Method to improve the producibility of a peptiligase enzyme

Abstract

The present invention relates to new peptiligases, which are characterized by sequences comprising two amino acid substitutions, one at position 2 and one at position 206 compared to the peptiligases in the prior art, wherein the numbering is according to the numbering of the subtilisin BPN' enzyme, as well as to methods of producing these new peptiligase variants. It further relates to a method of improving the producibility of a peptiligase in a bacillus subtilis, without impacting negatively on its activity in a coupling reaction of peptide fragments.

Description

Title: Method to improve the producibility of a peptiligase enzymeTechnical Field

[0001] The present invention relates to chemo-enzymatic synthesis, and its application in large scale production. New peptiligases are disclosed, which are enzymes that are used to synthesize polypeptides by coupling fragments thereof. They play an important role in the synthesis of active pharmaceutical ingredients. The peptiligase were derived from subtilisin BPN' enzyme. The enzymes are produced by recombinant methods. It further relates to a method of improving the producibility of a peptiligase in a bacillus subtilis, without impacting negatively on its activity in a coupling reaction of peptide fragments.Background of the Invention

[0002] Enzymatic coupling of peptide fragments with the help of peptiligases has been reported and debated academically since many years. The technology has been developed significantly since its early days, especially the tailoring of enzymes has gained much attention. For their industrial application these peptiligases need to be produced in a reproducible manner.

[0003] The peptiligases are originally derived from subtilisin BPN', which is why their amino acid sequence numbering still refers to this standard amino acid sequence, even though the peptiligases differ significantly from subtilisin BPN'. Subtilisin BPN' is an extracellular protease that depends upon calcium for its stability. When the calcium binding site (amino acids 75 to 83) of this BPN' protease was removed during development, the enzyme became instable. To stabilize it a disulfide bridge (C3- C206) was introduced by the group of Bryan and his coworkers. That disulfide bridge improved the half-life of the enzyme 17-fold. Other tested mutated variants did not have that striking effect (Strausberg et al. (1995) Nature Biotechnology, Vol 13, p 669-673 "Directed Evolution of a Subtilisin with Calcium-Independent Stability"). By replacing the serine at amino acid position 221 with a cysteine (or selenocysteine) and the proline at position 225 with a different amino acid, finally the synthesis over hydrolysis ratio (S / H ratio) could be altered to benefit its synthetic use, rather than its hydrolytic use, and the "peptiligases" were born.

[0004] In WO 2018 / 212658 further peptiligases (subtilisin BPN' mutants) were provided with one or more specific mutations in the penultimate pockets to the coupling site, i.e., in the S2' pocket and / or in the S2 pocket, thereby broadening the peptide substrate scope and improving coupling efficiency. Other mutations have been introduced to enable higher yields in specific ligating reactions (WO 2019 / 170918). Further modifications have been discovered which make these enzymes more resistant to chaotropic stress, and less temperature sensitive during the ligating reaction (PCT / EP2023 / 086713). The peptiligases that developed over the recent years show excellent performances in methods to produce peptides in an efficient way.

[0005] For successful application of the technology in commercial production of peptides, the supply of the employed enzymes which are now specifically tailored to the production of specific therapeutic peptides, such as liraglutide, for example, needs to be secure, environmentally friendly, reliable, controllable, and cost-efficient.

[0006] Generally, recombinant proteins are produced by cell culture, using either eukaryotic cells, such as mammalian cells, or prokaryotic cells, such as bacterial cells, engineered to produce the protein of interest by insertion of a recombinant plasmid containing the nucleic acid encoding the desiredprotein. The peptiligases are produced by Bacillus subtilis cultures, and were described as "very robust, easy to produce in Bacillus subtilis, and its purification is straightforward" (Toplak et al. (2016) Advanced Synthesis & Catalysis, Vol 358 (13) "Peptiligase, an Enzyme for Efficient Chemoenzymatic Peptide Synthesis and Cyclization in Water").Herein Toplak describes the term peptiligase as variant of a stable calcium-independent mutant of subtilisin BPN' (denoted as A 75-83), with the catalytic Ser221 mutated to Cys and Pro225 converted to Alanine (A).Accordingly, throughout the application a peptiligase is a mutant of subtilisin BPN' with the deletion of amino acids 75 to 83 (A 75-83), comprising mutations at S221C and P225A. The variants of the peptiligases according to the invention preferably carry a P225N mutation instead.All peptiligases disclosed in the art further contain the stabilizing mutations S3C and Q206C. Most do as well contain one or more of the additional mutations Q2K, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, N212G, Y217L, N218S, T254A and Q271E (if compared to BPN'). These latter (16) mutations may also be present in the peptiligase variants according to the invention, preferably they are.Peptiligases catalyse exceptionally efficient peptide coupling in water with a surprisingly high synthesis over hydrolysis (S / H) ratio.The term variant is used to describe enzymes that do differ in one or more amino acids in comparison to the enzyme the variant is compared with. A variant of an enzyme however keeps the same function. The sequence of a variant may differ from the comparator in a number of amino acids, often up to 5, 10, 15, or even 20%, without losing its functionality. These varying mutations may be evenly distributed along the sequence, but will not occur in the reactive sites, and will not change longer stretches entirely.According to the invention the claimed peptiligase variants may comprise additional mutations, if compared to the reference Sbtl49 (see page 19 description of sequences, and reference in Toplak 2016, and W02005017110, Figure 3). Preferably the peptiligase variant according to the invention is 80% identical to the sequence of Sbtl49, more preferably 90%, even more preferably 95% identical to the sequence of Sbtl49 (as provided at page 19), subject to the additional feature of having a S221C and a P225 substitution.If the amino acid numbering of the subtilisin BPN' is referred to in a peptiligase, the positions 75-83 do no longer occur, the numbers are skipped. If the numbering is adjusted, the S221 becomes a S212, and the P225 becomes a P216 (see Toplak et al. 2016). Throughout this application the numbering of the BPN' is used.

[0007] However, controlling the process of producing some of the most important peptiligases remained challenging. Bacillus subtilis normally secretes eight proteases. To produce a specific extracellular enzyme, you need to work with modified strains.

[0008] Attempts to increase yield of Bacillus excreted recombinant proteins comprise the modification of genes related to the biosynthetic pathway of the specific target enzyme on a chromosome of Bacillus subtilis (WO2020244527); or the more generic method to genetically alter the host cells to increase their capacity to produce expressed proteins by inactivating or deleting one or morechromosomal genes or even indigenous chromosomal regions from the corresponding wild-type Bacillus host chromosome (W02003083125). More specifically, to improve expression of proteins with disulfide bridges Kouwen in 2008 proposed genetic engineering of the Bacillus subtilis, by introduction of heterologous oxidases for proper disulfide bond formation, or by depletion of its intracellular reductase (TrxA). The latter has been tried on the formation of the peptiligase of interest but remained unsuccessful in this setting.

[0009] Accordingly, there is a need in the art to make these ligase production processes controllable, with a predictable outcome, and a shortened process time. There is also a need to improve the effectivity of the process, by gaining or obtaining higher amounts of the ligating enzymes at the same or less cost and time, as compared to the methods described today.

[0010] The method according to the invention solves or at least lessens some or all the above- mentioned problems. The ligases necessary to employ said method for specific applications are also provided.Summary of the Invention

[0011] It was surprisingly found that the disulfide bridge forming ability between amino acid residues C3 and C206 is not essential for the stability of the peptiligase. It turned out, that it can be removed without causing any significant losses in activity of these peptiligases. This discovery resulted in the development of a method which allowed for higher amounts of peptiligases to be obtained from the recombinant host cells. For this method peptiligase encoding sequences were generated and expressed in the host cell, which resulted in peptiligases without cysteine at residue positions 3 and 206 in the amino acid backbone of the peptiligases. The nucleic acid sequences encoding the peptiligase variants were altered such that the amino acid residues at position 3 and position 206 were no longer cysteines, but alternative amino acids, for example, serine and glutamine respectively, thereby enabling production thereof with significantly higher titers of the enzyme in the fermentation broth of Bacillus subtilis. The peptiligase enzyme can now be obtained therefrom as one form only, which appears to be a consequence of the enzyme's better processability, and most surprisingly without loss in activity of these ligases in their ligating reaction. Surprisingly, all this could be achieved without the need to genetically engineer the Bacillus subtilis.

[0012] Accordingly, the present invention generally relates to a method for preventing loss of obtainable amounts of the desired ligase, or for increasing obtainable amounts of a desired peptiligase variant expressed in a recombinant host cell, comprising the steps of transforming a host cell with an expression vector comprising at least one nucleic acid sequence encoding at least one peptiligase variant to produce a transformed host cell; and culturing the transformed host cell under conditions suitable for producing at least one peptiligase variant to produce at least one peptiligase variant, wherein said nucleic acid sequence is characterised in that it encodes an amino acid sequence of said peptiligase variant, wherein at position 3 and at position 206 of the amino acid sequence of said otherwise identical peptiligase, the amino acid residue is an alternative amino acid residue to cysteine, thereby affording the obtainment of an increased amount of peptiligase compared to the amount obtained when the amino acid residues are cysteines.

[0013] The invention further provides an isolated peptiligase variant, wherein said peptiligase variant comprises an amino acid substitution at the residue positions equivalent to residue position 3 and to residue position 206; wherein the amino acid positions of the peptiligase variant are numbered according to the numbering of corresponding amino acid positions in the amino acidsequence of subtilisin BPN' shown in SEQ. ID NO: 2. It is preferred that said peptiligase variant is a mature form having peptide ligating activity

[0014] According to a further aspect of the present invention there is provided a method for producing a variant of a peptiligase, comprising the steps of transforming a host cell with an expression vector comprising at least one nucleic acid sequence encoding at least one peptiligase variant according to the invention to produce a transformed host cell; and culturing the transformed host cell under conditions suitable for producing at least one peptiligase variant to produce at least one peptiligase variant. This method is characterized as producing a higher amount of peptiligase as compared to the otherwise identical method transforming the host cell with an expression vector comprising a nucleic acid sequence encoding the otherwise identical peptiligase variant with a cysteine at amino acid position 3 and at amino acid position 206.BRIEF DESCRIPTION OF THE DRAWINGSFigure 1: Figure 1 shows the "purified titer", obtained from a cultivation volume of 30 mL of the peptiligase variants that can be obtained from the culture medium, under otherwise identical conditions, with and without cysteine residues substituted at positions 3 and positions 206 of the amino acid sequence. The four pair of bars represent the results for four different peptiligases and their variants. The names of these peptiligase Ptl-079, Ptl-1613, Ptl-2502 and Ptl-400 are designated below the bars. Presented as the left bar of the pair is the amount obtained when the peptiligase has a cysteine residue at both positions, at positions 3 and 206. The right bar represents the amount of the enzymes according to the invention, wherein the cysteine residues are substituted with an alternative amino acid. Details can be found in Example 1.Figure 2: The figure shows two SDS page analysis results. The panel A shows the results for Ptl-400 with cysteine at positions 3 and 206 (lane 1, 2, 3, 5) and its variant without cysteine at positions 3 and 206 (lane 6 to 9). The panel B and shows the results for Ptl-2502 with cysteine at positions 3 and 206 (lane 1, 2, 4, 5) and its variant without Ptl-2502 (lane 6 to 9).Figure 3: The figures 3 and 4 show the enzymatic activity of four different peptiligases represented in a diagram of conversion rates given in percentage. Each peptiligase is represented as a pair of bars. The names of these peptiligase Ptl-079, Ptl-1613, Ptl-2502 and Ptl-400 are designated below the bars. In each pair the right bar represents the results of the substituted enzymes according to the invention, wherein the cysteine residues at positions 3 and 206 are substituted with an alternative amino acid, the unsubstituted ligase is presented as the left bar. The substitution in all cases is C3S and a C206Q. Figure 3 shows the results determined after 15 min, and Figure 4 shows the results determined after 240 min.Detailed Description of the preferred embodiments:The method for preventing loss of obtainable amounts of the desired ligase, and for increasing obtainable amounts thereof are described in more detail herein.Usually, to begin the production cycle, a small number of transformed recombinant host cells are allowed to grow in culture for several days. Once the cells have undergone several rounds of replication, they are transferred to a larger container where they are prepared to undergo fermentation. The media in which the cells are grown and the levels of oxygen, nitrogen and carbondioxide that exist during the production cycle may have a significant impact on the production process. Growth parameters are determined specifically for each cell line and these parameters are measured frequently to assure optimal growth and production conditions. When the cells grow to sufficient numbers and produce the recombinant protein, the recombinant protein can be harvested. Typically, the cells are engineered to secrete the polypeptide into the cell culture media, so the first step in the purification process is to separate the cells from the media. Typically, harvesting includes centrifugation and filtration to produce a Harvested Cell Culture Fluid (HCCF). The media may then be subjected to several additional purification steps that remove any cellular debris, unwanted proteins, salts, minerals or other undesirable elements.However when the recombinant plasmid containing the nucleic acid encoding a peptiligase according to the state of the art was inserted into the B.subtilis BH13, it repeatedly produced and secreted not only the one peptiligase with the desired activity, present as one band in the appropriate SDS-PAGE assay in its matured form, but secreted several different forms, presenting as several bands, which obviously was detrimental to the yield of the produced enzyme with the desired activity.The cultivation of several different peptiligase variants regularly resulted in low titers for purified enzymes. Especially, the production of Ptl-2502 (SEQ. ID NO 7), a peptiligase of significant importance for the production of the active pharmaceutically ingredient liraglutide, was low. Besides the low titer, for Ptl-2502, impaired processing of the pro-domain containing pro-enzyme was observed. During cultivation of Ptl-2502, mostly the 34 kDa pro-enzyme variant was obtained. The desired, active 30 kDa processed enzyme variant without pro-domain was only observed in a smaller portion.Herein the term pro-enzyme is meant to describe the precursor of an enzyme, requiring some change in formation or structure, usually the hydrolysis of an inhibiting fragment is required that masks an active grouping, to render it active.Herein the term matured form is meant to describe the active form of an enzyme, providing its final structure. This form is often only achieved outside of the host cell that is excreting the pro-enzyme.Apparently the intracellular and / or extracellular processing of the ligase was not efficient. First attempts have been made to control the Bacillus subtilis's production process, such as by increasing the temperature after separation from the cells to 37°C, over 40 h, or to 50°C, for 2h; or by adding external proteases to the crude excreted protein, to help it folding correctly; but these attempts remained unsuccessful. The amount of peptiligase that could be obtained remained less than desired.Instead, it was surprisingly found that the method for increasing obtainable amounts of the desired peptiligase variant expressed in a recombinant host cell as described below could solve the problem.The invention provides a method for increasing obtainable amounts of a desired peptiligase variant wherein a peptiligase is a mutant of subtilisin BPN', with the deletion of amino acids 75 to 83 (A 75- 83), comprising mutations S221C and P225A, expressed in a recombinant host cell, comprising the steps of transforming a host cell with an expression vector comprising at least one nucleic acid sequence encoding at least one peptiligase variant to produce a transformed host cell; and culturing the transformed host cell under conditions suitable for producing at least one peptiligase variant to produce at least one peptiligase variant, wherein said nucleic acid sequence is characterised in that it encodes an amino acid sequence of said peptiligase variant, wherein at position 3 and at position 206 of the amino acid sequence of said otherwise identical peptiligase, the amino acid residue is an alternative amino acid residue to cysteine, thereby affording the obtainment of an increased amount of peptiligase compared to the amount obtained when the amino acid residues are cysteines,LGenerally, a recombinant host cell may be either a eukaryotic cell, such as a mammalian cell, or a prokaryotic cell, such as a bacterial cell engineered to produce the protein of interest by insertion of a recombinant plasmid containing the nucleic acid encoding the desired protein. It is a preferred aspect of the invention that in the method the host cell is a cell of Bacillus subtilis, more preferably the cell is a cell of a Bacillus subtilis strain wherein the protease aprE is deleted, and even more preferably wherein the strain is a non-sporulating strain, and wherein the additional six proteases are also deleted; most preferably the cell is a cell of Bacillus subtilis strain BH13.The Bacillus strain which was used to express the peptiligases in the studies disclosed herein is B. subtilis BH13, wherein seven of the eight naturally occurring protease encoding genes are deleted, as described PCT / EP2023 / 086713 and in detail in Halmschlag (2020) Tailored poly-y-glutamic acid production with Bacillus subtilis 168. PhD Thesis. Rheinisch-Westfalische Technische Hochschule Aachen, Germany.The host cell is transformed with an expression vector. Usually a shuttle vector of the type pUB-110 based E. coll - B. subtilis is used, i.e., pBS42 or pBES. Said expression vector comprises at least one nucleic acid sequence encoding at least one peptiligase variant. The expression vector usually also contains a promoter. In the shuttle vector used in this study, the expression of the gene is under the control of the aprE promoter. The resulting plasmid may be propagated in E. coll and transformed into B. subtilis BH13 with the gene deletions as stated in the table below:In the method described above, the nucleic acid sequence encodes the amino acid sequence for the desired peptiligase enzyme, but which is characterized by a difference in its code as compared to the sequences known in the art, for two specific amino acid positions, i.e., position 3 and position 206 in the amino acid sequence of the peptiligase. The numbering of the amino acid sequences of the peptiligases remains the same as the numbering of the amino acid positions in the subtilisin BPN' (SEQ. ID NO 2), even though the peptiligases are characterized by a deletion of nine amino acids.Preferably in the method according to the invention the alternative amino acid residue to cysteine of the amino acid sequence of said otherwise identical peptiligase at position 3 and at position 206 is a natural amino acid, that is different from cysteine.The term residue refers to an amino-acid residue. Amino-acid residues are structures that lack a hydrogen atom of the amino group (-NH-CHR-COOH), or the hydroxyl moiety of the carboxyl group (NH2-CHR-CO-), or both (-NH-CHR-COO-); all units of a peptide chain are therefore amino-acid residues.In the more preferred method according to the invention the alternative amino acid residue of the amino acid sequence of said otherwise identical peptiligase at position 3 is selected from the group comprising serine, lysine and arginine, and the alternative amino acid at position 206 is selected from the group comprising glutamine, aspartic acid and glutamic acid. Even more preferably the alternative amino acid residue of the amino acid sequence of said otherwise identical peptiligase at position 3 is selected from the group consisting of serine, lysine and arginine, and the preferred alternative amino acid at position 206 is selected from the group consisting of glutamine, aspartic acid and glutamic acid. Most preferably, the alternative amino acid residue at position 3 is lysine or arginine (K, R), and the alternative amino acid residue at position 206 is aspartic acid (D), or the alternative amino acid residue at position 3 is serine (S) and the alternative amino acid residue at position 206 is glutamine (Q).Preferably the method as described above affords the obtainment of an increased amount of peptiligase from the cell culture medium, preferably after separation from the cells. It may then be further purified and used in the ligating reaction, preferably at large scale.By the replacement of these cysteine residues at position 3 and position 206 with alternative amino acids the amount of enzyme which can be obtained is increased, compared to the amount obtained when these amino acid residues are cysteines.Host cells of Bacillus subtilis strain BH13 producing the peptiligase variants without cysteine at positions 3 and 206 were constructed and investigated. The two cysteine residues at position 3 and 206 were first replaced with the amino acids present in the wildtype subtilisin BPN' variant serine and glutamine (C3S, C206Q). Further alternative amino acid substitutions at these two positions were tested as well, such as lysine (Lys, K) or arginine (Arg, R) at position 3 and aspartic acid (Asp, D) or glutamic acid (Glu, E) at position 206. When the cysteine residues at position 3 and 206 were substituted with the amino acids serine and glutamine respectively, the obtained amount of enzyme could be increased for the peptiligases Ptl-079, a peptiligase well suited to ligate fragments of, for example, calcitonin, and also for the peptiligase Ptl-2502, a peptiligase optimized to ligate fragments of liraglutide. Due to the observed increased production and processing of these variants with the cysteine replaced with alternative amino acids of Ptl-079 and Ptl-2502, the substitutions at position C3 and C206 were explored with further enzyme variants including Ptl-1613, a peptiligase optimized to ligate fragments of semaglutide and Ptl-400, a peptiligase well suited to ligate fragments of thymosin alpha.For all investigated enzymes an improved cultivation was obtained, resulting in an increased amount of obtainable enzyme, when the amino acid positions C3 and C206 were substituted, compared to enzyme production from ligases with cysteine in both positions. In these examples the alternative amino acid at position 3 was serine and the alternative amino acid at position 206 was glutamine.Accordingly, the invention further provides for an isolated peptiligase variant, wherein a peptiligase is a mutant of subtilisin BPN' with the deletion of amino acids 75 to 83 (A 75-83), comprising mutations at S221C and P225A, wherein said peptiligase variant comprises an amino acid substitution of the amino acid cysteine at the residue positions equivalent to residue position 3 and to residue position 206; wherein the amino acid positions of the peptiligase variant are numbered according to the numbering of corresponding amino acid positions in the amino acid sequence of subtilisin BPN1shown in SEQ ID NO: 2. It is preferred that said peptiligase variant is a mature form having peptide ligating activity.It is preferred that said peptiligase variant has the mutation P225N.Said variant shows an improved producibility performance in a method of recombinant production involving a bacillus subtilis strain, as compared to the otherwise identical peptiligase variant with cysteine residues at position 3 and 206.Preferably the substituted amino acid residue at position 3 is selected from the group comprising serine, lysine and arginine (S, K and R) and the substituted amino acid residue at position 206 is selected from the group comprising glutamine, aspartic acid and glutamic acid.Preferred pairwise combinations of amino acid substitutions at positions 3 and 206 are selected from the group comprising C3S and C206Q, C3K and C206D, C3K and C206E, C3R and C206D and C3R and C206E.It is further preferred that the peptiligase according to the invention is a variant of one of the peptiligases selected from the group comprising of Ptl-079 (SEQ ID NO: 5), Ptl-2502 (SEQ ID NO: 7), Ptl-1613 (SEQ ID NO:3) and Ptl-400 (SEQ ID NO: 13), wherein the amino acid substitution of the amino acid cysteine at the residue positions equivalent to residue position 3 and to residue position 206 are selected from the group comprising C3S and C206Q, C3K and C206D, C3K and C206E, C3R and C206D, and C3R and C206E.Specifically preferred isolated peptiligase variants are the variant of Ptl-1613 wherein the amino acid substitutions are C3S and C206Q (SEQ ID NO: 3), the variant Ptl-2502, wherein the amino acid substitutions are C3S and C206Q (SEQ ID NO: 7), the variant Ptl-400, wherein the amino acid substitutions are C3S and C206Q (SEQ ID NO: 13), and the variant Ptl-079, wherein the amino acid substitutions are C3S and C206Q (SEQ ID NO: 5).In another aspect of the invention the peptiligases according to the invention may differ in their sequence from the basic peptiligase also in other positions, which are of no significance to the peptiligases activity, i.e. which are not located at their active sites, not in the pockets as described in the art.The sequence identity of these peptiligase amino acid sequences to the amino acid sequence of peptiligase Ptl-1613 represented as SEQ ID NO: 3 preferably is 80%, 85%, 90%, 95% or 99%.Accordingly, preferably the variant according to the invention has a sequence identity of 80%, 85%, 90%, preferably 95% or most preferably 99% to SEQ ID NO:3.Surprisingly, this peptiligase variant has substantially the same ligating activity as the otherwise identical peptiligase variant without an amino acid substitution at the residue positions equivalent to residue position 3 and to residue position 206.Accordingly, it is a further aspect of the invention that the isolated peptiligase variant according to the description above, is characterised by having an increased, or at least even, ligating activity as compared to the otherwise identical peptiligase variant without an amino acid substitution at the residue positions equivalent to residue position 3 and to residue position 206.The specific peptiligases disclosed as SEQ ID NO: 15 and SEQ ID NO: 16 represent Ptl-1613 variants with the specific mutations C3S and C206Q (SEQ ID NO: 15) and P222H, H217R, D99R, S224V and C3S and C206Q (SEQ ID NO: 16). These may be disclaimed. It is a preferred embodiment of the invention wherein these two peptiligases are disclaimed.A further aspect of the invention is a DNA encoding a variant peptiligase as described above. A further aspect of the invention is an expression vector encoding said DNA. A further aspect of the invention is host cell transformed with said expression vector.Another aspect of the invention is a method for producing a variant of a peptiligase, wherein a peptiligase is a mutant of subtilisin BPN' with the deletion of amino acids 75 to 83 (A 75-83), comprising mutations at S221C and P225A comprising the steps of a) transforming a host cell with an expression vector comprising at least one nucleic acid sequence encoding at least one peptiligase variant according to the invention to produce a transformed host cell; b) culturing the transformed host cell under conditions suitable for producing at least one peptiligase variant to produce at least one peptiligase variant, wherein said peptiligase variant has substantially the same ligating activity as the same peptiligase variant without an amino acid substitution at the residue positions equivalent to residue position 3 and to residue position 206; wherein the method is characterized in producing a higher amount of peptiligase as compared to the method using an expression vector comprising a nucleic acid sequence encoding the same peptiligase variant without an amino acid substitution at the residue positions equivalent to residue position 3 and to residue position 206.It is preferred that the method comprises a further step of harvesting the produced peptiligase variant. Further it is preferred that in said method for producing a variant of a peptiligase the host cell is a Bacillus species, more preferably wherein the Bacillus species is Bacillus subtilis.In a further aspect the invention provides for a method on how to generate an improved peptiligase, said method is described as a method for improving the performance of a peptiligase in a coupling reaction of two suitable peptide fragments comprising the steps of(a) substituting the amino acid at position 3 in a selected peptiligase to produce a peptiligase wherein the substitution alters the expression rate and / or the processing of the peptiligase;(b) substituting the amino acid at position 206 in said selected peptiligase to produce a peptiligase wherein the substitution alters the expression rate and / or the processing of the peptiligase;(c) testing the variant with both positions altered to determine its activity compared to the selected peptiligase activity in an activity assay suitable to determine the activity in said coupling reaction of two suitable peptide fragments of said peptiligases,(d) repeating steps (a) to (c) as necessary to produce a peptiligase variant that is more, or at least, as active than the originally selected peptiligase.The method affords a new peptiligase that is easier to produce in a recombinant process, as described above. Any peptiligase may be selected as originally selected peptiligase.It is however preferred that in the method described above said originally selected peptiligase is a variant of the peptiligase selected from the group consisting of Ptl-079 (SEQ ID NO: 5), Ptl-2502 (SEQ ID NO: 7), Ptl-1613 (SEQ ID NO:3) and Ptl-400 (SEQ ID NO: 13) with an amino acid sequence identity of 80%. It is further preferred that said originally selected peptiligase is selected from the group consisting of Ptl-079 (SEQ ID NO: 5), Ptl-2502 (SEQ ID NO: 7), Ptl-1613 (SEQ ID NO:3) and Ptl-400 (SEQ ID NO: 13).ABBREVIATIONSSPPS Solid Phase Peptide SynthesisCTC 2-chloro-trityl chlorideAEEA 2-[2-(2-aminoethoxy)ethoxy]acetylCbz BenzyloxycarbonylFor FormylFmoc 9-FluorenylmethoxycarbonylBoo Tert-butyloxycarbonylSmoc 2.7-d isu If o-9-f I uorenyl methoxycarbonylAc AcetylPhAc PhenacetylTrt Trityl (triphenylmethyl) tBu Tert-butylPbf 2.2.4.6.7-Pentamethyl-dihydrobenzofuran-5-sulfonyl eq Equivalent h hour / s min minute / sHPLC High Performance Liquid ChromatographyDIPEA N,N-Diisopropylethyl amineTFA Trifluoroacetic acidTIS TriisopropylsilaneAC2O Acetic anhydrideDMF N,N-DimethylformamideDMA N,N-DimethylacetamideDCM DichloromethaneTHF TetrahydrofuranNMP N-Methyl-2-pyrrolidinoneMTBE Methyl-tert-butyletherMeOH MethanolDCC N,N‘-DicyclohexylcarbodiimideEDC N-(3-dimethylaminopropyl)-N'-ethylcarbodiimideHOBt 1-HydroxybenzotriazoleHOAt 1 - Hy d roxy-7-azabenzotri azol eTCEP tris(2-carboxyethyl)phosphineTricine N-(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)glycineOCam ester carboxamidomethyl esterExperimental SectionMethods:Production of peptide fragments (for use) according to the inventionUnless stated otherwise, chemicals were obtained from commercial sources and used without further purification. Analytical HPLC was performed on an HP1090 Liquid Chromatograph, using a reversed- phase column (Phenomenex, C18, 5 pm particle size, 150 x 4.6 mm) at 40°C. UV detection was performed at 220 nm using a UV-VIS 204 Linear spectrometer. The gradient program for 5+3 CEPS assay was: 0-1 5%B, followed by 1-8.5 isocratic 10%B, a gradient 8.5-8.6 min of 35% followed by 8.6- 10 min isocratic 95%B and from 10.1-12.3 5%B. The gradient program for Liraglutide CEPS was 0-1 5%B, followed by 1-8.5 isocratic 10%B, a gradient 8.5-8.6 min of 95% followed by 8.6-10 min isocratic 95%B and from 10.1-12.3 5%B (eluent A: 0.5 mL / L methane sulfonic acid (MSA) in H2O, eluent B 0.5 mL / L MSA in acetonitrile). The flow was 1 mL / min. Injection volumes were 20 pL.Preparative HPLC was performed on a Varian PrepStar system using a stationary-phase column (Pursuit XRs, C18, 10 pm particle size, 500 x 41.4 mm). LC-MS was performed on an Agilent 1200 series Liquid Chromatograph, using a reversed-phase column (Phenomenex, C18, 5 pm particle size, 150 x 4.6 mm) at 40°C. UV detection and gradient program were as described for analytical HPLC. The molecular weights were determined using an Agilent 6130 quadrupole LC / MS system.Protocol 1: Peptide-OCam-Leu-OH esters preparation1 g of Fmoc-Leu-Wang resin (with a loading of 0.72 mmol / g) was washed with DCM (2 x 2 min, 10 mL) and DMF (2 x 2 min, 10 mL) and Fmoc-deprotected using piperidine / DMF (1 / 4, v / v, 2 x 8 min, 10 mL). After washing with DMF (2 x 2 min, 10 mL), DCM (2 x 2 min, 10 mL) and DMF (2 x 2 min, 10 mL), iodoacetic acid (4 eq) was coupled to the resin using DCC (4 eq) and HOAt (4 eq) in DCM (45 min, 10 mL). After washing with DMF (2 x 2 min, 10 mL), DCM (2 x 2 min, 10 mL) and THF (2 x 2 min, 10 mL), the resin was loaded with a Fmoc-protected amino acid using 4 eq. Fmoc-XXX-OH and 10 eq. DIPEA in DMF / THF (1 / 1, v / v, 10 mL) at 50°C for 20h. Here and in other parts of this disclosure 'XXX' stands for one amino acid (variable depending on the target peptide, as indicated in the examples below).After washing with DMF (2 x 2 min, 10 mL), DCM (2 x 2 min, 10 mL) and DMF (2 x 2 min, 10 mL), standard SPPS protocols were followed to elongate the peptide. Cleavage from the resin and sidechain deprotection was performed using a mixture of TFA, TIS and water (95 / 2.5 / 2.5, v / v / v, 15 mL) for 120 min. The crude peptide was precipitated using MTBE / n-heptanes (1 / 1, v / v, 50 mL). The precipitated peptide was collected by centrifugation and washed twice with MTBE / n-heptanes (1 / 1, v / v, 50 mL) followed by lyophilization from acetonitrile / water (1 / 1, v / v, 50 mL).Protocol 2: C-terminal amide peptide nucleophiles preparation1 g of Rink resin (4-((2,4-dimethoxyphenyl)-(Fmoc-amino)methyl)-phenoxyalkyl linker, with a loading of 0.64 mmol / g) was washed with DCM (2 x 2 min, 10 mL) and DMF (2 x 2 min, 10 mL) and Fmoc- deprotected using piperidine / DMF (1 / 4, v / v, 2 x 8 min, 10 mL). Standard SPPS protocols were followed to elongate the peptide. Cleavage from the resin and side-chain deprotection was performed using a mixture of TFA / TIS / water (95 / 2.5 / 2.5, v / v / v, 15 mL) for 120 min. The crude peptide was precipitated using MTBE / n-heptanes (1 / 1, v / v, 50 mL). The precipitated peptide was collected by centrifugation, washed twice with MTBE / n-heptanes (1 / 1, v / v, 50 mL) and dried in vacuo. Prior to enzymatic ligation, the crude peptides were purified by preparative HPLC followed by lyophilization of the pure fractions.Protocol 3: C-terminal amide peptide nucleophiles preparationA preloaded Wang resin (Fmoc-Xxx-Wang-resin with a loading of 0,3 mmol / g) was washed with DCM (2 x 2 min, 10 mL) and DMF (2 x 2 min, 10 mL) and Fmoc-deprotected using piperidine / DMF (1 / 4, v / v, 2 x 8 min, 10 mL). Standard SPPS protocols were followed to elongate the peptide. Cleavage from the resin and side-chain deprotection was performed using a mixture of TFA / TIS / water (95 / 2.5 / 2.5, v / v / v, 15 mL) for 120 min. The crude peptide was precipitated using MTBE / n-heptanes (1 / 1, v / v, 50 mL). The precipitated peptide was collected by centrifugation, washed twice with MTBE / n-heptanes (1 / 1, v / v, 50 mL) and dried in vacuo. Prior to enzymatic ligation, the crude peptides were purified by preparative HPLC followed by lyophilization of the pure fractions.Protocol 4: N-Acetyl-protected peptide activated ester preparationAfter SPPS of the desired sequence according to one of the protocols 1 or 2, the resin bound peptide was Fmoc-deprotected using piperidine / DMF (1 / 4, v / v, 2 x 8 min, 10 mL). The resin was washed with DMF (2 x 2 min, 10 mL), DCM (2 x 2 min, 10 mL) and DMF (2 x 2 min, 10 mL) and the peptide N-terminal amine function was acetylated using a mixture of Ac2O (10 vol%), DIPEA (5 vol%), HOBt (0.2 wt%) in DMF (2 x 10 min, 10 mL). The resin was washed with DMF (3 x 2 min, 10 mL) and DCM (3 x 2 min, 10 mL). Cleavage from the resin and side-chain deprotection was performed using a mixture of TFA / TIS / water (95 / 2.5 / 2.5, v / v / v, 15 mL) for 120 min. The crude peptide was precipitated usingMTBE / n-heptanes (1 / 1, v / v, 50 mL). The precipitated peptide was collected by centrifugation, washed twice with MTBE / n-heptanes (1 / 1, v / v, 50 mL) and dried in vacuo. Prior to enzymatic ligation, the crude peptides were purified by preparative HPLC followed by lyophilization of the pure fractions.Production of enzymes (or enzyme variants) (for use) according to the inventionMutagenesis, Cloning and ExpressionThe plasmids (pBES, Takara) and genes for enzyme variants with the positions 3 and 206 substituted with alternative amino acids to cysteine and their parental genes were either ordered (Ranomics, GenScript) or constructed by PCR and isothermal DNA assembly as described previously for example in PCT / EP2023 / 086713.For Bacillus subtilis transformation, strain B. subtilis BH13 was grown in 5 mL LB culture in a culture tube at 37°C, 200 rpm shaking overnight. 1 mL of preculture was used to inoculate a 10 mL MC medium culture in a 100 mL shake flask. The MC medium contained per liter: 14.04 g K2HPO4x3H2O, 5.24 g KH2PO4, 20 g glucose, 8.8 g sodium citrate, 0.22 g ammonium ferric citrate, 3.35 mM MgSO4, 1 g casein hydrolysate, and 2 g potassium glutamate. The MC medium culture was incubated for 4 h at 37°C, 200 rpm shaking. Then, 400 mL of bacterial culture was mixed with the assembly reaction mix in a clean 1.5 mL tube. The tube containing the culture was incubated at 37°C, 200 rpm, shaking for an additional 2h. Afterward, the transformation mix was streaked on LB agar plates containing 10 pg / ml Kanamycin. The LB agar plates contained per liter: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, and 15 g agar. The agar plates were incubated at 37°C for 18h. Per the transformation plate, one bacterial colony was picked with a pipet tip and used to inoculate 5 mL of liquid LB medium containing 10 pg / ml Kanamycin. The liquid LB medium contained per liter: 10 g tryptone, 5 g yeast extract, and 10 g sodium chloride. The liquid culture was incubated overnight at 37°C, 200 rpm shaking. To create a glycerol stock of the created strain, 750 pl of the overnight culture was mixed with 250 pl of 60% v / v sterile glycerol in Milli-Q water. 4 ml of the overnight culture was used to isolate the plasmid using the plasmid isolation kit (available from Qiagen). The correctness of the plasmid was checked by DNA sequencing (Eurofins).Small scale enzyme generation:The small-scale enzyme generation was carried out in 100 mL shake flasks at 37°C, 200 rpm shaking for 72h. Per strain, 30 mL TB medium containing 10 pg / ml Kanamycin were inoculated with 400 pl of preculture. The preculture was performed as follows: 5 mL LB medium containing 10 pg / ml Kanamycin in a sterile 14 mL tube were inoculated from a glycerol stock of the corresponding strain by transferring cell material into the medium using a pipet tip. The preculture was incubate at 37°C, 200 rpm for 16-18h. The LB medium for precultures contained per liter: 10 g tryptone, 5 g yeast extract, and 10 g sodium chloride. The TB medium for main cultures contained per liter: 12 g tryptone, 24 g yeast extract, 4 g glycerol, 2.31 g KH2PO4, 12.54 g K2HPO4.Enzyme purification:For enzyme purification, the cells were separated by centrifugation (4°C, 3700 rpm, 90 min). The cell- free supernatant was transferred to a falcon tube containing 2 ml Co-NTA Agarose XL resin (Cube Biotech) in 2 ml buffer A. Buffer A contained 50 mM potassium phosphate buffer, 300 mM sodiumchloride, pH 7.5. The supernatant and resin mix was incubated at 4°C on an orbital shaker for lh. Afterwards, the liquid was transferred to Econo-Pac disposables columns (20 ml, BioRad, 1.5x12 cm; CAT# 7321010), left to drop out and the flow-through liquid was discarded. The resin was washed with 50 column volumes (CV) of buffer A to remove unbound impurities. Subsequently, each column was placed over a Vivaspinl5R centrifuge tube and the enzyme was eluted with 15 mL of buffer B. Buffer B contained 50 mM potassium phosphate buffer, 300 mM NaCI, and 500 mM imidazole, pH 7.5. Per sample, 100 pl of 100 mg / ml TCEP solution, pH 8, was added and the samples were incubated for 30 min at room temperature. Subsequently, the samples were centrifuged (4°C, 3700 rpm, 90 min) to concentrate to less than 1 mL. After centrifugation, 15 mL of buffer D were added. Buffer D contained 200 mM potassium phosphate buffer, pH 7.5. The samples in buffer D were centrifuged (4°C, 3700 rpm, 90 min) to concentrate to less than 1 mL. The addition of buffer D and subsequent centrifugation were repeated twice. Afterwards, the volume of the remaining liquid was adjusted to 500-1000 pl and the liquid containing the enzyme was transferred to a clean 1.5 mL tube. The protein concentration was determined with Nanodrop measurement using buffer D as blank and the program set to: 1 Absorbance unit = 1 mg / ml.Examples:Example 1: Production titer of variants with and without substitution of the cysteine residues at positions 3 and 206Cultivations were carried out as quadruplicates for each enzyme variant in batch cultivations at 37°C for 72h. Enzymes were purified using IMAC purification. Purified enzyme was stored in storage buffer. The enzyme concentration was determined with Nanodrop measurement and the purity was determined with SDS-PAGE. The titer was determined as obtained pure enzyme in mg / l.Table 2The titer is improved, i.e., the amount of enzyme obtained from the culture medium is increased, for the peptiligase variants without cysteine residues at positions 3 and 206 compared to those unsubstituted peptiligases, in fact a 3-8-fold titer improvement was observed. This result is illustrated in Figure 1.Example 2: Purity and processing of variants with and without substitution of the cysteine residues at positions 3 and 206For analysing the purity of the enzymes the SDS-PAGE technology was used, as is described below: The samples were diluted 2x to 20x with MQ water to obtain enzyme concentrations of 0.2 to 0.6 pg / pl. Per sample, 26 pl of sample were mixed with 4 pl of lOx reducing agent and 10 pl of 4x SDS loading buffer. The sample mix was denatured at 95°C for 5 min. The denatured samples were loaded onto a 4-12% Bis-Tris gel (available from Thermo Fisher) in lx MES running buffer. The MES running buffer was obtained by dilution of 20x concentrated purchased MES running buffer. 500 pl of Novex NuPAGE antioxidant were added to the gel chamber. The sample loading volume was adjusted depending on the protein concentration to obtain a protein loading of approx. 2 pg on the gel. 10 pl of Novex sharp were used as molecular weight standard. The gel was run at 200V for 35 min.For staining of the gel, the gel was incubated in 25 mL of Instant Blue staining for a minimum of lh on an orbital shaker. Afterwards, the staining solution was removed, and the gel was incubated in MQ water for a minimum of lh on an orbital shaker. The stained gel was analyzed using a densitometer GS900 (BioRad) and ImageLab analysis software. Protein bands were detected with 50% sensitivity.For determining the active enzymes concentration only the amount of the 30 kDa band is considered, i.e. the determined value is corrected by subtracting the respective amount of the 34kDa band.Regarding enzyme purity, the improvement of the enzyme's processing in the process of B.subtilis cultivation and harvesting of the enzyme is most striking for Ptl-2502: While cultivation and harvesting of the peptiligase variant of Ptl-2502 with the cysteine residues results in only 30% active enzyme of 30 kDa, properly processed, and the remaining 70% remain unprocessed 34 kDa pro-enzyme, the cultivation and harvesting of the peptiligase variant with the cysteine residues substituted with a different natural amino acid at positions 3 and 206 results in a fully processed enzyme only.This is illustrated in Figure 2. Panel B. The peptiligase with cysteine at positions 3 and 206 is displayed in lane 1, 2, 4 and 5, and peptiligase variants of Ptl-2502 without cysteine residues at positions 3 and 206 are displayed in lanes 6 to 9.This improved processing is also observed for Ptl-400 with substituted cysteine residues at positions 3 and 206. The 34 kDa band of unprocessed enzyme is only obtained if the variant is expressed which has a cysteine at both positions each (Ptl-400), at position 3 and at position 206. This result is illustrated in Figure 2, panel A. The peptiligase variant Ptl-400 with cysteine residues at positions 3 and 206 is displayed in lane 1, 2, 3 and 5, and the variant of Ptl-400 with the cysteine residues substituted according to the invention is displayed in lanes 6 to 9 of said SDS page analysis result. Lane 4 in panel A and lane 3 in panel B show the molecular weight standard.Example 3: Enzymatic activity with CEPS 5+3 assay:To determine the enzymatic activity of said peoptiligases, a CEPS assay with enzymatic coupling of a 5-amino acid ester (Ac-DFSKL-OCam-Leu-OH) and a 3-amino acid amine (H-ALR-NH2) was carried out. A coupling solution with 10 mM ester and 15 mM amine in MilliQ water was prepared and filtered with a 0.22 pm syringe filter. The enzyme samples were diluted to concentrations of 0.05 mg / ml with buffer D. Per enzyme reaction, 2.5 pl of enzyme (0.05 mg / ml), 2.5 pl Tris(2-carboxyethyl)phosphine (TCEP) solution (lOOmg / ml, pH 8.4 in MilliQ water), 70 pl of potassium phosphate buffer (IM, pH 8.5),and 25 pl of the prepared coupling solution were mixed. At time points 15 min, 60 min, 120 min, 180 min, and 240 min, 10 pl of sample were taken from the reaction mix and quenched in 190 pl of MilliQ water with 0.5 % v / v Methanesulfonic acid (MSA). The samples were analyzed using the Analytical HPLC as described above.The area ratio of the product peak, hydrolyzed ester peak, and ester peak with retention times of 6.9 min, 7.1 min, and 10.1 min, respectively, were determined by integration of the peaks. The conversion was determined as the area percentage of the product peak. The synthesis over hydrolysis ratio (S / H ratio) was determined by dividing the integrated product peak by the integrated hydrolyzed ester peak.Table 3The results of this table (%conversion) are illustrated in Figures 3 and 4.For all samples tested the initial activity of all peptiligase variants substituted according to the invention is increased. Even by a staggering 45% for substituted variants of Ptl-079 after 240 min. Moreover, the final activity is unexpectedly improved with accompanying improvements in the S / H ratio.Example 4: Testing different amino acid residues in position 3 and position 206 in variants of the peptiligase Ptl-2502To analyse the effect that the substituted amino acid has on the ligating activity of the peptliligase, the peptiligase Ptl-2502 was selected and its activity tested in a Liraglutide CEPS assay for the coupling of the 1-11 ester fragment (HAEGTFTSDVS-OCam-FK-NHj) and the 12-31 amine fragment (SYLEGQAAK(E-Palm)EEFIAWLVRGRG) was carried out. The peptide fragments were prepared by weighing in 1 mg of amine and 0.792 mg (=1.4 eq) of ester in a vial. The fragments were dissolved in 180 pl of 50 mM Tricine buffer with 220mM KTFA at pH 8.5. 0.5 pl of Tris(2-carboxyethyl)phosphine (TCEP) solution (lOOmg / ml, pH 8.4 in MilliQ) were added to the peptide fragment solution. The pH was adjusted to 7.8-8.1 using 3M KOH or 10% TFA in milliQ water. The enzyme samples (corrected for purity) were diluted to concentrations of 0.25 mg / ml with buffer D. The enzymatic reaction was started by adding 20 pl of diluted enzyme (0.25 mg / ml) to 180 pl of peptide fragment solution. At time points 15 min, 60 min, 120 min, 180 min, and 240 min, 10 pl of sample were taken from the reaction mix and quenched in 250 pl of MilliQ water with 0.5 % v / v Methanesulfonic acid (MSA). The quenched samples were analyzed with HPLC as described above.The cultures of Ptl-2502 variants wherein these have different alternative amino acids at positions 3 and 206 had higher titers than Ptl-2502 with cysteine and showed a higher percentage of the mature, processed 30 kDa enzyme.The ligating activity of the investigated Ptl-2502 and its variants, substituted at positions 3 and 206, was determined in CEPS reactions for Liraglutide synthesis. Although variations of the initial activity within the first hour are observed, the final conversion is comparable for all investigated variants.Table 4SequencesSEQ ID NO: 1: wild type gene encoding for subtilisin BPN' amino acids -107 to 275ENA | K02496 | K02496.1 B. Subtilisin BPN1Bacillus amyloliquefaciensGTGAGAGGCAAAAAAGTATGGATCAGTTTGCTGTTTGCTTTAGCGTTAATCTTTACGATGGCGTTCGGCAGCACATCCTCTGCCCAGGCGGCAGGGAAATCAAACGGGGAAAAGAAATATATTGTCGGGTTTAAACAGACAATGAGCACGATGAGCGCCGCTAAGAAGAAAGATGTCATTTCTGAAAAAGGCGGGAAAGTGCAAAAGCAATTCAAATATGTAGACGCAGCTTCAGCTACATTAAACGAAAAAGCTGTAAAAGAATTGAAAAAAGACCCGAGCGTCGCTTACGTTGAAGAAGATCACGTAGCACATGCGTACGCGCAGTCCGTGCCTTACGGCGTATCACAAATTAAAGCCCCTGCTCTGCACTCTCAAGGCTACACTGGATCAAATGTTAAAGTAGCGGTTATCGACAGCGGTATCGATTCTTCTCATCCTGATTTAAAGGTAGCAGGCGGAGCCAGCATGGTTCCTTCTGAAACAAATCCTTTCCAAGACAACAACTCTCACGGAACTCACGTTGCCGGCACAGTTGCGGCTCTTAATAACTCAATCGGTGTATTAGGCGTTGCGCCAAGCGCATCACTTTACGCTGTAAAAGTTCTCGGTGCTGACGGTTCCGGCCAATACAGCTGGATCATTAACGGAATCGAGTGGGCGATCGCAAACAATATGGACGTTATTAACATGAGCCTCGGCGGACCTTCTGGTTCTGCTGCTTTAAAAGCGGCAGTTGATAAAGCCGTTGCATCCGGCGTCGTAGTCGTTGCGGCAGCCGGTAACGAAGGCACTTCCGGCAGCTCAAGCACAGTGGGCTACCCTGGTAAATACCCTTCTGTCATTGCAGTAGGCGCTGTTGACAGCAGCAACCAAAGAGCATCTTTCTCAAGCGTAGGACCTGAGCTTGATGTCATGGCACCTGGCGTATCTATCCAAAGCACGCTTCCTGGAAACAAATACGGGGCGTACAACGGTACGTCAATGGCATCTCCGCACGTTGCCGGAGCGGCTGCTTTGATTCTTTCTAAGCACCCGAACTGGACAAACACTCAAGTCCGCAGCAGTTTAGAAAACACCACTACAAAACTTGGTGATTCTTTCTACTATGGAAAAGGGCTGATCAACGTACAGGCGGCAGCTCAGTAASEQ ID NO: 2: wild type subtilisin BPN' (mature)>SUBT_BACAM Subtilisin BPN1Bacillus amyloliquefaciens mature 1 to 275AQSVPYGVS QIKAPALHSQG YTGSNVKVAV IDSGIDSSHP DLKVAGGASM VPSETNPFQD 60NNSHGTHVAGTVAA LNNSIGVLGVAPSASLYAVKVLGADG SGQYSWIINGIEWAIANNMDVINMSLGGPS 130GSAALKAAVDKAVASGWWAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFS 190SVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPNWTNTQVRSSL 250ENTTTKLGDSFYYGKGLINVQAAAQThe sequence of SEQ ID NO 2 is the reference sequence that is used for the numbering.As mentioned before, peptiligases have been developed further, based on the subtilisin from B.amyloliquefaciens (BPN'). Bryan discloses the sequence that is referred to as Sbtl49 in the art and which is herein used as basic reference sequence to describe the peptiligases, in WO 2005 / 017110 in Figure 3, lane 1. Sbtl49 is characterised by the lack of the calcium binding site A 75-83, and the stabilizing mutations S3C and Q206C, as well as these additional mutations: Q2K, P5S, S9A, 13 IL, K43N, M50F, A73L, E156S, G166S, G169A, S188P, N212G, Y217L, N218S, T254A and Q271E.For illustrative purposes Sbtl49 has the following sequenceAKCVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSETNPFQD 60NNSHGTHVAGTVLAVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGP 129SGSAALKAAVDKAVASGVVVVAAAGNSGTSGSSSTVSYPAKYPSVIAVGAVDSSNQRAPF 189SSVGPELDVMAPGVSICSTLPGGKYGALSGTCMASPHVAGAAAULSKHPNWTNTQVRSS 249LENTATKLGDSFYYGKGUNVEAAAQ 275In the following all sequences are displayed with the His tag.The peptiligase Ptl-1613 which was analysed in this study in comparison to its improved versions can be described as based on Sbtl49+S221C with the following additional mutations known in the art M222P, L217H, P225N, F189W, S218D, S156K, S166D, N62A and L96I. It has the following sequence:SEQ ID NO: 3AKCVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSETNPFQDNASHGTHVAGTVLAVAPSASLYAVKVIGADGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNKGTSGSSSTVDYPAKYPSVIAVGAVDSSNQRAPWSSVGPELDVMAPGVSICSTLPGGKYGAHDGTCPASNHVAGAAALILSKHPNWTNTQVRSSLE NTATKLG DS FYYG KG LI N VE AAAQH H H H H HOne variant of this peptiligase according to the invention is provided in this sequence:SEQ ID NO: 4AKSVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSETNPFQDNASHGTHVAGTVLAVAPSASLYAVKVIGADGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNKGTSGSSSTVDYPAKYPSVIAVGAVDSSNQRAPWSSVGPELDVMAPGVSIQSTLPGGKYGAHDGTCPASNHVAGAAALILSKHPNWTNTQVRSSLE NTATKLG DS FYYG KG LI NVE AAAQH H H H H HThe peptiligase Ptl-079 which was analysed in this study in comparison to its improved versions can be described as a peptiligase of the type Sbtl49+S221C+M222P+L217H+P225N+F189W+ll07V has the following sequence:SEQ ID NO: 5AKCVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSETNPFQDNNSHGTHVAGTVLAVAPSASLYAVKVLGADGSGQYSWVINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNSGTSGSSSTVSYPAKYPSVIAVGAVDSSNQRAPWSSVGPELDVMAPGVSICSTLPGGKYGAHSGTCPASNHVAGAAALILSKHPNWTNTQVRSSLE NTATKLG DS FYYG KG LI NVE AAAQH H H H H HOne variant of this peptiligase according to the invention is provided in this sequence:SEQ ID NO: 6AKSVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSETNPFQDNNSHGTHVAGTVLAVAPSASLYAVKVLGADGSGQYSWVINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNSGTSGSSSTVSYPAKYPSVIAVGAVDSSNQRAPWSSVGPELDVMAPGVSIQSTLPGGKYGAHSGTCPASNHVAGAAALILSKHPNWTNTQVRSSLE NTATKLG DS FYYG KG LI NVE AAAQH H H H H HThe peptiligase Ptl-2502 which was analysed in this study in comparison to its improved versions can be described as based on Sbtl49+S221C with the following additional mutations known in the art M222H, L217R, P225N, F189W, S218D, S156K, S166D, N62A, L96I, S224V and D99R. It has the following sequence:SEQ ID NO: 7AKCVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSETNPFQDNASHGTHVAGTVLAVAPSASLYAVKVIGARGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNKGTSGSSSTVDYPAKYPSVIAVGAVDSSNQRAPWSSVGPELDVMAPGVSICSTLPGGKYGARDGTCHAVNHVAGAAALILSKHPNWTNTQVRSSLE NTATKLG DS FYYG KG LI NVE AAAQH H H H H HThe variants of this peptiligase according to the invention are:Ptl— 2502 substituted C3S-C206QSEQ ID NO: 8AKSVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSETNPFQDNASHGTHVAGTVLAVAPSASLYAVKVIGARGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNKGTSGSSSTVDYPAKYPSVIAVGAVDSSNQRAPWSSVGPELDVMAPGVSIQSTLPGGKYGARDGTCHAVNHVAGAAALILSKHPNWTNTQVRSSLE NTATKLG DS FYYG KG LI NVE AAAQH H H H H HPtl— 2502 substituted C3K-C206DSEQ ID NO: 9AKKVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSETNPFQDNASHGTHVAGTVLAVAPSASLYAVKVIGARGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNKGTSGSSSTVDYPAKYPSVIAVGAVDSSNQRAPWSSVGPELDVMAPGVSIDSTLPGGKYGARDGTCHAVNHVAGAAALILSKHPNWTNTQVRSSLE NTATKLG DS FYYG KG LI NVE AAAQH H H H H HPtl— 2502 substituted C3K-C206ESEQ ID NO: 10AKKVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSETNPFQDNASHGTHVAGTVLAVAPSASLYAVKVIGARGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNKGTSGSSSTVDYPAKYPSVIAVGAVDSSNQRAPWSSVGPELDVMAPGVSIESTLPGGKYGARDGTCHAVNHVAGAAALILSKHPNWTNTQVRSSLE NTATKLG DS FYYG KG LI NVE AAAQH H H H H HPtl-2502 substituted C3R-C206DSEQ ID NO: 11AKRVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSETNPFQDNASHGTHVAGTVLAVAPSASLYAVKVIGARGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNKGTSGSSSTVDYPAKYPSVIAVGAVDSSNQRAPWSSVGPELDVMAPGVSIDSTLPGGKYGARDGTCHAVNHVAGAAALILSKHPNWTNTQVRSSLE NTATKLG DS FYYG KG LI NVE AAAQH H H H H HPtl-2502 substituted C3R-C206ESEQ ID NO: 12AKRVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSETNPFQDNASHGTHVAGTVLAVAPSASLYAVKVIGARGSGQYSWIINGIEWAIANNMDVINIVISLGGPSGSAALKAAVDKAVASGVVVVAAAGNKGTSGSSSTVDYPAKYPSVIAVGAVDSSNQRAPWSSVGPELDVMAPGVSIESTLPGGKYGARDGTCHAVNHVAGAAALILSKHPNWTNTQVRSSLE NTATKLG DS FYYG KG LI NVE AAAQH H H H H HThe peptiligase Ptl-400 which was analysed in this study in comparison to its improved versions can be described as based on Sbtl49+S221C with the additional mutations P225N, M222G, L217R, S166D, F189W and S156NSEQ ID NO: 13AKCVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSETNPFQDNNSHGTHVAGTVLAVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNM DVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNNGTSGSSSTVDYPAKYPSVIAVGAVDSSNQRAPWSSVGPELDVMAPGVSICSTLPGGKYGARSGTCGASNHVAGAAALILSKHPNWTNTQVRSSLE NTATKLG DS FYYG KG LI NVE AAAQH H H H H HOne variant of this peptiligase according to the invention is:SEQ ID NO: 14AKSVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSETNPFQDNNSHGTHVAGTVLAVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNM DVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNNGTSGSSSTVDYPAKYPSVIAVGAVDSSNQRAPWSSVGPELDVMAPGVSIQSTLPGGKYGARSGTCGASNHVAGAAALILSKHPNWTNTQVRSSLE NTATKLG DS FYYG KG LI NVE AAAQH H H H H HSequence DISC 1SEQ ID NO: 15AKSVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSETNPFQD 60NASHGTHVAGTVLAVAPSASLYAVKVIGADGSGQYSWIINGIEWAIANNMDVINMSLGGPS 130(-9)GSAALKAAVDKAVASGWWAAAGNKGTSGSSSTVDYPAKYPSVIAVGAVDSSNQRAPWS 190(-9)SVGPELDVMAPGVSIQSTLPGGKYGAHDGTCPASNHVAGAAALILSKHPNWTNTQVRSS 250(-9)LENTATKLGDSFYYGKGLINVEAAAQHHHHHHSequence DISC 2SEQ ID NO: 16AKSVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSETNPFQD 60NASHGTHVAGTVLAVAPSASLYAVKVIGARGSGQYSWIINGIEWAIANNMDVINMSLGGPS 130(-9)GSAALKAAVDKAVASGWWAAAGNKGTSGSSSTVDYPAKYPSVIAVGAVDSSNQRAPWS 190(-9)SVGPELDVMAPGVSIQSTLPGGKYGARDGTCHAVNHVAGAAALILSKHPNWTNTQVRSS 250(-9)LENTATKLGDSFYYGKGLINVEAAAQHHHHHH

Claims

1. A method for increasing obtainable amounts of a desired peptiligase variant, wherein a peptiligase is a mutant of subtilisin BPN', with the deletion of amino acids 75 to 83 (A 75-83), comprising mutations S221C and P225A, expressed in a recombinant host cell, comprising the steps of transforming a host cell with an expression vector comprising at least one nucleic acid sequence encoding at least one peptiligase variant to produce a transformed host cell; and culturing the transformed host cell under conditions suitable for producing at least one peptiligase variant to produce at least one peptiligase variant, wherein said nucleic acid sequence is characterised in that it encodes an amino acid sequence of said peptiligase variant, wherein at position 3 and at position 206 of the amino acid sequence of said otherwise identical peptiligase variant the amino acid residue is an alternative amino acid residue to cysteine, thereby affording the obtainment of an increased amount of peptiligase compared to the amount obtained when the amino acid residues are cysteines.

2. The method according to claim 1 wherein the host cell is a cell of Bacillus subtilis, preferably a cell of a Bacillus subtilis strain wherein the protease aprE is deleted, more preferably wherein the strain is a non-sporulating strain, and wherein the additional six proteases are also deleted, most preferably a cell of Bacillus subtilis strain BH13.

3. The method according to claims 1 or 2 wherein the alternative amino acid residue of the amino acid sequence of said otherwise identical peptiligase at position 3 and at position 206 is a natural amino acid, that is different from cysteine.

4. The method according to any of the claims above wherein the alternative amino acid residue of the amino acid sequence of said otherwise identical peptiligase at position 3 is selected from the group comprising serine, lysine and arginine, and at position 206 is selected from the group comprising glutamine, aspartic acid and glutamic acid.

5. The method according to any of the claims above wherein the alternative amino acid residue of the amino acid sequence of said otherwise identical peptiligase at position 3 is selected from the group consisting of lysine and arginine, and the alternative amino acid at position 206 is aspartic acid.

6. The method according to any of the claims 1-5 wherein the alternative amino acid residue of the amino acid sequence of said otherwise identical peptiligase at position 3 is serine, and the alternative amino acid at position 206 is glutamine.

7. An isolated peptiligase variant, wherein a peptiligase is a mutant of subtilisin BPN' with the deletion of amino acids 75 to 83 (A 75-83), comprising mutations at S221C and P225A, wherein said peptiligase variant comprises an amino acid substitution of the amino acid cysteine at the residue positions equivalent to residue position 3 and to residue position 206, wherein the amino acid positions of the peptiligase variant are numbered according to the numbering of corresponding amino acid positions in the amino acid sequence of subtilisin BPN’ shown in SEQ ID NO: 2.

8. The peptiligase variant according to claim 7, wherein it has an improved producibility performance in a method of recombinant production involving a bacillus subtilis strain, as compared to the otherwise identical peptiligase variantwith cysteine residues at position 3 and 206.

9. The peptiligase variant according to claim 7 or claim 8 wherein the substituted amino acid residue at position 3 is selected from the group comprising serine, lysine and arginine, and the amino acid residue at position 206 is selected from the group comprising glutamine, aspartic acid and glutamic acid.

10. An isolated peptiligase variant according to claim 7, with an increased, or at least even, ligating activity as compared to the otherwise identical peptiligase variant without an amino acid substitution at the residue positions equivalent to residue position 3 and to residue position 206.

11. The isolated peptiligase variant according to any of claims 7 to 10 wherein the peptiligase variant has the mutation P225N.

12. The isolated peptiligase variant according to any of claims 7 to 11 wherein the peptiligase is further characterized ba having one or more of the stabilizing mutations of Q2K, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, N212G, Y217L, N218S, T254A and Q271E.

13. An isolated peptiligase variant according to claim 7 wherein the peptiligase variant is a variant of one of the peptiligases selected from the group comprising of Ptl-079 (SEQ ID NO: 5), Ptl-2502 (SEQ ID NO: 7), Ptl-1613 (SEQ ID NO:3) and Ptl-400 (SEQ ID NO: 13); and comprises an amino acid substitution of the amino acid cysteine at the residue positions equivalent to residue position 3 and to residue position 206.

14. An isolated peptiligase variant selected from the group consisting of the peptiligases represented by SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 14.

15. A DNA encoding a variant peptiligase of any one of claims 7 to 14.

16. An expression vector encoding the DNA of claim 15.

17. A host cell transformed with the expression vector of claim 16.

18. A method for producing a variant of a peptiligase, wherein a peptiligase is mutant of subtilisin BPN' with the deletion of amino acids 75 to 83 (A 75-83), comprising mutations at S221C and P225A comprising transforming a host cell with an expression vector comprising at least one nucleic acid sequence encoding at least one peptiligase variant according to any of claims 7 to 14 to produce a transformed host cell; culturing the transformed host cell under conditions suitable for producing at least one peptiligase variant to produce at least one peptiligase variant, wherein said peptiligase variant has substantially the same ligating activity as the same peptiligase variant without an amino acid substitution at the residue positions equivalent to residue position 3 and to residue position 206; wherein the method is characterized in producing a higher amount of peptiligase as compared to the method using an expression vector comprising a nucleic acid sequence encoding the same peptiligase variant without an amino acid substitution at the residue positions equivalent to residue position 3 and to residue position 206.

19. The method of claim 16, further comprising harvesting the produced peptiligase variant.

20. The method according to claim 18 or 19 wherein the host cell is a cell from a Bacillus species, preferably wherein the Bacillus species is Bacillus subtilis.

21. A method for improving the performance of a peptiligase in a coupling reaction of two suitable peptide fragments comprising the steps of(a) substituting the amino acid at position 3 in a selected peptiligase to produce a peptiligase wherein the substitution alters the expression rate and / or the processing of the peptiligase;(b) substituting the amino acid at position 206 in said selected peptiligase to produce a peptiligase wherein the substitution alters the expression rate and / or the processing of said peptiligase;(c) testing the variant with both positions altered to determine its activity compared to the selected peptiligase activity in an activity assay suitable to determine the activity in said coupling reaction of two peptide fragments suitable for said peptiligases.(d) repeating steps (a) to (c) as necessary to produce a peptiligase variant that is more or, at least, as active than the precursor peptiligase.

22. A method according to claim 21 wherein the selected peptiligase is selected the from the group consisting of Ptl-079 (SEQ ID NO: 5), Ptl-2502 (SEQ ID NO: 7), Ptl- 1613 (SEQ ID NO:3) and Ptl-400 (SEQ ID NO: 13).

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