“…The residues that generally interacted with PLP were also conserved in this group of decarboxylases (Fig. 1 b), indicating its similar PLP binding mechanism [ 35 , 40 , 41 ]. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Lysine (Lys349) binds to PLP and histidine (His235) coordinates the carboxyl group of the substrates. The leaving carboxyl group, in a perpendicular position to the plane formed by the PLP ring and the Schiff base moiety, is potentially stabilized by Arg510 [ 35 ].…”
Section: Resultsmentioning
confidence: 99%
“…After incubation at 37 °C, 1000 rpm for 10 min, the reaction was stopped by adding 20 μL SDS (10%). Samples were treated with o -phthalaldehyde for derivatization as described previously [ 35 ] and analyzed by high-performance liquid chromatography (HPLC) (Agilent, Palo Alto, CA, USA) using a C18 column (Welch Ultimate® AQ-C18, 4.6 × 250 mm, 5 μm) maintained at 35 °C with the mobile phase containing 50 mM NaAc buffer (45%) at pH 3.5 and Methanol (55%). One unit of ADC activity was defined as the amount of enzyme that catalyzes the reaction to produce 1 μmol of β-alanine per minute under the described conditions.…”
Background
β-Alanine is a precursor of many important pharmaceutical products and food additives, its market demand is continuously increasing nowadays. Whole-cell catalysis relying on the recombinant expression of key β-alanine synthesizing enzymes is an important method to produce β-alanine. Nevertheless, β-alanine synthesizing enzymes found so far have problems including easy inactivation, low expression or poor catalytic activity, and it remains necessary to develop new enzymes.
Results
Herein, we characterized an l-aspartate-α-decarboxylase, MpADC, from an aphid, Myzus persicae. It showed excellent catalytic activity at pH 6.0–7.5 and 37 °C. With the help of chaperone co-expression and N-terminal engineering guided by AlphaFold2 structure prediction, the expression and catalytic ability of MpADC in Escherichia coli were significantly improved. Using 50 g/L of E. coli cells expressing the MpADC-∆39 variant cultured in a 15-L fermenter, 232.36 g/L of β-alanine was synthesized in 13.5 h, with the average β-alanine yield of 17.22 g/L/h, which is best known so far.
Conclusions
Our research should facilitate the production of β-alanine in an environment-friendly manner.
“…The residues that generally interacted with PLP were also conserved in this group of decarboxylases (Fig. 1 b), indicating its similar PLP binding mechanism [ 35 , 40 , 41 ]. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Lysine (Lys349) binds to PLP and histidine (His235) coordinates the carboxyl group of the substrates. The leaving carboxyl group, in a perpendicular position to the plane formed by the PLP ring and the Schiff base moiety, is potentially stabilized by Arg510 [ 35 ].…”
Section: Resultsmentioning
confidence: 99%
“…After incubation at 37 °C, 1000 rpm for 10 min, the reaction was stopped by adding 20 μL SDS (10%). Samples were treated with o -phthalaldehyde for derivatization as described previously [ 35 ] and analyzed by high-performance liquid chromatography (HPLC) (Agilent, Palo Alto, CA, USA) using a C18 column (Welch Ultimate® AQ-C18, 4.6 × 250 mm, 5 μm) maintained at 35 °C with the mobile phase containing 50 mM NaAc buffer (45%) at pH 3.5 and Methanol (55%). One unit of ADC activity was defined as the amount of enzyme that catalyzes the reaction to produce 1 μmol of β-alanine per minute under the described conditions.…”
Background
β-Alanine is a precursor of many important pharmaceutical products and food additives, its market demand is continuously increasing nowadays. Whole-cell catalysis relying on the recombinant expression of key β-alanine synthesizing enzymes is an important method to produce β-alanine. Nevertheless, β-alanine synthesizing enzymes found so far have problems including easy inactivation, low expression or poor catalytic activity, and it remains necessary to develop new enzymes.
Results
Herein, we characterized an l-aspartate-α-decarboxylase, MpADC, from an aphid, Myzus persicae. It showed excellent catalytic activity at pH 6.0–7.5 and 37 °C. With the help of chaperone co-expression and N-terminal engineering guided by AlphaFold2 structure prediction, the expression and catalytic ability of MpADC in Escherichia coli were significantly improved. Using 50 g/L of E. coli cells expressing the MpADC-∆39 variant cultured in a 15-L fermenter, 232.36 g/L of β-alanine was synthesized in 13.5 h, with the average β-alanine yield of 17.22 g/L/h, which is best known so far.
Conclusions
Our research should facilitate the production of β-alanine in an environment-friendly manner.
“…Ma et al demonstrated that EDA can promote the expression of CSAD and speculated that CSAD might be a potential target of EDA therapy for stroke [32]. With the determination of the crystal structure of CSAD [33] and the in-depth study of its function and related mechanism, more regulatory factors will be discovered to regulate CSAD in the future, which might also provide more possibilities for the treatment of NAFLD.…”
Non-alcoholic fatty liver disease (NAFLD) is a chronic metabolic disease manifested in hepatic steatosis, inflammation, fibrosis, etc., which affects over one-quarter of the population around the world. Since no effective therapeutic drugs are available to cope with this widespread epidemic, the functional research of genes with altered expression during NAFLD helps understand the pathogenesis of this disease and the development of new potential therapeutic targets for drugs. In the current work, we discovered via the analysis of the Gene Expression Omnibus (GEO) dataset that cysteine sulfinic acid decarboxylase (CSAD) decreased significantly in NAFLD patients, which was also confirmed in multiple NAFLD mouse models (HFD-fed C57BL/6J, db/db and HFHFrHC-fed C57BL/6J mice). Next, CSAD’s function in the progression of NAFLD was explored using AAV-mediated liver-directed gene overexpression in an HFD-fed mouse model, where the overexpression of CSAD in the liver could alleviate NAFLD-associated pathologies, including body weight, liver/body weight ratio, hepatic triglyceride and total cholesterol, and the degree of steatosis. Mechanically, we found that the overexpression of CSAD could increase the expression of some genes related to fatty acid β-oxidation (Acad1, Ppara, and Acox1). Furthermore, we also detected that CSAD could improve mitochondrial injury in vitro and in vivo. Finally, we proposed that the effect of CSAD on lipid accumulation might be independent of the taurine pathway. In conclusion, we demonstrated that CSAD is involved in the development of NAFLD as a protective factor, which suggested that CSAD has the potential to become a new target for drug discovery in NAFLD.
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