Amplification and Expression of MBL Variants

In this study, we amplified and expressed MBL variants (β‐lactamase N1, BAA1717‐BLA‐2 and NDM‐1) and TEM‐1 (class A β‐lactamase) from S aureus, E coli strains and pET‐21a plasmid for use in further experiments. The protein‐coding genes were cloned into pET‐21a using the primers shown in Table 2. The combined plasmids were tested by next‐generation sequencing and then transformed into BL21 (DE3) competent cells. The BL21 (DE3) cells with the recombinant gene were cultured to OD_{600 nm} = 0.6 at 37°C and then were cultured with 0.2 mmol/L IPTG with shaking at 16°C for 8 hours. After centrifugation, the bacteria were resuspended in sterile phosphate buffer (pH = 7.2) and broken by ultrasound in an ice bath. Then, the mixtures were centrifuged at 4°C, and the supernatants were collected for the subsequent protein purification as described by Liu.15 The Gln242Ala and Ser369Ala mutants of β‐lactamase N1 were expressed and purified as described above, and the primers used for mutation are shown in Table 2.
A nitrocefin assay was used for the screening of potential effective inhibitors and further the determination of the inhibitory effect of TFDG on the hydrolysis activities of MBLs. Nitrocefin serves as an indicator whose colour changes from yellow to red with increased hydrolysis. β‐Lactamase N1 (500 ng/mL), BAA1717‐BLA‐2 (500 ng/mL) and NDM‐1 (250 ng/mL) were incubated with various concentrations of TFDG (0, 4, 8, 16 and 32 μg/mL) in phosphate buffer (pH = 7.2) at 37°C for 5 minutes, and then, 50 μg/mL of nitrocefin was added to the mixture. After 10 minutes of incubation, the samples were read at OD_{492 nm} to determine the level of nitrocefin hydrolysis. Additionally, the inhibitory effect of TFDG against β‐lactamase N1 in the presence of excess zinc ion was further evaluated as described above.