The process of producing xylanase enzyme from the hybrid yeast Pichia pastoris.

Abstract

This invention relates to the development of a process for producing xylanase enzyme from recombinant yeast. Pichia pastoris involves a process that includes batch fermentation to increase the number of hybrid yeast cells. P. pastoris with specific diet and induction of enzyme expression. Xylanase was added for 72 hours with the first addition of methanol at a concentration of 3-4%. The volume immediately increased to 50 when the decrease in dissolved oxygen during fermentation was measured. The percentage, or during the 12-24 hours of the fermentation process, is then reduced, and the cells are continued to be cultured until 24 hours are complete. After several hours, a second dose of methanol at a concentration of 1.5-2% by volume was added, and the cells were cultured. Continue for a full 24 hours, then add a third dose of methanol at a concentration of 0.75-1% by volume, using... A specific food formula according to this invention, which includes the following components: carbon, nitrogen, and... Amino acids, which are considered readily available and low-cost agricultural raw materials, are therefore... This helps reduce costs in the fermentation process, as the process described in this invention can produce enzymes. Xylanase from recombinant yeast cells P. pastoris containing the AOX1 promoter regulates the expression of genes involved in... The enzyme xylanase can effectively reduce the fermentation process by 10 hours. And it reduced the amount of methanol used to stimulate enzyme expression by 41.7 percent, including... The production of target enzymes was increased by up to 2.86 times compared to fermentation processes using different media. The traditional BGY (Bio-Glycerinated Yeast) process and recipe is therefore a specific type of formula that can be further developed. Towards large-scale production for the utilization of xylanase in various industries, including the food industry. Textiles, energy, and paper, etc.

Claims

1. The process of producing xylanase enzyme from the hybrid yeast Pichia pastoris involves several steps. too a. Batch fermentation to increase the number of recombinant yeast cells (P. pastoris) using a specific fermentation formula. Specifically, the initial cell concentration is determined by an absorption coefficient of 600 nm. The nanometer value is 0.2-0.

5. Then, the cells are cultured until the oxygen solubility value... The decrease returns to an increase of 50-100 percent, or the cell concentration reaches a value of... Absorbance of 600 nanometer light waves increases by 50–100 OD per milliliter, or until… The cell concentration remains constant, or the fermentation period of 12-24 hours has completed. B. Inducing the expression of slanase enzyme over a period of 72 hours by... Add methanol in the following order: First methanol addition. Methanol at a concentration of 3-4% by volume, immediately after measuring the solubility of... The oxygen levels that decreased during fermentation are increased again to 50 percent, or in... Hours 12-24 of the fermentation process. Second methanol addition Methanol at a concentration of 1.5-2% by volume after 24 hours. Add methanol for the first time. Third methanol addition. Methanol at a concentration of 0.75-1% by volume after 24 hours. Add methanol for the second time.

2. The process of producing xylanase enzyme from the hybrid yeast Pichia pastoris according to claim 1, which has... This step involves additionally cultivating a hybrid yeast starter culture, P. pastoris, before fermentation. The schedule is as follows: a. Culturing recombinant yeast cells (P. pastoris) in 10 ml of mixed YPD liquid medium. The antibiotic zucchini was administered in a 50 ml test tube under conditions of shaking 200-250 cycles. Per minute, the temperature will be 28-30 degrees Celsius for 12-24 hours. B. Culture the cells obtained from step A with 50 ml of YPD liquid medium mixed with antibiotics. Zeosin in a 250 ml test tube under shaking conditions of 200-250 rpm. The temperature will be 28-30 degrees Celsius for 12-24 hours.

3. The process of producing xylanase enzyme from the hybrid yeast Pichia pastoris according to claim 1, where the formula... This specific type of food contains the following ingredients: Carbon source concentration: 5-20 grams per liter. Available options include glycerol, glucose, fructose, lactic acid, molasses, and milk powder. Lactose, protein, and fat are the main components, either individually or in combination. Combined - Nitrogen source: Concentration of 10-30 grams per liter. The choice can be made from peptone, yeast extract, or a nitrogen-based yeast without amino acids. One or more combinations - Supplemental food source: Concentration 5-20 grams per liter. It is a composite amino acid containing glutamic acid as the main component, mixed with other amino acids. Choose from arginine, glutamine, phenylalanine, tyrosine, tryptophan, glycine, and alanine. Histidine, serine, polypropylene, aspartic acid, threonine, cysteine, methionine, leucine, asparagine. Isoleucine, varin, or a combination of these.

4. The process of producing xylanase enzyme from the hybrid yeast Pichia pastoris according to claim 3, where the formula... The most suitable specific diet includes the following components: Powdered milk contains 10-20 grams of lactose, protein, and fat per liter. 20-30 grams of yeast extract per liter. A composite amino acid containing glutamic acid as the main component, mixed with other select amino acids. Arginine, Glutamine, Phenylalanine, Tyrosine, Tryptophan, Lysine, Glycine, Alanine, Histidine, Serine Polyethylene, aspartic acid, thionine, cysteine, methionine, leucine, asparagine, isoleucine, varine. One or more of the following, at 5-10 grams per liter. And 100 millimolar potassium phosphate buffer, pH 6.

0.

5. The process of producing xylanase enzyme from the hybrid yeast Pichia pastoris according to claim 1, which states: The optimal time for the first methanol addition is 18 hours into the fermentation process.

6. The process of xylanase enzyme production from the hybrid yeast Pichia pastoris according to claim 1, whereby the The optimal methanol concentration for the first addition is 3% by volume.

7. The process of producing xylanase enzyme from the hybrid yeast Pichia pastoris according to claim 1, which states: The optimal conditions for inducing xylanase enzyme expression are under certain temperatures. The fermentation tank temperature is 30 degrees Celsius, the stirring speed is 500 revolutions per minute, and the aeration rate is... This equals 1.0 volume per volume per minute, and the pH is 6.

0.

8. The process of xylanase enzyme production from the hybrid yeast Pichia pastoris according to claim 1, where the shell... The aforementioned P. pastoris hybrid yeast contains xylanase-producing genes and a promoter. AOX1 controls the expression of these genes.

9. The process of producing xylanase enzyme from the hybrid yeast Pichia pastoris according to claim 8, where the gene... The enzyme xylanase was produced and isolated from the fungus *Aspergillus aculeatus*.