Modulation of a Loop Region in the Substrate Binding Pocket Affects the Degree of Polymerization of Bacillus subtilis Chitosanase Products.

The hydrolytic products of chitosanase from Streptomyces avermitilis (SaCsn46A) were found to be aminoglucose and chitobiose, whereas those of chitosanase from Bacillus subtilis (BsCsn46A) were chitobiose and chitotriose. Therefore, the sequence alignment between SaCsn46A and BsCsn46A was conducted, revealing that the structure of BsCsn46A possesses an extra loop region (194N-200T) at the substrate binding pocket. To clarify the impact of this loop on hydrolytic properties, three mutants, SC, TJN, and TJA, were constructed. Eventually, the experimental results indicated that SC changed the ratio of chitobiose to chitotriose hydrolyzed by chitosanase from 1:1 into 2:3, while TJA resulted in a ratio of 15:7. This experiment combined molecular research to unveil a crucial loop within the substrate binding pocket of chitosanase. It also provides an effective strategy for mutagenesis and a foundation for altering hydrolysate composition and further applications in engineering chitosanase.

The Roles of Cruciferae Glucosinolates in Disease and Pest Resistance.

With the expansion of the area under Cruciferae vegetable cultivation, and an increase in the incidence of natural threats such as pests and diseases globally, Cruciferae vegetable losses caused by pathogens, insects, and pests are on the rise. As one of the key metabolites produced by Cruciferae vegetables, glucosinolate (GLS) is not only an indicator of their quality but also controls infestation by numerous fungi, bacteria, aphids, and worms. Today, the safe and pollution-free production of vegetables is advocated globally, and environmentally friendly pest and disease control strategies, such as biological control, to minimize the adverse impacts of pathogen and insect pest stress on Cruciferae vegetables, have attracted the attention of researchers. This review explores the mechanisms via which GLS acts as a defensive substance, participates in responses to biotic stress, and enhances plant tolerance to the various stress factors. According to the current research status, future research directions are also proposed.

Multicomponent spectrofluorimetric method for the assay of carboxymethylflavin and its hydrolytic products: kinetic applications.

The simultaneous assay of carboxymethylflavin (CMF), an intermediate in the photolysis of riboflavin, and its hydrolytic side-chain cleavage products, lumichrome (LC) (acid solution) and LC and lumiflavin (LF) as well as isoalloxazine ring cleavage products, 1,2-dihydro-1-methyl-2-keto-3-quinoxaline carboxylic acid (KA) and 1,2,3,4-tetrahydro-1-methyl-2,3-dioxo-quinoxaline (DQ) (alkaline solution) has been carried out by a multicomponent spectrofluorimetric method. The method is based on the adjustment of pH of the degraded solutions to 2.0 and extraction of LC and LF with chloroform. The chloroform extract is evaporated to dryness under reduced pressure, the residue dissolved in pH 6.5 citro-phosphate buffer and LC and LF determined at their fluorescence maxima at 478 and 530 nm, respectively. The pH of the aqueous phase is re-adjusted to 6.5 and the solution used for the determination of CMF, KA and DQ at the wavelengths of 530, 443 and 420 nm, respectively. The proposed method has been validated according to ICH guidelines. The calibration curves for CMF and its hydrolytic products are linear in the concentration range of 0.5-5.0 × 10-6  M. The mean recovery ranges from 99.0-102.0% with relative standard deviation (RSD) of 0.19-0.99%. The limit of detection (LOD) and the limit of quantification (LOQ) are in the range of 1.17-1.78 × 10-7  M and 3.55-5.40 × 10-7  M, respectively. The uniformity of molar balance of CMF and degradation products during hydrolytic reactions indicates the accuracy of the proposed method for the spectrofluorimetric assay of the compounds. It has been applied to study the kinetics of hydrolytic reactions of CMF.

Multicomponent spectrofluorimetric method for the assay of formylmethylflavin and its hydrolytic products: Kinetic applications.

A multicomponent spectrofluorimetric method has been developed for the simultaneous assay of formylmethylflavin (FMF), an intermediate product in the photolysis of riboflavin (vitamin B2), and its side-chain hydrolytic products, lumichrome (LC) in acidic solution and LC and lumiflavin (LF) in the alkaline solution as well as its ring cleavage products, 1,2-dihydro-1-methyl-2-keto-3-quinoxaline carboxylic acid (KA) and 1,2,3,4-tetrahydro-1-methyl-2,3-dioxo-quinoxaline (DQ) in alkaline solution. The assay method also takes into account an oxidation product of FMF, i.e. carboxymethylflavin (CMF), in both acid and alkaline solutions. The method involves adjustment of the pH of hydrolysed solution to 2.0 to convert FMF to its protonated form, extraction of LC (acid solution) or LC and LF (alkaline solution) with chloroform and their simultaneous assay by fluorescence measurement at 478 and 530 nm, respectively. The aqueous phase is readjusted to pH 6.5, extracted with chloroform to remove undegraded FMF and used for the assay of CMF, KA and DQ at 530, 443 and 420 nm, respectively. The chloroform extract is used for the assay of FMF at 530 nm. The proposed method has been validated and applied to the study of the kinetics of a hydrolysis reaction of FMF at pH 11.0. The calibration curves for FMF and degradation products are linear in the range of 0.1-1.0 × 10-6 M. The limit of detection (LOD) and limit of quantification (LOQ) range from 2.54-5.75 × 10-8 M and 0.78-1.74 × 10-7 M, respectively, for these compounds. The mean recovery ranges from 99.3-102.1% with a RSD of 0.14-0.35%. Judging from the molar balance of FMF and the hydrolytic products, uniformity of analytical data during the reactions and linearity of kinetic plot, the method gives accurate results for the assay of FMF and all of its degradation products. It can be conveniently used for the assay of these compounds and for the kinetics and stability studies of FMF.

Rare constituents with anti-cancer activity from hydrolytic products in saponins of Panax quinquefolium stems and leaves / 中草药

Objective To study the chemical constituents of the acid hydrolytic products from the stems and leaves of Panax quinquefolium. Methods The stems and leaves of P. quinquefolium were hydrolyzed with acid;Isolation and purification were carried out by various chromatographic techniques,such as silica gel and so on. Compounds were identified and elucidated by spectral and chemical methods. Results Eight compounds were obtained from the acid hydrolytic products in the stems and leaves of P. quinquefolium and identified as 20 (S)-panaxadiol (Ⅰ),20 (S)-protopanaxadiol (Ⅱ),20 (R)-protopanaxadiol (Ⅲ),20 (S)-panaxatriol (Ⅳ),24 (R)-ocotillol (Ⅴ),20 (R)-protopanaxatriol (Ⅵ),20 (R)-dammarane-3?,12?,20,25-tetrol (Ⅶ),and 20 (R)-dammarane-3?,6?,12?,20,25-pentol (Ⅷ). Conclusion Compounds Ⅰ-Ⅷ are the derivates of the aglycones of ginsenosides,isolated from the acid hydrolytic products from the stems and leaves of P. quinquefolium for the first time. Among these compounds,Ⅳ,Ⅶ,and Ⅷ are discoverd from the roots,stems,leaves,fruits,and buds of P. quinquefolium for the first time. Compound Ⅶ is discovered and reported that it is the derivate of the aglycone of protopanoxadiol ginsenoside,with the conspicuous anticancer activity from the fruits of P. ginseng for the first time.