Highly efficient enzymatic synthesis of Z-aspartame in aqueous medium via in situ product removal

Liu, Keke; Zhu, Furong; Zhu, Ling; Chen, Guoguang; He, Bingfang

doi:10.1016/j.bej.2015.01.012

Cited by 8 publications

(6 citation statements)

References 21 publications

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“…Purified enzyme (0.9 μM) was added into the reaction mixture, and the reaction system was shaken at 37 °C and 180 rpm for 1 h. All samples were analyzed by highperformance liquid chromatography (HPLC) as previously reported. 6 The kinetic constants K M were calculated from Lineweaver−Burk plots.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…Additionally, extracellular expression may facilitate downstream processing and achieve higher production . Previously, we have demonstrated the expression of metalloprotease PT121 and its mutant Y114S with improved activity of Z-APM synthesis . However, expressed PT121 proteases were mainly obtained as intracellular form and the expression level remained low.…”

Section: Introductionmentioning

confidence: 98%

“…5 A previous study developed mutant Y114S, which can efficiently catalyze the synthesis of Z-APM, by site-directed mutagenesis of protease PT121 from Pseudomonas aeruginosa. 6 However, due to the limited expression level, these metalloproteases are one of the limiting factors in Z-APM enzymatic preparation.…”

Section: ■ Introductionmentioning

confidence: 99%

“…16 Previously, we have demonstrated the expression of metalloprotease PT121 and its mutant Y114S with improved activity of Z-APM synthesis. 6 However, expressed PT121 proteases were mainly obtained as intracellular form and the expression level remained low. Therefore, exploring an effective approach for improving extracellular expression is necessary.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Members of metalloprotease family M4 (also known as the thermolysin family) such as thermolysin and pseudolysin, as efficient biocatalysts, were utilized to catalyze the synthesis of Z-APM from N -carbobenzoxy- l -aspartic acid (Cbz-Asp) and l -phenylalanine methyl ester (Phe-OMe) . A previous study developed mutant Y114S, which can efficiently catalyze the synthesis of Z-APM, by site-directed mutagenesis of protease PT121 from Pseudomonas aeruginosa . However, due to the limited expression level, these metalloproteases are one of the limiting factors in Z-APM enzymatic preparation.…”

Section: Introductionmentioning

confidence: 99%

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Efficient Extracellular Expression of Metalloprotease for Z-Aspartame Synthesis

Zhu

Liu

et al. 2016

J. Agric. Food Chem.

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Metalloprotease PT121 and its mutant Y114S (Tyr114 was substituted to Ser) are effective catalysts for the synthesis of Z-aspartame (Z-APM). This study presents the selection of a suitable signal peptide for improving expression and extracellular secretion of proteases PT121 and Y114S by Escherichia coli. Co-inducers containing IPTG and arabinose were used to promote protease production and cell growth. Under optimal conditions, the expression levels of PT121 and Y114S reached >500 mg/L, and the extracellular activity of PT121/Y114S accounted for 87/82% of the total activity of proteases. Surprisingly, purer protein was obtained in the supernatant, because arabinose reduced cell membrane permeability, avoiding cell lysis. Comparison of Z-APM synthesis and caseinolysis between proteases PT121 and Y114S showed that mutant Y114S presented remarkably higher activity of Z-APM synthesis and considerably lower activity of caseinolysis. The significant difference in substrate specificity renders these enzymes promising biocatalysts.

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Section: ■ Materials and Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%