Difference between revisions of "Part:BBa K4979002"

Latest revision as of 11:56, 12 October 2023

alginate lyase AL2 +cellulase Bgls

Alginate lyase 2 (AL2) is an enzyme capable of degrading brown algal polysaccharide . According to a study (reference 1), AL2 is a protein secreted by the halophilic bacterium Alteromonas sp. L10. The study reported the cloning and expression of the AL2 gene and conducted in-depth research on its function in alginate degradation.The research results show that AL2 exhibits efficient alginate degradation activity, breaking down alginate into oligosaccharides and low molecular weight sugars. The degradation products of AL2 mainly include mannose, galactose, and glucose. Furthermore, the study demonstrated that AL2 maintains high catalytic activity at different pH values and temperatures.

On the other hand, β-glucosidase (bgls) is an enzyme involved in the hydrolysis of cellulose. According to a report from reference 2, bgls primarily participates in the terminal hydrolysis reaction of cellulose degradation pathways. The study identified a bacterial isolate from soil that efficiently produces bgls, belonging to the Flavobacterium genus.Bgls hydrolyzes the β-1,4-glycosidic bond of cellulose into glucose monomers. The research results showed that this bacterial isolate exhibited high bgls activity at different substrate concentrations. Additionally, bgls demonstrated some tolerance and maintained high enzymatic activity across a wide range of pH values and temperatures.

Usage and Biology

We initiated the transcription process using the T7 promoter and used the BBa_B0034 promoter to initiate the translation of genes encoding endoglucanase, exoglucanase, and β-glucosidase [1]. The cellulase Bgls gene sequence was cloned into the pET23b vector and expressed in Escherichia coli Rosetta.

Figure 1. The design of AL2 and bgls coexpresstion gene circuit.

Figure 2. (A) Gel electrophoresis of the AL2 and bgls. (B) Map of recombinant plasmid pET23b-AL2-bgls.

Characterization

Figure 3. Mechanism of Cellulase.

Figure 4. Experimental Results on AL2 .

The recombinant vector was transformed into E. coli Rosetta cells, the cell pellet was collected by centrifugation and resuspended in Tris-HCl (pH 7.4). The cells were then lysed through ultrasonication , yielding the cell lysate. The total protein concentration in the lysate was determined using the Bradford assay kit. To validate the function of the alginate lyase, 0.9 mL of substrate solution alginate, 50 mM Tris-HCI buffer, 200 mM NaCl, was mixed with 0.1 mL of cell lysate and incubated at 37°C for 30 minutes before sampling. The concentration of reducing sugars was determined using the 3,5-dinitrosalicylic acid (DNS) method. The absorbance at 540 nm was recorded to calculate the activity. The activity in the control group was measured at 5 U/mg. Under our experimental conditions, the specific activity of the alginate lyase crude extract was determined to be 323 U/mg in Figure4A. This means that each milligram of crude extract can release 323 micromoles of reducing sugar per minute. This result demonstrates the high efficiency of the alginate lyase in our engineered bacteria, providing strong support for further applications.

To determine the optimal reaction temperature for alginate lyase, we diluted the cell lysate with Tris-HCl buffer at pH 7.4 and mixed it with a 0.5% (w/v) solution of alginate. After incubating at 25°C, 37°C, and 45°C for 30 minutes, we measured the concentration of reducing sugar using the 3,5-dinitrosalicylic acid (DNS) method. To determine the optimal reaction pH for alginate lyase, we mixed the cell lysate with a 0.5% (w/v) solution of alginate using different buffer solutions. We used a 10 mM citrate buffer at pH 6.1, as well as Tris-HCl buffers at pH 7.4, 8.5, and 10.2. After incubating at 37°C for 30 minutes, we measured the concentration of reducing sugar using the 3,5-dinitrosalicylic acid (DNS) method. The result is shown in Figure 5B and C. The optimal reaction temperature for AL2 is 37°C, and the optimal pH condition is 7.4.

Figure 5. Experimental Results on bgls.

To evaluate the expression and activity of Bacillus subtilis cellulase Bgls in E. coli Rosetta, we first synthesized the bgls gene and cloned it into the pET23b vector. Engineered E. coli Rosetta was cultured in LB broth medium for 3 days at 37°C, and then centrifuged at 13,000 rpm for 5 minutes. Cell pellet was resuspended in PBS buffer (10 mM, pH 7.4) and then lysed through ultrasonication (150 W, 1s sonication with 3s intervals, for a total of 20 minutes). 1 ml of the supernatant (crude enzyme solution) was mixed with one ml of 1% carboxymethylcellulose (CMC, Sigma-Aldrich) solubilized in PBS buffer (10 mM, pH 7.4) and incubated at 37°C for 30 minutes under shaking (120 rpm). One ml of 3,5-dinitrosalicylic acid (DNS) reagent was added and the mixture was boiled for 5 minutes, then the absorbance was measured at 540 nm.

Under our experimental conditions, the specific activity of bgls was determined to be 11.45 U/mg in Figure 5A. Translation: To determine the optimal reaction temperature of cellulase, we mixed crude enzyme solution with a 1% CMC solution and incubated it at 25°C, 37°C, and 55°C for 30 minutes. After that, we used the 3,5-dinitrosalicylic acid (DNS) method to measure the concentration of reducing sugars. We found that the optimal reaction temperature for cellulase was 37°C, as shown in Figure 5B.

Furthermore, to determine the optimal reaction pH of cellulase, we mixed the crude enzyme solution with a 1% CMC solution under 37°C conditions. After incubation for 30 minutes at pH 5.8, 6.5, and 7.4 (in PBS buffer), we measured the concentration of reducing sugars using the DNS method. The optimal pH for Bgls was found to be 6.5, as shown in Figure 5C.

Figure 6. Digestion results of kelp using cellulase Bgls and alginase AL2.

To verify the digestion efficiency of AL2 and bgls, the kelp was thoroughly washed with purified water to remove any accumulated dirt and dried at 65°C for 2 hours. 1g of dried kelp was soaked in water for 2 hours and then cut into 1cm2 pieces. The crude enzyme solution of cellulase and brown algae enzyme was prepared using Tris-HCI (pH 7.4). 1g of dried kelp and 20 mL reaction mixture containing 10 mM Tris-HCI buffer, 10 mL brown algae enzyme solution, and varying volumes of cellulase solution were prepared. After incubation at 37°C and 180 rpm for 24 hours, samples were taken. The kelp was filtered, washed, and dried at 65°C until a constant weight was achieved. Degradation rates were calculated by comparing the weight of the kelp before and after treatment. As shown in Figure 6, we found that the degradation rate of the kelp increased progressively with increasing volume of the crude cellulase solution. When the volume of crude cellulase solution reached 10 mL, the degradation rate of the kelp reached 59.11%.

Alginate oligosaccharides have been shown to boost seed vitality and accelerate germination by promoting water uptake. Beside, the cellulase can hydrolyzes cellulose into monosaccharides, which serve as a source of energy for plants. To explore the potential of engineered bacterial degradation products as fertilizer, the kelp fermentation solution were collected and filtered using a 100 μm coarse mesh screen and 0.22-micron membrane to remove kelp residue and bacteria. The filtrate was collected and diluted to 10% with clean water to prevent adverse effects of high osmotic pressure on the seeds. Seeds in the test group were soaked in the filtered fermentation product for 24 hours. In contrast, the control group (CK) seeds were soaked in clean water. The treated seeds were then sown in seed cultivation trays for growth. On the third day, the germination status of each group of seeds was observed and recorded. Based on these observations, the germination rate for each group was calculated. We conducted a series of experiments to explore the impact of fermentation products on the germination rates of various seeds.

Table 1. Germination rate of different treatment groups.

The results revealed a significant increase in the germination rate of pine willow seeds after treatment with the fermentation products. In CK group, these seeds had a germination rate of 74%, but after the treatment, it rose to 90% (Table 1). Similarly, soybean seeds also showed a comparable trend, with their germination rate increasing from 76% to 84% (Table 1). In addition to these, we also tested the seeds of wheat, barley, and triticale. However, these seeds did not exhibit favorable germination in our experiments. Regardless of whether they were in the control group (CK) or the test group treated with fermentation products, the germination rates for these seeds were exceedingly low. We suspect that this might be due to issues related to our cultivation techniques or conditions. To address this issue, we plan to optimize and adjust our cultivation conditions in the future. We hope that by improving these conditions, we can enhance the germination rates of these seeds and further investigate the potential effects of the fermentation products on them.

Arabidopsis thaliana is a model organism in plant biology. To investigate the influence of fermentation products on Arabidopsis growth, we incorporated these products into the growth medium. Arabidopsis thaliana seeds were first disinfected to eliminate external contaminants by treating them with 70% alcohol, agitating gently for 1-2 minutes. The alcohol was then discarded, and the seeds were rinsed three times with sterile water to ensure the removal of any residual alcohol. Under sterile conditions, the MS solid growth medium was prepared with a 10% concentration of the fermentation products. Once the medium was set, the disinfected seeds were sown evenly across the plate using 1 mL pipette (test group). For comparative purposes, a control group (CK) was also established where seeds were sown on standard MS solid growth medium without the addition of fermentation products. The sown plates were then incubated under controlled conditions at room temperature with a 12-hour light-dark cycle to simulate a natural growth environment. On the 7th day post-sowing, seedlings were carefully picked using sterile tweezers. The root length of these seedlings was measured using a calibrated ruler. All data points were meticulously recorded for further analysis. Graphpad Prism software was employed to visualize the data in the form of bar graphs. To determine the statistical significance between the Test and CK groups, an unpaired student t-test was conducted. A p-value of less than 0.05 was considered indicative of a significant difference between the two groups.

Table 2. Arabidopsis thaliana images and root length statistics.

Potential application directions

Cellulase and brown algae enzymes have tremendous potential in the field of biomass degradation. Cellulase can effectively degrade cellulose, thus extracting renewable energy sources. Brown algae enzymes can break down brown algae, extracting organic fertilizers and bioactive substances. The applications of these two enzymes not only contribute to environmental protection and resource utilization but also bring sustainable solutions to the energy and agriculture industries.

Reference

1. Qin X, et al. (2016). Cloning and expression of a new alginate lyase gene from marine bacterium Alteromonas sp. L10 and functional identification of the enzyme. Appl Biochem Biotechnol, 179(8), 1378-1390. 2. Li S, et al. (2020). Purification and characterization of a β-glucosidase with significant transglycosylation activity from Flavobacterium sp. Curr Microbiol, 77, 1239-1247.

Sequence and Features