Driselase

Protocol for enzymatic hydrolysis of green tea residue by Driselase^[1]:

1. Powdered green tea (4 g) was extracted with 500 ml of hot water, and then with 100 ml of 70% ethanol three times each.
2. The residue was digested with Driselase (1 g), pectinase andproteases in 100 ml of a 0.1 M acetate buffer (pH 5.5) at 37℃ for 24 h.
3. To the digest, absolute ethanol was added to 70%, and the insoluble materials were removed by filtration.

Protocol for detailed protoplast isolation from hornwort tissue^[2]:

1. Incubate the hornwort tissue in 12 mL of 0.5 M mannitol in a Petri dish on an orbital shaker at 100 rpm for 30 min. In the meantime, prepare the Driselase (0.04 g/mL) in 0.5 M mannitol solution, incubate for 30 min on a rocking shaker, centrifuge at 3500 rpm for 1 min, and filter-sterilize.
2. Add a 4mL aliquot of the Driselase solution (end concentration 1% w/v) to the hornwort tissue, and cover the Petri dishes to prevent exposure to light during incubation on the orbital shaker for 10-12 h.
3. The protoplasts must be handled carefully during the following steps, and pipetting should be performed slowly as the protoplasts are sensitive to mechanical damage.
4. Used a 1mL pipette tip with the tip removed (ca. 2 mm cut off from the end) or a wide-neck glass pipette. Pipette the protoplast solution through a 70μm sieve into a 50mL Falcon tube by placing the pipette tip on the mesh rather than shooting the solution through. Wash the Petri dish twice with 3 mL of 0.5 M mannitol by tilting the Petri dish and rinsing it 3-4 times for each wash.
5. Divide the filtrate (ca. 10 mL each, topped up with 0.5 M mannitol if needed) into two glass tubes with screw caps then centrifuge at low speed for 15 min using the lowest ramp up and down speeds (600 rpm, acceleration 3, brake 3) to avoid damaging the protoplasts.
6. Discard the supernatant and carefully resuspend each pellet in 10 mL of 0.5 M mannitol by gently running the solution down the side of the tube. Slowly tilt the tube to a 40 angle, then gently roll the tube in the palm of your hands until all the protoplasts are resuspended and no longer form clumps.
7. Centrifuge the tubes at 600 rpm for 15 min and discard the supernatant, then carefully resuspend each pellet in 5 mL of 0.5 M mannitol and combine the samples from both tubes into one, either using cut pipette tips or by slowly allowing the sample to flow from one tube into the other.
8. Estimate the number of protoplasts using a hematocytometer, and then centrifuge at 600 rpm for 15 min. Discard the supernatant and resuspend the protoplasts.

Protocol for protoplasting from F. verticillioides strain 3693^[3]:

1. Culture and growth condition

(1)?? F. verticillioides strain 3693 was routinely grown on complete medium (CM). For conidiation, mycelial culture was inoculated on carboxy methyl cellulose medium (CMC) and incubated at 26℃ for 2 weeks.

1. Protoplasting media preparation

(1)?? Protoplasting medium consists of cell wall degrading enzymes and osmotic stabilizer.

(2)?? All the lyophilized enzyme powders except driselase were first dissolved in appropriate protoplasting media. The working stock of the driselase enzyme was prepared by dissolving the enzyme powder at 2 concentration in sterile water for 30 min using the magnetic stirrer. Then the enzyme solution is filtered through the 0.2micron filter to remove the un-dissolved residues. The 0.5 ml of filtered 2 driselase solution was taken in a test tube and to that 0.5 ml of 2 osmotic stabilizer was added so that the final 1 ml of the protoplasting medium contains 1 concentration of osmotic stabilizer and 1 concentration of driselase.

(3)?? Stock solution of lysing enzyme was prepared 20x concentration in water and incubated on ice for 10 min. The required amount of the lysing enzyme was added to the required amount of osmotic stabilizer to constitute the protoplasting medium.

(4)?? β-glucuronidase was added at 200 units/ml of the protoplasting medium. Different osmotic chemicals such as KCl, MgSO₄, NaCl, sorbitol and sucrose were used as osmotic stabilizers in the protoplasting medium. They were prepared in different concentrations viz., 0.6 M, 0.8 M, 1.0 M, 1.2 M, 1.4 M, etc. Appropriate amount of the salts was dissolved in water to achieve the required concentration and the pH was maintained at 5.6 to 5.8 and autoclave sterilized. Each treatment was replicated three times, and the experiment was repeated three times.

1. Protoplast preparation

（1）??? One ml of conidia of F. verticillioides at 10⁹ conidia/ml was inoculated in 100 ml of YPD medium and incubated at 28℃, 250 rpm.

（2）??? For harvesting different growth stages of fungi such as germinated conidia (2-10 μm length of protruded outgrowth from the conidia), germ tube (50-100 μm length of germ tube from the conidia) and well grown mycelia (germ tube grown beyond 200 μm length with two or three branched mycelia) for protoplasting, the inoculated conidia in YPD medium was incubated for 8, 12 and 24 h post-inoculation respectively.

（3）??? The germinated conidia, germ tube and mycelium were collected using miracloth and the adhering residual medium was removed by washing off with sterile water three times. The mycelium was collected and used for protoplasting or stored at 4℃ for a week.

（4）??? 25 mg of fungal material per ml of the protoplasting medium was taken in a test tube. The fungal material was mixed well into the protoplasting medium by gentle vortexing and incubated at 30℃ and 100 rpm for different time points. The protoplasts were counted using a hemocytometer.

This protocol only provides a guideline, and should be modified according to your specific needs.

参考文献:

[1]. ?Katsuno Y, Koyama Y, et,al. Apoptosis-inducing activity of a driselase digest fraction of green tea residue. Biosci Biotechnol Biochem. 2001 Jan;65(1):198-201. doi: 10.1271/bbb.65.198. PMID: 11272830.

[2]. Neubauer A, Ruaud S, et,al.Step-by-step protocol for the isolation and transient transformation of hornwort protoplasts. Appl Plant Sci. 2022 Feb 11;10(2):e11456. doi: 10.1002/aps3.11456. PMID: 35495192; PMCID: PMC9039799.

[3].Ramamoorthy V, Govindaraj L, et,al. Combination of driselase and lysing enzyme in one molar potassium chloride is effective for the production of protoplasts from germinated conidia of Fusarium verticillioides. J Microbiol Methods. 2015 Apr;111:127-34. doi: 10.1016/j.mimet.2015.02.010. Epub 2015 Feb 24. PMID: 25724844.