Limits...
The production and characterization of a new active lipase from Acremonium alcalophilum using a plant bioreactor.

Pereira EO, Tsang A, McAllister TA, Menassa R - Biotechnol Biofuels (2013)

Bottom Line: Microorganisms are the most proficient decomposers in nature, using secreted enzymes in the hydrolysis of lignocellulose.As such, they present the most abundant source for discovery of new enzymes.The enzyme is also highly active on xylose tetra-acetate and oat spelt xylan.

View Article: PubMed Central - HTML - PubMed

Affiliation: Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON N5V 4T3, Canada ; Department of Biology, The University of Western Ontario, London, ON N6A 5B7, Canada.

ABSTRACT

Background: Microorganisms are the most proficient decomposers in nature, using secreted enzymes in the hydrolysis of lignocellulose. As such, they present the most abundant source for discovery of new enzymes. Acremonium alcalophilum is the only known cellulolytic fungus that thrives in alkaline conditions and can be cultured readily in the laboratory. Its optimal conditions for growth are 30°C and pH 9.0-9.2. The genome sequence of Acremonium alcalophilum has revealed a large number of genes encoding biomass-degrading enzymes. Among these enzymes, lipases are interesting because of several industrial applications including biofuels, detergent, food processing and textile industries.

Results: We identified a lipA gene in the genome sequence of Acremonium alcalophilum, encoding a protein with a predicted lipase domain with weak sequence identity to characterized enzymes. Unusually, the predicted lipase displays ≈ 30% amino acid sequence identity to both feruloyl esterase and lipase of Aspergillus niger. LipA, when transiently produced in Nicotiana benthamiana, accumulated to over 9% of total soluble protein. Plant-produced recombinant LipA is active towards p-nitrophenol esters of various carbon chain lengths with peak activity on medium-chain fatty acid (C8). The enzyme is also highly active on xylose tetra-acetate and oat spelt xylan. These results suggests that LipA is a novel lipolytic enzyme that possesses both lipase and acetylxylan esterase activity. We determined that LipA is a glycoprotein with pH and temperature optima at 8.0 and 40°C, respectively.

Conclusion: Besides being the first heterologous expression and characterization of a gene coding for a lipase from A. alcalophilum, this report shows that LipA is very versatile exhibiting both acetylxylan esterase and lipase activities potentially useful for diverse industry sectors, and that tobacco is a suitable bioreactor for producing fungal proteins.

No MeSH data available.


Related in: MedlinePlus

Alignment of amino acid sequences of LipA from Acremonium alcalophilum with other lipases and feruloyl esterase from various microorganisms. The amino acid sequence of LipA was compared with the lipase amino acid sequences of Aspergillus niger (An_lipase), Thermomyces lanuginosus (Tl_lipase); and ferulic acid esterase amino acid sequence of Aspergillus niger (An_FAE). Identical residues among the four enzymes are marked with asterisks. Residues with conserved and semiconserved substitutions are indicated with colons and dots, respectively; the conserved GXSXG motif and putative residues of the catalytic triad are underlined in red.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750315&req=5

Figure 1: Alignment of amino acid sequences of LipA from Acremonium alcalophilum with other lipases and feruloyl esterase from various microorganisms. The amino acid sequence of LipA was compared with the lipase amino acid sequences of Aspergillus niger (An_lipase), Thermomyces lanuginosus (Tl_lipase); and ferulic acid esterase amino acid sequence of Aspergillus niger (An_FAE). Identical residues among the four enzymes are marked with asterisks. Residues with conserved and semiconserved substitutions are indicated with colons and dots, respectively; the conserved GXSXG motif and putative residues of the catalytic triad are underlined in red.

Mentions: With the recent genome sequencing of the alkaliphilic fungus Acremonium alcalophilum, over 200 genes encoding biomass-degrading enzymes were identified. Analysis in silico of the genome revealed a hypothetical protein (protein ID 1062717 of the A. alcalophilum v2.0 database) exhibiting a lipase_3 domain (IPR002921) and weak sequence identity with biochemically characterized fungal proteins. For example, pairwise amino acid sequence alignment using ClustalW2 (http://www.ebi.ac.uk) revealed 25% identity to the triglyceride lipase of Aspergillus niger (Accession no. DQ680030, [9]), 24% identity to the lipase of Thermomyces lanuginosus (Accession no. O59952, [10]), and 23% identity to the characterized A. niger feruloyl esterase (Accession no. O42807.1, [11]) (Figure 1). Therefore, this novel enzyme was provisionally designated LipA. However, LipA displays some sequence similarity to uncharacterized enzymes such as 57 and 61% identity to the putative lipases of Glomerella graminicola M1.001 (Accession no. EFQ29359.1), and Verticillium dahliae VdLs.17 (Accession no. EGY21687.1) respectively, and 40% identity to the putative feruloyl esterases of Trichophyton equinum CBS 127.97 (Accession no.EGE08976.1) and Arthroderma otae CBS 113480 (Accession no. XP 002843470.1). Taking into consideration the low sequence similarity to two different enzyme activities, and that sequence-based analysis only provides a presumptive compositional and functional blueprint of the gene analyzed, which in some cases may lead to erroneous assignment of substrate specificity [2], we assessed LipA for both lipase and carbohydrate esterase activity.


The production and characterization of a new active lipase from Acremonium alcalophilum using a plant bioreactor.

Pereira EO, Tsang A, McAllister TA, Menassa R - Biotechnol Biofuels (2013)

Alignment of amino acid sequences of LipA from Acremonium alcalophilum with other lipases and feruloyl esterase from various microorganisms. The amino acid sequence of LipA was compared with the lipase amino acid sequences of Aspergillus niger (An_lipase), Thermomyces lanuginosus (Tl_lipase); and ferulic acid esterase amino acid sequence of Aspergillus niger (An_FAE). Identical residues among the four enzymes are marked with asterisks. Residues with conserved and semiconserved substitutions are indicated with colons and dots, respectively; the conserved GXSXG motif and putative residues of the catalytic triad are underlined in red.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750315&req=5

Figure 1: Alignment of amino acid sequences of LipA from Acremonium alcalophilum with other lipases and feruloyl esterase from various microorganisms. The amino acid sequence of LipA was compared with the lipase amino acid sequences of Aspergillus niger (An_lipase), Thermomyces lanuginosus (Tl_lipase); and ferulic acid esterase amino acid sequence of Aspergillus niger (An_FAE). Identical residues among the four enzymes are marked with asterisks. Residues with conserved and semiconserved substitutions are indicated with colons and dots, respectively; the conserved GXSXG motif and putative residues of the catalytic triad are underlined in red.
Mentions: With the recent genome sequencing of the alkaliphilic fungus Acremonium alcalophilum, over 200 genes encoding biomass-degrading enzymes were identified. Analysis in silico of the genome revealed a hypothetical protein (protein ID 1062717 of the A. alcalophilum v2.0 database) exhibiting a lipase_3 domain (IPR002921) and weak sequence identity with biochemically characterized fungal proteins. For example, pairwise amino acid sequence alignment using ClustalW2 (http://www.ebi.ac.uk) revealed 25% identity to the triglyceride lipase of Aspergillus niger (Accession no. DQ680030, [9]), 24% identity to the lipase of Thermomyces lanuginosus (Accession no. O59952, [10]), and 23% identity to the characterized A. niger feruloyl esterase (Accession no. O42807.1, [11]) (Figure 1). Therefore, this novel enzyme was provisionally designated LipA. However, LipA displays some sequence similarity to uncharacterized enzymes such as 57 and 61% identity to the putative lipases of Glomerella graminicola M1.001 (Accession no. EFQ29359.1), and Verticillium dahliae VdLs.17 (Accession no. EGY21687.1) respectively, and 40% identity to the putative feruloyl esterases of Trichophyton equinum CBS 127.97 (Accession no.EGE08976.1) and Arthroderma otae CBS 113480 (Accession no. XP 002843470.1). Taking into consideration the low sequence similarity to two different enzyme activities, and that sequence-based analysis only provides a presumptive compositional and functional blueprint of the gene analyzed, which in some cases may lead to erroneous assignment of substrate specificity [2], we assessed LipA for both lipase and carbohydrate esterase activity.

Bottom Line: Microorganisms are the most proficient decomposers in nature, using secreted enzymes in the hydrolysis of lignocellulose.As such, they present the most abundant source for discovery of new enzymes.The enzyme is also highly active on xylose tetra-acetate and oat spelt xylan.

View Article: PubMed Central - HTML - PubMed

Affiliation: Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON N5V 4T3, Canada ; Department of Biology, The University of Western Ontario, London, ON N6A 5B7, Canada.

ABSTRACT

Background: Microorganisms are the most proficient decomposers in nature, using secreted enzymes in the hydrolysis of lignocellulose. As such, they present the most abundant source for discovery of new enzymes. Acremonium alcalophilum is the only known cellulolytic fungus that thrives in alkaline conditions and can be cultured readily in the laboratory. Its optimal conditions for growth are 30°C and pH 9.0-9.2. The genome sequence of Acremonium alcalophilum has revealed a large number of genes encoding biomass-degrading enzymes. Among these enzymes, lipases are interesting because of several industrial applications including biofuels, detergent, food processing and textile industries.

Results: We identified a lipA gene in the genome sequence of Acremonium alcalophilum, encoding a protein with a predicted lipase domain with weak sequence identity to characterized enzymes. Unusually, the predicted lipase displays ≈ 30% amino acid sequence identity to both feruloyl esterase and lipase of Aspergillus niger. LipA, when transiently produced in Nicotiana benthamiana, accumulated to over 9% of total soluble protein. Plant-produced recombinant LipA is active towards p-nitrophenol esters of various carbon chain lengths with peak activity on medium-chain fatty acid (C8). The enzyme is also highly active on xylose tetra-acetate and oat spelt xylan. These results suggests that LipA is a novel lipolytic enzyme that possesses both lipase and acetylxylan esterase activity. We determined that LipA is a glycoprotein with pH and temperature optima at 8.0 and 40°C, respectively.

Conclusion: Besides being the first heterologous expression and characterization of a gene coding for a lipase from A. alcalophilum, this report shows that LipA is very versatile exhibiting both acetylxylan esterase and lipase activities potentially useful for diverse industry sectors, and that tobacco is a suitable bioreactor for producing fungal proteins.

No MeSH data available.


Related in: MedlinePlus