Espersencrane4633

Earthworms are known as a source of a traditional medicine, and bioactive components have been reported. We have reported that a fraction (U3EE) with molecular mass under 3 kDa from the water extract of Eisenia fetida inhibits porcine pancreatic α-amylase (PPA) activity with a half-maximal inhibitory concentration (IC50) of 73.7 ± 4.0 mg/mL. Here we purified PPA-inhibitory components from U3EE by sequential procedures of 85 %-ethanol (EtOH) extraction, solid-phase extraction (SPE), and RP-HPLC. The water eluate from SPE of the 85 %-EtOH extract was a major inhibitory fraction, from which three components were separated by 2nd RP-HPLC and identified with MS, TLC, and UV spectroscopy as guanine (Gua), inosine (Ino), and guanosine (Guo). Kinetic analysis showed that Gua and Guo were non-competitive inhibitors and Ino a mixed-type one, suggesting a key role of the purine ring in inhibition. The inhibitor constants (Ki) of Gua and Guo were 0.28 ± 0.07 and 1.64 ± 0.14 mM, respectively, and Ki and Ki' of Ino in the EI and ESI complexes were 5.8 ± 1.1 and 59 ± 12 mM, respectively. U3EE might be useful for food supplements to prevent obesity and diabetes.The current chemical process for industrial indigo production puts a heavy burden on the environment. An attractive option would be to develop an alternative biotechnological process which does not rely on a petrochemical. This study describes a new biotransformation approach in which l-tryptophan is used as starting material. Its conversion to indigo can be achieved through recombinant overexpression of a bifunctional fusion enzyme, flavin-containing monooxygenase (FMO) fused to tryptophanase (TRP). First, TRP converts l-tryptophan into pyruvate, ammonia and indole. The formed indole serves as substrate for FMO, resulting in indigo formation, while pyruvate fuels the cells for regenerating the required NADPH. To optimize this bioconversion, different fusion constructs were tested. Fusing TRP to FMO at either the N-terminus (TRP-FMO) or the C-terminus (FMO-TRP) resulted in similar high expression levels of bifunctional fusion enzymes. Using whole cells and l-tryptophan as a precursor, high production levels of indigo could be obtained, significantly higher when compared with cells containing only overexpressed FMO. The TRP-FMO containing cells gave the highest yield of indigo resulting in full conversion of 2.0 g l-tryptophan into 1.7 g indigo per liter of culture. The process developed in this study provides an alternative biotransformation approach for the production of indigo starting from biobased starting material.'Candidatus Liberibacter asiaticus' ('Ca. L. asiaticus'), the suspected causative agent of citrus greening disease, is one of many phloem-restricted plant pathogens that have not been isolated and grown in an axenic culture. In this study, infected Asian citrus psyllids were used to prepare a host-free source of 'Ca. L. asiaticus'. Host-free mixed microbial cultures of 'Ca. L. asiaticus' were grown in the presence of various antibiotic treatments to alter the composition of the microbial communities. see more Our hypothesis was that the presence of selected antibiotics would enhance or reduce the presence of 'Ca. L. asiaticus' in a host-free culture composed of a mixed bacterial population through changes in the microbial community structure. We determined how 'Ca. L. asiaticus' growth changed with the various treatments. Treatment with vancomycin (50 μg/mL), streptomycin (0.02 μg/mL), or polymyxin B (4 μg/mL) was associated with an increased abundance of 'Ca. L. asiaticus' of 7.35 ± 0.27, 5.56 ± 0.15, or 4.54 ± 0.83 fold, respectively, compared to untreated mixed microbial cultures, while treatment with 100 μg/mL vancomycin; 0.5, 1, or 2 μg/mL streptomycin; or 0.5 μg/mL of polymyxin B was associated with reduced growth. In addition, the growth of 'Ca. L. asiaticus' was associated with the microbial community composition of the mixed microbial cultures. A positive relationship between the presence of the Pseudomonadaceae family and 'Ca. L. asiaticus' growth was observed, while the presence of 'Ca. L. asiaticus' was below the detection limit in cultures that displayed high abundances of Bacillus cereus. Our findings offer strategies for developing effective axenic culture conditions and suggest that enrichment of the Bacillaceae family could serve as a paratransgenic approach to controlling citrus greening disease.Prosaikogenin D, a rare secondary saponin in Radix Bupleuri, has much higher in vivo bioactivities than its original glycoside saikosaponin B2. Its preparation methods, such as conventional acid hydrolysis and column chromatograph, are unfriendly to environment with serious pollution and undesired products. The aim of this study was to establish an efficient and clean approach for convenient preparation of this rare steroid saponin based on the enzymatic hydrolysis. Cellulase was selected from four commercial enzymes due to its higher hydrolysis performance. Then the hydrolysis conditions were optimized by response surface methodology after preliminary investigation on affecting factors by single-factor experiments. The reaction system was constructed by 100 μg/mL of saikosaponin B2 and 8.00 mg/mL of cellulase, which was incubated in HAc-NaAc buffer (pH 4.7) at 60 °C for 33 h. Consequently, a high conversion ratio of the substrate has been achieved at 95.04 %. The newly developed strategy is an efficient and clean approach for the preparation of prosaikogenin D and it is a promising technology in industrial application.The microbial transglutaminase (mTGase) from Streptomyces mobaraense is widely used in the food industry. However, recombinant production of mTGase is challenging because the mTGase is synthesized as an inactive zymogen, and needs to be activated by proteolytic processing. In this study, self-cleaving intein Ssp DnaB was applied to activate the mTGase in Corynebacterium glutamicum. Premature cleavage of intein Ssp DnaB also occurred, but instead of suppressing premature cleavage, this phenomenon was used to produce active mTGase in C. glutamicum. Both SDS-PAGE analysis and mTGase activity assays indicated that the premature cleavage of intein Ssp DnaB activated the mTGase intracellularly in C. glutamicum. The subsequent N-terminal amino acid sequencing and site-directed mutagenesis studies further showed that the premature cleavage activated the mTGase intracellularly, in a highly specific manner. Moreover, the growth performance of C. glutamicum was not noticeably affected by the intracellular expression of active mTGase.