DOI:10.2298/ABS1003669X DETERMINATION OF TEA POLYSACCHARIDES IN CAMELLIA SINENSIS BY A MODIFIED PHENOL-SULFURIC ACID METHOD

Abstract- A direct procedure for the determination of total polysaccharides (TPS) in Camellia sinensis was set up based on the modified phenol-sulfuric acid method. The monosaccharide composition of TPS was analyzed by GC. Based on the results of GC, model monosaccharide mixtures were made which provided an adequate standard for this procedure. Through single-factor and orthogonal (L93 4) experiments, the experimental conditions such as the volume of phenol, the volume of concentrated sulfuric acid, the reaction time, and the incubation temperature, were optimized. The highest sensitivity of absorbance was obtained when the volume of concentrated sulfuric acid, the volume of phenol (6%), and the incubation temperature were 2.5 ml, 0.2 ml, and 50°C, respectively. Under optimum conditions, the prepared samples were determined satisfactorily, with the recovery from 100.2 % to 103.7%, and a relative standard deviation (RSD) of 2.1%. Overall, the modified method is easily operated, rapid, sensitive and accurate. A similar procedure can be applied to the determination of other plant polysaccharides as well

Programmable Polyproteams of Tyrosine Ammonia Lyases as Cross-Linked Enzymes for Synthesizing p-Coumaric Acid

Ideal immobilization with enhanced biocatalyst activity and thermostability enables natural enzymes to serve as a powerful tool to yield synthetically useful chemicals in industry. Such an enzymatic method strategy becomes easier and more convenient with the use of genetic and protein engineering. Here, we developed a covalent programmable polyproteam of tyrosine ammonia lyases (TAL-CLEs) by fusing SpyTag and SpyCatcher peptides into the N-terminal and C-terminal of the TAL, respectively. The resulting circular enzymes were clear after the spontaneous isopeptide bonds formed between the SpyTag and SpyCatcher. Furthermore, the catalytic performance of the TAL-CLEs was measured via a synthesis sample of p-Coumaric acid. Our TAL-CLEs showed excellent catalytic efficiency, with 98.31 ± 1.14% yield of the target product—which is 4.15 ± 0.08 times higher than that of traditional glutaraldehyde-mediated enzyme aggregates. They also showed over four times as much enzyme-activity as wild-type TAL does and demonstrated good reusability, and so may become a good candidate for industrial enzymes

Putting precision and elegance in enzyme immobilisation with bio-orthogonal chemistry

The covalent immobilisation of enzymes generally involves the use of highly reactive crosslinkers, such as glutaraldehyde, to couple enzyme molecules to each other or to carriers through, for example, the free amino groups of lysine residues, on the enzyme surface. Unfortunately, such methods suffer from a lack of precision. Random formation of covalent linkages with reactive functional groups in the enzyme leads to disruption of the three dimensional structure and accompanying activity losses. This review focuses on recent advances in the use of bio-orthogonal chemistry in conjunction with rec-DNA to affect highly precise immobilisation of enzymes. In this way, cost-effective combination of production, purification and immobilisation of an enzyme is achieved, in a single unit operation with a high degree of precision. Various bio-orthogonal techniques for putting this precision and elegance into enzyme immobilisation are elaborated. These include, for example, fusing (grafting) peptide or protein tags to the target enzyme that enable its immobilisation in cell lysate or incorporating non-standard amino acids that enable the application of bio-orthogonal chemistry.BT/Biocatalysi