Enzyme and Microbial Technology最新文献

The extensive utilization of conventional plastics has resulted in a concerning surge in waste. A potential solution lies in biodegradable polymers mostly derived from renewable sources. Cupriavidus necator DSM 545 is a microorganism capable, under stress conditions, of intracellularly accumulating Poly(3-hydroxybutyrate) (PHB), a bio-polyester. This study aimed to identify optimal conditions to maximize the intracellular accumulation of PHB and its global production using natural media obtained by processing lignocellulosic residues of cardoon, a low-cost feedstock. An intracellular PHB accumulation was observed in all of the tested media, indicating a metabolic stress induced by the lack of macronutrients. Increasing C/N ratios led to a significant decrease in cellular biomass and PHB production. Furthermore C. necator DSM 545 was incapable of consuming more than 25 g/L of supplied monosaccharides. Surprisingly, in the samples supplied with 60 % of the pentose-rich liquid fraction, complete consumption of xylose was observed. This result was also confirmed by subsequent tests using Medium 1 growth media containing xylose as the sole carbon source. Using a diluted medium with a C/N ratio of 5, a PHB production of 5.84 g/L and intracellular PHB accumulation of 77 % w/w were respectively achieved. Finally, comparative shelf-life tests conducted against conventional pre-packaging materials in PP suggested that PHB films performed similarly in preserve ready-to-eat products.

D-Mannose 2-epimerase (MEase) catalyzes the bioconversion between D-glucose and D-mannose. It is an important potential biocatalyst for large-scale production of D-mannose, a functional monosaccharide used in pharmaceutical and food industries. In this study, a new microbial MEase was characterized from Runella zeae DSM 19591. The enzyme was purified by one-step nickel-affinity chromatography and determined to be a dimeric protein with two identical subunits of approximately 86.1 kDa by gel filtration. The enzyme showed the highest activity at pH 8.0 and 40 °C, with a specific activity of 2.99 U/mg on D-glucose and 3.71 U/mg on D-mannose. The melting temperature (T_m) was 49.4 °C and the half-life was 115.14 and 3.23 h at 35 and 40 °C, respectively. The purified enzyme (1 U/mL) produced 115.7 g/L of D-mannose from 500 g/L of D-glucose for 48 h, with a conversion ratio of 23.14 %. It was successfully expressed in Bacillus subtilis WB600 via pP43NMK as the vector. The highest fermentation activity was 10.58 U/mL after fed-batch cultivation for 28 h, and the whole cells of recombinant B. subtilis produced 114.0 g/L of D-mannose from 500 g/L of D-glucose, with a conversion ratio of 22.8 %.

An immunoassay method based on penicillin-binding protein (PBP) was developed for the quantitative determination of 10 kinds of beta-lactam antibiotics (BLAs). First, two kinds of PBPs, which are named PBP1a and PBP2x, were expressed and purified, and they were characterized by SDS-PAGE and western blotting analysis. Then, the binding activity of PBP1a and PBP2x to template BLAs, cefquinome (CEFQ) and ampicillin (AMP), was determined. The effect of the buffer solution system, e.g., pH, ion concentration, and organic solvent, on the immune interaction efficiency between PBPs and BLAs was also evaluated. In the end, the PBP-based immunoassay method was developed and validated for the detection of 10 kinds of BLAs. Under optimal conditions, PBPs exhibited high binding affinity to BLAs. In addition, this method showed a high sensitivity for the detection of 10 kinds of BLAs with the limits of detection from 0.21 to 9.12 ng/mL, which are much lower than their corresponding maximum residual limit of European Union (4–100 ng/mL). Moreover, the developed PBP-immunoassay was employed for BLA detection from milk samples, and satisfactory recoveries (68.9–101.3 %) were obtained.

Fructooligosaccharides (FOS) are leading prebiotics that help keep the gut healthy and aid wellness by stimulating the growth and activity of beneficial intestinal bacteria. The best-studied FOS are inulin-type FOS, mainly oligosaccharides with β-Fruf-(2→1)-Fruf linkages, including 1-kestose [β-Fruf-(2→1)-β-Fruf-(2↔1)-α-Glcp] and nystose [β-Fruf-(2→1)-β-Fruf-(2→1)-β-Fruf-(2↔1)-α-Glcp]. However, the properties of other types of FOS—levan-type FOS with β-Fruf-(2→6)-Fruf linkages and neo-type FOS with β-Fruf-(2→6)-Glcp linkages—remain ambiguous because efficient methods have not been established for their synthesis. Here, using site-saturation mutation of residue His79 of β-fructofuranosidase from Zymomonas mobilis NBRC13756, we successfully obtained a mutant β-fructofuranosidase that specifically produces neo-type FOS. The H79G enzyme variant loses the native β-Fruf-(2→1)-Fru-transfer ability (which produces 1-kestose), and instead has β-Fruf-(2→6)-Glc-transfer ability and produces neokestose. Its hydrolytic activity specific to the β-Fruf-(2↔1)-α-Glcp bond of neokestose then yields blastose [β-Fruf-(2→6)-Glcp]. The enzyme produces 0.4 M blastose from 1.0 M sucrose (80 % of the theoretical yield). The production system for blastose established here will contribute to the elucidation of the physiological functions of this disaccharide.

Transaminases (EC 2.6.1.X, TAs) are important biocatalysts in the synthesis of chiral amines, and have significant value in the field of medicine. However, TAs suffer from low enzyme activity and poor catalytic efficiency in the synthesis of chiral amines or non-natural amino acids, which hinders their industrial applications. In this study, a novel TA derived from Paracoccus pantotrophus (ppTA) that was investigated in our previous study was employed with a semi-rational design strategy to improve its enzyme activity to 2-ketobutyrate. By using homology modeling and molecular docking, four surrounding sites in the substrate-binding S pocket were selected as potential mutational sites. Through alanine scanning and saturation mutagenesis, the optimal mutant V153A with significantly improved enzyme activity was finally obtained, which was 578 % higher than that of the wild-type ppTA (WT). Furthermore, the mutant enzyme ppTA-V153A also exhibited slightly improved temperature and pH stability compared to WT. Subsequently, the mutant was used to convert 2-ketobutyrate for the preparation of L-2-aminobutyric acid (L-ABA). The mutant can tolerate 300 mM 2-ketobutyrate with a conversion rate of 74 %, which lays a solid foundation for the preparation of chiral amines.