Improving the Oxygenation Performance of a Cyanobacterial Lipoxygenase by Oxygen Channel Engineering

Qi, Yi-Ke; Zheng, Yu‐Cong; Chen, Qi; He, Yiming; Zhang, Zhi Jun; Xu, Jian‐He

doi:10.1021/acssuschemeng.1c05117

Cited by 10 publications

(10 citation statements)

References 51 publications

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“…Potentially, if the gas channel was occupied by another nonsubstrate gas, the enzyme activity would lose rapidly and irreversibly. It has been reported that the oxidation performance of oxygenase could be improved by oxygen channel engineering . In Figure c, NOX was first bubbled with O 2 for 6.5 h, and the NOX activity decreased from 100 to 85%.…”

Section: Resultsmentioning

confidence: 84%

“…It has been reported that the oxidation performance of oxygenase could be improved by oxygen channel engineering. 20 In Figure 5c, NOX was first bubbled with O 2 for 6.5 h, and the NOX activity decreased from 100 to 85%. Following N 2 bubbling, the NOX activity decreased slowly from 85 to 14% after 28 h, and the activity loss was notably slower compared with Figure 5b, which might indicate that the oxygen channel of NOX was first occupied and saturated by O 2 ; therefore, the dissolved N 2 would not take over the active site of NOX, and only the N 2 −water interface affected the deactivation of NOX.…”

Section: ■ Results and Discussionmentioning

confidence: 93%

“…For example, the initial reaction rate of a lipoxygenase to convert linoleic acid into linoleic acid hydroperoxide has no significant decrease when the initial dissolved oxygen concentration was adjusted from 10 to 80% of saturation, which indicates a low K MO for lipoxygenase . Therefore, the increasing oxygen concentration may not result in high enzyme activity if the K MO of an oxidase is low, and the high activity and stability can be achieved through protein engineering alone. , However, a gas phase with high oxygen concentration may decrease the reaction rate because of fast enzyme deactivation . It was also reported that hydrogenases are sensitive to oxygen concentration since their activity decreases as the oxygen partial pressure increases. , Therefore, an oxidant with air or pure oxygen is a critical issue that may have different effects on enzyme activity and stability.…”

Section: Introductionmentioning

confidence: 99%

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Effect of Nitrogen, Air, and Oxygen on the Kinetic Stability of NAD(P)H Oxidase Exposed to a Gas–Liquid Interface

Wang

Erdem

Woodley

2023

Org. Process Res. Dev.

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Biocatalytic oxidation is an interesting prospect for the selective synthesis of active pharmaceutical intermediates. Bubbling air or oxygen is considered as an efficient method to increase the gas−liquid interface and thereby enhance oxygen transfer. However, the enzyme is deactivated in this process and needs to be further studied and understood to accelerate the implementation of oxidative biocatalysis in larger production processes. This paper reports data on the stability of NAD(P)H oxidase (NOX) when exposed to different gas−liquid interfaces introduced by N 2 (0% oxygen), air (21% oxygen), and O 2 (100% oxygen) in a bubble column. A pH increase was observed during gas bubbling, with the highest increase occurring under air bubbling from 6.28 to 7.40 after 60 h at a gas flow rate of 0.15 L min −1 . The kinetic stability of NOX was studied under N 2 , air, and O 2 bubbling by measuring the residual activity, the deactivation constants (k d1 ) were 0.2972, 0.0244, and 0.0346 with the corresponding half-lives of 2.2, 28.6, and 20.2 h, respectively. A decrease in protein concentration of the NOX solution was also observed and was attributed to likely enzyme aggregation at the gas−liquid interface. Most aggregation occurred at the air−water interface and decreased greatly from 100 to 14.16% after 60 h of bubbling air. Furthermore, the effect of the gas−liquid interface and the dissolved gas on the NOX deactivation process was also studied by bubbling N 2 and O 2 alternately. It was found that the N 2 −water interface and O 2 −water interface both had minor effects on the protein concentration decrease compared with the air−water interface, whilst the dissolved N 2 in water caused serious deactivation of NOX. This was attributed not only to the NOX unfolding and aggregation at the interface but also to the N 2 occupying the oxygen channel of the enzyme and the resultant inaccessibility of dissolved O 2 to the active site of NOX. These results shed light on the enzyme deactivation process and might further inspire bioreactor operation and enzyme engineering to improve biocatalyst performance.

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Section: Resultsmentioning

confidence: 84%

Section: ■ Results and Discussionmentioning

confidence: 93%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Effect of Nitrogen, Air, and Oxygen on the Kinetic Stability of NAD(P)H Oxidase Exposed to a Gas–Liquid Interface

Wang

Erdem

Woodley

2023

Org. Process Res. Dev.

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“…Changing the enzyme specificity from 9R-to almost exclusively 13S-dioxygenation or vice versa was achieved in some bacterial LOXs by exchanging the Coffa site residue for a bulkier amino acid residue (Andreou et al, 2008;Newie et al, 2016;Yi et al, 2020). Improving the enzyme activity by enlarging the oxygen channel through structure-guided engineering (Ala324Gly/ Ser392Gly double substitution) has been reported recently (Qi et al, 2021).…”

Section: Enzyme Improvement Approachesmentioning

confidence: 99%

Bacterial lipoxygenases: Biochemical characteristics, molecular structure and potential applications

Chrisnasari

Hennebelle

Vincken

et al. 2022

Biotechnology Advances

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“…Lipoxygenase (EC. 1.13.11.12, LOX) mainly catalyzes the dioxygenation of polyunsaturated fatty acids with one or more cis , cis -1, 4-pentadiene units to produce hydroperoxy fatty acids [ 1 ], which are used in the food, chemical, and textile industries [ 2 , 3 ]. Presently, most of the applied research is based on LOX extracted form soybean [ 4 ].…”

Section: Introductionmentioning

confidence: 99%

Enhancing extracellular production of lipoxygenase in Escherichia coli by signal peptides and autolysis system

et al. 2022

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Background Lipoxygenase (LOX) is a non-heme iron containing dioxygenase that is widely used to improve food quality and produce active drug intermediates and biodiesel. Escherichia coli is one of the most widely used host microorganisms for recombinant protein expression; however, its weak extracellular secretion ability precludes its effective production of recombinant proteins into the extracellular environment. To facilitate subsequent characterization and application of LOX, improving its secretion efficiency from E. coli is a major challenge that needs to be solved. Results Several strategies were adopted to improve the extracellular secretion of LOX based on the signal peptides and cell wall permeability of E. coli. Here, we studied the effect of signal peptides on LOX secretion, which increased the secretory capacity for LOX marginally. Although surfactants could increase the permeability of the cell membrane to promote LOX secretion, the extracellular LOX yield could not meet the requirements of industrialization production. Subsequently, an autolysis system was constructed in E. coli based on the bacteriophage lysis gene ΦX174-E to enhance the production of extracellular proteins. Thus, the extracellular production of LOX was achieved and the content of inclusion bodies in the cell was reduced by optimizing cell lysis conditions. The extracellular LOX yield reached 368 ± 1.4 U mL−1 in a 5-L bioreactor under optimized lysis conditions that is, an induction time and temperature, and arabinose concentration of 5 h, 25 °C, and 0.6 mM, respectively. Conclusions In this study, the different signal peptides and cell autolysis system were developed and characterized for extracellular LOX production in E. coli. Finally, the cell autolysis system presented a slight advantage on extracellular LOX yield, which also provides reference for other protein extracellular production.

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Improving the Oxygenation Performance of a Cyanobacterial Lipoxygenase by Oxygen Channel Engineering

Cited by 10 publications

References 51 publications

Effect of Nitrogen, Air, and Oxygen on the Kinetic Stability of NAD(P)H Oxidase Exposed to a Gas–Liquid Interface

Effect of Nitrogen, Air, and Oxygen on the Kinetic Stability of NAD(P)H Oxidase Exposed to a Gas–Liquid Interface

Bacterial lipoxygenases: Biochemical characteristics, molecular structure and potential applications

Enhancing extracellular production of lipoxygenase in Escherichia coli by signal peptides and autolysis system

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