Carrageenan-Based Pickering Emulsion Gels Stabilized by Xanthan Gum/Lysozyme Nanoparticle: Microstructure, Rheological, and Texture Perspective.

In this study, Pickering emulsion gels were prepared by the self-gel method based on kappa carrageenan (kC). The effects of particle stabilizers and polysaccharide concentrations on the microstructure, rheological characteristics, and texture of Pickering emulsion gels stabilized by xanthan gum/lysozyme nanoparticles (XG/Ly NPs) with kC were discussed. The viscoelasticity of Pickering emulsion gels increased significantly with the increase of kC and XG/Ly NPs. The results of temperature sweep showed that the gel formation mainly depended on the kC addition. The XG/Ly NPs addition could accelerate the formation of Pickering emulsion gels and increase its melting temperature (Tmelt), which is helpful to improve the thermal stability of emulsion gels. Cryo-scanning electron microscope (Cryo-SEM) images revealed that Pickering emulsion gel has a porous network structure, and the oil droplets were well wrapped in the pores. The hardness increased significantly with the increase of XG/Ly NPs and kC. In particular, the Pickering emulsion gel hardness was up to 2.9 Newton (N) when the concentration of kC and XG/Ly NPs were 2%. The results showed that self-gelling polysaccharides, such as kC, could construct and regulate the structure and characteristics of Pickering emulsion gel. This study provides theoretical support for potential new applications of emulsion gels as functional colloids and delivery systems in the food industry.

Biosynthesis of phenylpyruvic acid from l-phenylalanine using chromosomally engineered Escherichia coli.

The efficiency of whole-cell biotransformation is often affected by the genetic instability of plasmid-based expression systems, which require selective pressure to maintain the stability of the plasmids. To circumvent this shortcoming, we constructed a chromosome engineering strain for the synthesis of phenylpyruvic acid (PPA) from l-phenylalanine. First, l-amino acid deaminase (pmLAAD) from Proteus myxofaciens was incorporated into Escherichia coli BL21 (DE3) chromosome and the copy numbers of pmLAAD were increased by chemically induced chromosomal evolution (CIChE). Fifty-nine copies of pmLAAD were obtained in E. coli BL8. The PPA titer of E. coli BL8 reached 2.22 g/L at 6 h. Furthermore, the deletion of lacI improved PPA production. In the absence of isopropyl-ß-d-thiogalactopyranoside, the resulting strain, E. coli BL8△recA△lacI, produced 2.65 g/L PPA at 6 h and yielded a 19.37% increase in PPA production compared to E. coli BL8△recA. Finally, the engineered E. coli BL8△recA△lacI strain achieved 19.14 g/L PPA at 24 h in a 5-L bioreactor.

Overexpression and biochemical characterization of a carboxyspermidine dehydrogenase from Agrobacterium fabrum str. C58 and its application to carboxyspermidine production.

BACKGROUND: Carboxyspermidine (C-Spd) is a potentially valuable polyamine carboxylate compound and an excellent building block for spermidine synthesis, which is a critical polyamine with significant implications for human health and longevity. C-Spd can also be used to prepare multivalent cationic lipids and modify nucleoside probes. Because of these positive effects on human health, C-Spd is of considerable interest as a food additive and pharmaceutical target. RESULTS: A putative gene afcasdh from Agrobacterium fabrum str. C58, encoding carboxyspermidine dehydrogenase with C-Spd biosynthesis activity, was synthesized and transformed into Escherichia coli BL21 (DE3) for overexpression. The recombinant AfCASDH was purified and fully characterized. The optimum temperature and pH for the recombinant enzyme were 30 °C and 7.5, respectively. The coupled catalytic strategy of AfCASDH and various NADPH regeneration systems were developed to enhance the efficient production of C-Spd compound. Finally, the maximum titer of C-Spd production successfully achieved 1.82 mmol L-1 with a yield of 91% by optimizing the catalytic conditions. CONCLUSION: A novel AfCASDH from A. fabrum str. C58 was characterized that could catalyze the formation of C-Spd from putrescine and l-aspartate-ß-semialdehyde (L-Asa). A whole-cell catalytic strategy coupled with NADPH regeneration was established successfully for C-Spd biosynthesis for the first time. The coupled system indicated that AfCASDH might provide a feasible method for the industrial production of C-Spd. © 2021 Society of Chemical Industry.

A novel feruloyl esterase with high rosmarinic acid hydrolysis activity from Bacillus pumilus W3.

A novel feruloyl esterase (BpFae12) with rosmarinic acid (RA) hydrolysis activity was isolated from Bacillus pumilus W3 and expressed in Escherichia coli BL21 (DE3). With RA as a substrate, the optimal pH and temperature of BpFae12 were pH 8.0 and 50 °C, respectively. The specific enzyme activity was 12.8 U·mg-1. BpFae12 showed the highest activity and substrate affinity toward RA (Vmax of 13.13 U·mg-1, Km of 0.41 mM). Moreover, it also presented strong hydrolysis performance against chlorogenic acid (190.17 U·mg-1). RA was effectively Hydrolyzed into more bioactive caffeic acid and 3,4-dihydroxyphenyllactic acid by BpFae12, which have potential applications in the food industry.

Biosynthesis of D-danshensu from L-DOPA using engineered Escherichia coli whole cells.

D-Danshensu (D-DSS), a traditional Chinese medicine, is used to treat cardiovascular and cerebrovascular diseases. However, current isolation protocols for D-DSS both natural and synthetic are not ideal; therefore, in this study, we have developed a whole-cell biotransformation method to produce D-DSS from L-DOPA. This was done by co-expressing L-amino acid deaminase (aadL), D-lactate dehydrogenase (ldhD), and glucose dehydrogenase (gdh). To begin to optimize the production of D-DSS, varying copy number plasmids were used to express each of the required genes. The resulting strain, Escherichia coli ALG7, which strongly overexpressed aadL, ldhD, and weakly overexpressed gdh, yielded a 378% increase in D-DSS production compared to E. coli ALG1. Furthermore, the optimal reaction conditions for the production of D-DSS were found to be a pH of 7.5, temperature at 35 °C, and 50 g/L wet cells for 12 h. Under these optimized conditions, the D-DSS amount achieved 119.1 mM with an excellent ee (> 99.9%) and a productivity of 9.9 mM/h.

Redox self-sufficient biocatalyst system for conversion of 3,4-Dihydroxyphenyl-L-alanine into (R)- or (S)-3,4-Dihydroxyphenyllactic acid.

We developed an efficient multi-enzyme cascade reaction to produce (R)- or (S)-3,4-Dihydroxyphenyllactic acid [(R)- or (S)-Danshensu, (R)- or (S)-DSS] from 3,4-Dihydroxyphenyl-L-alanine (L-DOPA) in Escherichia coli by introducing tyrosine aminotransferase (tyrB), glutamate dehydrogenase (cdgdh) and D-aromatic lactate dehydrogenase (csldhD) or L-aromatic lactate dehydrogenase (tcldhL). First, the genes in the pathway were overexpressed and fine-tuned for (R)- or (S)-DSS production. The resulting strain, E. coli TGL 2.1 and E. coli TGL 2.2, which overexpressed tyrB with the stronger T7 promoter and cdgdh, csldhD or tcldhL with the weaker Trc promoter, E. coli TGL 2.1 yielded 57% increase in (R)-DSS production: 59.8 ± 2.9 mM. Meanwhile, E. coli TGL 2.2 yielded 54% increase in (S)-DSS production: 52.2 ± 2.4 mM. The optimal concentration of L-glutamate was found to be 20 mM for production of (R)- or (S)-DSS. Finally, L-DOPA were transformed into (R)- or (S)-DSS with an excellent enantiopure form (enantiomeric excess > 99.99%) and productivity of 6.61 mM/h and 4.48 mM/h, respectively.

One-pot, three-step cascade synthesis of D-danshensu using engineered Escherichia coli whole cells.

D-danshensu (D-DSS), extracted from the plant Salvia miltiorrhiza (Danshen), is widely used to treat cardiovascular and cerebrovascular diseases. Here we engineered Escherichia coli strains to produce D-DSS from catechol, pyruvate and ammonia by one-pot biotransformation. Tyrosin-phenol lyase (TPL), L-amino acid deaminase (aadL), D-lactate dehydrogenase (ldhD) and glucose dehydrogenase (gdh) genes were overexpressed in Escherichia coli strain. First, the expression of genes was regulated by different copy number plasmids combination, the result of E. coli TALG6, with strong overexpression of TPL, aadL, ldhD and moderate overexpression of gdh, exhibited 253.7% increase D-DSS production compared to E. coli TALG1. Second, the optimum concentration of catechol was found to be 50 mM. Finally, a fed-batch biotransformation strategy was proposed, namely the amount of catechol was added to 50 mM every 2 h. The total production of D-DSS reached 55.35 mM within 14 h, which was 1.7 times that without feeding.