Stable protease variants

Abstract

The present invention relates to a protease variant which is at least 90% identical to the full length amino acid sequence of a Kumamolisin AS backbone as set forth in any of SEQ ID NOs 1-3, while maintaining proteolytic activity, or a fragment, fraction or shuffled variant thereof maintaining proteolytic activity, which protease variant demonstrates altered or improved stability compared to the Kumamolisin AS wildtype as set forth in SEQ ID NO 4, or the Kumamolisin AS backbone as set forth in any of SEQ ID NOs 1-3.

Claims

1. A protease variant comprising an amino acid sequence which is at least 90% identical to SEQ ID NO:1, or a fragment, or shuffled variant thereof maintaining proteolytic activity, which protease variant has at least one or more amino acid substitutions, wherein at least one amino acid substitution occurs at the residue position of SEQ ID NO:1 corresponding to A517 of SEQ ID NO:4, wherein the protease variant has increased thermostability compared to wild type Kumamolisin Alicyclobacillus sendaiensis (AS).

2. The protease variant of claim 1, which protease variant demonstrates at least one altered or improved stability compared to the Kumamolisin AS wildtype as set forth in SEQ ID NO 4, or the Kumamolisin AS backbone as set forth in any of SEQ ID NOs: 1-3.

3. The protease variant of claim 1, which protease variant has at least the amino acid substitution as compared to the Kumamolisin AS as set forth in SEQ ID NO:1.

4. The protease variant of claim 1, which protease variant has at least 2 amino acid substitutions compared to the Kumamolisin AS backbone as set forth in SEQ ID NO:1.

5. The protease variant of claim 1, which protease variant has at least one, at least two, at least three, at least four, at least five, or at least six amino acid substitutions that occur at the residue position of SEQ ID NO:1 corresponding to residue positions of SEQ ID NO:4 selected from the group consisting of D447S, A449Y, A517T, N510H, E360L, E360V, E360C, V502C, E453W, A514T, A514Y, A514D, A514S, A460W, and A386I.

6. The protease variant of claim 1, which protease variant has a set of substitutions at selected residues in the Kumamolisin AS backbone as set forth in SEQ ID NO:1, which set corresponds to at least one of the following residue positions of SEQ ID NO:4: a) 360, 447, 449 and 510 b) 447, 449 and 514, and/or c) 447, 449, 453, and 517.

7. The protease variant of claim 1, wherein said improved stability is improved thermostability (IT50) of either the activated enzyme or the zymogen.

8. The protease variant of claim 1, which protease variant has an IT50 of between >75° C. and <105° C.

9. A composition comprising the protease variant or protease of claim 1, which composition has a pH of >5.

10. A feed additive, feed ingredient, feed supplement, and/or feedstuff comprising protease variant or protease of claim 1.

11. The protease variant of claim 1, further comprising one or more amino acid substitutions at one or more residue positions in SEQ ID NO:1 selected from the group of residue positions corresponding to residue positions in SEQ ID NO:4 consisting of A449, A517, N510, V502, E453, E360, A514, and/or A460.

12. The protease variant of claim 11, wherein the one or more substitutions at A449, A517, N510, V502, E453, E360, A514, and/or A460 are selected from the group consisting of A449Y, A517T, N510H, E360L, E360V, E360C, V502C, E453W, A514T, A514Y, A514D, A514S, A460W as compared to the corresponding residue positions of Kumamolisin AS as set forth in SEQ ID NO:1.

13. A method comprising combining the protease variant of claim 1 with an animal feed, wherein the protease variant of claim 1 improves digestibility of the animal feed.

Description

SHORT DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the distribution of mutations in variants optimized for thermal stability of the zymogen and the activated enzyme.

(2) FIG. 2 shows the effects of the ionic strength on stability and performance for the WT and top variants #1 to #7 from table 4.

(3) FIG. 3-5 show the occurrence of substitutions at AA position in different sets of distinct clones and combinatorial clones.

EXAMPLE 1: PROTEASE ACTIVITY ASSAY

(4) Protease activity assays were carried out in microtiter plates

(5) a) AAPF Assay 96 Well Formate

(6) Assay buffer: 200 mM Sodium Acetate, 1 mM CaCl.sub.2, 0.01% Triton X-100 at pH 3 depending on the experiment Substrate stock solution: 100 mM in water free DMSO Substrate working solution: Substrate Stock solution diluted 1:50 in assay buffer,

(7) Execution: Load 50 μL of the diluted sample into the wells of a Nunc 96 clear flat bottom plate. Dilution is made in water containing 0.01% Triton-X100 corresponding to the volumetric activity of the sample. Start reaction by adding 50 μL of substrate working solution. Measure kinetics at 37° C. by monitoring the increase in adsorption at 410 nm as a measure for enzymatic activity. The activity was calculated by building a calibration curve with a reference enzyme preparation of the backbone with known proteolytic activity measured by a reference method.

(8) For assaying the protease activity at different pH values the following buffers were used, each 200 mM: glycine/HCL between pH 2.0-3.0, trisodium citrate/citric acid between 3.0 and 6.0 and Tris/maleic acid between 6.0 and 7.5.

(9) b) IT.sub.50

(10) IT.sub.50 defines the temperature where 50% of the activity is inactivated under the conditions described above. Although not equivalent to, it is a measure for the thermal stability in the application, e.g. pelleting conditions or conditions in a detergent application, either dish washing or the cleaning of a fabric or hard surface and other technical applications.

(11) The screening of enzyme variants under predictive conditions is essential. For proteases like those described herein, screening for thermally more stable variants by methods as also described herein can be affected by the self-hydrolysis of the protease. As already described in patent application EP16176044 Example 9, screening for variants with higher thermal stability under conditions where the protease is active results in a large number of false positives, as the result of a mixed effect of thermal inactivation and self-hydrolysis. The same applications teaches to circumvent this problem in the absence of small molecule reversible enzyme inhibitors, as is the case for the class of acid protease described herein, by executing the test for thermal stability of the enzyme and enzyme variants in the form of the inactive enzyme zymogen in the way described below.

(12) Assay buffers: 50 mM sodium phosphate, 0.25 mM CaCl.sub.2) pH6.5

(13) 800 mM glycine/HCl pH2.8

(14) Thermal inactivation execution: Samples were diluted corresponding to the volumetric activity in potassium phosphate buffer. The pH of the final solution was checked to be above pH 6.3. The samples were transferred in replicates, 20 μL per well, into a 384 well PCR plate according to the direction of the temperature gradient of the PCR machine. The plates were sealed with an adhesive or hot melting cover foil and incubated on a thermal gradient cycler with a temperature gradient of +/−12° C. around the expected IT50 value for 10 minutes. The samples were cooled to 8° C. before measuring the residual activity of the samples with AAPF-pNA as followed. Samples, 15 μL each from the temperature incubation plate were transferred into a 384 well greiner clear flat bottom PS-microplate and 9 μL of glycine buffer was added to activate the protease during an incubation of 1 hour at 37° C. After the activation of the protease the assay was started by adding 24 μL of an AAPF-pNA solution (2 mM AAPF-pNA in water with 0.01% Triton-X100) and activity was measured by following the kinetics at 37° C. The normalized experimental data for residual activity at the inactivation temperatures were fitted to a four parameter logistics function to evaluate the IT50.

(15) c) IT50 without Propeptide—Activated Enzyme Protein:

(16) Enzyme activation prior to thermal inactivation execution. Samples were diluted corresponding to the volumetric activity in glycine buffer pH 2.8 as described in 2b) and pH was checked to be equal or lower than pH 4.0. Samples were activated by an incubation for 1 hour at 37° C. After the incubation pH was set to above 7.0 by diluting the samples 1:3 in 50 mM sodium phosphate buffer pH 8.0. Thermal inactivation of activated enzyme protein execution. Aliquots of the activated enzyme protein were transferred in replicates, 20 μL per well, into a 384 well PCR plate according to the direction of the temperature gradient of the PCR machine. The plates were sealed with an adhesive or hot melting cover foil and incubated on a thermal gradient cycler with a temperature gradient of +/−12° C. around the expected IT50 value for 10 minutes. The samples were cooled to 8° C. before measuring the residual activity of the samples with AAPF-pNA as followed. Samples, 15 μL each from the temperature incubation plate were transferred into a 384 well greiner clear flat bottom PS-microplate and 9 μL of glycine/HCl buffer was added to adjust the pH to 3.0. The assay was started by adding 24 μL of an AAPF-pNA solution (2 mM AAPF-pNA in water with 0.01% Triton-X100) and activity was measured by following the kinetics at 37° C. The normalized experimental data for residual activity at the inactivation temperatures were fitted to a four parameter logistics function to evaluate the IT50.

(17) d) pH-Profile—Activated Enzyme Protein

(18) Undiluted bacterial supernatant containing enzyme protein was titrated with 1 M HCl to pH 4 and enzyme was activated at 37° C. for 60 min. 20 μl of sample were added to 200 μL Britton Robinson buffer with pH 1.8-7.0 (adjusted to conductivity of 15 mS/cm with NaCl). 20 μL were then transferred into a 384-well Greiner flat bottom PS-microplate plus 20 μl substrate solution (2 mM AAPF-pNA in water with 0.01% Triton-X100) and activity was measured by monitoring the kinetics at 410 nm and 37° C. as described in example 1a). Each kinetic experiment was run in quadruplet.

(19) e) pH/Pepsin-Resistance

(20) Undiluted bacterial supernatant containing enzyme protein was titrated with 1 M HCl to pH 2.5. 90 μl were then transferred to a Nunc 96-well clear flat bottom microtiter plate. 10 μl of a 250 μg/mL Pepsin stock solution in pH 2.5 buffer (final concentration in assay 25 μg/mL) or pH 2.5 buffer were added to each well and then incubated at 37° C. for 30 min. Finally, 5 μl of a 100 μM Pepstatin A solution (final concentration 5 μM) was added to each well to stop the pepsin reaction. 25 μl of the sample were transferred in 175 μl glycine/HCl buffer pH 3.0 in a new Nunc 96-well clear flat bottom microtiter plate. 20 μl were then transferred into a 384-well Greiner flat bottom PS-microplate plus 20 μl substrate solution (2 mM AAPF-pNA in water with 0.01% Triton-X100) and activity was measured by monitoring the kinetics at 410 nm and 37° C. as described in example 1a). Each kinetic experiment was run in quadruplet.

(21) f) Conductivity Dependency

(22) 20 μl undiluted bacterial supernatant was diluted in 180 μL glycine/HCl buffer pH 3.0 adjusted with NaCl to conductivity of 2, 4, 6, 10, 20, 30, 40, 50 mS/cm in a Nunc 96-well clear flat bottom microtiter plate. The samples were incubated at 37° C. for 20 min and then 20 μL sample were then transferred into a 384-well Greiner flat bottom PS-microplate plus 20 μl substrate solution (2 mM AAPF-pNA in water with 0.01% Triton-X100) and activity was measured by monitoring the kinetics at 410 nm and 37° C. as described in example 1a). Each kinetic experiment was run in quadruplet.

(23) g) BBI/KTI Hydrolysis—Functional Trypsin Assay

(24) Bowman-Birk and Kunitz-type inhibitors (BBI/KTI) are strong inhibitors of serine proteases which are widely spread in seed of legumes and cereal grains. The assay principle is that a proteolytic degradation of the BBI/KTI by protease activity recovers the natural trypsin activity on Benzyl-Arginine-pNA (Bz-R-pNA) substrate without inhibitors. 90 μL of bacterial supernatant containing enzyme protein was diluted in glycine/HCl buffer to pH 3.0 and then incubated at 37° C. for 30 min. 20 μl of the sample was then mixed with 20 μl inhibitor solution (KTI: 8 μg/mL; BBI: 16 μg/mL; KTI/BBI: 4/8 μg/mL diluted in glycine buffer pH 3.0) and further incubated at 37° C. for 60 min. 15 μl of the sample were transferred into a 384-well Greiner flat bottom PS-microplate and then 15 μl trypsin solution in pH 8.0 (final trypsin concentration 1 μg/mL; final pH 7.0 or pH 7.5) was added to each well and the plate was incubated at 37° C. for 10 min. Finally, 30 μL substrate solution (2 mM Bz-R-pNA in water with 0.01% Triton-X100) was added to each well and activity was measured by monitoring the kinetics at 410 nm and 37° C. as described in example 1a). Each kinetic experiment was run in quadruplet.

EXAMPLE 2: GENERATION OF GENETIC DIVERSITY

(25) Initial genetic diversity was introduced by randomizing each position of the active enzyme core sequence of SEQ ID NO 1. Mutant enzyme single site saturation libraries were introduced in the gene carried on an E. coli/Bacillus shuttle vector using mutagenesis methods as described in Green & Sambrook (eds), Molecular Cloning, 4.sup.th edition, CSHL and suitable mutagenic PCR methods as disclosed in Cadwell and Joyce (PCR Methods Appl. 3 [194], 136-140. Protease enzyme variants were characterized after heterologous expression in Bacillus subtilis and phenotypically optimized variants selected by the screening procedure outlined in Example 3.

(26) In general, methods to mutagenize a protein, like an enzyme, to obtain a library of mutated proteins members of which may have altered characteristics, are well established. Methods to mutagenize a protein encompass site directed mutagenesis and others, as described e.g. in Hsieh & Vaisvila (2013), content of which incorporated herein by reference for enablement purposes.

(27) Such methods are sometimes called “directed evolution”, namely when the established library is then screened for particular features. Packer & Liu (2015) provide an overview of the respective methodology, content of which incorporated herein by reference for enablement purposes.

EXAMPLE 3: PHENOTYPICALLY SCREENING FOR ENZYME VARIANTS WITH INCREASED THERMAL STABILITY

(28) The generated genetic diversity either in the initial stage in form of single site saturation libraries or in the subsequent stage in the form of recombination libraries or distinct clones was screened for variants with an optimized phenotype, i.e. increased thermal stability using the method as described in example 1b) with adaptations required to run them in a fully automated robotic workstation at high throughput. These were mainly adaptation in incubation times, volumes, substrate and the main adaptation was to select optimized variants not by the thermal inactivation profile on a temperature gradient but by the residual activity after incubation at a single temperature, the temperature which was set to discriminate optimized variants from the average of the genetic diversity. Protease variants were derived which differed in one or more amino acid positions from SEQ ID NO 2, including two positions, three positions, n positions. Appropriate iterative rounds of the procedures described herein were performed to satisfy the demands of the application

EXAMPLE 4

(29) The following individual mutations which increase the IT50 compared to the used backbone were identified. The IT50 was analyzed as described above and compared to the IT50 of the used backbone (=wildtype with missing N-terminal methionine) characterizing the variant by the corresponding ΔIT50. The backbone has an IT50 of 79.6° C.+/−0.4° C. (n=46) as zymogen and an IT50 of 59° C.+/−1° C. (n=10) as activated enzyme.

(30) TABLE-US-00005 TABLE 1 Kumamolisin AS single amino acid substitutions relative to SEQ ID NO 1, and their ΔIT50 compared to the backbone for the zymogen and the activated enzyme ΔIT50 ΔIT50 activated Position Mutation Zymogen Enzyme A190 D 1.5 0.8 T196 S 0.7 0.3 D199 E 0.5 1.0 Q202 D 0.4 −0.3 I219 L 1.1 0.8 E228 Q 0.7 0.1 A229 W 0.2 n.d. S230 D 2.8 −0.8 A242 S 0.3 −0.4 Q244 C 0.5 −3.6 Q244 G 0.7 1.5 D251 S 0.8 −0.3 S262 C 0.9 −0.3 G266 A 1.7 0.0 E269 M 2.4 −0.1 E269 T 2.6 −0.1 E269 C 2.1 −1.1 E269 H 4.0 −0.5 E269 Q 2.0 −1.4 V274 I 1.8 1.3 G281 R 2.0 5.4 K283 L 0.6 −0.2 Y287 K 0.2 5.2 N291 T 0.7 0.5 N291 S −0.2 1.0 D293 Y 0.8 1.0 D293 F 1.1 1.3 L297 T 1.2 0.2 T301 S 0.6 7.6 T301 C 0.8 1.0 T301 M 0.7 0.5 H305 F 0.4 −0.4 H305 W 0.1 −2.7 D306 S 0.3 −0.5 T308 C 0.5 −0.8 V314 M 0.6 0.3 V314 L 2.5 0.7 S315P P 0.8 3.0 G320 A 3.0 −0.2 G320 Q 3.6 1.5 G320 S 1.0 0.6 S324 L 0.1 1.3 S324 R 0.7 2.0 W325 K −0.3 2.7 T326 R 1.7 1.2 T326 W 0.9 0.2 T326 L 1.7 1.6 T326 K 1.9 1.2 S327 F 1.2 0.6 S327 L 1.5 1.1 S327 W 2.0 1.0 A328 W 0.6 0.5 A328 D 1.3 1.1 A328 R 1.1 0.1 A328 Y 1.5 0.8 A329 Q 2.8 0.2 A329 H 2.1 0.3 A329 T 1.0 0.9 I330 L 1.1 0.8 A331 F 2.0 0.6 A331 Y 1.3 0.6 M333 I 2.5 −0.7 M333 Y 0.3 1.0 M333 L 2.4 −1.0 L338 R −0.5 1.5 A342 R −0.6 3.9 A351 S 1.3 −0.9 S354 E 1.6 3.3 S354 Q 2.0 0.3 D358 G −2.0 0.7 E360 L 1.4 3.1 E360 V 2.4 2.9 E360 C 2.3 2.3 Q361 C 0.9 1.5 Q361 L 0.2 0.1 A372 S 2.4 −0.7 A378 G 1.5 1.5 A386 I 3.6 0.5 A386 L 2.7 1.3 A386 V 2.1 1.2 A386 M 1.7 0.0 G388 C 0.6 −3.5 I391 W 1.7 0.6 A392 V 2.8 0.7 A392 L 3.0 0.9 A392 I 3.7 2.4 A392 M 2.3 2.0 Q393 S 0.9 0.2 D399 S 2.3 2.1 D402 E 0.6 1.7 R412 Q 0.5 2.4 R412 M 1.5 2.9 R412 E 1.8 4.4 R412 D 0.4 3.5 A418 W 2.8 0.2 E421 R 1.0 0.5 A423 V 1.1 0.8 A433 G 1.4 1.9 S434 G 1.9 0.7 S435 I 1.7 1.6 S435 R 1.8 0.5 S435 T 2.5 4.7 S435 V 1.6 2.1 L442 W 1.4 0.3 L442 W −0.7 2.4 D447 S 4.0 3.2 D447 C 3.0 1.4 D447 A 1.6 1.3 A449 Y 1.7 0.7 A449 L 0.8 0.3 A449 M 1.9 −0.9 A449 E 1.6 0.4 A449 N 1.6 3.3 E453 W 2.4 0.0 E453 Y 2.6 0.7 E453 F 1.1 −0.5 V455 I 1.2 0.3 V455 L 1.8 0.7 E459 W 0.9 −0.3 A460 W 2.6 0.5 A460 R 2.0 −0.6 T461 V 1.2 0.0 T461 C 1.2 0.6 A470 V 0.6 2.3 A475 V −0.3 3.7 A478 L 1.2 0.2 K483 A 1.5 0.7 A487 Q 0.0 1.6 Y490 W 1.5 0.3 Q497 Y 1.8 1.2 Q497 M 0.8 0.8 Q497 D 0.3 1.0 Q497 R 0.6 0.2 V502 C 2.3 1.9 V502 T 1.5 1.6 T507 L 0.2 1.0 N510 H 2.4 7.9 A514 T 2.2 1.3 A514 Y 1.3 −1.2 A514 D 1.5 1.2 A514 S 2.4 0.5 N515 G 2.0 −0.2 R516 L 0.5 1.2 R516 E 1.1 3.5 R516 I 1.2 4.3 A517 T 1.3 3.9 A517 S 0.3 7.7 Q518 G 1.6 4.1 L540 V 0.7 0.5 Q542 H 0.9 −0.2 Q542 D 1.1 0.4 Q542 S 0.4 0.5 A548 S 0.2 n.d. P551 N 0.9 −0.4 P551 R 0.6 0.3 P553 K 0.5 0.3 P553 L 0.8 0.2 R166 I 1.0 0.7 D265 T 1.7 n.d.

(31) Quite a few distinct clones and combinatorial clones as shown in Table 3 have substitutions in these positions, leading to synergistic effects in thermal stabilization, when two or more residues thereof are mutated simultaneously.

EXAMPLE 5

(32) Distinct variants were generated by introducing selected distinct mutations into the Kumamolisin AS wild-type sequence via site-directed mutagenesis. Suitable mutagenic PCR methods known in the art and standard cloning techniques as described in Green & Sambrook (eds), Molecular Cloning, 4.sup.th edition, CSHL were used. Protease enzyme variants were characterized after heterologous expression in Bacillus subtilis and phenotypically analysis using the methods described above.

(33) Combinatorial libraries, combining mutations identified in the examples provided above and outlined in Table 1 were generated by well-known PCR methods as described in Yolov and Shabarova (1990) and standard cloning techniques as described in Green & Sambrook (eds), Molecular Cloning, 4th edition, CSHL were used. Combinatorial libraries were screened for optimized variants as described in example 3.

EXAMPLE 6

(34) Distinct clones and combinatorial clones comprising two or more mutations from Table 1 were identified, the IT50 analyzed as described above and compared to the IT50 of the used backbone (=wildtype with missing N-terminal methionine) characterizing the variant by the corresponding ΔIT50. As the IT50 of the backbone was determined in the same experiment as the variant the measured IT50 of the backbone can be slightly different from the average value. Results are shown in the following Table 2a (FIG. 3 shows results in graphic form):

(35) TABLE-US-00006 TABLE 2a Distinct clones comprising selected combinations of mutations from table 1, and their ΔIT50 compared to the wildtype # Mutations in distinct clones and selected combinatorial clones 1 E360L A392V 2 T301S E360V A386I 3 E360L A386I A392V 4 E360L A392I 5 E360V A386I A392I 6 T301S G320A E360L 7 T301S E360L A3861 A3921 8 T301S E360V A392I 9 E360V A392V 10 E360L A386I 11 T301S E360L A392I 12 T301S E360L A386I 13 E360L A386I A392I 14 E360V A392V 15 E360L A386I 16 T301S E360L 17 T301S E360L A392V 18 T301S E360V A386I 19 E360V A386I 20 T301S E360V A392I 21 D199E E360V 22 E360L A386I 23 E360L A386I A392I 24 E360V A392V 25 E269T E360V A386I 26 T301S E360L A392V 27 E360L A392V 28 E360V A392I 29 T301S E360L 30 E360L A386I A392I 31 T301S E360L A3861 A392V 32 E360V A392I 33 T301S E360L A392I 34 E360V 35 E360L A386I A392V 36 T301S E360V A386I 37 E360L A386I 38 T301S E360L A3861 A392V 39 T301S E360V A392V 40 T301S E360L A3861 A392V ctd': Distinct clones comprising selected combinations of mutations from table 1, and their ΔIT50 compared to the wildtype # Mutations in distinct clones and selected combinatorial clones 1 D447S A449Y A460W V502C N510H 2 D447S A449Y E453W A460W V502C N510H 3 D447S A449Y E453W A460W V502C N510H 4 D447S A449Y A460W V502C N510H 5 D447S A449Y E453W A460W V502C N510H 6 D447S A449Y E453W A460W V502C N510H 7 D447S A449Y E453W V502C N510H 8 D447S A449Y E453W A460W V502C N510H 9 D447S A449Y E453W A460W N510H 10 D447S A449Y E453W A460W V502C N510H 11 D447S A449Y E453W A460W V502C N510H 12 D447S A449Y E453W A460W V502C N510H 13 D447S A449Y E453W A460W V502C N510H 14 D447S A449Y A460W V502C N510H 15 D447S A449Y E453W A460W V502C N510H 16 D447S A449Y A460W V502C N510H 17 D447S A449Y E453W A460W V502C N510H 18 D447S A449Y E453W A460W V502C N510H 19 D447S A449Y E453W A460W V502C N510H 20 D447S A449Y E453W A460W V502C N510H 21 D447S A449Y A460W V502C N510H 22 D447S A449Y A460W V502C N510H 23 D447S A449Y E453W A460W V502C N510H 24 D447S A449Y E453W A460W V502C N510H 25 D447S A449Y V502C N510H 26 D447S A449Y E453W A460W V502C N510H 27 D447S A449Y E453W A460W V502C N510H 28 D447S A449Y A460W V502C N510H 29 D447S A449Y E453W A460W V502C N510H 30 D447S A449Y E453W A460W V502C N510H 31 D447S A449Y E453W A460W V502C N510H 32 D447S A449Y E453W A460W V502C N510H 33 D447S A449Y E453W A460W V502C N510H 34 D447S A449Y E453W A460W V502C N510H 35 D447S A449Y E453W V502C N510H 36 D447S A449Y E453W A460W V502C N510H 37 D447S A449Y E453W A460W V502C N510H 38 D447S A449Y E453W A460W V502C N510H 39 D447S A449Y E453W A460W V502C N510H 40 D447S A449Y E453W A460W V502C N510H ctd': Distinct clones comprising selected combinations of mutations from table 1, and their ΔIT50 compared to the wildtype Mutations in distinct clones and selected IT50 ΔIT50 IT50 active ΔIT50 active # combinatorial clones Zymogen Zymogen enzyme enzyme 1 A517T 95.5 17.0 90.1 30.6 2 A517T >95 >17 90.1 30.6 3 A517T 99.5 21.0 89.2 29.7 4 A517T 97.3 18.8 89.1 29.6 5 A517T 99.4 20.9 88.8 29.3 6 A517T 96.4 17.9 88.6 29.1 7 A517T 96.4 17.9 88.5 29.0 8 A517T 99.1 20.6 88.5 29.0 9 A517T Q518G 97.8 19.3 88.5 29.0 10 A517T 98.4 19.9 88.4 28.9 11 A517T 97.7 19.2 88.4 28.9 12 A517T 98.6 20.1 88.3 28.8 13 A517T 99.5 21.0 88.2 28.7 14 A517T >95 >17 88.2 28.7 15 A517T 98.3 19.8 88.1 28.6 16 A517T 95.8 17.3 88.0 28.5 17 A517T 97.2 18.7 88.0 28.5 18 A517T 97.6 19.1 87.8 28.3 19 A517T 98.5 20.0 87.8 28.3 20 A517T Q518G 97.0 18.5 87.8 28.3 21 A517T >95 >17 87.8 28.3 22 A517T >95 >17 87.8 28.3 23 A517T 97.1 18.6 87.8 28.3 24 A517T 99.0 20.5 87.8 28.3 25 A517T 94.0 16.0 87.7 27.0 26 A517T 97.4 18.9 87.7 28.2 27 A517T Q518G 98.0 19.5 87.7 28.2 28 A517T >95 >17 87.6 28.1 29 A517T 96.5 18.0 87.6 28.1 30 A517T 99.0 20.2 87.5 28.0 31 A517T 98.1 19.6 87.5 28.0 32 A517T Q518G 97.9 19.4 87.4 27.9 33 A517T Q518G 97.1 18.6 87.4 27.9 34 A517T 95.6 17.1 87.4 27.9 35 A517T 98.2 19.7 87.4 27.9 36 A517T 98.5 20.0 87.4 27.9 37 A517T Q518G 97.9 19.4 87.4 27.9 38 A517T >95 >17 87.3 27.8 39 A517T 96.2 17.7 87.2 27.7 40 A517T Q518G >95 >17 87.1 27.6 ctd': Distinct clones comprising selected combinations of mutations from table 1, and their ΔIT50 compared to the wildtype # Mutations in distinct clones and selected combinatorial clones 41 T301S E360L A386I 42 E360V A392I 43 E360V A386I A392I 44 E360L A392V 45 D199E E360V 46 T301S E360L A386I A392I 47 D199E G266A E360V A392V 48 G266A E360V A392V 49 E360L A392I 50 T301S E360V A386I 51 E360L A386I 52 E360L A386I 53 D199E G266A E360V A392V 54 E360V A386I A392I 55 D199E G266A E269H E360V A392L 56 E360V A386I 57 E360V A392V 58 T301S E360L A386I A392I 59 D199E E360V A386I 60 E360V A386I A392I 61 D199E E360V A386I 62 E360V A386I 63 D199E G266A T301S E360L 64 D199E G266A E269T G320A E360V A392L 65 E360L A386I 66 G266A E360V A392V 67 E360L A386I 68 D199E E360V 69 D199E E360V A386I 70 D199E E360L 71 D199E G266A E269H T301S E360L 72 D199E E360L 73 D199E G266A E360V 74 D199E G266A E269H E360V A392L 75 D199E E360V 76 T301S E360L A392I 77 D199E E360V 78 D199E E360L 79 E360V A386I 41 D447S A449Y E453W A460W V502C N510H 42 D447S A449Y A460W N510H 43 D447S A449Y E453W V502C N510H 44 D447S A449Y E453W A460W N510H 45 D447S A449Y E453W A460W V502C N510H 46 D447S A449Y E453W A460W V502C N510H 47 D447S A449Y E453W A460W V502C N510H 48 R412E D447S A449Y E453W A460W 49 D447S A449Y E453W V502C N510H 50 D447S A449Y A460W V502C N510H 51 D447S A449Y E453W V502C N510H 52 D447S A449Y A460W V502C N510H 53 D447S A449Y E453W A460W V502C N510H 54 D447S A449Y E453W A460W V502C N510H 55 D447S A449Y E453W A460W V502C N510H 56 D447S A449Y E453W V502C N510H 57 D447S A449Y E453W A460W V502C N510H 58 D447S A449Y A460W V502C N510H 59 D447S A449Y V502C N510H 60 D447S A449Y E453W A460W V502C N510H 61 D447S A449Y E453W V502C N510H 62 D447S A449Y A460W V502C N510H 63 D447S A449Y E453W V502C N510H 64 D447S A449Y E453W A460W V502C N510H 65 D447S A449Y V502C N510H 66 D447S A449Y E453W A460W 67 D447S A449Y E453W V502C N510H 68 D447S A449Y E453W V502C N510H 69 D447S A449Y E453W A460W V502C N510H 70 D447S A449Y A460W V502C N510H 71 D447S A449Y E453W V502C N510H 72 D447S A449Y E453W V502C N510H 73 D447S A449Y E453W V502C N510H 74 D447S A449Y E453W A460W V502C N510H 75 D447S A449Y A460W V502C N510H 76 D447S A449Y E453W A460W N510H 77 D447S A449Y E453W V502C N510H 78 D447S A449Y E453W V502C N510H 79 D447S A449Y V502C N510H ctd': Distinct clones comprising selected combinations of mutations from table 1, and their ΔIT50 compared to the wildtype Mutations in distinct clones and selected IT50 ΔIT50 IT50 active ΔIT50 active # combinatorial clones Zymogen Zymogen enzyme enzyme 41 A517T Q518G 98.4 19.9 87.0 27.5 42 A517T 92.7 14.2 86.9 27.4 43 A517T >95 >17 86.9 27.4 44 A517T 96.9 18.4 86.8 27.3 45 A517T 93.5 15.1 86.7 26.0 46 A517T Q518G 97.4 18.9 86.7 27.2 47 A517T Q518G 101.5 23.0 86.6 27.1 48 A517T Q518G 100.3 21.8 86.6 27.1 49 A517T Q518G >95 >17 86.6 27.1 50 A517T Q518G 94.7 16.2 86.5 27.0 51 A517T >95 >17 86.5 27.0 52 A517T >95 >17 86.4 26.9 53 A517T Q518G P553K 102.2 23.7 86.4 26.9 54 A517T Q518G >95 >17 86.4 26.9 55 A517T Q518G P553K 101.7 23.2 86.3 26.8 56 A517T 93.1 14.6 86.2 27.0 57 A517T Q518G >95 >17 86.2 26.7 58 A517T Q518G >95 >17 86.2 26.7 59 A517T 92.2 14.1 86.1 25.4 60 A517T Q518G 97.2 18.7 86.0 26.5 61 A517T 93.2 14.9 85.9 26.2 62 A517T 92.4 13.9 85.9 26.6 63 A517T 95.7 17.2 85.8 26.3 64 A517T Q518G P553K 100.1 21.6 85.8 26.8 65 A517T 94.0 15.9 85.8 25.1 66 R5161 A517T Q518G 100.1 21.6 85.7 26.2 67 A517T >95 >17 85.7 26.2 68 A517T Q518G >95 >17 85.7 26.2 69 A517T 94.9 16.6 85.4 25.7 70 A517T −10.0 −10.0 85.4 25.9 71 A517T 95.8 17.3 85.4 25.9 72 A517T Q518G >95 >17 85.4 25.9 73 A517T Q518G >95 >17 85.4 25.9 74 A517T Q518G 100.4 21.9 85.3 25.8 75 A517T >95 >17 85.3 25.8 76 A517T Q518G 95.1 16.6 85.2 25.7 77 A517T 94.9 16.4 85.1 25.5 78 A517T >95 >17 85.1 25.6 79 A517T 93.0 15.0 85.0 25.7

(36) TABLE-US-00007 TABLE 3 Some preferred substitutions and their key characteristics G320 A 3.0 −0.2 2 186 Q 3.6 1.5 46 S 1.0 0.6 35 T326 R 1.7 1.2 11 W 0.9 0.2 L 1.7 1.6 1 6 K 1.9 1.2 1 1 T461 V 1.2 0.0 1 26 C 1.2 0.6 48 Q244 C 0.5 −3.6 46 G 0.7 1.5 1 1 D293 Y 0.8 1.0 1 24 F 1.1 1.3 A487 Q 0.0 1.6 1 24 V274 I 1.8 1.3 104 A372 S 2.4 −0.7 82 K283 L 0.6 −0.2 68 T308 C 0.5 −0.8 30 A418 W 2.8 0.2 12 H 1.1 1.3 16 I391 W 1.7 0.6 21 A423 V 1.1 0.8 18 A331 F 2.0 0.6 7 Y 1.3 0.6 9 S327 F 1.2 0.6 L 1.5 1.1 16 W 2.0 1.0 I219 L 1.1 0.8 16 M333 I 2.5 −0.7 16 A329 Q 2.8 0.2 5 H 2.1 0.3 3 T 1.0 0.9 7 N515 G 2.0 −0.2 13 A378 G 1.5 1.5 12 S434 G 1.9 0.7 12 E421 R 1.0 0.5 1 11 A433 G 1.4 1.9 11 S230 D 2.8 −0.8 9 Q393 S 0.9 0.2 3 D399 S 2.3 2.1 4 Y490 W 1.5 0.3 2 G281 R 2.0 5.4 Y287 K 0.2 5.2 R516 I 1.2 4.3 E 1.1 3.5 L 0.5 1.2 A475 V −0.3 3.7 S354 E 1.6 3.3 S315P P 0.8 3.0 W325 K −0.3 2.7 L442 W −0.7 2.4 W 1.4 0.3 A470 V 0.6 2.3 S324 R 0.7 2.0 S324 L 0.1 1.3 Q361 C 0.9 1.5 Q361 L 0.2 0.1 A190 D 1.5 0.8 T196 S 0.7 0.3 Q202 D 0.4 −0.3 E228 Q 0.7 0.1 A229 W 0.2 n.d. A242 S 0.3 −0.4 D251 S 0.8 −0.4 S262 C 0.9 −0.3 N291 T 0.7 0.5 N291 S −0.2 1.0 L297 T 1.2 0.2 H305 F 0.4 −0.4 H305 W 0.1 −2.7 D306 S 0.3 −0.5 V314 M 0.6 0.3 V314 L 2.5 0.7 A328 W 0.6 0.5 A328 D 1.3 1.1 A328 R 1.1 0.1 A328 Y 1.5 0.8 I330 L 1.1 0.8 M333 Y 0.3 1.0 M333 L 2.4 −1.0 L338 R −0.5 1.5 A342 R −0.6 3.9 A351 S 1.3 −0.9 S354 Q 2.0 0.3 D358 G −2.0 0.7 G388 C 0.6 −3.5 D402 E 0.6 1.7 V455 I 1.2 0.3 V455 L 1.8 0.7 E459 W 0.9 −0.3 A478 L 1.2 0.2 K483 A 1.5 0.7 Q497 Y 1.8 1.2 Q497 M 0.8 0.8 Q497 D 0.3 1.0 Q497 R 0.6 0.2 V502 T 1.5 1.6 T507 L 0.2 1.0 L540 V 0.7 0.5 Q542 H 0.9 −0.2 Q542 D 1.1 0.4 Q542 S 0.4 0.5 A548 S 0.2 n.d. P551 N 0.9 −0.4 P551 R 0.6 0.3 P553 L 0.8 0.2 R166 I 1.0 0.7 D265 T 1.7 n.d.

(37) It is further to be understood that the mutations can have positive or negative effects on other enzyme parameters, as the producibility in fermentative microbial production systems or the stability against pH-conditions or endogenous proteases of the animal, like pepsin. Testing the stability of feed enzymes at low pH and in the presence of pepsin is a standard for feed enzymes and was performed in this study as outlined in example 1e. The stability against higher ionic strength is not a standard test for feed enzymes though high ion concentrations can interfere with the enzyme stability and with the enzyme performance under such conditions and can be found for example in the gut. The secretion of acid in the gut and the feed ingredients translate to an increased ionic strength.

(38) FIG. 2 shows that the wildtype suffers from combined effects of stability and performance reduction in the presence of higher ionic strength. FIG. 2 also shows the effect of ionic strength on the top variants also shown in table 4, variants #1 to #7.

(39) The performance and stability in high ionic strength was tested as described in example 1d. The pH profile was a control parameter and tested as described in example 1f. The digestion of proteinaceous antinutritive factors like the Trypsin/chymotrypsin inhibitors BBI and KTI (Bowman-Birk inhibitors and Kunitz-type inhibitors) is a potential beneficial performance characteristic of a protease which was tested as described in example 1 g.

(40) From the 651 individual combinatorial and distinct variants tested in detail, Table 4 describes the variants consolidating a multitude of performance and stability parameters (FIG. 5 shows results in graphic form).

(41) All variants shown in table 4 are better or equally well produced in a microbial production system than the wildtype and have no relevant changes in their pH activity profile tested as described in example 1d. Table 4 ranks these variants based on the thermal stability of the activated enzyme, the pH/pepsin stability and the stability against and the performance under higher ionic strength.

(42) It was further found that the best variants can hydrolyze BBI and KTI (Bowman-Birk inhibitors and Kunitz-type inhibitors) as tested in a functional trypsin inhibition assay, which differentiates these variants from the parent enzyme, beside the high thermal stability engineered into these variants.

(43) TABLE-US-00008 TABLE 4 Some distinct and combinatorial clones with particularly good performance # mutant code Mutations in distinct clones and selected combinatoral clones  1 GIN_3B2_08.42a T301S E360V A392I D447S A449Y E453W A460W  2 Gin_48a5xa D199E G266A E360V A392V D447S A449Y E453W A460W  3 Gin_85b5xa D199E G266A E269H E360V A392L D447S A449Y E453W A460W  4 Gin_24b5xa D199E G266A E269T G320A E360V A392L D447S A449Y E453W A460W  5 GIN_3B2_08.34a E360V A386I D447S A449Y E453W A460W  6 GIN_3B2_08.58a E360L A392V D447S A449Y E453W A460W  7 Gin_48a4xa D199E G266A E360V A392V D447S A449Y E453W A460W  8 GIN_48a1xbxe G266A E360V A392V D447S A449Y E453W A460W  9 GIN_3B2_08.85c E360V A392V D447S A449Y E453W A460W 10 GIN_3B2_08.47a E360L A386I D447S A449Y E453W A460W 11 GIN_3B3_05.84c T301S E360L A392V D447S A449Y E453W A460W 12 GIN_3B2_10.87a T301S E360V A392I D447S A449Y E453W A460W 13 GIN_3B3_03.04c E360L A386I A392I D447S A449Y E453W A460W 14 GIN_3B3_01.11a E360V A392V D447S A449Y E453W A460W 15 GIN_3B3_02.55a E360V A392I D447S A449Y E453W A460W 16 GIN_3B2_09.09c E360L A386I A392V D447S A449Y E453W 17 CIN_3B3_05.09c E360L A386I D447S A449Y E453W A460W 18 GIN_3B2_09.31a T301S E360L A386I D447S A449Y E453W A460W 19 GIN_3B3_01.52a E360L A392V D447S A449Y E453W A460W 20 GIN_48a1xbxd G266A E360V A392V R412E D447S A449Y E453W A460W 21 GIN_3B3_01.21b T301S E360L A386I A392I D447S A449Y E453W A460W 22 Gin_28a5x2xf D199E G266A T301S E360L D447S A449Y E453W 23 Gin_28a5x3xa D199E G266A E269H T301S E360L D447S A449Y E453W 24 Gin_85b4xa D199E G266A E269H E360V A392L D447S A449Y E453W A460W 25 Gin_28a5x1xf D199E T301S E360L D447S A449Y E453W A460W 26 Gin_28a4xa5x D199E E360V A392V D447S A449Y E453W 27 GIN_48a1xbxg G266A E360V A392V R412D D447S A449Y E453W A460W 28 Gin_28a4xa6x D199E E360L A392V D447S A449Y E453W 29 Gin_28a5x1xg D199E T301S E360L D447S A449Y E453W 30 GIN_3B3_05.20b E360L A392V D447S A449Y E453W A460W 31 GIN_3B2_08.81c T301S E360L A386I D447S A449Y E453W A460W 32 Gin_28a5x2xd D199E G266A E269T T301S E360L D447S A449Y E453W 33 Gin_48a3xa D199E G266A A392V D447S A449Y E453W A460W pH/ pepsin Stability/ IT50 IT50 Stability perfor- (° C.) (° C.) % mance Inhibitor Mutations in distinct clones and selected Zymo- acti- residual at 25 mS hydrolysis # combinatoral clones gen vated activity cm-1 BBI KTI  1 V502C N510H A514Y A517T 99.10 88.48  93 62 48% 41%  2 V502C N510H A514Y A517T Q518G P553K 102.15 86.37  96 87 58% 46%  3 V502C N510H A514Y A517T Q518G P553K 101.73 86.34  92 62 60% 47%  4 V502C N510H A514Y A517T Q518G P553K 100.13 85.82  95 77 68% 65%  5 V502C N510H A514Y A517T 98.48 87.82  99 68 50% 48%  6 V502C N510H A514Y A517T Q518G 98.00 87.65  91 48 45% 40%  7 V502C N510H A514Y R516I A517T Q518G 101.45 86.59  92 81 58% 52%  8 A514Y A517T Q518G 100.10 85.73  88 98 75% 56%  9 N510H A514Y A517T Q518G 97.78 88.46  93 33 41% 40% 10 V502C N510H A514Y A517T 98.33 88.10  92 38 pos. pos. 11 V502C N510H A514Y A517T 97.18 87.96  90 32 pos. pos. 12 V502C N510H A514Y A517T 96.99 87.81  81 29 pos. pos. 13 V502C N510H A514Y A517T 97.06 87.79  68 24 pos. pos. 14 V502C N510H A514Y A517T 99.01 87.75 102 49 pos. 40% 15 V502C N510H A514Y A517T Q518G 97.88 87.42  92 32 pos. pos. 16 V502C N510H A514Y A517T 98.23 87.38  80 18 pos. pos. 17 V502C N510H A514Y A517T Q518G 97.85 87.35  68 32 pos. pos. 18 V502C N510H A514Y A517T Q518G 98.41 86.97  79 29 pos. pos. 19 N510H A514Y A517T 96.85 86.75  61 39 pos. pos. 20 A514Y A517T Q518G 100.30 86.59  90 89 58% 41% 21 V502C N510H A514Y A517T Q518G 98.50 86.59  94 34 pos. pos. 22 V502C N510H A514Y A517T 95.73 85.83  74 51 pos. pos. 23 V502C N510H A514Y A517T 95.81 85.38  75 34 pos. pos. 24 V502C N510H A514Y A517T Q518G 100.40 85.34  89 57 pos. pos. 25 V502C N510H A517T 94.77 84.81  95 35 55% 40% 26 V502C N510H A514Y A517T Q518G P553K 98.90 84.73  95 52 55% pos. 27 A514Y A517T Q518G 96.46 84.30  94 70 50% pos. 28 V502C N510H A514Y A517T Q518G P553K 99.08 84.08 112 33 43% pos. 29 V502C N510H A514Y A517T >97 83.84 n.d. 32 pos. pos. 30 N510H A514Y A517T >97 83.75  80 30 54% 48% 31 V502C N510H A514Y A517T 96.61 83.73  73 28 46% 46% 32 V502C N510H A517T 94.68 83.66  81 44 pos. pos. 33 V502C N510H A514Y A517T Q518G 97.99 82.38 101 42 50% pos.

(44) The following Table 5 shows the frequency of occurrence of given mutations preferred combinatorial and distinct variants. The frequency of occurrence is a measure for the role and importance of a given mutation.

(45) TABLE-US-00009 TABLE 5 Frequency of occurrence of given mutations in preferred combinatorial and distinct variants. Frequency of occurrence is a measure for the role and importance of a given mutation. Frequency of occurence Frequency of occurence in preferred in preferred combinatorial and Frequency of occurence in preferred combinatorial and distinct variants distinct variants zymogen combinatorial and distinct variants Mutation (activated enzyme (IT50 > 80° C.)) Mutation (IT50 > 85° C.) Mutation zymogen (IT50 > 85° C.) D447S 127 D447S 365 A331F 7 A449Y 127 A449Y 353 A331Y 9 A517T 127 A517T 256 A329Q/H/T 15 N510H 125 N510H 175 S435R/I 11 E360L/V 125 E360V/L 262 V274I 104 V502C 120 V502C 185 A372S 82 E453W 92 E453W 320 K283L 66 A514Y/T 84 A514Y/T 265 Q244C 46 A460W 72 A460W 308 T380C 30 A386I 56 A386I 178 A418W 28 A392V/I 50 A392V/I 392 I391W 21 T301S 44 T301S 79 A423V 18 D199E 43 D199E 30 T326L 16 Q518G 36 Q518G 250 I219L 16 P553K 20 P553K 6 S327L 16 E269T/H 12 E269T/H 133 M333I 16 G266A 19 G266A 133 N515G 13 D293Y 1 D293Y 24 A378G 12 G320A 1 G320A 265 S434G 12 R412Q 1 R412Q 74 A433G 10 E421R 1 E421R 11 S230D 9 A487Q 1 A487Q 24 Q393S 3 T461V 1 T461V 26 D399S 3 T461C 48 Y490W 3

(46) The following Table 6 shows the impact of single mutations on ΔIT50 of the zymogen or the activated form. Again, the amount of impact of a single mutation on ΔIT50 is a measure for the role and importance of a said mutation.

(47) TABLE-US-00010 TABLE 6 Impact of single mutations on ΔIT50 of the zymogen (left) or the activated form (right). The amount of impact of a single mutation on ΔIT50 is a measure for the role and importance of a said mutation. ΔIT50 ΔIT50 ΔIT50 ΔIT50 activated activated Mutation Zymogen Mutation Zymogen Mutation Enzyme Mutation Enzyme D447S 4.8 L297T 1.2 A517S 7.7 D358G 0.7 E269H 4.0 S327F 1.2 N510H 7.6 A331Y 0.6 A392I 3.7 V455I 1.2 T301S 7.6 S327F 0.6 G320Q 3.6 T461V 1.2 G281R 5.4 T461C 0.6 A386I 3.6 T461C 1.2 Y287K 5.2 G320S 0.6 G320A 3.0 A478L 1.2 S435T 4.7 A386I 0.5 A392L 3.0 R516I 1.2 R412E 4.4 A460W 0.5 D447C 3.0 I219L 1.1 R516I 4.3 A514S 0.5 S230D 2.8 D293F 1.1 Q518G 4.1 S435R 0.5 A329Q 2.8 A328R 1.1 A517T 3.9 A190D 0.5 A392V 2.8 I330L 1.1 A475V 3.7 E421R 0.5 A418W 2.8 A423V 1.1 R516E 3.5 N291T 0.5 A386L 2.7 E453F 1.1 R412D 3.5 T301M 0.5 E269T 2.6 R516E 1.1 A342R 3.4 L540V 0.5 E453Y 2.6 Q542D 1.1 D447S 3.3 A328W 0.5 A460W 2.6 G320S 1.0 S354E 3.3 Q542S 0.5 V314L 2.5 A329T 1.0 A449N 3.3 A449E 0.4 M333I 2.5 E421R 1.0 E360L 3.1 Q542D 0.4 S435T 2.5 R166I 1.0 S315PP 3.0 A329H 0.3 E269M 2.4 V410I 1.0 E360V 2.9 S354Q 0.3 M333L 2.4 S262C 0.9 R412M 2.9 Y490W 0.3 E360V 2.4 T326W 0.9 W325K 2.7 L442W 0.3 A372S 2.4 Q361C 0.9 A392I 2.4 V455I 0.3 E453W 2.4 Q393S 0.9 R412Q 2.4 A449L 0.3 N510H 2.4 E459W 0.9 L442W 2.4 T196S 0.3 A514S 2.4 Q542H 0.9 E360C 2.3 V314M 0.3 E360C 2.3 P551N 0.9 A470V 2.3 P551R 0.3 A392M 2.3 D251S 0.8 D399S 2.1 P553K 0.3 D399S 2.3 D293Y 0.8 S435V 2.1 A329Q 0.2 V502C 2.3 T301C 0.8 A392M 2.0 A418W 0.2 A514T 2.2 S315P 0.8 V502C 1.9 L297T 0.2 E269C 2.1 A449L 0.8 A433G 1.9 A478L 0.2 A329H 2.1 Q497M 0.8 S324R 1.9 T326W 0.2 A331F 2.1 P553L 0.8 D402E 1.7 Q393S 0.2 A386V 2.1 T196S 0.7 T326L 1.6 P553L 0.2 E269Q 2.0 E228Q 0.7 S435I 1.6 Q497R 0.2 G281R 2.0 Q244G 0.7 V502T 1.6 A331F 0.1 S327W 2.0 N291T 0.7 A487Q 1.6 A328R 0.1 S354Q 2.0 T301M 0.7 G320Q 1.5 E228Q 0.1 A460R 2.0 L540V 0.7 A378G 1.5 Q361L 0.1 N515G 2.0 K283L 0.6 Q361C 1.5 E453W 0 T326K 1.9 T301S 0.6 Q244G 1.5 G266A 0 S434G 1.9 V314M 0.6 L338R 1.5 A386M 0 A449M 1.9 S324R 0.6 D447C 1.4 I391W 0 V274I 1.8 A328W 0.6 A386L 1.3 T461V 0 R412E 1.8 G388C 0.6 A514T 1.3 E269T −0.1 S435R 1.8 D402E 0.6 V274I 1.3 E269M −0.1 V455L 1.8 A470V 0.6 D447A 1.3 Q542H −0.1 Q497Y 1.8 Q497R 0.6 D293F 1.3 G320A −0.2 G266A 1.7 P551R 0.6 S324L 1.3 N515G −0.2 T326R 1.7 D199E 0.5 A386V 1.2 K283L −0.2 T326L 1.7 Q244C 0.5 T326K 1.2 S262C −0.3 A386M 1.7 T308C 0.5 Q497Y 1.2 E459W −0.3 I391W 1.7 R412Q 0.5 T326R 1.2 D251S −0.3 S435I 1.7 R516L 0.5 A514D 1.2 Q202D −0.3 A449Y 1.7 P553K 0.5 R516L 1.2 P551N −0.4 D265T 1.7 Q202D 0.4 S327L 1.1 H305F −0.4 S354E 1.6 H305F 0.4 A328D 1.1 A242S −0.4 S435V 1.6 R412D 0.4 S327W 1.0 E269H −0.5 D447A 1.6 Q542S 0.4 D293Y 1.0 E453F −0.5 A449E 1.6 A242S 0.3 T301C 1.0 D306S −0.5 A449N 1.6 D306S 0.3 D199E 1.0 A460R −0.6 Q518G 1.6 M333Y 0.3 M333Y 1.0 M333I −0.7 A190D 1.5 Q497D 0.3 Q497D 1.0 A372S −0.7 S327L 1.5 A517S 0.3 T507L 1.0 S230D −0.8 A328Y 1.5 A229W 0.2 N291S 1.0 T308C −0.8 A378G 1.5 Y287K 0.2 A392L 0.9 A449M −0.9 R412M 1.5 Q361L 0.2 A329T 0.9 A351S −0.9 K483A 1.5 T507L 0.2 V455L 0.8 M333L −1.0 Y490W 1.5 A548S 0.2 A328Y 0.8 E269C −1.1 A514D 1.5 S324L 0.1 I330L 0.8 E269Q −1.4 E360L 1.4 A487Q 0 A423V 0.8 H305W −2.7 A433G 1.4 N291S −0.2 Q497M 0.8 G388C −3.5 L442W 1.4 W325K −0.3 A392V 0.7 Q244C −3.6 A328D 1.3 A475V −0.3 E453Y 0.7 D265T n.d. A331Y 1.3 L338R −0.5 V314L 0.7 R166I n.d. A351S 1.3 A342R −0.6 S434G 0.7 V410I n.d. A514Y 1.3 L442W −0.7 A449Y 0.7 A229W n.d. A517T 1.3 D358G −2 K483A 0.7 A548S n.d.

(48) It is further to be understood that some mutations of Table 1 and Table 6 can interchangeably be used to engineer thermostability in Kumamolisin As. Table 7 shows a set of variants based on variant #1 of Table 7. In the course of engineering the mutations at position 502 and 510 seemed to change the activity at extrem acidic pH, below pH 2.

(49) Excluding mutations at 502 and 510 reduced the thermostability significantly below the targeted temperature stability for the activated enzyme, as for example in Table 7, clone #2 which has a 7.8° C. reduction in thermal stability compared to clone #1. A set of distinct variants were constructed by a rational approach taking advantage of the mutations identified and shown in Tables 1 and 6 to compensate for the effect of 502 and 510. With the exception of D399S substitutions can gradually or fully compensate the effect of mutations at 502 and 510.

(50) TABLE-US-00011 TABLE 7 A set of variants based on variant #1 # Mutations in distinct clones and selected combinatorial clones 1 G266A E360V A392V D447S A449Y 2 G266A E360V A392V D447S A449Y 3 G266A E360V A392V R412E D447S A449Y 4 G266A E360V A392V R412D D447S A449Y 5 G266A E360V A392V R412Q D447S A449Y 6 G266A E360V A392V S435I D447S A449Y 7 G266A E360V A392V A433G D447S A449Y 8 G266A T326L E360V A392V D447S A449Y 9 G266A E360V A392V R412M D447S A449Y 10 G266A E360V A392V S435T D447S A449Y 11 G266A E360V A392V D447S A449Y 12 G266A T326K E360V A392V D447S A449Y 13 G266A E360V A392V D447S A449Y 14 Q244G G266A E360V A392V D447S A449Y 15 G266A E360V A392V S435V D447S A449Y 16 G266A E360V A392V D399S D447S A449Y A set of variants based on variant #1 IT50 [° C.] IT50 [° C.] # Mutations in distinct clones and selected combinatorial clones Zymogen activated 1 E453W A460W V502C N510H A514Y A517T Q518G 101.5 86.6 2 E453W A460W A514Y A517T Q518G >95 78.8 3 E453W A460W A514Y A517T Q518G 100.3 86.6 4 E453W A460W A514Y A517T Q518G 96.5 84.3 5 E453W A460W A514Y A517T Q518G 97.7 81.8 6 E453W A460W A514Y A517T Q518G 97.4 81.4 7 E453W A460W A514Y A517T Q518G 98.1 81.3 8 E453W A460W A514Y A517T Q518G 98.7 81.1 9 E453W A460W A514Y A517T Q518G 98.3 81.0 10 E453W A460W A514Y A517T Q518G 99.2 81.0 11 E453W A460W A487Q A514Y A517T Q518G 96.3 81.0 12 E453W A460W A514Y A517T Q518G 97.2 80.5 13 E453W A460W A514Y A517T Q518G 96.2 80.4 14 E453W A460W A514Y A517T Q518G 96.3 80.2 15 E453W A460W A514Y A517T Q518G 98.6 80.1 16 E453W A460W A514Y A517T Q518G 87.90 71.92

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