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laccase

Degrading antibiotics

Sequence and Features

Profile

Origin: Pleurotus ostreatus（P. ostreatus HAUCC 162）
Properties: Degradation of sulfadiazine

Usage and Biology

Laccase is an oxidoreductase that uses oxygen as an electron acceptor. It can oxidize a variety of aromatic compounds and produce water as a by-product. In the 19th century, humans first discovered laccase in Japanese sumac. Because of its outstanding application value in biotechnology applications, scientists have gradually deepened their research on laccase. They discovered that laccase is also present in animals and microorganisms. Laccases derived from different hosts have different functions in the body. Among them, fungal laccases derived from white rot fungi, Trametes, Pleurotus ostreatus and other fungal laccases occupy the mainstream of the current laccase application process. This type of laccase has a relatively wide range of substrate specificity during the application process, and does not require hydrogen peroxide. With the participation of, a variety of phenolic compounds and their derivatives can be directly oxidized with oxygen as the electron acceptor.

In recent years, the main methods for removing antibiotic residues from different sources are traditional methods such as high-temperature composting, fermentation; oxidation, adsorption, electrochemical treatment, membrane method, and microbial degradation. However, these methods have the disadvantages of low removal efficiency, high cost of use, and the possibility of secondary pollution to the environment. Moreover, these methods cannot fundamentally solve the problem of antibiotic residues in poultry. Therefore, it is necessary to find a direct, effective and low-cost method to remove antibiotic residues in poultry. At present, many studies have shown that fungal laccase plays an important role in the process of treating antibiotic residues in wastewater. Three laccase-producing genes LACC6, LACC9, and LACC10 obtained from Pleurotus ostreatus were heterologously expressed in Pichia pastoris and found that the efficiency of degrading sulfa antibiotics reached more than 97%.

Heterologous expression and application advantages of fungal laccase

Studies have shown that fungal laccase is a multi-copper oxidase that can deliver 4 copper ions at the same time, so it can catalyze a variety of phenolic compounds and aromatic compounds. Based on its wide range of substrates, laccase has become a useful biocatalyst in the application of biotechnology, especially in the field of bioremediation, including the degradation of lignin, the decolorization of synthetic dye wastewater, and the degradation and detoxification of environmental pollutants. However, the laccase secreted by wild strains is limited in yield and difficult to purify, on the other hand, its activity is susceptible to environmental factors. In recent years, microbial fermentation engineering has gradually emerged, and the fermentation production of most laccases mainly stays at the level of shake flasks and fermentation tanks. Ryan et al. used an air-lift fermentor to ferment Trametes villus, and the laccase activity in the fermentation broth could reach 11.8 U/ml. At this stage, although the production and application of some fungal laccases can be realized by using the fermentation process, the culture conditions of microorganisms such as temperature, pH, humidity, and the length of the fermentation cycle will affect the production of laccase during the fermentation process. Therefore, most laccases are fermented The process only stays at the laboratory scale. Based on the above shortcomings, it is very important to develop an efficient, low-cost, and environmentally friendly method for producing laccase.

At this stage, researchers have used molecular biology methods to heterologously express laccase genes on the basis of overcoming many shortcomings of traditional laccase purification processes and fermentation processes, in order to obtain laccase with higher yield and enzymatic activity. At present, according to production requirements, laccases from different sources have been successfully expressed in various host cells such as bacteria, fungi, and insect rod cells. At present, in industrial applications, fungal cells such as Pichia pastoris, Saccharomyces cerevisiae and Kluyveromyces lactis dominate the heterologous expression process of laccase. These fungal cells have the following advantages in the application process: first, high-density fermentation can increase the production of laccase, second, the engineered strain has higher enzyme activity, and third, most fungal host cells have been confirmed to be directly edible by humans.

In recent years, with the deepening of research, scientists have found that laccase also has many shortcomings in fungal heterologous expression. When the engineered strain is fermented, it is found that the fermentation cycle is longer and usually takes about one week, and the strain culture consumes a lot. In their research, Maestre et al. found that host yeast can inactivate laccase by interfering with the glycosylation metabolism of laccase. Laccase-producing bacteria from the same source often have multiple laccase genes at the same time, and the existence of these genes ultimately leads to a more complex and diverse isozyme system of laccase. Therefore, when the researchers performed heterologous expression of these isoenzymes, they found that not all fungal expression systems can maximize the productivity of laccase, and the laccase activity produced by heterologous expression in yeast cells is far less than that of wild-type laccase. Strains are also susceptible to factors such as pH. Compared with wild strains, the half-life of laccase in some expression systems has also changed. Comprehensive analysis of the heterologous expression of laccase in fungi, the bacterial expression system has been gradually applied to the direction of heterologous expression of laccase due to its easy operation, short culture period, and strong reproductive ability.

The ability of fungal laccase to degrade sulfadiazine

EcN-Lacc6 refers to the fungal laccase gene expressed in the cell

This experiment mainly uses HPLC to quantitatively analyze the residues of sulfadiazine. The process involves two parts: sample pretreatment and HPLC on-machine detection. The specific experimental method is as follows;

(1) Sample pretreatment. First, EcN-IL and the control wild-type EcN, the unloaded EcN and the intracellular expression strain EcN-Lacc6 were inoculated into LB medium at 37°C overnight at an inoculum of 1%. After centrifugation to remove the supernatant, the bacteria were collected (6000 rpm, 10min). The bacteria were washed twice with the same amount of PBS (pH=7.0) buffer solution and prepared into a bacterial suspension with a cell concentration of 1.0 × 109 CFU/mL. Take 10 mL of the cell suspension and add SDZ sodium salt with a concentration of 30, 50, and 100 mg/L into it, with three replicates in each group. Incubate at room temperature for 3 hours and then centrifuge (10000 r/min, 10 min), collect the supernatant into a 10mL PE tube, and finally filter it into a special brown sample bottle for HPLC using a 0.22μm filter membrane.

(2) HPLC on-machine detection conditions selection. HPLC loading conditions selection: select VWD detector, detection wavelength is set to 270 nm, methanol and ultrapure water are organic phase, mobile phase: methanol: water=25:75, detection column Waters XTERRA MS C18 column (250× 4.6 mm), the column temperature is set to 30°C, and the mobile phase flow rate is 1.0 mL/min. HPLC detection data is calculated using external standard method.

a, b, c show the degradation rate analysis of EcN-IL to sulfadiazine at different concentrations (30, 50 and 100 mg/L). Compared with the control EcN group, the degradation rate of EcN-IL to SDZ can reach up to 30 ±2.3%.

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.