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A spectrophotometric method for high-throughput screening of ?-l-rhamnosidase activity on rutin coupled with a ?-d-glucosidase assay.

ABSTRACT: ?-l-Rhamnosidase may biotransform rutin into isoquercetin with better bioavailability and bioactivity. To date, the high-throughput screening for the activity of ?-l-rhamnosidases on rutin could not be achieved. Herein, based on the spectral differences between rutin and its aglycone quercetin in alkaline pH 10.0, we have developed a novel and simple spectrophotometric method for high-throughput screening of ?-l-rhamnosidase activity on rutin by combining with a highly active ?-d-glucosidase. Quercetin showed the maximum absorbance at 320 nm in alkaline pH 10.0, and could be considered as the characteristic peak of quercetin because rutin had low absorption at 320 nm. Meanwhile, rutin exhibited the maximum absorption at 400 nm and quercetin showed low absorption at 400 nm in pH 10.0. With this novel spectrophotometric method, the relative abilities of nine different ?-l-rhamnosidases on rutin had been evaluated by monitoring the absorption values of the reaction mixture in alkaline pH 10.0 at 320 nm and 400 nm, and the trend in the activity on rutin was consistent with that obtained by HPLC. Moreover, the library from site-directed saturation mutagenesis at the residue Val338 in the ?-l-rhamnosidase BtRha78A from Bacteroides thetaiotaomicron was constructed for high-throughput screening by this novel spectrophotometric method, and the mutant V338S with improved activity on rutin was obtained. The conversion rate of the mutant V338S on rutin increased by 21.7% and 16.8% than wild type when using whole cells and purified enzymes, respectively. Our findings demonstrated that this novel spectrophotometric method coupled with the ?-d-glucosidase assay might be applied for high-throughput screening of different ?-l-rhamnosidases and a great number of mutants from semi-rational design and directed evolution for ?-l-rhamnosidase.

SUBMITTER: Li BC

PROVIDER: S-EPMC6529497 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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A spectrophotometric method for high-throughput screening of α-l-rhamnosidase activity on rutin coupled with a β-d-glucosidase assay.

Li Bin-Chun BC Peng Bo B Zhang Tian T Li Yan-Qin YQ Ding Guo-Bin GB

3 Biotech 20190521 6

α-l-Rhamnosidase may biotransform rutin into isoquercetin with better bioavailability and bioactivity. To date, the high-throughput screening for the activity of α-l-rhamnosidases on rutin could not be achieved. Herein, based on the spectral differences between rutin and its aglycone quercetin in alkaline pH 10.0, we have developed a novel and simple spectrophotometric method for high-throughput screening of α-l-rhamnosidase activity on rutin by combining with a highly active β-d-glucosidase. Qu ...[more]

PMID: 31139542

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Project description:Background2'-5' oligoadenylate synthetases (OAS) are interferon inducible enzymes that polymerizes ATP to 2'-5'-linked oligomers of adenylate (2-5As). As part of the innate immune response, these enzymes are activated by viral double stranded RNA or mRNAs with significant double stranded structure. The 2-5As in turn activate RNaseL that degrade single stranded RNAs. Three distinct forms of OAS exist in human cells (OAS1, 2 and 3) with each form having multiple spliced variants. The OAS enzymes and their spliced variants have different enzyme activities. OAS enzymes also play a significant role in regulating multiple cellular processes such as proliferation and apoptosis. Moreover, Single nucleotide polymorphisms that alter OAS activity are also associated with viral infection, diabetes and cancer. Thus detection of OAS enzyme activity with a simple spectrophotometric method in cells will be important in clinical research.ResultsHere we propose a modified coupled spectrophotometric assay to detect 2-5 oligoadenylate synthetase (OAS) enzyme activity in prostate cell lines as a model system. The OAS enzyme from prostate cancer cell lysates was purified using Polyinosinic: polycytidylic acid (poly I:C) bound activated sepharose beads. The activated OAS enzyme eluted from Sepharose beads showed expression of p46 isoform of OAS1, generally considered the most abundant OAS isoform in elutes from DU14 cell line but not in other prostate cell line. In this assay the phosphates generated by the OAS enzymatic reaction is coupled with conversion of the substrate 2-amino-6-mercapto-7-methylpurine ribonucleoside (methylthioguanosine, a guanosine analogue; MESG) to a purine base product, 2-amino-6-mercapto-7-methylpurine and ribose1-phosphate via a catalyst purine nucleoside phosphorylase (phosphorylase) using a commercially available pyrophosphate kit. The absorbance of the purine base product is measured at 360 nm. The higher levels of phosphates detected in DU145 cell line indicates more activity of OAS in this prostate cancer cell line.ConclusionThe modified simple method detected OAS enzyme activity with sensitivity and specificity, which could help in detection of OAS enzymes avoiding the laborious and radioactive methods.

Project description:Quantitative high throughput screening (qHTS) assays use cells or tissues to screen thousands of compounds in a short period of time. Data generated from qHTS assays are then evaluated using nonlinear regression models, such as the Hill model, and decisions regarding toxicity are made using the estimates of the parameters of the model. For any given compound, the variability in the observed response may either be constant across dose groups (homoscedasticity) or vary with dose (heteroscedasticity). Since thousands of compounds are simultaneously evaluated in a qHTS assay, it is not practically feasible for an investigator to perform residual analysis to determine the variance structure before performing statistical inferences on each compound. Since it is well-known that the variance structure plays an important role in the analysis of linear and nonlinear regression models it is therefore important to have practically useful and easy to interpret methodology which is robust to the variance structure. Furthermore, given the number of chemicals that are investigated in the qHTS assay, outliers and influential observations are not uncommon. In this article we describe preliminary test estimation (PTE) based methodology which is robust to the variance structure as well as any potential outliers and influential observations. Performance of the proposed methodology is evaluated in terms of false discovery rate (FDR) and power using a simulation study mimicking a real qHTS data. Of the two methods currently in use, our simulations studies suggest that one is extremely conservative with very small power in comparison to the proposed PTE based method whereas the other method is very liberal. In contrast, the proposed PTE based methodology achieves a better control of FDR while maintaining good power. The proposed methodology is illustrated using a data set obtained from the National Toxicology Program (NTP). Additional information, simulation results, data and computer code are available online as supplementary materials.

Dataset Information

A spectrophotometric method for high-throughput screening of ?-l-rhamnosidase activity on rutin coupled with a ?-d-glucosidase assay.

Publications

A spectrophotometric method for high-throughput screening of α-l-rhamnosidase activity on rutin coupled with a β-d-glucosidase assay.

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