Characterization and identification of Lactobacillus fermentum 4,6-α-glucosyltransferase and its products / 生物工程学报

4,6-α-glucosyltransferases (4,6-α-GTs), which converts amylose into α(1-6) bonds-containing α-glucan, possesses great application potential in enzymatic synthesis of dietary fiber. Primers were designed according to the conserved motifs existing in the amino acid sequence of 4,6-α-GTs, and used to amplify a putative GTFB-Like 4,6-α-GTs gene (named as gtf16) from the genomic DNA of Lactobacillus. The gtf16 gene was cloned into the plasmid pET15b, expressed in Escherichia coli BL21(DE3), followed by purification and characterization. The optimum pH and the optimum temperature of the purified enzyme were 5.0 and 40 °C, respectively. The biotransformation product of this enzyme was systematically characterized by thin-layer chromatography, NMR spectroscopy, and hydrolysis reaction. The Gtf16-catalyzed product shows a similar structure to that of the isomalto/malto-polysaccharide (IMMP), which is the amylose-derived product catalyzed by GtfB from Lactobacillus reuteri 121. Moreover, The Gtf16-catalyzed product contains up to 75% of α(1-6) bonds and has an average molecular weight of 23 793 Da. Furthermore, the content of the anti-digestive components was 88.22% upon hydrolysis with digestive enzymes.

Deletion of a dynamic surface loop improves thermostability of (R)-selective amine transaminase from Aspergillus terreus / 生物工程学报

Chiral amines are important building blocks for the synthesis of pharmaceutical products and fine chemicals. Highly stereoselective synthesis of chiral amines compounds through asymmetric amination has attracted more and more attention. ω-transaminases (ω-TAs) are a promising class of natural biocatalysts which provide an efficient and environment-friendly access to production of chiral amines with stringent enantioselectivity and excellent catalytic efficiency. Compared with (S)-ω-TA, the research focused on (R)-ω-TA was relatively less. However, increasing demand for chiral (R)-amines as pharmaceutical intermediates has rendered industrial applications of (R)-ω-TA more attractive. Improving the thermostability of (R)-ω-TA with potential biotechnological application will facilitate the preparation of chiral amines. In this study, the dynamic surface loop with higher B-factor from Aspergillus terreus (R)-ω-TA was predicted by two computer softwares (PyMOL and YASARA). Then mutant enzymes were obtained by deleting amino acid residues of a dynamic surface loop using site-directed mutagenesis. The results showed that the best two mutants R131del and P132-E133del improved thermostability by 2.6 ℃ and 0.9 ℃ in T₅₀¹⁰ (41.1 ℃ and 39.4 ℃, respectively), and 2.2-fold and 1.5-fold in half-life (t1/2) at 40 ℃ (15.0 min and 10.0 min, respectively), compared to that of wild type. Furtherly, the thermostability mechanism of the mutant enzymes was investigated by molecular dynamics (MD) simulation and intermolecular interaction analysis. R131del in the loop region has lower root mean square fluctuation (RMSF) than the wild type at 400 K for 10 ns, and mutant enzyme P132-E133del increases four hydrogen bonds in the loop region. In this study, we obtain two stability-increased mutants of (R)-ω-TA from A. terreus by deleting its dynamic surface loop and also provide methodological guidance for the use of rational design to enhance the thermal stability of other enzymes.

8-Isoprostane in Exhaled Breath Condensate of Patients with Asthma / 中国呼吸与危重监护杂志

Objective To invesitgate the relationship between 8-iseprostane (8-iso-PG) level in exhaled breath condensates (EBCs) and severity of asthma and explore the role of 8-iso-PG in asthma evaluation and monitoring.Methods Fifty-nine patients with asthma were enrolled.In which 15 eases were acute exacerbation, 13 eases were mild intermittent, 15 eases were mild persistent, and 16 eases were mederate-to-severe persistent.Thirteen healthy volunteers were recruited as control.EBCs were collected using EeoSereen system.The 8-iso-PG levels in EBCs were measured by a specific enzyme immunoassay.The patients with mild intermittent asthma were treated with inhaled corticosteroid (ICS) for one month and their EBCs were recollected for 8-iso-PG measurement.Results Exhaled 8-iso-PG levels were obviously increased in the patients with acute asthma compared with those chronic asthmatics [(47.2±6.8) pg/mL vs (24.5±12.0) pg/mL, P < 0.01].In the chronic persistent asthma, the levels were significantly higher in patients with mild persistent and moderate-to-severe asthma [(17.9±1.2) pg/mL and (39.7±4.0) pg/mL, P <0.01].While 8-iso-PG level did not differ significantly in intermittent asthma [(13.5±1.1) pg/mL] compared with the control subjects (P > 0.05).After one-month ICS treatment the 8-iso-PG level in the patients with mild intermittent asthma did not change significantly although the ACT score improved.Conclusions 8-iso-PG levels in EBC are associated with the severity of asthma,implicating 8-iso-PG may be useful in monitoring airway oxidative stress in asthma.ICS treatment is incapable of decreasing the 8-iso-PG,suggesting the ICS has minor impact on oxidative stress.

Association between clara cell protein 10 and airway insufflation pressures in guinea pig asthma models / 中国实用内科杂志

0.05).The CC10 levels in lung homogenate of the allergic asthma group were significantly decreased than that of control group(P0.05).Conclusion The CC10 levels in lung homogenate is decreased,and has negative correlation with AIPs,which suggests that CC10 may take part in the onset of asthma,and work as an important endogenous anti-inflammatory factor.