Part:BBa_K863020
bhal laccase from Bacillus halodurans with T7 promoter, RBS and His-tag
bhal laccase from Bacillus halodurans with T7 and HIS
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 190
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
We aimed to express this protein in E. coli to study the laccase activity on synthetic substrates at different pH levels. E. coli BL21Star cells were transformed with these parts, each of which contained a T7 promoter and termination sequence flanking the coding sequences. Expression was induced with IPTG and protein expression was performed, after which cells were harvested followed by sonication in protein extraction buffer. A pET160-GW/CAT construct expressing the chloramphenicol acetyltransferase (CAT) protein was used as a positive control for induction. The soluble fraction was analyzed on a 12% SDS-PAGE gel followed by Coomassie staining (Fig. 1). We observed expression of the positive control CAT protein (~32 kDa), but no evidence of tthl or bhal expression (Fig 2.)
Fig 1. SDS PAGE of laccase proteins.
Nevertheless, we assumed there would be some functional laccase present, and proceeded with decolorisation assays.
We tested the laccases on the decolorization of bromophenol blue. We first experimented with a standard solution of laccase purified from Trametes versicolor by incubating bromophenol blue in a buffer containing copper sulphate with various quantities of Trametes laccase.
Next, we assayed the E. coli protein extracts for laccase activity at different pH levels. The positive control, from Trametes, worked very well as a positive control, but only at low pH. Aside from that, there was no significant difference between the absorbance of the laccases and the negative control (Fig 3.).
Fig 3. Absorbance values of laccase proteins (Trametes = Positive control; CAT = Negatice control)
Added by LZU-HS-China-C
We think that this laccase may degrade antibiotics, but we are worried that the degradation antibiotic products of this laccase will have an adverse effect on the human body, so we studied the sulfadiazine degradation products of this laccase.
Speculate the degradation pathway of sulfadiazine
In the experiment, liquid-mass spectrometry (HPLC-MS) was used in the resting state to explore the degradation pathway of SDZ. First, EcN-IL was inoculated into LB medium at 37°C overnight at 1% inoculum. After centrifugation to remove the supernatant, the bacteria were collected (6000 r/min, 10 min). The bacteria were washed twice with deionized water and then prepared into a bacterial suspension with a cell concentration of 1.0×109 CFU/mL, and 5 mL of the cell suspension was taken. Solution, add SDZ sodium salt with a final concentration of 5 mg/L to it, three replicates for each group. Incubate at 35°C for 3 hours and then centrifuge (10000 r/min, 10 min), collect the supernatant into a 2mL PE tube, and finally filter it into a special brown sample bottle for HPLC using a 0.22 μm organic filter membrane. The chromatographic analysis column is a C18 column (2.1 mm×100 mm; particle size: 3 μm) with a column temperature of 30°C; mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is methanol, flow rate is 0.3 mL/min, and sample volume 20 μL, electrospray ion source (ESI+). Elution program
Laccase first breaks the S-N bond of sulfadiazine itself to divide it into two parts, and then undergoes a series of oxidation reactions to finally form an ion with m/z of 174.07 (C6H7NO3S) sulfanilic acid and a mass-to-charge ratio of 127.09.
//function/degradation
//proteindomain/degradation
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