一种β-内酰胺类抗生素的酶热检测方法

The penicillinase thermistor biosensor（Penicillinase sensor） was developed for the rapid monitoring of blood penem antibiotics concentration and rapid identification of extraneous penicillinase in milk on site.However, the wide application of the penicillinase thermistor biosensor was limited due to its intrinsic poor activity to hydrolyze cephem and carbapenem antibiotics.The recently identified carbapenemase New Delhi metallo-beta-lactamase 1（NDM-1） is able to hydrolyze all commercially available β-lactam antibiotics in high efficacy.We coupled the NDM-1 and the enzymatic thermistor biosensor to develop a NDM-1 thermistor biosensor（NDM-1 sensor） by the installment of the enzymatic thermistor with an enzyme column loaded with NDM-1 conjugated CPG beads.The NDM-1 sensor shows high response activity to Piperacillin（PIP）,Ceftriaxone（CTRX）, and Meropenem（MEM）, and the response activity of the NDM-1 sensor to these three β-lactam antibiotics are all Zn2+ dependent.Moreover, the response activity of the NDM-1 sensor to Penicillin G（P）, PIP, Cefazolin（CZO）, CTRX, Cefepime（FEP） and MEM all linearly correlated with antibiotic concentration from 31.25 to 1 000 mg/L.Within pH from 6.0 to 8.0, the optimal response activity of the NDM-1 sensor to P,PIP, CZO, CTRX and FEP are found at pH 6.5, while the optimal response activity of the NDM-1 sensor to MEM is found at pH8.0.These data indicate that the featured activity of NDM-1 was well maintained after conjugation on CPG beads, and NDM-1 sensor is capable to quantitate three classes of β-lactam antibiotics including penem, cephem and carbapenem within a wide concentration range

氨曲南竞争性抑制NDM-1对β-内酰胺类抗生素的水解

We investigated the effect of Aztreonam on hydrolysis of β-lactam antibiotics by MBL using New Delhi Metallo-β-lactamase-1(NDM-1) as a model. The results showed that Aztreonam significantly inhibited hydrolysis of Nitrocefin and Meropenem by soluble NDM-1, but also inhibited hydrolysis of Penicillin G by CPG beads immobilized NDM-1(NDM-1 beads). Moreover, in spite of extensive washing for multiple times, the activity to hydrolyze Penicillin G by the Aztreoman pre-treated NDM-1 beads was just partially recovered. These data suggest that Aztreonam can covalently and stably bind on NDM-1, thus efficiently inhibiting hydrolysis of other kinds of β-lactam antibiotics by NDM-1 in a competitive way

Role of the NC-Loop in Catalytic Activity and Stability in Lipase from <em>Fervidobacterium changbaicum</em>

<div><p>Flexible NC-loops between the catalytic domain and the cap domain of the α/β hydrolase fold enzymes show remarkable diversity in length, sequence, and configuration. Recent investigations have suggested that the NC-loop might be involved in catalysis and substrate recognition in many enzymes from the α/β hydrolase fold superfamily. To foster a deep understanding of its role in catalysis, stability, and divergent evolution, we here systemically investigated the function of the NC-loop (residues 131–151) in a lipase (FClip1) from thermophilic bacterium <em>Fervidobacterium changbaicum</em> by loop deletion, alanine-scanning mutagenesis and site-directed mutagenesis. We found that the upper part of the NC-loop (residues 131–138) was of great importance to enzyme catalysis. Single substitutions in this region could fine-tune the activity of FClip1 as much as 41-fold, and any deletions from this region rendered the enzyme completely inactive. The lower part of the NC-loop (residues 139–151) was capable of enduring extensive deletions without loss of activity. The shortened mutants in this region were found to show both improved activity and increased stability simultaneously. We therefore speculated that the NC-loop, especially the lower part, would be a perfect target for enzyme engineering to optimize the enzymatic properties, and might present a hot zone for the divergent evolution of α/β hydrolases. Our findings may provide an opportunity for better understanding of the mechanism of divergent evolution in the α/β hydrolase fold superfamily, and may also guide the design of novel biocatalysts for industrial applications.</p> </div

Design of the NC-loop deletion mutagenesis of FClip1.

<p>(A) Detailed structural conformation of the NC-loop. The upper part of the NC-loop (Asp131–Ser138), which is buried inside the protein, is shown in yellow; the lower part (Glu139–Lys151), which is exposed in the solvent, is shown in red. (B) The design of systematic deletion of the NC-loop.</p

Thermodynamic parameters of the wild type FClip1 and the NC-loop deletion mutants.

a<p>Numbers in brackets indicate the values relative to wild type.</p

Structural comparison of the wild type FClip1 and the CΔ13 mutant.

<p>(A) Structural differences between the wild type FClip1 and the CΔ13 mutant near the NC-loop region. The NC-loop of the wild type FClip1 is shown in green and that of the CΔ13 mutant is shown in yellow. The catalytic triads are shown in cyan, thick lines. The residues subjected to site-directed mutagenesis are shown in ball and sticks. (B) The entrance of the substrate binding pocket of the wild type FClip1 (left) and the CΔ13 mutant (right). The surface of NC-loop is shown in red, and the catalytic residue Ser107 is shown in blue.</p

Specific activities and kinetic parameters of the wild type FClip1 and the deleted mutants.

<p>Specific activities were measured in 50 mM phosphate buffer (pH 8.0) at 75°C using <i>p</i>NPC4 and <i>p</i>NPC12 as the substrates, respectively. Kinetic parameters were obtained in 50 mM phosphate buffer (pH 8.0) at 75°C using <i>p</i>NPC4 as the substrate. The fitting curves for kinetic parameters are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046881#pone.0046881.s006" target="_blank">Figure S6</a>.</p>a<p>Numbers in brackets indicate the values relative to wild type.</p

Specific activities and kinetic parameters of the wild type FClip1 and its mutants.

<p>Enzyme assays were performed in 50 mM phosphate buffer (pH 8.0) at 75°C using <i>p</i>NPC4 as the substrate. The fitting curves for kinetic parameters are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046881#pone.0046881.s006" target="_blank">Figure S6</a>.</p>a<p>Numbers in brackets indicate the values relative to wild type.</p

Robust Anticancer Efficacy of a Biologically Synthesized Tumor Acidity-Responsive and Autophagy-Inducing Functional Beclin 1

As a potent autophagy inducer, Beclin 1 is essential for the initiation of autophagic cell death, and triggering extensive autophagy by targeted delivery of Beclin 1 to tumors has enormous potential to inhibit tumor growth. Yet, the therapeutic application of Beclin 1 is hampered by its inability to internalize into cells and nonselective biodistribution in vivo. To tackle this challenge, we employed a novel Beclin 1 delivery manner by constructing a functional protein (Trx-pHLIP-Beclin 1, TpB) composed of a thioredoxin (Trx) tag, a pH low insertion peptide (pHLIP), and an evolutionarily conserved motif of Beclin 1. This protein could effectively transport Beclin 1 to breast and ovarian cancer cell lines under weakly acidic conditions (pH 6.5), markedly inhibit tumor cell growth and proliferation, and induce obvious autophagy. Furthermore, the in vivo antitumor efficacy of the functional Beclin 1 against an SKOV3 xenograft tumor mouse model was tested via intravenous injection. TpB preferentially accumulated in tumors and exhibited a significantly higher tumor growth inhibition than the nontargeted Beclin 1 control, whereas no overt side effects were observed. Taken together, this study sheds light on the potential application of TpB as a highly efficient yet safe antitumor agent for cancer treatment