Overproduction and characterization of a lytic polysaccharide monooxygenase in Bacillus subtilis using an assay based on ascorbate consumption

Yu, Mi-Ji; Yoon, Sunhee; Kim, Young-Wan

doi:10.1016/j.enzmictec.2016.08.014

Cited by 21 publications

(17 citation statements)

References 34 publications

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“…After 48 h of shake-flask fermentation, bands corresponding to the target proteins were clearly visible on SDS-PAGE gels (Figure 2A). The apparent molecular weights of BcChiA1 and BatLPMO10 in this study were approximately 73 and 20 kDa, which was in agreement with previous reports (Watanabe et al, 1990;Yu et al, 2016). The results indicated that all signal peptides led to successful BcChiA1 secretion, but the noSP group performed best.…”

Section: Efficient Secretory Expression Of Bcchia1 and Batlmpo10 In Bsupporting

confidence: 93%

High-Efficiency Secretion and Directed Evolution of Chitinase BcChiA1 in Bacillus subtilis for the Conversion of Chitinaceous Wastes Into Chitooligosaccharides

Wang

et al. 2020

Front. Bioeng. Biotechnol.

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Limitations of enzyme production and activity pose a challenge for efficient degradation of chitinaceous wastes. To solve this problem, we engineered a system for high-yielding extracellular secretion of chitinase A1 from Bacillus circulans (BcChiA1) in B. subtilis. Furthermore, an innovative chitinase high-throughput screening method based on colloidal chitin stained with Remazol Brilliant Blue R (CC-RBB) was established and used to identify three mutants with improved chitinase activity: Y10A/R301A/E327A (Mu1), Y10A/D81A/E327A (Mu2), and F38A/K88A/R301A (Mu3). Their highest specific activity reached 1004.83 ± 0.87 U/mg, representing a 16.89-fold increase in activity compared to native BcChiA1. Additionally, we found that there is a synergistic effect between BcChiA1 and a lytic polysaccharide monooxygenase from Bacillus atrophaeus (BatLPMO10), which increased the chitin processing efficiency by 50% after combining the two enzymes. The yield of chitooligosaccharide (COS) production using the mutant Mu1 and BatLPMO10 reached 2885.25 ± 2.22 mg/L. Taken together, the results indicated that the CC-RBB high-throughput screening method is a useful tool for chitinase screening, and evolution of BcChiA1 in collaboration with BatLPMO10 has tremendous application potential in the biological treatment of chitinaceous wastes for COS production.

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Section: Efficient Secretory Expression Of Bcchia1 and Batlmpo10 In Bsupporting

confidence: 93%

High-Efficiency Secretion and Directed Evolution of Chitinase BcChiA1 in Bacillus subtilis for the Conversion of Chitinaceous Wastes Into Chitooligosaccharides

Wang

et al. 2020

Front. Bioeng. Biotechnol.

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“…158 Similarly, efforts by Yu et al to measure the activity of chitin-active LPMOs rely on monitoring the consumption of ascorbic acid. 174 All of these methods are dependent on a tight coupling to other activities and require that those other activities are not limiting. Although not readily available, a direct measure of activity will not have these limitations.…”

Section: Industrial Use Of Lpmosmentioning

confidence: 99%

Oxygen Activation by Cu LPMOs in Recalcitrant Carbohydrate Polysaccharide Conversion to Monomer Sugars

et al. 2017

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Natural carbohydrate polymers such as starch, cellulose, and chitin provide renewable alternatives to fossil fuels as a source for fuels and materials. As such, there is considerable interest in their conversion for industrial purposes, which is evidenced by the established and emerging markets for products derived from these natural polymers. In many cases, this is achieved via industrial processes that use enzymes to break down carbohydrates to monomer sugars. One of the major challenges facing large-scale industrial applications utilizing natural carbohydrate polymers is rooted in the fact that naturally occurring forms of starch, cellulose, and chitin can have tightly packed organizations of polymer chains with low hydration levels, giving rise to crystalline structures that are highly recalcitrant to enzymatic degradation. The topic of this review is oxidative cleavage of carbohydrate polymers by lytic polysaccharide monooxygenases (LPMOs). LPMOs are copper-dependent enzymes (EC 1.14.99.53–56) that, with glycoside hydrolases, participate in the degradation of recalcitrant carbohydrate polymers. Their activity and structural underpinnings provide insights into biological mechanisms of polysaccharide degradation.

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“…In order to explore different approaches, we developed a range of vectors for producing LPMOs in three different cellular compartments of E. coli : The cytoplasm, the periplasm, and attached to the surface of the cell. The LyGo vectors were tested with four different LPMOs: Thermobifida fusca TfLPMO10A 22 , Streptomyces coelicolor ScLPMO10B 23 , BatLPMO10 from Bacillus atrophaeus 24 , and LsAA9A from Lentinus similis 25 . TfLPMO10A, ScLPMO10B, and BatLPMO10 were chosen because they previously were produced successfully in E. coli 22,23,24 .…”

Section: Resultsmentioning

confidence: 99%

“…The LyGo vectors were tested with four different LPMOs: Thermobifida fusca TfLPMO10A 22 , Streptomyces coelicolor ScLPMO10B 23 , BatLPMO10 from Bacillus atrophaeus 24 , and LsAA9A from Lentinus similis 25 . TfLPMO10A, ScLPMO10B, and BatLPMO10 were chosen because they previously were produced successfully in E. coli 22,23,24 . LsAA9A was chosen as an example of a fungal enzyme and because it had the added benefit of being compatible with a simple chromogenic assay (based on AZCL-HEC) available 26 .…”

Section: Resultsmentioning

confidence: 99%

“…The current vector collection consists of 14 vectors in total (Table 1), and we established a simple abstraction and nomenclature, in order to easily recognize plasmids and translate their names into meaningful concepts. LyGo vectors are designated: "pLyGo" followed by two letters indicating the 24 , and LsAA9A from Lentinus similis 25 . TfLPMO10A, ScLPMO10B, and BatLPMO10 were chosen because they previously were produced successfully in E. coli 22,23,24 .…”

Section: Nomenclature For Lygo Vectorsmentioning

confidence: 99%

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LyGo: A platform for rapid screening of lytic polysaccharide monooxygenase production

Hernández-Rollán

Falkenberg

Rennig

et al. 2020

Preprint

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Environmentally friendly sources of energy and chemicals are essential constituents of a sustainable society. An important step towards this goal is the utilization of non-edible biomass as supply of building blocks for future biorefineries. Lytic polysaccharide monooxygenases (LPMOs) are enzymes that play a critical role in breaking the chemical bonds in the most abundant polymers found in recalcitrant biomass, such as cellulose and chitin. Predicting optimal strategies for producing LPMOs is often non-trivial, and methods allowing for screening several strategies simultaneously are therefore needed. Here, we present a standardized platform for cloning LPMOs. The platform allows users to combine gene fragments with different expression vectors in a simple 15-minute reaction, thus enabling rapid exploration of several gene contexts, hosts and expression strategies in parallel. The open-source LyGo platform is accompanied by easy-to-follow online protocols for both cloning and expression. As a demonstration, we utilize the LyGo platform to explore different strategies for expressing several different LPMOs in Escherichia coli, Bacillus subtilis, and Komagataella phaffii.

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Overproduction and characterization of a lytic polysaccharide monooxygenase in Bacillus subtilis using an assay based on ascorbate consumption

Cited by 21 publications

References 34 publications

High-Efficiency Secretion and Directed Evolution of Chitinase BcChiA1 in Bacillus subtilis for the Conversion of Chitinaceous Wastes Into Chitooligosaccharides

High-Efficiency Secretion and Directed Evolution of Chitinase BcChiA1 in Bacillus subtilis for the Conversion of Chitinaceous Wastes Into Chitooligosaccharides

Oxygen Activation by Cu LPMOs in Recalcitrant Carbohydrate Polysaccharide Conversion to Monomer Sugars

LyGo: A platform for rapid screening of lytic polysaccharide monooxygenase production

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