Organic food waste stream sample was collected from local food street rich with food waste materials. For further analysis, the samples were transported to the laboratory in a sterile container. The collected samples were diluted in sterile 0.9 g/L saline solution of 100 mL in 250 mL conical flask and agitated at 150 rpm for 30 min in a water bath shaker at 37oC. One mL of dilution was plated onto nutrient agar plates. The inoculated plates were then incubated at 37o C for 48 h. The colonies were isolated from nutrient agar plates and streaked in fresh plates until single uniform colonies were obtained.
Screening of proteolytic bacteria
Skim milk agar medium [10.0g/L Skim milk, 1.0g/L Peptone, 5.0g/L NaCl and 20.0g/L agar] [pH 7.0] was prepared and sterilized at 121°C for 15 minutes. TAS-2 colonies were picked from nutrient agar plate and streaked the Skim milk agar Petri plates. The inoculated plates were incubated at 37°C for 72 hours at static chamber. A clear zone on skim milk agar plates were examined for qualitative detection of proteolytic activities exhibited by the TAS-2. TAS-2 isolate was biochemically characterized using standard procedure and protocols of Bergey's Manual of Determinative Bacteriology [12]. The TAS-2 colony was tested for gram staining, spore staining, catalase test, methyl red test, indole test, ogesproskauer test, citrate utilization test, nitrate test, urease test, and hydrolysis of casein. Approximately 6 mL of sterile nutrient agar was placed in a sterile test tube and placed on an inclined level to solidify at room temperature. A sterile loop was used to transfer TAS-2 colonies in test tubes, and then incubated at a temperature of 37 °C for 24 h and stored in a refrigerator at a temperature of 4 °C for subsequent experiments.
Selection and molecular identification of target strain
To find out the strain that releases protease, 50 ml of nutrient broth [pH 7.0] was inoculated with TAS-2 and incubated at 37°C for 24 hours in shaking incubator set at 150 rpm. After that, the culture broth was centrifuged for 10 minutes at 4°C at 10,000 rpm. Pure genomic DNA was isolated from 24h old TAS-2 culture cells. Polymerase chain reaction [PCR] was performed for 16S rRNA genes with the following universal
Table 1: Polymerase chain reaction [PCR] reaction and cycle conditions
Primer F1
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[5’AGAGTTTGATCCTGGCTCAG3’]
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Primer R1
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[5’ACGG(H)TACCTTGTTACGACTT3’]
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Initial denaturation
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95°C for 5 minutes
|
Cycle denaturation
|
95°C for 30 sec
|
Cycle annealing
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52°C for 30 sec
|
Cycle extension
|
72°C for 30 sec
|
Finale extension
|
72°C for 5 minutes
|
Total cycles
|
30
|
After completion of PCR, 2 μL reaction was loaded on to the 1% electrophoresis gel to confirm PCR band. The amplified 16 sDNA sequence was sequenced by Automated DNA sequencing method. The obtained sequence was analyzed using the BLAST resource website from NCBI, and similar species sequences were used for construction on Neighbor joining trees [13].
Production of alkaline protease
For protease production, a colony of TAS-2 from stored slant was transferred into 100 mL of TSB [1.7% tryptone, 0.3 % peptone, 0.25% glucose, 0.5% NaCl] under Laminar airflow aseptic condition. Prior to sterilization at 121°C for 15 minutes, the medium's pH was adjusted to 7.0± 0.2. In a water bath shaker, the inoculated broth was kept for 24 hours at 37°C at 150 rpm. Subsequently, 1% (v/v) of this inoculum was used to inoculate a 250 mL flask containing sterile 100 mL of basal medium [pH 7.0± 0.2] [1.0g/L K2HPO4, 1.0g/L MgSO4.7H2O, 5.0g/L NaCl, 1.0g/L Peptone, 1.0g/L Yeast extract]. The medium was incubated at 37°C in water bath shaker at 150 rpm for 120h. At the harvesting points, the fermented broth was centrifuged at 10000 rpm at 4oC for 10min. The supernatant was used for protease activity.
Protease assay
Protease was assayed as previously described. Protease activity mixture contains 0.5 mL of 0.5% casein, and 1 mL culture-free filtrate. Following ten minutes of incubation at 37°C, 5 mL of 100 mmol/l trichloroacetic acid (TCA) was added to the reaction mixture in order to stop the enzyme reaction. The supernatant was collected after the reaction mixture was centrifuged for 20 minutes at 10000 rpm. To 2 millilitres of the supernatant, add 5 millilitres of an alkaline solution (500 mmol/l Na2CO3) and 1 millilitre of a 25% Folin phenol reagent at room temperature. Protease activity was measured spectrophotometrically at 660 nm after 30 minutes. Control sample reaction comprise of 1 mL heat-inactivated enzyme, and 0.5 mL of 0.5% casein. The amount of enzyme needed to release one 1 µmol of tyrosine per millilitre per minute is known as one unit of protease activity. BSA was used as a standard to calculate the samples' total protein content using the methodology outlined by Lowry [14].
Protease activity was calculated from the formula.
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)
The calculation of the sample's specific activity involved dividing the enzyme units (U) by the amount of protein present.
![](data:image/png;base64,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)
TE= total enzyme, TP= total proteins
This is the percentage of the activity of a sample relative to the sample in which the maximum activity was obtained.
![](data:image/png;base64,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)
AS= activity of sample in [u], MEA= maximum enzyme activity [u]
This represents the percentage of an enzyme's activity in a sample compared to the activity of the control (the sample that was not treated).
![](data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAeAAAABTCAYAAABH/dT6AAAVtUlEQVR4Ae2dPa4URxSF2YLFDrDECkziFCSzApyRYcmpReTIqSVEbAnnLACxAZwjx0hOIXHGAsb6nnzGl+tb3dX9pqd7pk9JQ3VX3d9TP6er37zHnYOLETACRsAIGAEjcHYE7pzdox0aASNgBIyAETACBxOwJ4ERMAJGwAgYgRUQMAGvALpdGgEjYASMgBEwAXsOGAEjYASMgBFYAQET8Aqg26URMAJGwAgYAROw54ARMAJGwAgYgRUQMAGvALpdGgEjYASMgBEwAXsOGAEjYASMgBFYAQET8Aqg26URMAJGwAgYAROw54ARMAJGwAgYgRUQMAGvALpdGoFzIfD06dPDnTt3vvg8fPjw8Pnz53OFYD//IvD+/fvD3bt3vxgLxubFixfGaKcImIB3OvBOex8IiIDfvHkzOWGI4d69e4ePHz9263748OHw+PHjI8lAOD/99NPh06dPBwjo1atX3bZuK8hDxrt37w7Pnz8/3L9//9D74EGMIkrq169fj4YyR4cxMQGPQnvVAibgqx5eJ7d3BOYSMKQL+UIQveSNHIQF0UHEFEjwl19+ORLyuU57+H3y5Mnhhx9+OJJpDwGDFzm8ffv2Jn5q7ofinqODcRPwDcS7/scEvOvhd/LXjsBcAoZw9OoaG2NFhN06Ma9JNspljIAll8lW7dWDiPqm6AjLNTFRDK7XRcAEvC7+9m4EFkVgDgHr9Pj999/fkHCLVGPgIpMhsqYvE1W0sdS1SHKIgIceIPSz24zDHJ2YozBbA5MYh6/XQ8AEvB729mwEFkdgDgFDDJAVPz/l9WvPa2iRXCapmCB21yAbxTZEwEMyPJCgm3GYo5PxwOYamMQ4fL0eAibg9bC3ZyOwOAJzCBgdyJIi/SHyQk6nOQjlwYMHx58BxwTHvoQViQ47+oigdBJV+1hM8j1ElJJRntS5xLhi/xydaFuYKb/Y5+t9IGAC3sc4O8udIiCSEKGOwQDJQaC8XqWIJDgJ09cqkaQgSORfvnw569ed+NaxSDYSnuLBNl/swmdPGSNgvUrGZ4sMhaNIf45OjlXYtnxmed9fHwIm4OsbU2dkBI4IiDh6CRj5SAg9RCNnmYQhNH79p+fXeGRDtUgTGzF2fqUpxif5oVq2RJ5ZtidH4Sgbc3SyXxNwRmR/9ybg/Y25M94RAiKOSGKt9CGVR48eHU+/kpMNkY/aqxoS5uSrnx3rJNt6LV3ZUJv86ufKEOlU8sWWCViIut4aAibgrY2I4zECJ0RAJNZDwBAV8rnopJZPo1ku3vN7wM+ePTu+SkYXUu6JQ3biiZqTNKffOcUEPAc165wDARPwOVC2DyOwEgK9BBzJTqfWqq4Ieig1iJjTr2zpNDukE/viF6/mnH6xdUoCVv5TXkFLJ+bFtR5s5uaV7fn+8hAwAV/emDliI9CNQC8BQwZDr5hFYi0CRX/odCt9iHgK4UDA33777ZHAh3y0QJHvVn7x4aMiy9iv2GNbr06Oj1ym4pFt+P6yETABX/b4OXojMIhALwEjN0Ru8SRaydEmcqoCioQ1JBd1OWXyx0CoRaKtB4Col6+l2yJg5Idk4mk35j5HJ8aGLRNwRGR/1ybg/Y25M94RAj0EDLnyd5MhyVaJBFoRGWQyRo6KBeIaK/gjJmKjjPkfsjdElNLTA0aVg/py3mqfoiN/1CbgiMY+r03A+xx3Z70TBER68eSWU0emhxRFGJzasj31tb4oJQKtyCrHw33160bxJErMvaWHgLEluZxbq32ujuIWZj3YS8f1dSFgAr6u8XQ2RuALBMYIWH/0oocEREQQMESq//EIhyIT+vjSlU6u9CHH6bHnW9AQNX9kA9loQ0kpn95Xt9iTTo5ZNlXHhwTlNobPHB35E2Y92EvH9XUhYAK+rvF0NkbgCwREPvlUJ+KAyPRpkV4lKx3sU7DPB1n+C7/8fwLzK0kitS8CTDeR5PGhuFsx5NfC0ZxyV6yqh07h+NEDAPI8TOi/Joy24/UcHfTJDR8m4Ijmvq5NwPsab2e7MwREQiKynaW/6XRNwJsenrMEZwI+C8x2YgTWQcAEvA7uPV5NwD0oXbeMCfi6x9fZ7RwBE/B2J4AJeLtjc67ITMDnQtp+jMAKCJiAVwC906UJuBOoKxa7OAL+5ptvDnzGyl9//XX48ccfD1999dWY6K36//jjjxs/fJliC6UXn1as4AZmP//8c0vk5O36cozqX3/99eQ+yIs/6rBW+e67745fdiJP7s9RRMDClnroi0vniGmvPvR7w3EsuN77l7DYa9hzWKN7K92skTeQOIkAj80NMlq69BAMA8kmTlzEuVT5888/b/6rNWGxlJ8pdnvwGbK3FgFn0uVeuMY6E1cll22R72+//Tbrv8WrsIrxxOve+U8OOY/Kj9uMQAuBOO+Gri9hnm2ZgNlPl8RwEjv9/fffh6+//vpmY4ybDZubyG7O//3ZmmS3bQc4JufS5Vx+Yh56yIhtS18ztnHcT+WPMapIE/s82NHPvGP+VYV2xoDF0pIZ6qtsjrUx57Xxjf2aSrZFrHxcjMBtENDvKLf2OObo1uYZbyUvpejhfkkMJ7MTwTDgeSPWhgQRb6Uo1qXjOZefmAcTuUVaUe6U15BgHvdT2Gc+tXLRJjO2cNFv2eBhZYnXzyLgqRgwX/i4GIHbIjA2B9mXt1JYh8R7CYV9Tg//S67VyWiIbPJGLHABOPetBbhiXdr/ufwoD5FSi3Akd8oaAlxqbLHbyoW5NNSvHIcImE0IzE5diIvP1MJ84eNiBG6LwNw5eFu/U/V5M8VbqDnrZaqv28orVmriXXKtTt49RDaZZCMB87PRXEiGTVyvqhmM/OoOPQ0Spy2eQOLGjE/ZGLNPnLKFrDbyPGGxr7YMNPHJBjJVzNgWJjmmfI8vvcLHHvlVWEEW8gte/IwE/CgxXsUtjCp8FFuWxVbs03jim7giFlFOdqo25dvCWv25xqZyyH2y1eqXPP0tGbAUftTCF3mdjomBsdF4IKOxIlfpyx+1sIhtPdfY4+NiBG6LwNAcbK0H1pROd+izp/KRfFzbaiNO+aLWfkE7a0P7Mn3sWdxrzbDGtJaiDXTpY3+jP9oULviXLjX3sivfPGCzxulj/Sp+auxPLWAjbiLeJdfqyQiY5AkWkHIBMADS6xBAEcFE0Bk4Nj4K7QKce4AFFA1g9CH72MQ290yALEu7BjPqy24EGn/okxcFu+gSYy4a8Nwe78kde8qXmnsGOxYmo/KgXbiSj4p0FRvtLXzIWYsN27ngK2IufxELdNSu+GlTTmCCn1jAFLxye5TRdcRZbaqrXNUXa+KLeKiPcYsYY09zg3auaQMD4iBmrsEqylbYIc9nagHbjG+2oTklH0N1lXe25/vrREDzImfH2qzmBXM77rNxL47yWtuxjbWsecnaUGEdsW6wRUGHuLARi3TVhj3tE8hHm8ggH/dCxUSbCjra3+J6lixtUwrxxLVOXMSxVJm8ewhEgcXGrw2NgeU+FwYkEgj96MfkdB83bMCIEwA9+Y8+AKwiASYFPmKp9OmPsXCveGI+mlixDdmWzegXXWKMBb3YJp/UKkxqZOJEklzGBp0qFmzk/JDVgpIv1ZWsco+xIa/Jr8UnG8jnBai+XOOvygW5oVyjHfQrG8SgBwzJy2ZcaPRpHjPvYqnwoJ92PlMLY8THxQjcFgHNwarO60H7QGs9RHmtkdhGrNzjK+4DzOW4PyGHTG6r9qaWTWLERt5XtEZjXIo1r2eIGhu9BV/EGDkI/SXXan90/2YhEAksfgAkBh6TFhBRPl4jiy5tDFoEPQKNnPxH+xBUBVIlW7VhC9+VDfqIh4HHD3Jx8rViivHla/RFXNhT0eTSfavWhMvYIN/KT7YjthU5YaPCAl9V7tijHfsqjGXv6Vf+qlzoG8pV/qjRr2ww9/K8bNls5Vjhobjpm1oYo9Zcm2qrJU9c/lwWBq2xHGrXGGcZ1nZeDxBUNV+r9VC14aO1RuQf4tR+n+d4a2+qbGIjHk5kn8MPObC/qLRibfmTXq6Rz4crfOU8st5t7ifvHkqKpCkCe+i0QxJ5MlRBc/IQybGhR7KQvPzrvgU+/Vm21UZ7BTQbN3Ew2EwsTWDlrhgqP+qLNYOLLB9yzXr5PurG66k5oyudSJSMXVUqLKpFIl1soqPJC1bRj+Ra9dD8UNxj84f+LMP8yU/hxNCy2cqxwgM7tPOZWjQHhvQ0F+RjqM55D9l133UhoHlRZZXnheZUlq3WQ9WGXmuNsJ+xT7LuRZL4i6Xlv7JJXkP7E/0qrVhb/qQXa2KoOAw/OY+od9vr/7LotKSkSJrCJifSZOOtCkn0bsiQHmC0bMq//Aj8yn6WRadqoz0Drbyi3WqiDNlUjNRMUHxEjHIs+T7qx2vlTDy5DNnQwxIYE0+lj72MBW2t3OkDq6jDQqStt6DbikW5ktdQQT8vIO4j3tKXzeyzlWPMTTaoaecztZDLWD5TbVp+nwhMmYOtvaFaD1UbCFdrhIMJ+7UewJGr1kzLf2VzKK/c14o1+5Mf6atWvLpv1eifukzePZQUSasIAAKPg6B+Nn4GKL8KpD9vmtJBNhKG2uVf93raqp6Wsiw6VRvtxE6fCsRLW4xZAxhzH7IpW9SQUo4xxyKfkGMukUiEdzUhss1oBxvkhB5yLZLMWGCjlbvsK3YWY8RR/UO1YqpklGvGLsty0s3jgk4cP+nIZsavlWOFB7Zo5zNUqvkNPlMxGvLhvv0i0DMHNc+Zc8jnPbpaD1UbKOc1oodv1n0s+MlzXP6jXGWTNvbLKlb6su1WrNkfcsSfP4ohtytXYuEa/VOX4d2j8KakcjAKNj8JKTlAY0OMmz42dMLkOpIMejo1xgkj/zE0DVbWr2QVZ4xDJI68inTjBn6bV9DKX/apwYN2FeVLPtEv18IJWbBCj1xyUdy5XffYZoyiPfWpxnbEgnbhlsddOlqI6FYPEJKr6lYuktX4xnmgPmraySkW4qlePyPTwq+VY4UHdmjn0yr4MQG30HH7KRAYm4PxLRDXyOd1ofa8nyCb9wl0adc+oLUUCZj9Cpm8h7T2pmrdqS371z4T93rFkONv+ZuCe5XHFP0x2fbuUWiSvDbDDAziGhw2QwASiVCLbJQQ98hJBhClhy3asYecCm3IYCNuxiIu2hkEbFFLlglGG4W4kMO25KSPPHr4EdlKjnyVA/Y02K2YFLNq6aKHP+wKS+41oWgnPvqQJQ6uY77ErRzQnRILsugKD8WnWrbxSW4q0qMmVsWrfmowQm9qIR7lUOlqfLDNtQrzET3GLbbTzxhVMapP+MkWtbCPelrw+OBaRZsWdiJO9HOPjawjXTaGvDmpz7UR6EVgaA5ig/44B5mX2odYS6wd1iyfag0igz5zmfXFXhT3Rdqwia7kkNU6whcxaO8SIcqW1pPakY1FsaodedqINxbyIIbYTlzET7v8R53ea/SXXKvdBCyQCCh+4kYeB1gy6qcPgBgo+hgkDQBgIAe46kcGefTUL5uqZZt+BlWAU3NPzPiJcshqEiGnzRa/DKRiwq9yJi4GEZv4pp1+7CoW1dioCvqKj5jQVxzUsXAvHJCtJhDYjMWS88YHfsmnKprIyoVaRXq05Xglg74Wi9p6amy2cJM+GJCzMEQHjGir8CFHYs5FY4q+PsjoWjVyFR7VmEsn18RaFWzzcTECcxHIc611n9c6a0JrgPnJnqY5nddgtWchgz56KnrYZD1qb8Av99pfkcUebbE9xx1jINa4FxJv7Memcol2lE9sy3qKfazGxpJr9b8ddiwS9xuBEQRYIBXpjajdkN/cBTJme4v9LOglF/UWc3ZM20VAhLWnNbiV0TABb2UkLjwOTr6cRucUnjL3tPhNwHNmiXWWQsAEvBSy43ZNwOMYWaKBAEQCeep105zTL6ZNwA2AL7D56dOn/3ud//Dhw8Pnz583lc379+8Pd+/e/V+sL1682FSc5wiGh2fWID/ucjkvAibg8+J9Vd74+QwLFyKufg7bmyw24ucaT8N6WFGe3F9jEQG/efNmcnqQ37179w4fP36crAvBv3z58nD//v0v5tLjx49vfl5J/6tXrw4Qb1WIl7HZGwHneQkGLudDwGifD2t7MgJXj8BcAoZ0IV8IYCp5Q6ycZh88eHAkWwHNl4Vo14NPy/ZeCVg4uV4HARPwOrjbqxG4SgTmEjAnT5EkNnqL/A295ub0KzkTcC+yljsHAibgc6BsH0ZgJwiMEV0FAwT55MmT4++P9r6GFmn3yOuEbQKuRsBtayFgAl4Lefs1AleIwBwChhQ5wb579+74xagWUQqy+CWq3p/bIteSxR8n8Fa//Lo2AqdEwAR8SjRtywjsHIE5BIyOCFf6Q6+UgRiihDD52W/ri1V5KJDj58VVMQFXqLhtaQRMwEsjbPtGYEcIiEBFqGOpQ4p8SUrffBYRDhErr6whaAi45/XzWAz0y69PwD1oWeZUCJiAT4Wk7RgBIzD6ZacMEYQdSU8/qx16HRxlxk7K2V/r3gTcQsbtSyJgAl4SXds2AjtDYMoJGCJ99OjR8fQrqGSjRa7x578tGdnqrU3AvUhZ7pQImIBPiaZtGYGdIyDyhNDGCidf5HMRGXIKruz4BJwR8/2lImACvtSRc9xGYIMI9BJw/DkuRNv6VARtAt7gwDukWQiYgGfBZiUjYAQqBHoJmJPt0Otjfcu59SUr+Wn1V7ENtenUHX8ePSTvPiNwCgRMwKdA0TaMgBG4QUDEWL06jhAhNyQTf85byYkwh76sFf2NXcueCXgMKfefEgET8CnRtC0jsHMEeggYcuUvX/EaulXiK+rWSVm+ek/B2Pz9999LlybgEhY3LoyACXhhgG3eCOwJAZFidWoVDsj0nDRFiqf4Mhbk+/z58/9941oxyVdPXNJxbQRui4AJ+LYIWt8IGIEjAmME/Pr16+4/+QgZ6stZnHI/fPhw9KOLeFLmj3fwXxJ++vRJ3TfXtI2duE3AR8h8cUYETMBnBNuujMC1I9Ai4EiUItXWX7uqZKWD/arwWvvZs2fHvyWNPPb1/wFXOrHNBBzR8PW5EDABnwtp+zECO0CgRcBbT90EvPURus74TMDXOa7OygisgoAJeBXY7fRCETABX+jAOWwjsEUETMBbHBXHtFUETMBbHRnHZQQuEAET8AUOmkNeDQET8GrQ27ERuD4ERMD60hR16/d418w+/qGPGKt/DWnNUdmfbxPw/sbcGRsBI2AEjMAGEDABb2AQHIIRMAJGwAjsDwET8P7G3BkbASNgBIzABhAwAW9gEByCETACRsAI7A8BE/D+xtwZGwEjYASMwAYQMAFvYBAcghEwAkbACOwPARPw/sbcGRsBI2AEjMAGEDABb2AQHIIRMAJGwAjsDwET8P7G3BkbASNgBIzABhAwAW9gEByCETACRsAI7A+BfwCCI6r2y6N7YQAAAABJRU5ErkJggg==)
AS= activity of sample in [u], AC= activity of control [u]
Factors optimization for protease expression
Various parameters influencing enzyme production during submerged fermentation were optimized. The strategy was to optimize each parameter individually and subsequently combine the optimal conditions to obtain an increase in the yield. Various parameters optimized for maximal enzyme production, in sequential order are as follows: incubation period, pH, temperature, agitation speed, size and age of inoculum, substrate concentration, fermentor volume, different nitrogen sources, and different carbon sources.
Incubation time
A 250 ml conical flask containing 100 mL of TSB [17 g/L tryptone, 3 g/L soy peptone, 2.5 g/L glucose, 5 g/L NaCl, and 2.5 g/L K2HPO4] was used to investigate the impact of incubation period on protease production. All the flasks were autoclaved at 121 °C and 15 lb pressure for 20 min. The media were cooled down and then evenly distributed inoculums were added. The incubation period was set at 37 ± 1°C for 24, 48, 72, 96, and 120 hours. Afterward, the culture filtrate was collected, and the enzyme activity was assessed.
pH
To observe the effect of pH on enzyme production, 100 mL of TSB was taken in 250 ml conical flask. The initial pH of the medium was adjusted to 6-11 before autoclaving at 121°C for 15 min. All the flasks were inoculated with equal quantity of fresh inoculum and incubated at 37o C. At the harvesting points, the fermented broth was centrifuged at 10,000 rpm at 4oC for 10min.The protease activity was measured using cell free filtrate.
Temperature
Equal amounts of culture were added to a series of 250 ml conical flasks containing 100 ml of TSB in order to find out the effect of temperature on protease production. To find the ideal temperature for protease production, each flask was incubated at 30, 40, 45, 50, 55, 60, and 70°C.
Agitation
To examine the impact of speed on protease synthesis, different agitation speeds (100, 125, 150, 175 and 200 rpm) were used during flask fermentation. In the harvesting points, the fermented broth was centrifuged for ten minutes at 4°C at 10,000 rpm. A cell-free filtrate was used to measure protease activity.
Substrate Concentrations and Nitrogen Sources
A range of casein concentrations (1-5% w/v) was used to examine the impact of substrate concentration on protease generation. Several nitrogen sources were added to the medium, including peptone, beef extract, meat extract, casein, gelatin, urea, sodium nitrate, and ammonium chloride at a concentration of 1% (w/v). After that, the mixture was incubated. Upon completion of fermentation, enzyme activity was determined.
Carbon Sources
To determine the effect of various carbon sources on protease production by TAS-2, the production medium was enriched with different carbon sources such as glucose, lactose, fructose, sucrose, xylose, maltose, raffinose, and starch. These sources were added at a concentration of 0.5% (w/v), and incubated. The enzyme activity was then measured at the end of the fermentation process. After that, the ideal carbon source for maximum enzyme production was identified by modifying its concentration to 0.5%, 1%, 1.5%, 2%, and 2.5% (w/v).
Protease Purification
The 500 mL basic culture medium [1.0g/L K2HPO4, 1.0g/L MgSO4·.7H2O, 5.0g/L NaCl] was combined with all the ideal parameters in a 1000 mL vessel, and the mixture was fermented for 72 hours under ideal conditions. A cell-free filtrate was used to measure the enzyme activity of the fermentation broth containing crude TAS-2 protease at harvest time. The broth was centrifuged at 10,000 rpm for 10 min at 4°C.Centrifugation was used to separate the culture supernatant from the growth medium, and then ammonium sulfate precipitation (at 60,70 and 80% saturation) was used. The precipitate was obtained by centrifuging it at 7500 rpm for 20 minutes at 4°C, and the pellet that was produced was then resuspended in Tris-HCl (0.1M, pH 8.0). By dialyzing against a Tris-HCl (0.1M, pH 8.0), the ammonium sulfate was eliminated. A DEAE-cellulose column from Sigma-Aldrich in Missouri, USA, was used to bind the dialyzed product, and a solution of 0.5 M NaCl and Tris-HCl (0.1M, pH 8.0), was used to elute the protease at a flow rate of 0.8 mL/min. Using the Bradford assay technique, the protein concentration was determined [15]. The samples having the highest protease activity were combined and applied at a flow rate of 0.5 mL/min to a Sephadex G-200 column (Sigma-Aldrich) that had been pre-equilibrated with Tris-HCl (0.1M, pH 8.0) [14].
Enzyme Characterization
The physico-chemical characteristics of the partially purified protease were assessed.
Effect of Temperature on Protease Activity
The study aimed to evaluate the effect of temperature on the activity of proteases. The reaction mixtures were exposed to different temperatures, ranging from 25 to 60ºC, in Tris-HCl buffer. The substrate used was casein (1%), and the relative activity was measured under standard assay conditions.
Effect of pH on Protease Activity
For pH effect on protease activity, the pH levels were adjusted from 4.0 to 11.0 using different buffers, including sodium acetate (pH 4.0-5.5), sodium phosphate (pH 6.5-7.5), Tris–HCl (pH 7.2-9.0), and glycine-NaOH (pH 8.6-11.0). The reaction mixture contained 1% casein substrate and was incubated at 37 degrees Celsius for 30 minutes. After incubation, the relative activity of the enzyme was assessed.
Effect of Inhibitors on Enzyme Activity
To test effect of inhibitors on the partly purified enzyme, at optimum conditions, the partly purified protease was first pre-incubated and then incubated with different inhibitors, such as PMSF, EDTA, and DTT, at a final concentration of 5 mM for 60 minutes at 60 degrees Celsius. The residual activity of the enzyme was then measured and compared to the control group (without inhibitors).
Effect of Surfactants on Enzyme Activity
The experiment aimed to examine the effect of different surfactants, such as SDS, Triton X-100, CTAB, and Tween-60, on enzyme activity. The enzyme was pre-incubated with each surfactant separately at a final concentration of 5 mM for 1 hour at 60°C. After that, the residual activity of the enzyme was measured, and compared to the enzyme's activity without any surfactants, which was considered 100%.
Effect of Metal ions on Enzyme Activity
The effects of metal ions such as Ca2+, Mn2+, Fe2+, Zn2+, and Mg2+ on enzyme stability at the final concentration of 5mMwere investigated. The enzyme was pre-incubated with different metal ions at 5mM concentration for 1h at 60o C and then the residual activities were measured and compared with the control (without metal ions).
Effect of Temperature on Protease Stability
To determine the thermal stability of the partially purified enzyme, its residual activity was measured every 30 minutes under standard assay conditions. The enzyme was pre-incubated for 1 hour at different temperatures ranging from 55 to 70 degrees Celsius. A control group with 100% enzyme activity was used for comparison, which consisted of the enzyme that was not subjected to any heat treatment.
Effect of pH on Protease Stability
To study the effect of pH on protease stability, the partially purified protease's activities was evaluated under different pH conditions (ranging from 7 to 12) by pre-incubating it at 45°C for an hour in buffers of varying pH levels (0.1M). The buffers used included Dipotassium hydrogen phosphate [K2HPO4] (pH 6.0-7.5), Tris-HCl (pH 8.0-9.0), and Glycine-NaOH (pH 9.0-12.0). We measured the residual activity of the enzyme under standard assay conditions, with the enzyme's pre-incubation activity (100%) serving as the control.