Secretion, purification, and characterization of a recombinant Aspergillus oryzae tannase in Pichia pastoris

Zhong, Xiaofen; Peng, Lisheng; Zheng, Shan Suo; Sun, Zhizhi; Ren, Yufeng; Dong, Meiling; Xu, Anlong

doi:10.1016/j.pep.2004.04.016

Cited by 71 publications

(29 citation statements)

References 11 publications

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“…These results suggest a carbohydrate content of 31.2% -higher than that of the A. oryzae tannase (22.7%) but close to the level found for the recombinant form of the A. oryzae enzyme produced in Pichia pastoris (Aguilar et al, 2001;Zhong et al, 2004).…”

Section: Discussionsupporting

confidence: 79%

“…For example, the Paecilomyces variotii tannase is a monomeric enzyme with a molecular mass of only 45 kDa (Mahendran et al, 2006), whereas the A. oryzae tannase is an octamer, consisting of four identical heterodimeric units (Hatamoto et al, 1996) and the same tannase produced by P. pastoris forms single heterodimers (Zhong et al, 2004). The molecular mass of the native, glycosylated A. adeninivorans tannase was estimated here to be 320 kDa, implying that it comprises four subunits with molecular sizes of about 80 kDa.…”

Section: Discussionmentioning

confidence: 81%

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

Böer¹,

Bode²,

Mock³

et al. 2009

Yeast

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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.

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Section: Discussionsupporting

confidence: 79%

Section: Discussionmentioning

confidence: 81%

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

Böer¹,

Bode²,

Mock³

et al. 2009

Yeast

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“…The expression levels of the hCPM in Pichia obtained in the present study was much higher (around 10-fold) when compared to the published expression system using baculovirus-infected insect cells, about 30-40 mg/L of recombinant mutant CPM in the culture medium (17). Some investigators, using the same system, have also described a similar recovery for the enzyme tannin acyl hydrolase (25), and even higher recoveries for the enzyme lipase/acyltransferase from Candida parapsilosis (EC 3.1.1.3) (26), and for the tetanus toxin fragment C (24,27), highlighting the potential of this technique to express high yield of heterologous proteins.…”

Section: Purification Of Human Carboxypeptidase Msupporting

confidence: 56%

“…In addition, the great success of P. pastoris is related to the fact that this organism is a eukaryote, capable to express proteins with post-translational modifications, sometimes essential for proper function. A further benefit of the P. pastoris system is that strong promoters are available to drive the expression of the foreign gene(s), thus enabling production of large amounts of the target protein(s) with relative technical ease and at a lower cost than most other eukaryotic systems (25).…”

Section: Purification Of Human Carboxypeptidase Mmentioning

confidence: 99%

<a name="home"></a>High expression of human carboxypeptidase M in Pichia pastoris: Purification and partial characterization

Craveiro

Ramalho

Chagas

et al. 2006

Braz J Med Biol Res

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Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidylinositol-anchored membrane glycoprotein, which removes the Cterminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. The enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein -1 min -1 ). This purification protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa), suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes

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“…Bacteria (Kumar et al, 2010), yeasts (Zhong et al, 2004) and filamentous fungi (Battestin and Macedo, 2007;Hamdy, 2008) are able to degrade hydrolysable tannins and produce tannase. However, Aspergillus and Penicillum are the best tannase producers during submerged (SmF) and solid state fermentation (Aguilar et al, 2007;Kumar et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Catalytic properties of immobilized tannase produced from Aspergillus aculeatus compared with the free enzyme

El-Tanash

Sherief

Nour

2011

Braz. J. Chem. Eng.

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-Aspergillus aculeatus tannase was immobilized on several carriers by entrapment and covalent binding with cross-linking. Tannase immobilized on gelatin with cross-linking agent showed the highest activity and immobilization yield. The optimum pH of the immobilized enzyme was shifted to a more acidic range compared with the free enzyme (from pH 5.5 to pH 5.0). The optimum temperature of the reaction was determined to be 50°C for the free enzyme and 60°C for the immobilized form. The thermal stability, as well as stability over a wide range of pH, was significantly improved by the immobilization process. The calculated K m of the immobilized tannase (11.8 mg ml -1 ) is higher than that of the free tannase (6.5 mg ml -1 ), while V max of the immobilized enzyme (0.32 U (µg protein) -1 ) is lower than that of the free tannase (2.7 U (µg protein) -1 ). The immobilized enzyme was able to retain 84 % of the initial catalytic activity after 5.0 cycles.

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Secretion, purification, and characterization of a recombinant Aspergillus oryzae tannase in Pichia pastoris

Cited by 71 publications

References 11 publications

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

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

<a name="home"></a>High expression of human carboxypeptidase M in Pichia pastoris: Purification and partial characterization

Catalytic properties of immobilized tannase produced from Aspergillus aculeatus compared with the free enzyme

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