Protease enzyme production process using molasses-urea media enriched with yeast extract

Abstract

This invention relates to a protease enzyme production process. Specifically, the production of protease enzymes by fermentation using the Bacillus megaterium DSM 319 bacterial strain by making a preculture, a 100ml culture, a 1L culture in a shaker flask using liquid LB media incubated in an incubator shaker for 3-4 hours at 35-40°C with an agitation speed of 150 rpm, until an OD600 of 0.8-1.0 is obtained. The 10L inoculum starter, 100L starter production, and 1000L production culture were grown in the fermenter using 2.8% molasses (wv), 0.5% urea (wv), and 0.05% yeast extract (wv) media at 35-40°C, an aeration rate of 1 vvm, a pO2 of at least 30%, a pH of 7.5, an agitation speed of 150-200 rpm for the starter inoculum and an incubation time of 6-8 hours for the starter production. Meanwhile, for the production culture, the agitation speed is 75-150 rpm, and the incubation time for protease harvest is 14-18 hours, or at the time of protease activity of 20-100 U / ml. The ratio of the volume of inoculant culture to the total culture volume between one stage and the next stage is 1:10.

Description

[0001] Description

[0002] PROTEASE ENZYME PRODUCTION PROCESS USING MOLASSES-UREA MEDIA

[0003] ENRICHED WITH YEAST EXTRACT

[0004] Technical Field of the Invention

[0005] This invention relates to a protease enzyme production process . Speci fically, the production process of protease enzymes by fermenting the Bacill us mega teri um DSM 319 bacterial strain in a molasses and urea fermentation medium enriched with a yeast extract . The process occurs in a fermenter to increase protease enzyme activity .

[0006] Background of the Invention

[0007] Biocatalysts , more commonly known as enzymes , are important industrial commodities . Microorganisms produce most of the enzymes used in industrial processes through fermentation processes . Most enzymes are used as biocatalysts in chemical trans formations . There are about 400 known enzyme types , and about 200 are commercially used .

[0008] Protease is a widely used enzyme type because of its high economic value in various industrial processes , including in detergents , leather, textile , food, and milk processing, as well as pharmaceutical and industrial waste processing (Raz zaq et al . , 2019 ) . Proteases constitute one of the three largest enzyme groups in the enzyme industry, constituting 65% of the total enzymes traded worldwide (Yuniati et al . , 2015 ) .

[0009] Several factors , such as temperature , pH, source of inorganic salts , carbon, nitrogen, and substrate type , influence the production of protease enzymes from microorganisms . Current protease production processes still use raw materials with high protein content as a nitrogen source , such as peptone , beef extract , nutrient broth, skim milk, and casein, making the process relatively expensive . In addition, the process requires other media ingredients , such as NaCl , citric acid, CaC12 , glutamic acid, yeast extract, KC1, xylose, glucose, galactose, and more.

[0010] Several studies on protease production generally use raw media materials as the main media or an additional component to increase microbial productivity if producing protease from waste media materials. For example, protease production from Bacillus lichen! formis Fll. 1 uses peptone media, yeast extract, and NaCl (Sitti Nur Imiah et al., 2018) ; protease production from Geobacillus thermoglucosidasius SKF4 uses casein media and yeast extract (Suleiman, A.D. et al., 2020) ; protease production from Bacillus subtilis PL4 uses peptone and xylose media materials (Ibrahim Ahmed Shaikh et al., 2023) ; protease production from Bacillus subtilis B22 uses agricultural waste media enriched with casein, peptone, and yeast extract (Elumalai, P. et al., 2020) ; protease production from Bacillus mojavensis uses a media made from eggshell membranes enriched with peptone, yeast extract, and glucose (Mumecha, T.K. et al, 2021) ; and protease production from Bacillus gibsonii 6BS15-4 uses household wastewater media enriched with skim milk, glucose, and yeast extract (Polson Mahakhan et al., 2023) ,

[0011] If raw materials are used as the main media component or as an additional media component in commercial-scale protease production, it will most likely incur high costs. Therefore, an invention is needed to find a relatively cheap but suitable media material to produce protease enzymes without reducing the quality of the resulting product. Additionally, if used on a commercial scale, the invention should reduce the procurement costs of media materials.

[0012] The invention of protease production using relatively cheap media has been disclosed in the Indonesian patent IDP0023785, which is related to the protease production process using molasses-urea media from the bacteria Bacillus megaterium DSM 319. However, the molasses-urea media has limitations regarding its amino acids, peptides, vitamins, and fat content, which are required for stimulating microbial growth and productivity, such that the protease activity produced in the invention is relatively low.

[0013] Patent ID0000821S discloses the production process of protease from cloned Bacillus subtilis DB104 bacteria using tofu liquid waste media supplemented with full-cream milk and calcium salt or with synthetic media containing soy flour, calcium salt, phosphate salt, skim milk, and starch. However, the invention still has several limitations; among others, the method disclosed in ID0000821S still uses full-cream milk media, soy flour, and starch, which are relatively expensive food commodities .

[0014] In contrast to the above inventions, the present invention reformulates molasses-urea media by adding yeast extract to overcome the limited source of amino acids, peptides, vitamins, and fats, stimulating cell growth and increasing protease activity .

[0015] Summary of the Invention

[0016] This invention aims to improve the efficiency of the fermentation process to produce proteases with higher activity. In addition, this invention provides a relatively inexpensive fermentation media using molasses and urea enriched with yeast extract .

[0017] Stages of the protease production process comprise growing preculture isolates of Bacillus megaterium DSM 319 in a whipped flask containing a sterile liquid LB medium of pH 7.5 with a preculture volume of 1:10 of the total culture volume. The preculture is then incubated in an incubator shaker for 3-4 hours at 35-40°C at an agitation speed of 150 rpm until the bacterial cell reaches an Optical Density (OD60o) of 0.8-1.0.

[0018] The preculture that has reached ODeoo of 0.8-1.0 cells are inoculated into a shaker flask containing a sterile liquid LB medium of pH 7.5, with the ratio of inoculant volume to the total culture volume being 1:10, for a total culture volume of 100 ml. Then, it is incubated in an incubator shaker for 3-4 hours at 35-40°C and at an agitation speed of 150 rpm.

[0019] The culture that has reached ODeoo of 0.8-1.0 cells is then inoculated into a shaker flask containing a sterile liquid LB medium pH 7.5 with a ratio of inoculant volume to the total culture volume being 1:10, for a total culture volume of IL. It is then incubated in an incubator shaker for 3-4 hours at 35- 40°C and at an agitation speed of 150 rpm.

[0020] The culture that has reached OD6oo of 0.8-1.0 is then inoculated into the fermenter containing sterile starter inoculum media consisting of 2.8% molasses (w / v, ) 0.5% urea (w / v) and 0.05% yeast extract (w / v) , with the ratio of inoculant volume to total culture volume being 1:10, for a total culture volume of 10 L. Then, the culture is fermented in a fermenter for 6-8 hours at 35-40°C, with an agitation speed of 200 rpm, an aeration rate of 1 vvm, aPO2 of at least 30%, and a pH of 7.5. The operating conditions are maintained throughout the process.

[0021] The starter inoculum that has reached ODeoo of 0.8-1.0 is then inoculated into the fermenter containing sterile production starter media consisting of 2.8% molasses (w / v, ) 0.5% urea (w / v) and 0.05% yeast extract (w / v) , with the ratio of inoculant volume to total culture volume being 1:10, for a total culture volume of 100 L. Then, the culture is fermented in a fermenter for 6-8 hours at 35-40°C, with an agitation speed of 200 rpm, an aeration rate of 1 vvm, aPO2 of at least 30%, and a pH of 7.5. The operating conditions are maintained throughout the process.

[0022] The culture that has reached ODeoo of 0.8-1.0 is then inoculated into the fermenter containing sterile production media consisting of 2.8% molasses (w / v, ) 0.5% urea (w / v) , and 0.05% yeast extract (w / v) , with the ratio of inoculant volume to total culture volume being 1:10, for a total culture volume of 1000 L. Then, the culture is fermented in a fermenter at 35- 40°C, with an agitation speed of 75-150 rpm, an aeration rate of 1 vvm, aPC>2 of at least 30%, and a pH of 7.5. The operating conditions of the production process are maintained until protease is produced. The protease production process in the 1000L production culture media can last for 14-18 hours before being harvested, or protease can be harvested when the protease activity reaches 20-100 U / ml.

[0023] The protease production process stages described above includes the steps for making the preculture, the 100ml culture, the IL culture, the 10L starter inoculum, the 100L production starter, and the 1000L production culture media. They have a total volume ratio of 1:10 between one stage and the next.

[0024] Brief Description of the Figure

[0025] Figure 1 exhibits the flow of the protease production process using a molasses-urea-yeast extract media.

[0026] Description of the Invention

[0027] This invention aims to improve the efficiency of the fermentation process to produce proteases with higher activity. In addition, this invention provides a relatively inexpensive fermentation media using molasses and urea enriched with yeast extract .

[0028] Deden R.W. et al., with the patent number IDP0023785 of 2000, disclose a protease production process using relatively cheap media. The patent discloses a method to produce proteases from the Bacillus megaterium DSM 319 bacteria, using molasses- urea media comprising 1.5-2.0% molasses (wv) and 1-2% urea (wv) . However, each stage of the production process requires a relatively long time of about 8-24 hours. Moreover, the long incubation time for the culture inoculation between stages could still produce insufficient bacterial vegetative cells. The molasses-urea media used also has limitations regarding the amino acids, peptides, vitamins, and fat content required to stimulate microbial growth and productivity, such that the resulting protease has a relatively low activity of about 4.98- 8.566 U / ml . Meanwhile , Maggy T . Suhartono ' s invention with the patent number ID0000821S of 2000 discloses the production process o f protease from cloned Bacill us subtili s DB104 bacteria using tofu liquid waste media supplemented with full-cream and calcium salt or a synthetic media containing soy flour, calcium salt , phosphate salt , skim milk, and starch . However, this invention ' s use of tofu liquid waste still has limitations . One is the need for full-cream milk, soy flour, and starch, which are relatively expensive food commodities for commercial processes .

[0029] The protease production process in the present invention is a reasonably ef ficient and relatively more economical process because it uses relatively inexpensive media, namely molasses as a source of carbon and urea as a source of nitrogen . Additionally, the media is enriched with yeast extract as a source of amino acids , peptides , vitamins , and fats , which stimulates bacterial cell growth and increases protease production, resulting in 5- 10 times higher protease activity than previous inventions , with the protease activity obtained reaching 20- 100 U / ml . This invention has also been tested for protease production on a volume scale of 1000 liters . In addition to Figure 1 , this invention can be described in detail as follows :

[0030] This invention uses microbes from the Bacill us mega teri um DSM 319 bacterial strain as microbial protease producers . These microbes are used for fermentation of protease production in molasses-urea media enriched with yeast extract conducted under speci fic compositions , production processes , and operating conditions .

[0031] The preculture and culture media uses a Lysogeny Broth / LB standard media with its composition listed in Table 1 . Meanwhile , the starter inoculum media, the production starter, and the production culture were developed from patent number IDP0023785 of 2000 by reformulating the composition of the molasses-urea media enriched with yeast extract . The composition of molasses-urea-yeast extract media is listed in Table 2 . Table 1: The Composition of the Lysogeny Broth / LB Media

[0032] Ingredients (%) Volume (% wv)

[0033] Bacto Peptone 1%

[0034] Yeast Extract 0.5%

[0035] NaCl 0.5%

[0036] (Demineralized water is used to dissolve the LB media ingredients according to the medium's required volume. The pH is set to 7.7 by adding an alkaline or acid (pH ~ 7.5 after sterilization) . The media is sterilized in an autoclave at 121 °C, 1 atm, for 15 minutes.

[0037] Table 2: Composition of the Molasses-Urea-Yeast Extract

[0038] Ingredients (%) Volume (% wv)

[0039] Molasses 2.8

[0040] Urea 0.5

[0041] Yeast extract / YE 0.05

[0042] (Demineralized water is used to dissolve the media ingredients mentioned in Table 1 according to the medium's required volume. The pH is set to 7.7 by adding an alkaline or acid (pH of ~ 7.5 after sterilization) . The media is sterilized in an autoclave at 121 °C, 1 atm, for 15 minutes.

[0043] The Bacillus megaterium DSM 319 isolate is grown as a preculture in a shaker flask containing a sterile liquid LB medium with a pH of 7.5 (The medium's composition is stated in Table 1) with a preculture volume of 1:10 of the total culture volume. The preculture is then incubated in a shaker incubator at 35-40°C and an agitation speed of 150 rpm for 3-4 hours.

[0044] The preculture that has reached ODHOO of 0.8-1.0 cells is inoculated into a shaker flask containing a sterile liquid LB medium of pH 7.5, with the ratio of inoculant volume to the total culture volume being 1:10, for a total culture volume of 100 ml. Then, it is incubated in an incubator shaker for 3-4 hours at 35-40°C and an agitation speed of 150 rpm.

[0045] The culture that has reached Optical Density (OD60o) of 0.8 1.0 is then inoculated into a shaker flask containing a sterile liquid LB medium culture of pH 7.5, with the ratio of inoculant volume to the total culture volume being 1:10, for a total culture volume of 1 L. It is then incubated in an incubator shaker at 35-40°C and an agitation speed of 150 rpm for 3-4 hours.

[0046] The culture that has reached OD6oo of 0.8-1.0 is then inoculated into a fermenter containing a sterile molasses-urea- yeast extract starter inoculum medium (The medium's composition is stated in Table 2) with the ratio of inoculant volume to the total culture volume being 1:10, for a total starter inoculum volume of 10 L. It is then fermented in a fermenter for 6-8 hours at 35-40°C, stirring at 200 rpm, aeration of 1 vvm, aPO2 of at least 30%, and a pH of 7.5. The operating conditions are maintained throughout the process.

[0047] The inoculum starter that has reached ODeoo of 0.8-1.0 is then inoculated into a fermenter containing a sterile molasses- urea-yeast extract starter culture medium (The medium's composition is stated in Table 2) with the ratio of inoculant volume to the total culture volume being 1:10, for a total starter inoculum volume of 10 L. It is then fermented in a fermenter for 6-8 hours at 35-40°C, stirring at 150-200 rpm, with an aeration rate of 1 vvm, aPO2 of at least 30%, and a pH of 7.5. The operating conditions are maintained throughout the process .

[0048] The production starter that has reached ODeoo of 0.8 - 1.0 is then inoculated into a fermenter containing a sterile molasses- urea-yeast extract starter culture medium (The medium's composition is stated in Table 2) with the ratio of inoculant volume to the total culture volume being 1:10, for a total production culture volume of 1000 L. The fermenter's operating conditions for protease production are stated in Table 3. The operating conditions of the fermenter are maintained during the production process until protease is harvested at about 14-18 hours or when the protease activity reaches 20-100 U / ml.

[0049] Table 3: Protease production process conditions

[0050] Parameters Value pH of the media 7.5

[0051] Inoculant Vol. : 1:10

[0052] Tot .Vol . Culture*

[0053] Temperature 35-40°C

[0054] Agitation Speed 75-150 rpm

[0055] Aeration 1 vvm

[0056] PO230% <

[0057] Fermentation Duration 14-18 hours *Inoculant Vol. : Volume of the inoculated culture

[0058] Tot . Vol . Culture : Total volume of the culture after inoculation Protease activity is measured periodically during the protease production process for the 1000L scale by a modified method of the Amano K Enzyme Assay to determine harvest time.

[0059] The protease production process stages described above includes the stages for making the preculture, the 100ml culture, the IL culture, the 10L starter inoculum, the 100L production starter, and the 1000L production culture media. They have a total volume ratio of 1:10 between one stage and the next .

Claims

Claims1. A protease enzyme production process using a molasses-urea medium enriched with yeast extract comprising the following stages : a. growing a preculture of the Bacillus megaterium DSM 319 isolate in a shaker flask containing sterile liquid LB medium with a pH of 7.5 with a preculture volume of 1 10 of the total culture volume, then incubated in an incubator shaker for 3-4 hours at 35-40°C with an agitation speed of 150 rpm until the bacterial cells reach Optical Density (OD6oo) 0.8-1.0; b. inoculating the preculture obtained from stage a) into a shaker flask containing sterile liquid LB culture medium with a ratio of inoculant volume to total culture volume of 1 10, for a total culture volume of 100 ml, then incubated in an incubator shaker for 3-4 hours at 35-40°C with an agitation speed of 150 rpm until an ODeoo of 0.8- 1.0 is obtained; c. inoculating the culture obtained from stage b) into a shaker flask containing sterile liquid LB media with a ratio of inoculant volume to total culture volume of 1 10, for a total culture volume of 1 L, then incubated in an incubator shaker for 3-4 hours at 35-40°C with an agitation speed of 150 rpm until an ODeoo of 0.8-1.0 is obtained; d. inoculating the culture obtained from stage c) into a fermenter containing a molasses-urea-yeast extract sterile liquid medium with a ratio of inoculant volume to total culture volume being 1:10, for a total culture volume of 10L, then fermented for 6-8 hours at 35-40°C with an agitation speed of 200 rpm, an aeration rate of 1 vvm, a pC>2 of at least 30%, and a pH of 7.5, the fermenter's operating conditions are maintained throughout the process until an OD eoo of 0.8-1.0 is obtained;e. inoculating the inoculum starter obtained from stage d) into a fermenter containing a molasses-urea-yeast extract sterile liquid medium with a ratio of inoculant volume to total culture volume being 1:10, for a total culture volume of 100 L, then fermented for 6-8 hours at 35-40°C, with an agitation speed of 150-200 rpm, an aeration rate of 1 vvm, aPO2 of at least 30%, and a pH of 7.5, the fermenter's operating conditions are maintained throughout the process until an CD6oo of 0.8-1.0 is obtained; f. inoculating the culture obtained from stage e) into a fermenter containing a molasses-urea-yeast extract sterile liquid medium with a ratio of inoculant volume to total culture volume of 1:10, for a total culture volume of 1000L, then fermented for 6-8 hours at 35-40°C with an agitation speed of 75-150 rpm, an aeration rate of 1 vvm, aPO2 of at least 30%, and a pH of 7.5, the fermenter's operating conditions are maintained throughout the process until protease is produced.

2. A protease enzyme production process using a molasses-urea medium enriched with yeast extract of Claim 1, where the molasses-urea-yeast extract medium comprises 2.8% molasses (wv) , 0.5% urea (wv) , and 0.05% yeast extract (wv) .

3. A protease enzyme production process using a molasses-urea medium enriched with yeast extract of Claim 1, where the ratio between the inoculant culture volume with the total culture volume in one stage and the next stage is 1:10.

4. A protease enzyme production process using molasses-urea media enriched with yeast extract of Claim 1, where the protease is ready to be harvested at 14-18 hours or when protease activity reaches 20-100 U / ml.

Images