Efficient Extracellular Expression of Metalloprotease for Z-Aspartame Synthesis

Abstract

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.

Considerations on the Rational Design of Covalently Conjugated Cell-Penetrating Peptides (CPPs) for Intracellular Delivery of Proteins: A Guide to CPP Selection Using Glucarpidase as the Model Cargo Molecule

“…The stability of the mRNA structure affects the expression level of proteins. A more positive ΔG value of the mRNA secondary structure in the translation initiation region correlates with a higher probability of the translation of mRNA into protein [49]. The obtained ΔG values from the mfold server indicated that 74% of the N-terminal conjugates had higher ΔG values compared with the unconjugated CPG2.…”

“…Crotamin, a toxin found in snake venom, has two nuclear localization domains, named crot (2–18) and crot (27–39). Various derivatives of Crot (27–39) CPP with the original amino acid sequence of “KMDCRWRWKCCKK” are developed using amino acid substitution, deletion, or addition [49]. “Crot (27–39) derivative 7” from our candidate list belongs to this family.…”

Introducing a delivery system for melanogenesis inhibition in melanoma B16F10 cells mediated by the conjugation of tyrosine ammonia‐lyase and a TAT‐penetrating peptide

“…To investigate whether the addition of TAT sequence to the N‐terminal of TAL has any serious unfavorable effects on the enzyme expression, stability, and function further in silico analyses were employed. The minimum free energy of mRNA translation initiation region and secondary structure have been shown to affect the level of translation 35 . The start codon of TAT‐RsTAL in PET 28a was positioned on the loop, which means that the initiating codon will be more exposed.…”

“…The minimum free energy of mRNA translation initiation region and secondary structure have been shown to affect the level of translation. 35 The start codon of TAT-RsTAL in PET 28a was positioned on the loop, which means that the initiating codon will be more exposed. Therefore, higher levels of translation compared with a start codon positioned on the stem can be expected.…”

A comprehensive in silico characterization of bacterial signal peptides for the excretory production of Anabaena variabilis phenylalanine ammonia lyase in Escherichia coli

“…In this study, the ΔG and the secondary structure of pET28a linked to mRNA of SP-AvPAL or AvPAL were retrieved. The higher ΔG (the more positive ΔG) indicates the higher expression probability (Zhu et al 2016). According to the results retrieved from the Quickfold server, addition of SP did not always lead to an increase in expression.…”