High-level expression and characterization of a stereoselective lipase from Aspergillus oryzae in Pichia pastoris

Zheng, Jingwu; Lan, Xing; Li, Xiaojun; Huang, Lijuan; Zhang, Yinjun; Zhao, Wenjing

doi:10.1016/j.pep.2018.10.012

Cited by 20 publications

(7 citation statements)

References 31 publications

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“…Metal ions may influence the enzyme activity by interacting with disulfide bonds to change the structure of the enzyme or reacting with the substrate ( Zheng et al, 2019 ). Therefore, the effects of metal ions on AoNagase activity were tested.…”

Section: Resultsmentioning

confidence: 99%

A Novel GH Family 20 β-N-acetylhexosaminidase With Both Chitosanase and Chitinase Activity From Aspergillus oryzae

Zhang

Qin

et al. 2021

Front. Mol. Biosci.

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Aminooligosaccharides possess various biological activities and can exploit wide applications in food, pharmaceutical and cosmetic industries. Commercial aminooligosaccharides are often prepared by the hydrolysis of chitin and chitosan. In this study, a novel GH family 20 β-N-acetylhexosaminidases gene named AoNagase was cloned from Aspergillus oryzae and expressed in Pichia pastoris. The purified AoNagase had maximal activity at pH 5.5 and 65°C. It exhibited good pH stability in the range of pH 6.0–7.5 and at temperatures below 50°C. AoNagase was capable of hydrolyzing not only colloidal chitosan (508.26 U/mg) but also chitin (29.78 U/mg). The kinetic parameters (Km and Vmax) of AoNagase were 1.51 mM, 1106.02 U/mg for chitosan and 0.41 mM, 40.31 U/mg for colloidal chitin. To our knowledge, AoNagase is the first GH family 20 β-N-acetylhexosaminidase capable of hydrolyzing both chitosan and chitin. AoNagase is an endo-type β-N-acetylhexosaminidases and can potentially be used for the manufacturing of aminooligosaccharides.

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Section: Resultsmentioning

confidence: 99%

A Novel GH Family 20 β-N-acetylhexosaminidase With Both Chitosanase and Chitinase Activity From Aspergillus oryzae

Zhang

Qin

et al. 2021

Front. Mol. Biosci.

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“…In fed-batch cultivations, the development of inclusion bodies may be observed by tracking shifts in intrinsic light dispersion via cytometric flow [ 143 ]. This enables the growth conditions to be controlled in real time as soon as inclusion bodies are observed even at low rates, and the formation of such bodies may be prevented theoretically [ 144 ]. Protein folding involves the presence of a particular cofactor.…”

Section: Soluble and Insoluble Expressionmentioning

confidence: 99%

Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds

Lee

Park

2020

Antibiotics

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Bacteria can produce recombinant proteins quickly and cost effectively. However, their physiological properties limit their use for the production of proteins in their native form, especially polypeptides that are subjected to major post-translational modifications. Proteins that rely on disulfide bridges for their stability are difficult to produce in Escherichia coli. The bacterium offers the least costly, simplest, and fastest method for protein production. However, it is difficult to produce proteins with a very large size. Saccharomyces cerevisiae and Pichia pastoris are the most commonly used yeast species for protein production. At a low expense, yeasts can offer high protein yields, generate proteins with a molecular weight greater than 50 kDa, extract signal sequences, and glycosylate proteins. Both eukaryotic and prokaryotic species maintain reducing conditions in the cytoplasm. Hence, the formation of disulfide bonds is inhibited. These bonds are formed in eukaryotic cells during the export cycle, under the oxidizing conditions of the endoplasmic reticulum. Bacteria do not have an advanced subcellular space, but in the oxidizing periplasm, they exhibit both export systems and enzymatic activities directed at the formation and quality of disulfide bonds. Here, we discuss current techniques used to target eukaryotic and prokaryotic species for the generation of correctly folded proteins with disulfide bonds.

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“…Among them, a chitinase from filamentous fungi has received more attention due to its advantages in the efficiency of chitin hydrolysis. Several species of the chitinase have been identified biochemically, such as chitinases from A. oryzaeIn [ 36 ], P.oxalicum k10 [ 30 ]. It is well known that the genomes of A. fumigatus are rich in abundance of chitinase genes.…”

Section: Introductionmentioning

confidence: 99%

The Discovery, Enzymatic Characterization and Functional Analysis of a Newly Isolated Chitinase from Marine-Derived Fungus Aspergillus fumigatus df347

Wang

Yang

et al. 2022

Marine Drugs

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In order to discover a broad-specificity and high stability chitinase, a marine fungus, Aspergillus fumigatus df347, was identified in the sediments of mangrove wetlands in Qinzhou Bay, China. The chitinase gene (AfChi28) from A. fumigatus df347 was cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme AfChi28 was purified and characterized. AfChi28 is an acido-halotolerant- and temperature-resistant bifunctional enzyme with both endo- and exo-cleavage functions. Its enzymatic products are mainly GlcNAc, (GlcNAc)2, (GlcNAc)3 and (GlcNAc)4. Na+, Mg2+, K+, Ca2+ and Tris at a concentration of 50 mM had a strong stimulatory effect on AfChi28. The crude enzyme and pure enzyme exhibited the highest specific activity of 0.737 mU/mg and 52.414 mU/mg towards colloidal chitin. The DxDxE motif at the end of strand β5 and with Glu154 as the catalytic residue was verified by the AlphaFold2 prediction and sequence alignment of homologous proteins. Moreover, the results of molecular docking showed that molecular modeling of chitohexaose was shown to bind to AfChi28 in subsites −4 to +2 in the deep groove substrate-binding pocket. This study demonstrates that AfChi28 is a promising chitinase for the preparation of desirable chitin oligosaccharides, and provides a foundation for elucidating the catalytic mechanism of chitinases from marine fungi.

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High-level expression and characterization of a stereoselective lipase from Aspergillus oryzae in Pichia pastoris

Cited by 20 publications

References 31 publications

A Novel GH Family 20 β-N-acetylhexosaminidase With Both Chitosanase and Chitinase Activity From Aspergillus oryzae

A Novel GH Family 20 β-N-acetylhexosaminidase With Both Chitosanase and Chitinase Activity From Aspergillus oryzae

Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds

The Discovery, Enzymatic Characterization and Functional Analysis of a Newly Isolated Chitinase from Marine-Derived Fungus Aspergillus fumigatus df347

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