Substrate (chemistry)

About: Substrate (chemistry) is a research topic. Over the lifetime, 35902 publications have been published within this topic receiving 740722 citations. The topic is also known as: enzyme substrate.

Purification and characterization of (2S)-flavanone 3-hydroxylase from Petunia hybrida.

Abstract: (2S)-Flavanone 3-hydroxylase from flowers of Petunia hybrida catalyses the conversion of (2S)-naringenin to (2R,3R)-dihydrokaempferol. The enzyme could be partially stabilized under anaerobic conditions in the presence of ascorbate. For purification, 2-oxoglutarate and Fe2+ had to be added to the buffers. The hydroxylase was purified about 200-fold by a six-step procedure with low recovery. The Mr of the enzyme was estimated by gel filtration to be about 74,000. The hydroxylase reaction has a pH optimum at pH 8.5 and requires as cofactors oxygen, 2-oxoglutarate, Fe2+ and ascorbate. With 2-oxo[1-14C]glutarate in the enzyme assay dihydrokaempferol and 14CO2 are formed in a molar ratio of 1:1. Catalase stimulates the reaction. The product was unequivocally identified as (+)-(2R,3R)-dihydrokaempferol. (2S)-Naringenin, but not the (2R)-enantiomer is a substrate of the hydroxylase. (2S)-Eriodictyol is converted to (2R,3R)-dihydroquercetin. In contrast, 5,7,3',4',5'-pentahydroxy-flavanone is not a substrate. Apparent Michaelis constants for (2S)-naringenin and 2-oxoglutarate were determined to be respectively 5.6 mumol X l-1 and 20 mumol X l-1 at pH 8.5. The Km for (2S)-eriodictyol is 12 mumol X l-1 at pH 8.0. Pyridine 2,4-dicarboxylate and 2,5-dicarboxylate are strong competitive inhibitors with respect to 2-oxoglutarate with Ki values of 1.2 mumol X l-1 and 40 mumol X l-1, respectively.

Hydrogel-based microreactors as a functional component of microfluidic systems.

Abstract: A simple two-step method for fabricating poly(ethylene glycol) (PEG) hydrogel-based microreactors and microsensors within microfluidic channels is described. The intrachannel micropatches contain either a dye, which can report the pH of a solution within a fluidic channel, or enzymes that are able to selectively catalyze specific reactions. Analytes present within the microfluidic channel are able to diffuse into the micropatches, encounter the enzymes, and undergo conversion to products, and then the products interact with the coencapsulated dye to signal the presence of the original substrate. The micropatches are prepared by photopolymerizing the PEG precursor within the channel of a microfluidic system consisting of a poly(dimethylsiloxane) mold and a glass plate. Exposure takes place through a slit mask oriented perpendicular to the channel, so the size of the resulting micropatch is defined by the channel dimensions and the width of the slit mask. Following polymerization, the mold is removed, leaving behind the micropatch(es) atop the glass substrate. The final microfluidic device is assembled by irreversibly binding the hydrogel-patterned glass slide to a second PDMS mold that contains a larger channel. Multiple micropatches containing the same or different enzymes can be fabricated within a single channel. The viability of this approach is demonstrated by sensing glucose using micropatches copolymerized with glucose oxidase, horseradish peroxidase, and a pH-sensitive dye.

Solid Culturing of Bacillus amyloliquefaciens for α-Amylase Production

Abstract: Summary Fourteen different agroresidues were screened for alpha amylase production using Bacillus amyloliquefaciens ATCC 23842. Among them, wheat bran (WB) and groundnut oil cake (GOC) in mass ratio of 1:1 was proved as the best substrate source. Supplementation with 0.01 M KH2PO4 and 1 % soluble starch enhanced the enzyme yield considerably. Maximum enzyme recovery from the solid mass was obtained when extracted with 0.1 M acetate buffer, pH=5.0. Maximum enzyme titer expressed as units per mass of dry substrate obtained was 62 470 U/g after 72 hours of fermentation at 37 °C by using the above solid substrate mixture (5 g) with the initial moisture of 85 % and inoculated with Bacillus amyloliquefaciens of 2·10 9 CFU/mL.