Biocatalytic access to β-alanine by a two-enzyme cascade synthesis / 生物工程学报

Enzymatic synthesis is an important way to produce β-alanine, but the biological method is expensive generally because of the high price of substrate. In this paper, a two-step enzymatic cascade system was developed, combining L-aspartase from Escherichia coli DH5α and L-aspartate α-decarboxylase from Corynebacterium glutamicum. This system catalyzes Fumarate and ammonia to β-alanine. The optimal ratio of AspA and PanD was 1:80 (W/W), and the concentration of AspA was 10 μg/mL, at 37 ℃ and pH 7.0. When the concentration of Fumarate was 100 mmol/L, the reaction reached its equilibrium after 8 h, and the yield of β-alanine was 90 mmol/L (7 g/L). The yield of β-alanine can reach 126 mmol/L (9.8 g/L) when the concentration of Fumarate increased to 200 mmol/L. Extending reaction time, the conversion rate did not change obviously. Using this two-step enzymatic cascade system, β-alanine from cheaper substrate Fumarate can be obtained.

A comparative study on cell disruption methods for release of aspartase from E. coli K-12.

Applicability of different mechanical cell disruption techniques namely sonication, bead milling and French press for the release of aspartase from E. coli K-12 was compared. Various operating parameters of each technique were optimized to obtain maximum aspartase release. The efficiency of aspartase release and cell disruption by all the methods was also compared under optimal conditions. The maximum release of aspartase (98.22%) and maximum cell breakage (84.25%) was observed using French press, while 92% of aspartase release was obtained by both sonication and bead milling. The order of cell disruption constant (k) for aspartase release by these methods was French press > bead milling > sonication. Disruption of cells using French press also demonstrated maximum protein release (14.12 mg/mL). The crude enzyme preparations can be further used for purification and its applications.

Observation for the process of Vibrio vulnificus inducing dendritic cell apoptosis / 中华微生物学和免疫学杂志

Objective To observe the process of Vibrio vulnificus inducing dendritic cell strain apoptosis.Methods We established the mixed culture model of mouse dendritic cell ( DC 2.4 strain) and Vibrio vulnificus( Vv1.1758 strain ),analyzed morphological characteristics of cell apoptosis by DAPI fluorescence staining,detect DNA fragmentation level of apoptosis cells by DNA Ladder assay,analyze DC2.4 apoptosis rate by Annexin V FITC/PI staining,determine activities of caspase-3 and caspase-8 by means of spectrophotometric method and detect changes of mitochondrial transmembrane potential ( △ Ψm ) by JC-1 fluorescence labeling.Results After Vv1.1758 strain and DC2.4 cell were mixed and cultured for 4 h,DAPI fluorescence staining showed typical apoptosis characteristics-chromatin condensation and marginalization; DNA agarose gel electrophoresis showed apoptosis band; apoptosis rates at 2,4 and 6 h were respectively (37.8±9.8) ％,(54.3 ± 12.7 ) ％ and ( 68.2± 14.6 ) ％ ; Mitochondrial transmembrane potentials (△Ψm) at 1 h,2 h and 4 h reduced by 7.1％,16.1％ and 46.7％ respectively; caspase-8 activity increased at 1.5 h and reached the peak at 2 h [ (2.48±0.19) U/μg],while caspase-3 activity started to increase at 3h and reached the peak at 4 h [ ( 1.91 ±0.16) U/μg ].Conclusion Vibrio vulnificus could induce dendritic cells by two pathways: reducing mitochondrial transmembrane potential and activating caspase-8 promoter and finally activate effector caspase-3 to promote apoptosis.