Cloning, characterization, and heterologous expression of a dextranase gene from Penicillium pinophilum SMCU3-14

Rerngsamran, Panan; Temjitpukdee, Pimonrut; Assavasirijinda, Nilnate; Chareonpornwattana, Supat; Thaniyavarn, Suthep

doi:10.2306/scienceasia1513-1874.2014.40.405

Cited by 6 publications

(5 citation statements)

References 24 publications

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“…This is similar to the CBB-stained gel, but with the difference being that the enzyme migrated less. Other authors report the same MW (CBB staining and dextran blue zymogram) for recombinant dextranases expressed in Escherichia coli [31,41]. Similar results were obtained from the analysis in gels for the DEX protein produced by Aspergillus allahabadii X26 and Catenovulum agarivorans MNH15 [35,42].…”

Section: Detection In Polyacrylamide Gels Of the Shaker Produced R-tm...supporting

confidence: 71%

Constitutive High Expression Level of a Synthetic Deleted Encoding Gene of Talaromyces minioluteus Endodextranase Variant (r–TmDEX49A–ΔSP–ΔN30) in Komagataella phaffii (Pichia pastoris)

et al. 2022

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In the sugar industry, dextran generates difficulties in the manufacturing process. Using crude dextranase (EC 3.2.1.11) to eliminate dextran in sugar is an effective practice. In this study, a synthetic dextranase-encoding gene of the filamentous fungus Talaromyces minioluteus, lacking its putative native signal peptide (1–20 amino acids) and the next 30 amino acids (r–TmDEX49A–ΔSP–ΔN30), was fused to the Saccharomyces cerevisiae prepro α–factor (MFα–2) signal sequence and expressed in Komagataella phaffii under the constitutive GAP promoter. K. phaffii DEX49A–ΔSP–ΔN30, constitutively producing and secreting the truncated dextranase, was obtained. The specific activity of the truncated variant resulted in being nearly the same in relation to the full-length mature enzyme (900–1000 U·mg−1 of protein). At shaker scale (100 mL) in a YPG medium, the enzymatic activity was 273 U·mL−1. The highest production level was achieved in a fed-batch culture (30 h) at 5 L fermenter scale using the FM21–PTM1 culture medium. The enzymatic activity in the culture supernatant reached 1614 U·mL−1, and the productivity was 53,800 U·L−1·h−1 (53.8 mg·L−1·h−1), the highest reported thus far for a DEX49A variant. Dextran decreased r–TmDEX49A–ΔSP–ΔN30 mobility in affinity gel electrophoresis, providing evidence of carbohydrate–protein interactions. K. phaffii DEX49A–ΔSP–ΔN30 shows great potential as a methanol-free, commercial dextranase production system.

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Section: Detection In Polyacrylamide Gels Of the Shaker Produced R-tm...supporting

confidence: 71%

Constitutive High Expression Level of a Synthetic Deleted Encoding Gene of Talaromyces minioluteus Endodextranase Variant (r–TmDEX49A–ΔSP–ΔN30) in Komagataella phaffii (Pichia pastoris)

et al. 2022

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“…This is like the CBB stained gel, but with the difference being that the enzyme migrated less. Other authors report the same MW (CBB staining and dextran blue zymogram) for recombinant dextranases expressed in Escherichia coli [30,40]. Similar results were obtained from the analysis in gels for the DEX protein produced by Aspergillus allahabadii X26 and Catenovulum agarivorans MNH15 [34,41].…”

Section: Detection In Polyacrylamide Gels Of the Shaker Produced R-tm...supporting

confidence: 71%

Constitutive High Expression Level of A Synthetic Deleted Encoding Gene of Talaromyces minioluteus Endodextranase Variant (r-TmDEX49A-ΔSP-ΔN30) in Komagataella phaffii (Pichia pastoris)

Aristicas¹,

Valdés²,

Gámez³

et al. 2022

Preprint

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In the sugar industry, dextran generates difficulties in the manufacturing process. Crude dextranase (EC 3.2.1.11) to eliminate dextran in sugar is an effective practice. In this study, a synthetic dextranase encoding gene of the filamentous fungus Talaromyces minioluteus, lacking its putative native signal peptide (1-20 amino acids) and the next 30 amino acids (r-TmDEX49A-ΔSP-ΔN30), was fused to the Saccharomyces cerevisiae prepro -factor (MF-2) signal sequence and expressed in Komagataella phaffii under the constitutive GAP promoter. K. phaffii DEX49A-ΔSP-ΔN30, constitutively producing and secreting the truncated dextranase was obtained. The specific activity of the truncated variant resulted nearly the same in relation to the full-length mature enzyme (900-1000 U.mg-1 of protein). At shaker scale (100 mL) in YPG medium, the enzymatic activity was 273 U.mL-1. The highest production level was achieved in a fed-batch culture (30 h) at 5 L fermenter scale using the FM21-PTM1 culture medium. The enzymatic activity in the culture supernatant reached 1614 U.mL-1 and the productivity was 53800 U.L-1.h-1 (53.8 mg.L-1.h-1), the highest reported so far for a DEX49A variant. Dextran decreased r-TmDEX49A-ΔSP-ΔN30 mobility in affinity gel electrophoresis, providing evidence of carbohydrate-protein interactions. K. phaffii DEX49A-ΔSP-ΔN30 shows great potential as a methanol-free, commercial dextranase production system.

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“…P. pinophilum , like other penicillia, is known for producing bioactive metabolites [ 36 , 37 ]. Enzymes involved in the degradation of lignocellulosic biomass have been identified from various isolates [ 38 – 42 ], and a gene encoding a dextranase was recently cloned from a marine isolate [ 43 ].…”

Section: Resultsmentioning

confidence: 99%

Growth of marine fungi on polymeric substrates

et al. 2016

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BackgroundMarine fungi are a diverse group of opportunistic and obligate organisms isolated from marine environments. These fungi are now often included in screens for novel metabolites, while less attention has been given to their production of hydrolytic enzymes. Most enzymes derived from marine microorganisms have been obtained from marine bacteria. The enzymes produced by marine fungi may have different properties than those derived from bacteria or from terrestrial fungi. Here we assess the growth of six filamentous marine fungi on a wide range of polymeric substrates as an indication of their general capacity to produce hydrolytic enzymes.ResultsCalcarisporium sp. KF525, Tritirachium sp. LF562, Bartalinia robillardoides LF550, Penicillium pinophilum LF458, Scopulariopsis brevicaulis LF580 and Pestalotiopsis sp. KF079 all grew on both casein and gelatin as N-source, indicating secretion of proteases. All species also grew on starch, laminarin, xylan, pectin and oil, indicating production of amylases, glucanases, xylanases, pectinases and lipases. Growth on cellulose occurred but was weaker than on xylan. All strains also grew to some extent on sulphated arabinogalactan, although only LF562 could utilise arabinose. Four strains grew on the sulphated ulvans, whereas only KF525 grew on agar or carrageenan. KF525 and LF562 showed limited growth on alginate. Although fucose was used as carbon source by several species, fucoidan did not support biomass production.ConclusionsMarine fungi could be excellent sources of a wide range of hydrolytic enzymes, including those able to hydrolyse various seaweed polymers. Although the native hosts may secrete only small amounts of these enzymes, the genes may provide a rich source of novel enzymes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0233-5) contains supplementary material, which is available to authorized users.

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Cloning, characterization, and heterologous expression of a dextranase gene from Penicillium pinophilum SMCU3-14

Cited by 6 publications

References 24 publications

Constitutive High Expression Level of a Synthetic Deleted Encoding Gene of Talaromyces minioluteus Endodextranase Variant (r–TmDEX49A–ΔSP–ΔN30) in Komagataella phaffii (Pichia pastoris)

Constitutive High Expression Level of a Synthetic Deleted Encoding Gene of Talaromyces minioluteus Endodextranase Variant (r–TmDEX49A–ΔSP–ΔN30) in Komagataella phaffii (Pichia pastoris)

Constitutive High Expression Level of A Synthetic Deleted Encoding Gene of <i>Talaromyces minioluteus</i> Endodextranase Variant (r-TmDEX49A-ΔSP-ΔN30) in <i>Komagataella phaffii</i> (<i>Pichia pastoris</i>)

Growth of marine fungi on polymeric substrates

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