1.1 LB Luquid Media

Solid media supplemented with 15–20 g/L agar

1.2 YPD Luquid Media

Solid media supplemented with 15–20 g/L agar

1.3 BMGY Luquid Media

1.4 BMMY Luquid Media

1.5 MD Culture media

2.1 PCR System and Reaction Procedure:

2.2 Primer

(1) lipases:

(2) Promoters：

(3) Signal peptides：

3.1 Calculation of Homologous Recombination System Usage

Amount of Linearized Vector and Insert Fragment

(1) Entry cloning and single-fragment homologous recombination reaction:

Optimal amount of cloning vector = [0.02 × cloning vector base pairs] ng (0.03 pmol)

Optimal amount of insert fragment = [0.04 × insert fragment base pairs] ng (0.06 pmol)

(2) Multi-fragment homologous recombination reaction (2–5 fragments):

Optimal amount of cloning vector = [0.02 × cloning vector base pairs] ng (0.03 pmol)

Optimal amount of each fragment = [0.02 × each fragment base pairs] ng (0.03 pmol)

3.2 Preparation of Homologous Recombination System

Prepare the mixture according to the above system based on the actual amount required, then incubate at 50°C for 30 minutes.

4.1 Agarose gel electrophoresis

(1) Weigh agarose powder and add it to electrophoresis buffer (TAE). Heat in a microwave until the solution becomes completely transparent.

(2) Add nucleic acid stain (YeaGreen).

(3) Pour the gel solution into the casting tray, insert a comb, and allow it to solidify at room temperature for 15 minutes.

(4) After solidification, remove the comb and place the gel into the electrophoresis tank. Add buffer until the gel is submerged by 1–2 mm.

(5) Load the PCR-amplified DNA samples into the wells, with DNA marker added to the first well.

(6) Run the electrophoresis at 180 V for 15 minutes, ensuring correct electrode orientation.

(7) After electrophoresis, place the gel under a blue-light gel cutter to observe the bands.

4.2 DNA Gel Extraction

4.2.1 Materials: Binding column, collection tube, Buffer B2, wash solution, elution buffer

4.2.2 Procedure:

(1) Excise the gel slice containing the target DNA band and weigh it.

(2) Add 3–6 volumes of Buffer B2 (relative to the gel weight) and incubate at 50 °C for 5–10 minutes to dissolve the gel.

(3) For fragments <500 bp, add isopropanol equivalent to 1/3 volume of Buffer B2.

(4) Transfer the dissolved gel solution into the binding column and centrifuge at 8,000 × g for 30 seconds. Discard the flow-through.

(5) Add 500 μl of wash solution and centrifuge at 9,000×g for 30 seconds. Discard the flow-through.Repeat Step 5 once.

(6) Centrifuge the empty column at 9,000×g for 1 minute to dry.

(7) Transfer the column to a clean 1.5 ml centrifuge tube. Apply 15–40 μl of elution buffer to the center of the column membrane. Incubate at room temperature for 1 minute, then centrifuge for 1 minute. Collect and store the eluted DNA solution.

5.1 One-Step Preparation of Competent E. coli Cells

Pre-treatment: Mix 2× BT Buffer T and sterile distilled water in equal volume and pre-chill on ice. Use 100 μl of the diluted 1× BT Buffer T per 1 ml of bacterial culture.

(1) Pick a single colony from a plate or use frozen glycerol stock to inoculate 2–5 ml of appropriate medium. Incubate overnight at 37°C.

(2) Dilute the overnight culture 1:100 into pre-warmed fresh LB medium. Shake at 250 rpm and 37°C until early log phase (OD₆₀₀ = 0.3–0.4) is reached, typically 1.5–3 hours.

(3) Centrifuge the culture at 3,500–5,000 rpm and 4°C for 5–10 minutes to harvest the cells.

(4) Resuspend the pellet thoroughly in the pre-chilled 1× BT Buffer T (100 μl per ml of original culture). Incubate on ice for 10 minutes. The competent cells can be used directly for transformation or aliquoted, flash-frozen in liquid nitrogen, and stored at -70°C.

5.2 Plasmid Transformation

(1) Add 10 μl of the recombinant product to 100 μl of competent cells. Mix gently.

(2) Incubate on ice for 30 minutes to allow DNA adsorption.

(3) Apply heat shock at 42°C for 90 seconds to facilitate DNA uptake.

(4) Immediately return the tube to ice for 2 minutes.

(5) Add 900 μl of LB broth (without antibiotic) and incubate at 37°C with shaking for 45–60 minutes for recovery.

(6) Spread appropriate volumes onto selective plates under sterile conditions

6.1 Materials

Binding column, collection tube, Buffer S, Buffer SP1, Buffer SP2, Buffer SP3, Buffer DW1, Wash Solution, Elution Buffer, RNase A

6.2 Procedure

(1) Preparation:

Ensure RNase A has been added to Buffer SP1.

Confirm that absolute ethanol has been added to the Wash Solution.

Check for precipitation in Buffers SP2 and SP3; redissolve if necessary.

(2) Column equilibration: Add 500 µl of Buffer S to the binding column (placed in a collection tube). Centrifuge at 12,000 × g for 1 minute. Discard the flow-through and reassemble the column with the same collection tube.

(3) Harvest 1.5–5 ml of overnight bacterial culture by centrifugation at 8,000 × g for 2 minutes. Discard the supernatant completely.

(4) Resuspend the pellet thoroughly in 250 µl of Buffer SP1.

(5) Add 250 µl of Buffer SP2 and immediately mix by inverting the tube gently 5–10 times. Incubate at room temperature for 2–4 minutes.

(6) Add 350 µl of Buffer SP3 and mix immediately by inverting the tube gently 5–10 times.

(7) Centrifuge at 12,000 × g for 5–10 minutes. Transfer the supernatant to the equilibrated binding column and centrifuge at 8,000 × g for 30 seconds. Discard the flow-through.

(8) Add 500 µl of Buffer DW1 and centrifuge at 9,000 × g for 30 seconds. Discard the flow-through.

(9) Add 500 µl of Wash Solution and centrifuge at 9,000 × g for 30 seconds. Discard the flow-through.

(10) Repeat Step 9 once.

(11) Centrifuge the empty column at 9,000 × g for 1 minute to dry.

(12) Transfer the column to a clean 1.5 ml microcentrifuge tube. Apply 50–100 µl of Elution Buffer to the center of the membrane. Incubate at room temperature for 1 minute, then centrifuge at 9,000 × g for 1 minute. Collect and store the eluted DNA solution

(1) Set up the reaction according to the following system and incubate at 37 °C for 1 hour.

(2) Verify linearization by agarose gel electrophoresis.

(3) Purify the linearized vector using a gel extraction kit.

8.1 Preparation of Yeast Competent Cells

(1) Strain activation: Streak glycerol stock of P. pastoris stored at -80 °C onto a YPD agar plate. Incubate inverted at 30 °C for 2–4 days to activate the strain.

(2) Inoculation: Pick a single fresh yeast colony using a sterile loop or tip and inoculate into 10 ml of YPD liquid medium.

(3) Culture: Incubate at 28–30 °C with shaking at 250 rpm overnight.

(4) Subculture: Transfer the overnight culture into 50 ml of fresh YPD liquid medium to an initial OD₆₀₀ of 0.1–0.2. Culture at 30 °C, 250–300 rpm for 4–6 hours until OD₆₀₀ reaches 0.6–0.8 (mid-log phase).

(5) When OD₆₀₀ reaches 0.6–0.8, centrifuge the culture at 3,000 × g for 3 minutes at room temperature. Discard supernatant and retain pellet.

(6) Resuspend the pellet in 5 ml of Buffer A. Centrifuge at 3,000 × g for 3 minutes at room temperature. Discard supernatant.

(7) Resuspend the pellet in 400 μl of Buffer A and aliquot 100 μl into sterile 1.5 ml microcentrifuge tubes.

(8) Add 5.5 μl of DMSO to each tube and mix gently. The competent cells can be used directly for transformation or slowly frozen using a programmed freezing container at -80 °C for long-term storage.

8.2 Pichia pastoris Transformation — LiCl Method

(1) Mix 3 μg of linearized plasmid DNA (in 10 μl) with 5 μl of heat-denatured Carrier DNA by pipetting. Add the mixture to 50 μl of competent cells. The total volume of DNA added should not exceed 10 μl.

(2) Incubate in a 37 °C water bath for 5 minutes. Invert tube gently 1–2 times during incubation until frozen cells are fully thawed.

(3) Remove from water bath and add 750 μl of Buffer B. Mix gently by inverting the tube.

d. Incubate in a 30 °C water bath for 1 hour. Gently mix by inverting or flicking the tube every 15 minutes to improve transformation efficiency.

(4) Centrifuge at 2,000 × g for 10 minutes at room temperature. Discard supernatant and retain pellet. Resuspend the pellet in 750 μl of Buffer C.

(5) Centrifuge at 2,000 × g for 10 minutes. Discard supernatant and resuspend the pellet gently in 200 μl of Buffer C.

(6) Spread the entire resuspension evenly onto solid selective medium with appropriate antibiotics. Incubate inverted at 30 °C for 3–4 days until single colonies appear.

Pick single colonies from the transformation plate and prepare the reaction mixture according to the following system. Run the corresponding PCR program.

10.1 Yeast Culture

(1) Inoculate the strain into YPD liquid medium and incubate overnight. Then transfer 1% of the culture into 3 mL of BMGY medium and incubate overnight until OD600 reaches approximately 10.

(2) Harvest the cells by centrifugation at 4,000 rpm for 5 minutes. Resuspend the pellet in 5 mL of BMMY induction medium and retain 100 µL as the initial sample.

(3) Culture at 30 °C with shaking at 250 rpm. Take 100 µL samples every 24 hours and supplement with methanol to a final concentration of 1% each time after sampling.

10.2 SDS-PAGE for Protein Expression Analysis

(1) Assemble the gel-casting apparatus. Begin by preparing the lower gel (for a 1.50 mm mini-gel).

(2) Mix 4 mL of lower gel solution (2X) with 4 mL of lower gel buffer (2X) in a gel preparation cup.

(3) Add 80 µL of modified polymerization accelerator to the mixture from step 2. Stir gently to avoid bubbles.

(4) Pour the mixed lower gel solution into the gel mold until the liquid is 1.5 cm below the top of the glass plates. Carefully overlay with water or alcohol to flatten the surface and minimize exposure to air.

(5) Let it stand at room temperature (25 °C) for 6–10 minutes. A visible interface between the gel and overlay indicates polymerization.

(6) Prepare the upper gel (for a 1.50 mm mini-gel).

(7) Slowly remove the overlay solution. Mix 1.0 mL of upper gel solution (2X) with 1.0 mL of colored upper gel buffer (2X) in a new cup.

(8) Add 20 µL of modified polymerization accelerator to the mixture from step 7. Mix gently without introducing bubbles.

(9) Pour the upper gel solution onto the polymerized lower gel until full. Slowly insert the comb, avoiding bubbles.

(10) Allow the upper gel to polymerize for 10–15 minutes. Carefully remove the comb and rinse the wells with electrophoresis buffer using a pipette tip or syringe. Proceed with SDS-PAGE electrophoresis.

(1) Label the protein sample before binding to the resin as "S".

(2) Incubate the protein sample with Ni-NTA resin on ice with gentle shaking for 1 hour.

(3) After binding, centrifuge the mixture at 1,000 × g for 10 seconds. Transfer the supernatant to a new tube and label as "FT".

(4) Add 50 µl of Wash Buffer to the resin, centrifuge at 1,000 × g for 10 seconds, and transfer the supernatant to a new tube labeled "W".

(5) Repeat Step 4 and combine the supernatant with the previous wash fraction.

(6) Add 30 µl of Elution Buffer to the resin, centrifuge at 1,000 × g for 10 seconds, and transfer the supernatant to a new tube labeled "E".

(7) Repeat Step 6 and combine the eluate with the previous elution fraction.

(8) Prepare samples for SDS-PAGE by mixing 20 µl of each sample (S, FT, W, E) with 5 µl of 5× SDS-PAGE Loading Buffer.

(9) Denature the samples in a PCR instrument at 98°C for 5 minutes. Store temporarily at -20°C.

12.1 Reagent Preparation

(1) Calculate the total volume of BCA working solution required using the following formula:

Total volume of BCA working solution = (Number of standard points + Number of samples) × Number of replicates × Volume of BCA working solution required per sample.

(2) Based on the calculated total volume, pipette Solution A and Solution B at a ratio of 50:1. Mix well to prepare the BCA working solution.

(3) Dilute a certain amount of the BSA standard solution to a concentration of 500 μg/ml using 1X PBS buffer.

(4) Label 16 microcentrifuge tubes, setting up for 8 duplicate pairs. Process the tubes in parallel according to the table below. Dilute the samples with 1X PBS to an appropriate concentration for the assay.

12.2 Protein Concentration Assay

(1) Select 20 wells on the microplate, divided into standard and sample groups. For the standard group (16 wells), add 20 μl of the corresponding concentration of standard protein solution to each well, with one replicate per concentration. For the remaining 4 wells (sample group), assign them into 2 duplicate pairs (wells with the same ID are replicates). Add 20 μl of differently diluted sample solutions to the wells with different IDs.

(2) Add 200 μl of the BCA working solution to each well and mix thoroughly immediately.

(3) Incubate the plate at 37°C in a water bath for 30 minutes.

(4) After cooling to room temperature, measure the absorbance (A₅₆₂) of each well using a microplate reader.

(5) Generate a standard curve by plotting the average A₅₆₂ value for each standard concentration (y-axis) against the corresponding protein concentration (x-axis). Calculate the protein concentration of the diluted samples from the standard curve using the average A562 value of the duplicate measurements. Select the dilution factor that yields a reading within the linear range of the standard curve. Multiply the calculated concentration by the dilution factor to determine the final protein concentration of the original sample.

13.1 Standard Curve for Enzyme Activity

(1) p-Nitrophenol (p-NP) Stock Solution (2 mmol/L)

20 mmol/L: Weigh 0.014 g p-nitrophenol (p-NP) and dissolve in 5 mL PB buffer (pH 8.0, containing 1% Triton X-100). Store in a brown bottle at 4°C.

2 mmol/L: Dilute the 20 mmol/L stock solution 10-fold using the same PB buffer. Store in a brown bottle at 4°C.

(2) Standard Curve Preparation (in triplicate)

Take 0.01, 0.02, 0.03, 0.04, 0.06, and 0.08 mL of the 2 mmol/L p-NP stock solution and dilute each to 2 mL with PB buffer (pH 8.0, 1% Triton X-100). Measure the absorbance at 410 nm.

Plot a standard curve with p-NP concentration (0.01, 0.02, 0.03, 0.04, 0.06, 0.08 mmol/L) on the x-axis and absorbance (y) on the y-axis.

13.2 Lipase Activity Measurement

(1) Solution Preparation

Solution A (3 mg/mL): Dissolve 30.0 mg p-nitrophenol palmitate (p-NPP) in 10.00 mL isopropanol. Store at 4°C in a brown bottle or a foil-wrapped 15 mL tube.

Solution B: Add 1 drop of Triton X-100 to 100 mL PB buffer (pH 8.0). Mix well and store at 4°C.

(2) Activity Assay (in triplicate, using a microplate reader)

Mix 1 mL Solution A with 9 mL PB buffer (pH 8.0, 1% Triton X-100) in a tube. Incubate at 37 °C for 5 minutes. Add 190 µL Reaction Mix to each well of a 96-well plate. Then add:10 µL sample, 10 µL PB buffer, 10 µL heat-inactivated enzyme (boiled for 5 min). Incubate at 37 °C for 10 minutes and measure absorbance at 410 nm. Perform each measurement in triplicate and calculate the average.

(3) Definition of Lipase Activity Unit (U):

One unit (U) of lipase activity is defined as the amount of enzyme required to release 1 μmol of p-nitrophenol (p-NP) per minute from p-nitrophenol palmitate (p-NPP) under assay conditions (410 nm). The calculation formula is:

Activity (U/mL)

Where:

y: Absorbance value of the sample

b: Absorbance of the blank control

k: Slope of the standard curve (from p-NP standard curve)

n: Dilution factor (n = 1 if undiluted)

V: Total reaction volume (in mL). *Note: Here the total reaction volume in the well is 0.2 mL (190 µL Reaction Mix + 10 µL sample)*

1000: Conversion factor from mmol to μmol

t: Reaction time (in minutes)

14.1 Solution Preparation

(1) Solution A: Dissolve 30.0 mg of p-nitrophenol palmitate (p-NPP) in 10.00 mL of isopropanol. Store at 4°C in a brown bottle or a foil-wrapped 15 mL centrifuge tube.

(2) Solution B: Prepare 100 mL of phosphate buffer at each target pH (6-10). Add one drop of Triton X-100 to each, mix well, and store at 4°C.

14.2 Lipase Activity Measurement

(1) Mix 1 mL of Solution A with 9 mL of Solution B (at a specific pH) in a centrifuge tube. Incubate at 37°C for 5 minutes to form the Reaction Mix.

(2) Add 190 µL of the Reaction Mix to each well of a 96-well plate. Then add:10 µL of enzyme sample, 10 µL of phosphate buffer at corresponding pH.

(3) Incubate at 37°C for 10 minutes.

(4) Measure the absorbance at 410 nm. Perform each measurement in triplicate and calculate the average value.

(5) Record absorbance values at different pH, and plot an enzyme activity-pH profile.

15.1 Solution Preparation

Refer to the previous section for solution preparation. Use a buffer adjusted to the experimentally determined optimal pH.

15.2 Lipase Activity Measurement

(1) Add 1 mL of Solution A to 9 mL of Solution B (at optimal pH) in a centrifuge tube. Mix thoroughly and incubate at 37 °C for 5 minutes to form the Reaction Mix.

(2) Add 190 µL of the Reaction Mix to each well of a 96-well plate. Then add:10 µL of enzyme sample, 10 µL of phosphate buffe

(3) Incubate at the different temperatures for 10 minutes (20-70 °C).

(4) Measure the absorbance at 410 nm. Perform each measurement in triplicate and calculate the average value.

(5) Record the absorbance values at each temperature and plot an enzyme activity-temperature profile.