A method for efficient expression of Pseudomonas aeruginosa alginate lyase in Pichia pastoris

Abstract Background Lytic polysaccharide monooxygenases (LPMOs) are attracting large attention due their ability to degrade recalcitrant polysaccharides in biomass conversion and to perform powerful redox chemistry. Results We have established a universal Pichia pastoris platform for the expression of fungal LPMOs using state-of-the-art recombination cloning and modern molecular biological tools to achieve high yields from shake-flask cultivation and simple tag-less single-step purification. Yields are very favorable with up to 42 mg per liter medium for four different LPMOs spanning three different families. Moreover, we report for the first time of a yeast-originating signal peptide from the dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1 (OST1) form S. cerevisiae efficiently secreting and successfully processes the N-terminus of LPMOs yielding in fully functional enzymes. Conclusion The work demonstrates that the industrially most relevant expression host P. pastoris can be used to express fungal LPMOs from different families in high yields and inherent purity. The presented protocols are standardized and require little equipment with an additional advantage with short cultivation periods.

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Expression of the Klebsiella pneumoniae alginate lyase gene in Pseudomonas aeruginosa - effect on alginate structure

Biochemical Society Transactions ◽

10.1042/bst024407s ◽

1996 ◽

Vol 24 (3) ◽

pp. 407S-407S

Author(s):

Peter J. Tatnell ◽

Joanna B. Goldberg ◽

Peter Gacesa

Keyword(s):

Pseudomonas Aeruginosa ◽

Klebsiella Pneumoniae ◽

Alginate Lyase

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Synergistic Effect of Acetylalginate Esterase and Alginate Lyase on the Degradation of Acetylalginate from Pseudomonas aeruginosa ATCC 39324

Journal of Life Science ◽

10.5352/jls.2013.23.12.1420 ◽

2013 ◽

Vol 23 (12) ◽

pp. 1420-1427 ◽

Cited By ~ 1

Author(s):

Hee Sook Kim

Keyword(s):

Pseudomonas Aeruginosa ◽

Synergistic Effect ◽

Alginate Lyase

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Efficient expression and isolation of recombinant human interleukin-11 (rhIL-11) in Pichia pastoris

Protein Expression and Purification ◽

10.1016/j.pep.2018.01.012 ◽

2018 ◽

Vol 146 ◽

pp. 69-77 ◽

Cited By ~ 4

Author(s):

Kuo-Ming Yu ◽

Johnson Yiu-Nam Lau ◽

Manson Fok ◽

Yuk-Keung Yeung ◽

Siu-Ping Fok ◽

...

Keyword(s):

Pichia Pastoris ◽

Efficient Expression ◽

Interleukin 11

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Expression and Characterization of an Alginate Lyase and Its Thermostable Mutant in Pichia pastoris

Marine Drugs ◽

10.3390/md18060305 ◽

2020 ◽

Vol 18 (6) ◽

pp. 305

Author(s):

Suxiao Yang ◽

Zhemin Liu ◽

Xiaodan Fu ◽

Changliang Zhu ◽

Qing Kong ◽

...

Keyword(s):

Pichia Pastoris ◽

Degradation Products ◽

Alginate Lyase ◽

Maximal Activity ◽

Degree Of Polymerization ◽

Molecular Weights ◽

Industrial Preparation ◽

Alginate Oligosaccharides ◽

Elimination Mechanism ◽

G Ratio

Alginate is one of the most abundant polysaccharides in algae. Alginate lyase degrades alginate through a β-elimination mechanism to produce alginate oligosaccharides with special bioactivities. Improving enzyme activity and thermal stability can promote the application of alginate lyase in the industrial preparation of alginate oligosaccharides. In this study, the recombinant alginate lyase cAlyM and its thermostable mutant 102C300C were expressed and characterized in Pichia pastoris. The specific activities of cAlyM and 102C300C were 277.1 U/mg and 249.6 U/mg, respectively. Both enzymes showed maximal activity at 50 °C and pH 8.0 and polyG preference. The half-life values of 102C300C at 45 °C and 50 °C were 2.6 times and 11.7 times the values of cAlyM, respectively. The degradation products of 102C300C with a lower degree of polymerization contained more guluronate. The oligosaccharides with a polymerization degree of 2–4 were the final hydrolytic products. Therefore, 102C300C is potentially valuable in the production of alginate oligosaccharides with specific M/G ratio and molecular weights.

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