Team:SZ SHD/Measurement

Measurements

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Measurements

The measurement of keratinase activity was progressive since either the concentration of the enzyme or the substrate was unspecified, so was the effect. The experiments were all done in triplicates and with proper blank and controls.

It is concluded that among the 4 keratinases, KU3 was testified to possess the best performance. In terms of activity, there is a common feature that the keratinase activity is increasing with a comparably fast speed within the first 30 min, which is perfect for reactions when it comes to depilatory because of the time requirements. After reaching an optimum point of activity, it becomes decreasing, of which the optimum point is usually within 12 h. Summarizing results can be found in Proof of Concept section.



1. The effect of different concentrations of keratinases on a fixed concentration of substrates is compared to obtain the best.

An increase in 0.01 OD was considered as 1 unit of enzyme activity. The concentration of protein was verified via BCA before tests as mentioned in other sections.

Enzyme concetration (mg/ml) Correction coefficient (to 100 mg/ml)
KerBIMKU3 ori 94.5 0.95
KerAVDZ50 ori 139.73 1.4
KerBteQ7 ori 91.6 0.92
KerBIER15 ori 191.55 1.92


Test of the keratinase activity assay of KU3 on an artificial substrate: 0.1% azocasein

Concentration gradient: 0.51, 0.25, 0.125 (1, 1/2, 1/4) mg/mL.

Figure 1. Comparison of azocasein samples treated with different concentrations of KU3, before and after.


KU3   blank (0.1% azocasein+tris) 1: 1 NaOH
Sample Concentration (mg/mL) 0.51 0.25 0.125 /
OD (A440) after 1h of incubation 2.1162
2.4868
1.9620 		1.7770
1.5310
1.5429 		1.2339
1.0938
1.2115 		0.4614


Keratinase activity assay of KU3, Z50 and R15 on an artificial substrate: 0.1% azocasein (0.1g/100ml).

Concentration gradient of keratinase (1, 1/2, 1/4, 1/8, 1/16)


Figure 2. Comparison of azocasein samples treated with different concentrations of KU3, Z50, and R15, before and after.



30min KU3 average Z50 average R15 average
16 times dilution 0.4191 0.4182 0.4132 0.416833 0.4323 0.4335 0.4197 0.4285 0.4862 0.4397 0.4173 0.447733
8 times dilution 0.4765 0.4704 0.4433 0.4634 0.4521 0.4785 0.4605 0.4637 0.499 0.4491 0.4593 0.469133
4 times dilution 0.4932 0.5188 0.4999 0.503967 0.4935 0.4884 0.4774 0.486433 0.5155 0.4607 0.426 0.4674
2 times dilution 0.5532 0.536 0.5663 0.551833 0.4962 0.5019 0.4901 0.496067 0.5371 0.4795 0.4719 0.496167
Original concentration 0.6791 0.6292 0.6328 0.647033 1.0797 0.9255 0.8164 0.940533 0.9895 0.6044 0.5412 0.7117
60min KU3 average Z50 average R15 average
16 times dilution 0.4931 0.4934 0.4948 0.493767 0.4555 0.4529 0.433 0.447133 0.5419 0.5607 0.5442 0.548933
8 times dilution 0.5393 0.5854 0.5586 0.5611 0.4724 0.5017 0.4943 0.489467 0.5762 0.5726 0.5662 0.571667
4 times dilution 0.5847 0.5858 0.5952 0.588567 0.4967 0.5287 0.5105 0.511967 0.6528 0.6513 0.656 0.653367
2 times dilution 0.6266 0.601 0.6096 0.6124 0.5217 0.5418 0.5327 0.532067 0.7632 0.7071 0.6657 0.712
Original concentration 0.8886 0.8807 0.7392 0.836167 1.3083 1.3317 1.2249 1.2883 0.7519 0.816 0.7082 0.7587

Keratinase activity assay of KU3, Z50, Q7 and R15 on 0.1% cacein .
Blank Control KU3 Z50 Q7 R15
500ul of 100mM Glycine-NaOH buffer
2.5ml Na2CO3
500ul Folin reagent 		500ul of 100mM Glycine-NaOH buffer with 0.1% cacein
2.5ml Na2CO3
500ul Folin reagent 		Ori
1/2
1/4
1/10
1/100 		Ori
1/2
1/4
1/10
1/100 		Ori
1/2
1/4
1/10
1/100 		Ori
1/2
1/4
1/10
1/100

casein Gelatin BSA
KU3, Z50 Q7 R15 control KU3, 0.1% casein Z50, 0.1% casein Q7, 0.1% casein R15, 0.1% casein Control KU3 Z50 Control KU3 on 1% gelatin Z50 on 1% gelatin Q7, 1% gelatin R15 1% gelatin KU3 Z50 Q7 R15 control KU3, 0.1% BSA Z50, 0.1% BSA Q7, 0.1% BSA R15, 0.1% BSA
100 (Original concentration) 0.8874 0.8894 1.016 0.9692 0.1352 54.11511 53.87143 81.55556 43.4375 1.383 1.507 0.2418 82.10072 90.37143 52.76852 30.375 1.3925 0.9537 1.2078 1.3747 0.1336 90.56835 58.57857 99.46296 64.64063
50 (2 times dilute) 0.807 0.5874 0.8163 0.8066 0.1352 48.33094 32.3 63.06481 34.96875 0.8183 1.227 0.2418 41.47482 70.37143 40.89815 22.29167 1.2432 0.6687 0.9284 0.9545 0.1336 79.82734 38.22143 73.59259 42.75521
25 (4 times dilute) 0.5402 0.5145 0.7388 0.6594 0.1352 29.13669 27.09286 55.88889 27.30208 0.4853 0.4917 0.2418 17.51799 17.85 25.83333 20.44271 0.9109 0.4448 0.4272 0.3725 0.1336 55.92086 22.22857 27.18519 12.44271
10 (10 times dilute) 0.3288 0.1915 0.4293 0.5491 0.1352 13.92806 4.021429 27.23148 21.55729 0.4006 0.4904 0.2418 11.42446 17.75714 19.52778 17.56771 0.5214 0.3549 0.403 0.1919 0.1336 27.89928 15.80714 24.94444 3.036458
1 (100 times dilute) 0.2501 0.1365 0.2457 0.2688 0.1352 8.266187 0.092857 10.23148 6.958333 0.1352 0.3019 0.4527 0.2418 4.323741 15.06429 18.88889 7.166667 0.3252 0.1699 0.1776 0.1847 0.1336 13.78417 2.592857 4.074074 2.661458

Figure 3. Comparison of casein samples treated with different concentrations of keratinases, OD (A660) examined using Folin-phenol method, before and after.

Figure 4. Comparison of gelatin samples treated with different concentrations of keratinases, OD (A660) examined using Folin-phenol method, before and after.

Figure 5. Comparison of protease activity of different concentrations of keratinases on 0.1% casein, OD (A660) examined using Folin-phenol method.

Figure 6. Comparison of protease activity of different concentrations of keratinases on 1% gelatin, OD (A660) examined using Folin-phenol method.

Figure 7. Enzyme activity of KU3 on casein, gelatin and BSA.

Figure 8. Enzyme activity of Q7 on casein, gelatin and BSA.

Figure 9. Enzyme activity of R15 on casein, gelatin and BSA.

Figure 10. Enzyme activity of Z50 on casein, gelatin and BSA.

Figure 11. Protease activity of 4 keratinases on different natural substrates at 30 min of incubation, pH 7.5. The degree of action of keratinase on the substrate is in the order of BSA, casein, and gelatin, OD (A660) detected via Folin-Ciocalteu's phenol reagent. KU3, Z50 and Q7 all showed a relatively high enzyme activity among 4 keratinases at a higher concentration (100mg/ml) on the three substrates (casein, BSA and gelatin). Whereas, the activity of Z50 is relatively lower at a lower concentration (<50 mg/ml), compared with KU3 and Q7.


2. Measurement of different concentrations of substrates under the same concentration of keratinases to predict the optimum of enzyme activity.

KU3 and Q7 on azocasein

Blank： buffer + same procedure as sample.
Controls：azocasein only.

casein Gelatin BSA
KU3, Z50 Q7 R15 control KU3, 0.1% casein Z50, 0.1% casein Q7, 0.1% casein R15, 0.1% casein Control KU3 Z50 Control KU3 on 1% gelatin Z50 on 1% gelatin Q7, 1% gelatin R15 1% gelatin KU3 Z50 Q7 R15 control KU3, 0.1% BSA Z50, 0.1% BSA Q7, 0.1% BSA R15, 0.1% BSA
100 (Original concentration) 0.8874 0.8894 1.016 0.9692 0.1352 54.11511 53.87143 81.55556 43.4375 1.383 1.507 0.2418 82.10072 90.37143 52.76852 30.375 1.3925 0.9537 1.2078 1.3747 0.1336 90.56835 58.57857 99.46296 64.64063
50 (2 times dilute) 0.807 0.5874 0.8163 0.8066 0.1352 48.33094 32.3 63.06481 34.96875 0.8183 1.227 0.2418 41.47482 70.37143 40.89815 22.29167 1.2432 0.6687 0.9284 0.9545 0.1336 79.82734 38.22143 73.59259 42.75521
25 (4 times dilute) 0.5402 0.5145 0.7388 0.6594 0.1352 29.13669 27.09286 55.88889 27.30208 0.4853 0.4917 0.2418 17.51799 17.85 25.83333 20.44271 0.9109 0.4448 0.4272 0.3725 0.1336 55.92086 22.22857 27.18519 12.44271
10 (10 times dilute) 0.3288 0.1915 0.4293 0.5491 0.1352 13.92806 4.021429 27.23148 21.55729 0.4006 0.4904 0.2418 11.42446 17.75714 19.52778 17.56771 0.5214 0.3549 0.403 0.1919 0.1336 27.89928 15.80714 24.94444 3.036458
1 (100 times dilute) 0.2501 0.1365 0.2457 0.2688 0.1352 8.266187 0.092857 10.23148 6.958333 0.1352 0.3019 0.4527 0.2418 4.323741 15.06429 18.88889 7.166667 0.3252 0.1699 0.1776 0.1847 0.1336 13.78417 2.592857 4.074074 2.661458

Figure 12. Enzyme activity of 100 mg/ml of KU3 on different concentrations of azocasein.

Figure 13. Enzyme activity of 100 mg/ml of Q7 on different concentrations of azocasein.


KU3 and Q7 on casein and gelatin

Blank： buffer + same procedure as sample.
Controls：Casein only; Gelatin only; KU3 only; Q7 only.

OD (A660)
KU3 0min 10min 20min 30min 12h 24h 36h
Casein (w/v) sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv
1.50% 0.5 0.5123 0.489 0.500433 0.011656 0.6018 0.6038 0.628 0.6112 0.014584 0.6283 0.6266 0.6238 0.626233 0.002272 0.9107 0.9461 0.9718 0.942867 0.030678 1.109 1.105 1.147 1.120333 0.02318 0.8203 0.8499 0.856 0.842067 0.019096 0.7367 0.7321 0.7326 0.7338 0.002524
1% 0.4823 0.5304 0.5227 0.5118 0.025836 0.5223 0.5239 0.5435 0.5299 0.011805 0.5581 0.5638 0.5693 0.563733 0.0056 0.6361 0.6426 0.6157 0.631467 0.014036 0.5302 0.5902 0.5944 0.5716 0.035915 0.7499 0.7549 0.7607 0.755167 0.005405 0.5467 0.6116 0.596 0.584767 0.033877
0.50% 0.4509 0.428 0.4586 0.445833 0.015917 0.5069 0.4915 0.4965 0.4983 0.007856 0.5106 0.5087 0.5127 0.510667 0.002001 0.5868 0.5582 0.5734 0.5728 0.014309 0.455 0.4703 0.4897 0.471667 0.01739 0.5894 0.6024 0.6058 0.5992 0.008656 0.435 0.4456 0.4418 0.4408 0.00537
0.10% 0.3043 0.3024 0.3036 0.303433 0.000961 0.3313 0.3336 0.3307 0.331867 0.001531 0.3691 0.3679 0.3839 0.373633 0.008911 0.4396 0.4442 0.4379 0.440567 0.003259 0.332 0.3439 0.3385 0.338133 0.005958 0.3416 0.3403 0.344 0.341967 0.001877 0.2829 0.2931 0.2898 0.2886 0.005205
Activity (U/mg)
KU3 0min 10min 20min 30min 12h 24h 36h
Casein (w/v) sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv
1.50% 52.91005 54.21164 51.74603 52.95591 1.233444 63.68254 63.89418 66.45503 64.67725 1.543233 66.48677 66.30688 66.01058 66.26808 0.240455 96.37037 100.1164 102.836 99.77425 3.246356 117.3545 116.9312 121.3757 118.5538 2.452958 86.80423 89.93651 90.58201 89.10758 2.020702 77.95767 77.4709 77.52381 77.65079 0.267078
1% 51.03704 56.12698 55.31217 54.15873 2.733991 55.26984 55.43915 57.51323 56.07407 1.249215 59.0582 59.66138 60.24339 59.65432 0.592624 67.31217 68 65.15344 66.82187 1.485269 56.10582 62.45503 62.89947 60.48677 3.800518 79.3545 79.8836 80.49735 79.91182 0.571951 57.85185 64.71958 63.06878 61.88007 3.584856
0.50% 47.71429 45.29101 48.5291 47.17813 1.684314 53.64021 52.01058 52.53968 52.73016 0.831345 54.03175 53.83069 54.25397 54.0388 0.211728 62.09524 59.06878 60.67725 60.61376 1.514226 48.14815 49.7672 51.82011 49.91182 1.840246 62.37037 63.74603 64.10582 63.40741 0.91594 46.03175 47.15344 46.75132 46.6455 0.568285
0.10% 32.20106 32 32.12698 32.10935 0.101683 35.0582 35.30159 34.99471 35.11817 0.161989 39.0582 38.93122 40.62434 39.53792 0.943007 46.51852 47.00529 46.33862 46.62081 0.344904 35.13228 36.39153 35.82011 35.78131 0.630526 36.14815 36.01058 36.40212 36.18695 0.19863 29.93651 31.01587 30.66667 30.53968 0.550773
OD (A660)
Q7 0min 10min 20min 30min 12h 24h 36h
Casein (%w/v) sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv
1.50% 0.5271 0.5209 0.5452 0.531067 0.012626 0.5779 0.5732 0.5709 0.574 0.003568 0.5735 0.5852 0.5731 0.577267 0.006873 0.6227 0.6164 0.6094 0.616167 0.006653 0.4416 0.4513 0.4636 0.452167 0.011026 0.3283 0.3306 0.3233 0.3274 0.003732 0.2437 0.2336 0.2254 0.234233 0.009166
1% 0.4908 0.4842 0.4984 0.491133 0.007106 0.5478 0.5041 0.5391 0.530333 0.023131 0.5383 0.536 0.5491 0.541133 0.006995 0.5405 0.5415 0.5551 0.5457 0.008156 0.3574 0.3633 0.3706 0.363767 0.006612 0.295 0.2877 0.2846 0.2891 0.005339 0.238 0.2438 0.2504 0.244067 0.006204
0.50% 0.3848 0.3974 0.412 0.398067 0.013612 0.445 0.4039 0.4339 0.4276 0.021262 0.4562 0.4397 0.4394 0.4451 0.009614 0.446 0.4506 0.446 0.447533 0.002656 0.2358 0.2314 0.2401 0.235767 0.00435 0.2302 0.2204 0.2283 0.2263 0.005197 0.1927 0.2194 0.2321 0.214733 0.02011
0.10% 0.2239 0.2063 0.2153 0.215167 0.008801 0.2284 0.2284 0.2326 0.2298 0.002425 0.2535 0.2807 0.2643 0.266167 0.013696 0.3576 0.3677 0.3769 0.3674 0.009653 0.2274 0.2226 0.2132 0.221067 0.007223 0.2024 0.1899 0.1981 0.1968 0.006351 0.2133 0.2089 0.2107 0.210967 0.002212
Activity (U/mg)
Q7 0min 10min 20min 30min 12h 24h 36h
Casein (%w/v) sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv
1.50% 57.54367 56.86681 59.51965 57.97671 1.378417 63.08952 62.57642 62.32533 62.66376 0.38951 62.60917 63.88646 62.5655 63.02038 0.750369 67.98035 67.29258 66.52838 67.2671 0.726318 48.20961 49.26856 50.61135 49.36317 1.203666 35.84061 36.0917 35.29476 35.74236 0.407455 26.6048 25.50218 24.60699 25.57132 1.000701
1% 53.58079 52.86026 54.41048 53.61718 0.77575 59.80349 55.03275 58.85371 57.89665 2.525266 58.76638 58.51528 59.94541 59.07569 0.763594 59.00655 59.11572 60.60044 59.57424 0.890391 39.01747 39.66157 40.45852 39.71252 0.721874 32.20524 31.4083 31.06987 31.56114 0.582912 25.98253 26.61572 27.33624 26.64483 0.677325
0.50% 42.00873 43.38428 44.97817 43.45706 1.486053 48.58079 44.09389 47.369 46.68122 2.321172 49.80349 48.00218 47.96943 48.5917 1.049569 48.68996 49.19214 48.68996 48.85735 0.289936 25.74236 25.26201 26.21179 25.73872 0.474901 25.131 24.06114 24.92358 24.70524 0.567371 21.03712 23.95197 25.33843 23.4425 2.195445
0.10% 24.44323 22.52183 23.50437 23.48981 0.960781 24.9345 24.9345 25.39301 25.08734 0.264724 27.67467 30.6441 28.85371 29.0575 1.495168 39.0393 40.14192 41.14629 40.10917 1.053875 24.82533 24.30131 23.27511 24.13392 0.788549 22.09607 20.73144 21.62664 21.48472 0.693296 23.28603 22.80568 23.00218 23.0313 0.241494
OD (A660)
KU3 0min 10min 20min 30min 12h 24h 36h
Gelatin (w/v) sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv
1.50% 0.2159 0.2189 0.2249 0.2199 0.004583 0.3927 0.3865 0.3972 0.392133 0.005372 0.6626 0.7121 0.6791 0.6846 0.025204 0.7154 0.7663 0.718 0.733233 0.028666 0.7761 0.7713 0.7976 0.781667 0.014006 0.7234 0.768 0.7546 0.748667 0.022884 0.6038 0.5848 0.6181 0.602233 0.016705
1% 0.2064 0.1927 0.1934 0.1975 0.007716 0.2195 0.2324 0.2253 0.225733 0.006461 0.3396 0.3308 0.3397 0.3367 0.00511 0.3491 0.3397 0.349 0.345933 0.005398 0.6878 0.7007 0.7036 0.697367 0.008411 0.7287 0.7072 0.7218 0.719233 0.010977 0.5445 0.5519 0.5585 0.551633 0.007004
0.50% 0.1874 0.1891 0.1787 0.185067 0.005579 0.2104 0.2186 0.2152 0.214733 0.00412 0.2404 0.2333 0.2366 0.236767 0.003553 0.2521 0.2467 0.2504 0.249733 0.002761 0.4821 0.4933 0.4804 0.485267 0.007009 0.6245 0.6142 0.6132 0.6173 0.006255 0.3953 0.4062 0.3953 0.398933 0.006293
0.10% 0.1386 0.1376 0.1465 0.1409 0.004875 0.2092 0.2004 0.2093 0.2063 0.00511 0.2225 0.223 0.2138 0.219767 0.005173 0.2235 0.2444 0.2303 0.232733 0.01066 0.2936 0.2939 0.2911 0.292867 0.001537 0.3458 0.3457 0.3393 0.3436 0.003724 0.2445 0.2462 0.242 0.244233 0.002113
Activity (U/mg)
KU3 0min 10min 20min 30min 12h 24h 36h
Gelatin (w/v) sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv
1.50% 22.84656 23.16402 23.79894 23.26984 0.484929 41.55556 40.89947 42.03175 41.49559 0.568514 70.1164 75.3545 71.86243 72.44444 2.667108 75.7037 81.08995 75.97884 77.59083 3.033446 82.12698 81.61905 84.40212 82.71605 1.482099 76.55026 81.26984 79.85185 79.22399 2.421624 63.89418 61.8836 65.40741 63.7284 1.767745
1% 21.84127 20.39153 20.46561 20.89947 0.816462 23.22751 24.59259 23.84127 23.88713 0.683694 35.93651 35.00529 35.94709 35.62963 0.540719 36.9418 35.94709 36.93122 36.6067 0.571265 72.78307 74.14815 74.45503 73.79541 0.890043 77.11111 74.83598 76.38095 76.10935 1.16163 57.61905 58.40212 59.10053 58.3739 0.741144
0.50% 19.83069 20.01058 18.91005 19.58377 0.590352 22.26455 23.13228 22.77249 22.7231 0.435965 25.43915 24.68783 25.03704 25.05467 0.375972 26.67725 26.10582 26.49735 26.42681 0.292173 51.01587 52.20106 50.83598 51.35097 0.741672 66.08466 64.99471 64.88889 65.32275 0.661947 41.83069 42.98413 41.83069 42.21517 0.665938
0.10% 14.66667 14.56085 15.50265 14.91005 0.51592 22.13757 21.20635 22.14815 21.83069 0.540719 23.54497 23.59788 22.62434 23.25573 0.547442 23.65079 25.86243 24.37037 24.62787 1.128081 31.06878 31.10053 30.80423 30.99118 0.162679 36.59259 36.58201 35.90476 36.35979 0.3941 25.87302 26.05291 25.60847 25.8448 0.223562
Q7 OD (A660)
Gelatin (w/v) 0min 10min 20min ave sdv 12h ave sdv 24h ave sdv 36h ave sdv
1.50% 0.3274 0.2911 0.3179 0.312133 0.018825 0.6939 0.6792 0.6349 0.669333 0.030713 1.112 0.9042 0.8449 0.9537 0.140262 0.1889 0.191 0.191 0.1903 0.001212 0.3353 0.3521 0.4559 0.3811 0.065321 0.4923 0.4823 0.4896 0.488067 0.005173 0.5405 0.4091 0.4072 0.452267 0.076418
1% 0.2386 0.2217 0.222 0.227433 0.009672 0.5893 0.5927 0.537 0.573 0.031223 0.784 0.8536 0.764 0.800533 0.047032 0.1516 0.1469 0.1323 0.1436 0.010064 0.3642 0.3673 0.3614 0.3643 0.002951 0.5202 0.5671 0.5011 0.529467 0.033962 0.4768 0.3899 0.3804 0.4157 0.053127
0.50% 0.2025 0.184 0.1845 0.190333 0.01054 0.4673 0.5309 0.4957 0.497967 0.031861 0.7397 0.7774 0.8034 0.7735 0.032029 0.1345 0.1437 0.1096 0.129267 0.017642 0.3545 0.3723 0.3421 0.3563 0.01518 0.392 0.4062 0.3295 0.3759 0.040806 0.237 0.2376 0.2442 0.2396 0.003995
0.10% 0.1517 0.1506 0.1587 0.153667 0.004394 0.3274 0.2911 0.3179 0.312133 0.018825 0.325 0.3134 0.3278 0.322067 0.007635 0.1358 0.1097 0.1361 0.1272 0.015156 0.193 0.2232 0.2074 0.207867 0.015105 0.2159 0.2189 0.2309 0.2219 0.007937 0.1676 0.1572 0.1569 0.160567 0.006093
Activity (U/mg)
Q7 0min 10min 20min 30min 12h 24h 36h
Gelatin (w/v) sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv sample 1 sample 2 sample 3 ave sdv
1.50% 35.74236 31.77948 34.70524 34.07569 2.055081 75.75328 74.14847 69.31223 73.07132 3.352903 121.3974 98.71179 92.23799 104.1157 15.31239 20.62227 20.85153 20.85153 20.77511 0.132362 36.6048 38.43886 49.77074 41.6048 7.131119 53.74454 52.65284 53.44978 53.28239 0.564774 59.00655 44.66157 44.45415 49.37409 8.3426
1% 26.04803 24.20306 24.23581 24.82897 1.055871 64.33406 64.70524 58.62445 62.55459 3.408649 85.58952 93.18777 83.40611 87.39447 5.134549 16.55022 16.03712 14.44323 15.67686 1.098722 39.75983 40.09825 39.45415 39.77074 0.322191 56.79039 61.91048 54.70524 57.80204 3.707619 52.0524 42.5655 41.52838 45.3821 5.799882
0.50% 22.10699 20.08734 20.14192 20.77875 1.150612 51.01528 57.95852 54.11572 54.36317 3.478224 80.75328 84.869 87.70742 84.44323 3.49657 14.68341 15.68777 11.96507 14.11208 1.925992 38.70087 40.6441 37.34716 38.89738 1.657233 42.79476 44.34498 35.97162 41.03712 4.454804 25.87336 25.93886 26.65939 26.15721 0.436135
0.10% 16.56114 16.44105 17.32533 16.77584 0.479646 35.74236 31.77948 34.70524 34.07569 2.055081 35.48035 34.21397 35.78603 35.16012 0.833516 14.82533 11.97598 14.85808 13.88646 1.654606 21.06987 24.36681 22.64192 22.69287 1.649062 23.56987 23.89738 25.20742 24.22489 0.866512 18.29694 17.16157 17.12882 17.52911 0.665163

Velocity of keratinases KU3 and Q7 on different substrates

Azocasein (artificial substrates):

0-10 min azocasein
KU3 OD A440 Substrate concentration 0.4237mM 0.2119mM 0.0424mM 0.0212mM KU3 OD A440 Substrate concentration/mM 0.4237 0.2119 0.0424 0.0212
10min 1.891 0.5933 0.4026 0.0071 Time
/10 (V0) 0.1891 0.0593 0.0403 0.0007 30min 2.2223 0.8273 0.5001 0.0898
1/V0 5.288207298 16.85393258 24.83649309 1401.869159 /30 (V0) 0.4445 0.1655 0.1 0.018
1/S 2.36 4.72 23.6 47.2 Calculated V0 using the formula -1.34415172 0.524229916 0.043267106 0.020152664
Q7 OD A440 Substrate concentration 0.4237mM 0.2119mM 0.0424mM 0.0212mM Q7 OD A440 Substrate concentration/mM 0.4237 0.2119 0.0424 0.0212
10min 0.711 0.0422 0.0512 0.0472 Time
/10 (V0) 0.071103333 0.004216667 0.00512 0.004723333 30min 2.798 1.6365 1.0393 0.9419
1/V0 14.06403825 237.1541502 195.3125 211.7148906 /30 (V0) 0.559593333 0.327306667 0.207866667 0.188373333
1/S 2.36 4.72 23.6 47.2 Calculated V0 using the formula 0.394737045 0.370408564 0.248116343 0.175627451
a b Vmax Km R^2
1.124 -3.3969 -0.2944 -0.3309 0.8747 V0 = -0.2944[S]/-0.3309+[S]
0.0705 2.3671 0.4225 0.0298 0.8957 V0 = 0.4225[S]/0.0298+[S]
10-30 min azocasein
KU3 OD A440 Substrate concentration 0.4237mM 0.2119mM 0.0424mM 0.0212mM KU3 OD A440 Substrate concentration/mM 0.4237 0.2119 0.0424 0.0212
10-30min 0.3313 0.234 0.0975 0.0827 Time
/20 (V0) 0.0166 0.0117 0.0049 0.0041 30min 2.2223 0.8273 0.5001 0.0898
1/V0 60.36217304 85.47008547 205.1282051 241.8379686 /30 (V0) 0.4445 0.1655 0.1 0.018
1/S 2.36 4.72 23.6 47.2 Calculated V0 using the formula -1.34415172 0.524229916 0.043267106 0.020152664
Q7 OD A440 Substrate concentration 0.4237mM 0.2119mM 0.0424mM 0.0212mM Q7 OD A440 Substrate concentration/mM 0.4237 0.2119 0.0424 0.0212
10-30min 1.5126 1.0622 0.2986 0.2342 Time
/10 (V0) 0.075628333 0.05311 0.014928333 0.011711667 30min 2.798 1.6365 1.0393 0.9419
1/V0 13.22255768 18.82884579 66.9867143 85.38494379 /30 (V0) 0.559593333 0.327306667 0.207866667 0.188373333
1/S 2.36 4.72 23.6 47.2 Calculated V0 using the formula 0.394737045 0.370408564 0.248116343 0.175627451
a b Vmax Km R^2
1.124 -3.3969 -0.2944 -0.3309 0.8747 V0 = -0.2944[S]/-0.3309+[S]
0.0705 2.3671 0.4225 0.0298 0.8957 V0 = 0.4225[S]/0.0298+[S]

Casein & Gelatin (natural substrates):


0-10 min casein & gelatin 10-30 min casein & gelatin
KU3 KU3
Casein (%w/v) 0 10 Difference /10 1/V0 1/S Casein (%w/v) 10 30 Difference /20 1/V0 1/S
0.015 0.500433333 0.6112 0.110766667 0.011076667 90.27986759 160 0.015 0.6112 0.942866667 0.331666667 0.016583333 60.30150754 160
0.01 0.5118 0.5299 0.0181 0.00181 552.4861878 240 0.01 0.5299 0.631466667 0.101566667 0.005078333 196.9149984 240
0.005 0.445833333 0.4983 0.052466667 0.005246667 190.5972046 480 0.005 0.4983 0.5728 0.0745 0.003725 268.4563758 480
0.001 0.303433333 0.331866667 0.028433333 0.002843333 351.6998828 2400 0.001 0.331866667 0.440566667 0.1087 0.005435 183.9926403 2400
Q7 Q7
Casein (%w/v) 0 10 Casein (%w/v) 10 30
0.015 0.531066667 0.574 0.042933333 0.004293333 232.9192547 160 0.015 0.574 0.616166667 0.042166667 0.002108333 474.3083004 160
0.01 0.491133333 0.530333333 0.0392 0.00392 255.1020408 240 0.01 0.530333333 0.5457 0.015366667 0.000768333 1301.518438 240
0.005 0.398066667 0.4276 0.029533333 0.002953333 338.6004515 480 0.005 0.4276 0.447533333 0.019933333 0.000996667 1003.344482 480
0.001 0.215166667 0.2298 0.014633333 0.001463333 683.3712984 2400 0.001 0.2298 0.3674 0.1376 0.00688 145.3488372 2400
KU3 KU3
Gelatin (w/v) 0 10 Gelatin (w/v) 10 30
0.015 0.2199 0.392133333 0.172233333 0.017223333 58.06077027 36.66666667 0.015 0.392133333 0.733233333 0.3411 0.017055 58.63383172 36.66666667
0.01 0.1975 0.225733333 0.028233333 0.002823333 354.1912633 55 0.01 0.225733333 0.345933333 0.1202 0.00601 166.3893511 55
0.005 0.185066667 0.214733333 0.029666667 0.002966667 337.0786517 110 0.005 0.214733333 0.249733333 0.035 0.00175 571.4285714 110
0.001 0.1409 0.2063 0.0654 0.00654 152.9051988 550 0.001 0.2063 0.232733333 0.026433333 0.001321667 756.6204288 550
Q7 Q7
Gelatin (w/v) 0 10 Gelatin (w/v) 10 30
0.015 0.312133333 0.669333333 0.3572 0.03572 27.99552072 36.66666667 0.015 0.669333333 0.1903 -0.47903333 -0.02395167 -41.750748 36.66666667
0.01 0.227433333 0.573 0.345566667 0.034556667 28.93797627 55 0.01 0.573 0.1436 -0.4294 -0.02147 -46.5766185 55
0.005 0.190333333 0.497966667 0.307633333 0.030763333 32.50623036 110 0.005 0.497966667 0.129266667 -0.3687 -0.018435 -54.2446433 110
0.001 0.153666667 0.312133333 0.158466667 0.015846667 63.10475389 550 0.001 0.312133333 0.1272 -0.18493333 -0.00924667 -108.14708 550
Casein %w/v mmol Gelatin %w/v mmol Casein %w/v mmol Gelatin %w/v mmol
0.015 0.00625 0.015 0.027272727 0.015 0.00625 0.015 0.027272727
0.01 0.004166667 0.01 0.018181818 0.01 0.004166667 0.01 0.018181818
0.005 0.002083333 0.005 0.009090909 0.005 0.002083333 0.005 0.009090909
0.001 0.000416667 0.001 0.001818182 0.001 0.000416667 0.001 0.001818182

Figure 14-17: Velocity curves, plotted based on OD and calculation in the table.


Figure 18. Enzyme activity of 100 mg/ml of KU3 on different concentrations of casein.

Figure 19. Enzyme activity of 100 mg/ml of Q7 on different concentrations of casein.

Figure 20. Enzyme activity of 100 mg/ml of KU3 on different concentrations of gelatin.

Figure 21. Enzyme activity of 100 mg/ml of Q7 on different concentrations of gelatin.