highly repressed by compounds with reduced nitrogen (6), and maybe other factors, such as a lower entA gene dosage or deficient MF␣1 sentA recognition by the A. adeninivorans G1212EA Sec machinery, would explain the low antimicrobial activity and undetectable production of secreted EntA.…”

<b><i>Arxula adeninivorans</i></b> Recombinant Urate Oxidase and Its Application in the Production of Food with Low Uric Acid Content

“…Transgenic A. adeninivorans producer strains were constructed that accumulated high levels of recombinant urate oxidase. The Xplor ® 2 transformation/expression platform [Böer et al, 2009b], previously used to transform A. adeninivorans and other yeasts to synthesize biotechnologically interesting enzymes such as Alip1p [Böer et al, 2005], Atan1p [Böer et al, 2009a] and RR-ADH [Giersberg et al, 2012], was used to express the AUOX gene in A. adeninivorans . The AUOX gene was integrated into the genome of the yeast as described in Böer et al [2009bBöer et al [ , 2011.…”

“…The endogenous urate oxidase was subsequently purified to homogeneity by DEAE-Sepharose column chromatography and gel filtration on a Superdex TM 200 column [Böer et al, 2009a].…”

“…Western analyses were performed as described by Towbin et al [1979] and treated with anti-HisTagspecific antibodies as described by Böer et al [2009a]. The dyebinding method of Bradford [1976] was used for protein quantification using bovine serum albumin (BSA) as standard.…”

PRODUCTION & PURIFICATION OF A LOW MOLECULAR WEIGHT EXTRACELLULAR TANNASE FROM ASPERGILLUS TPF-4

“…Tannase from A. niger ATCC 16620 has a single monomeric unit of 168 kDa (Sabu et al, 2005). [24] The tannases with a molecular weight of 45 kDa from Paecilomyces variotii, [23] 59 kDa from Selenomonas ruminantium, [25] 63 kDa from Aspergillus oryzae, [26] 101 kDa from Aspergillus awamori MTCC 9299, [22] 180 kDa from Aspergillus tamarii, [16] 310 kDa for tannase from Penicillium variable, [27] 320 kDa from Arxula adeninivorans, [28] etc. have been documented in earlier studies.…”