A quantitative, high-throughput urease activity assay for comparison and rapid screening of ureolytic bacteria

Abstract Background The AA9 (auxiliary activities) family of lytic polysaccharide monooxygenases (AA9 LPMOs) are ubiquitous and diverse group of enzymes amongst the fungal kingdom. They catalyze the oxidative cleavage of glycosidic bonds in lignocellulose and exhibit great potential for secondary biorefinery applications. Screening of AA9 LPMOs for desirable properties is crucial for biorefinery industrial applications. However, robust, high-throughput and direct method for AA9 LPMO activity assay, which is prerequisite for screening of LPMOs with excellent properties, is still lacking. Here, we have described a gluco-oligosaccharide oxidase (GOOX) based horseradish peroxidase (HRP) colorimetric method for AA9 LPMO activity assay. Results We cloned and expressed a GOOX gene from Sarocladium strictum in Trichoderma reesei, purified the recombinant SsGOOX, validated its properties, and set up a SsGOOX based HRP colorimetric method for cellobiose concentration assay. Then we expressed two AA9 LPMOs from Thielavia terrestris, TtAA9F and TtAA9G in T. reesei, purified the recombinant proteins, and analyzed their product profiles and regioselectivity towards phosphoric acid swollen cellulose (PASC). TtAA9F was characterized as a C1 type (class 1) LPMO, while TtAA9G was characterized as a C4 type (class 2) LPMO. Finally, the SsGOOX based HRP colorimetric method was used to quantify the total concentration of reducing lytic products from LPMO reaction, and consequently, the activities of both C1 and C4 types of LPMOs were analyzed. These LPMOs could be effectively analyzed with limits of detection (LoDs) lower than 30 nmol/L, and standard curves between A515 and LPMO concentrations with determination coefficients greater than 0.994 were obtained. Conclusions A novel, sensitive and accurate assay method that directly targets the main activity of both C1 and C4 type of AA9 LPMOs was established. This method is easy to use and could be performed on a microtiter plate ready for high-throughput screening of AA9 LPMOs with high properties.

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High-Throughput Lipolysis in 96-Well Plates for Rapid Screening of Lipid-Based Drug Delivery Systems

Journal of Pharmaceutical Sciences ◽

10.1016/j.xphs.2016.12.026 ◽

2017 ◽

Vol 106 (4) ◽

pp. 1183-1186 ◽

Cited By ~ 3

Author(s):

Mette D. Mosgaard ◽

Philip J. Sassene ◽

Huiling Mu ◽

Thomas Rades ◽

Anette Müllertz

Keyword(s):

Drug Delivery ◽

High Throughput ◽

Drug Delivery Systems ◽

Delivery Systems ◽

Rapid Screening

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Emerging Capabilities for the High-Throughput Characterization of Structural Materials

Annual Review of Materials Research ◽

10.1146/annurev-matsci-080619-022100 ◽

2021 ◽

Vol 51 (1) ◽

Author(s):

Daniel B. Miracle ◽

Mu Li ◽

Zhaohan Zhang ◽

Rohan Mishra ◽

Katharine M. Flores

Keyword(s):

High Throughput ◽

Materials Science ◽

Structural Materials ◽

Rapid Screening ◽

Annual Review ◽

Publication Date ◽

Synthesis Methods ◽

Alloy Development ◽

Composition Gradients ◽

Future Vision

Structural materials have lagged behind other classes in the use of combinatorial and high-throughput (CHT) methods for rapid screening and alloy development. The dual complexities of composition and microstructure are responsible for this, along with the need to produce bulk-like, defect-free materials libraries. This review evaluates recent progress in CHT evaluations for structural materials. High-throughput computations can augment or replace experiments and accelerate data analysis. New synthesis methods, including additive manufacturing, can rapidly produce composition gradients or arrays of discrete alloys-on-demand in bulk form, and new experimental methods have been validated for nearly all essential structural materials properties. The remaining gaps are CHT measurement of bulk tensile strength, ductility, and melting temperature and production of microstructural libraries. A search strategy designed for structural materials gains efficiency by performing two layers of evaluations before addressing microstructure, and this review closes with a future vision of the autonomous, closed-loop CHT exploration of structural materials. Expected final online publication date for the Annual Review of Materials Science, Volume 51 is August 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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A Direct Fluorescent Activity Assay for Glycosyltransferases Enables Convenient High‐Throughput Screening: Application to O ‐GlcNAc Transferase

Angewandte Chemie International Edition ◽

10.1002/anie.202000621 ◽

2020 ◽

Vol 59 (24) ◽

pp. 9601-9609 ◽

Cited By ~ 1

Author(s):

Matthew G. Alteen ◽

Christina Gros ◽

Richard W. Meek ◽

David A. Cardoso ◽

Jil A. Busmann ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Activity Assay ◽

Glcnac Transferase

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Development of a dual-fluorescence reporter system for high-throughput screening of L-aspartate-α-decarboxylase

Acta Biochimica et Biophysica Sinica ◽

10.1093/abbs/gmaa134 ◽

2020 ◽

Vol 52 (12) ◽

pp. 1420-1426

Author(s):

Mingyue Fei ◽

Xudan Mao ◽

Yiyang Chen ◽

Yalan Lu ◽

Lin Wang ◽

...

Keyword(s):

High Throughput ◽

Industrial Production ◽

High Throughput Screening ◽

Relative Activity ◽

Expression Level ◽

Rapid Screening ◽

Dual Fluorescence ◽

Reporter System ◽

The Cost ◽

Chemical Synthesis Route

Abstract β-Alanine (3-aminopropionic acid) holds great potential in industrial application. It can be obtained through a chemical synthesis route, which is hazardous to the environment. It is well known that l-aspartate-α-decarboxylase (ADC) can convert l-aspartate to β-alanine in bacteria. However, due to the low activity of ADC, industrial production of β-alanine through the green biological route remains unclear. Thus, improving the activity of ADC is critical to reduce the cost of β-alanine production. In this study, we established a dual-fluorescence high-throughput system for efficient ADC screening. By measuring the amount of β-alanine and the expression level of ADC using two different fluorescence markers, we can rapidly quantify the relative activity of ADC variants. From a mutagenesis library containing 2000 ADC variants, we obtained a mutant with 33% increased activity. Further analysis revealed that mutations of K43R and P103Q in ADC significantly improved the yield of β-alanine produced by the whole-cell biocatalysis. Compared with the previous single-fluorescence method, our system can not only quantify the amount of β-alanine but also measure the expression level of ADC with different fluorescence, making it able to effectively screen out ADC variants with improved relative activity. The dual-fluorescence high-throughput system for rapid screening of ADC provides a good strategy for industrial production of β-alanine via the biological conversion route in the future.

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