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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1025—Acyltransferases (2.3)
  • C12N9/104—Aminoacyltransferases (2.3.2)
  • C12N9/1044—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y203/00—Acyltransferases (2.3)
  • C12Y203/02—Aminoacyltransferases (2.3.2)
  • C12Y203/02013—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII

Definitions

• Transglutaminases are u. a. used for the covalent crosslinking of proteins.
• a microbial transglutaminase MMG [Ando et al., 1990]
• MMG microbial transglutaminase
• Attempts to produce the enzyme recombinantly and to achieve expression in soluble form in E. coli have failed so far. Insoluble inclusion bodies were always obtained. Genetic modifications of the enzyme were impossible for this reason.
• the commercially available enzyme preparation Activa WM was used (Table 1).
• the purified, recombinantly produced and sequence-modified enzyme (FRAP-MTG-HiS 6 ) has a lower stability at higher temperatures than the commercial enzyme preparation Activa WM.
• the latter contains 99% maltodextrin for stabilization. Removing the maltodextrin significantly reduces the stability (Table 1, column "MTG from Activa WM after purification").
• Transglutaminase in the form of Activa WM is mainly used in food technology.
• the enzyme also outside of the food sector such as in the pharmaceutical and cosmetics sector as well as the polymer synthesis It would be beneficial to be able to carry out the crosslinking reactions at higher temperatures and thus produce more temperature-stable and more active transglutaminases.
• the object of the invention was therefore to produce transglutaminases which have a higher thermal stability.
• the problem was solved by the use of random mutagenesis by means of error prone PCR to produce modified transglutaminases. These were identified in a screening for thermostability and the improved enzymes were subsequently characterized.
• the gene sequences of the transglutaminases, which had improved properties, were analyzed and the corresponding altered amino acid sequence derived. With the aid of this sequence information and the known crystal structures, it has been possible to identify those regions in the protein which have an increased temperature stability compared to the wild-type enzyme or the altered histidine-tagged enzyme (hot spots).
• Example 1 Cultivation of E. coli in microtiter plates (MTP) and expression of the pro- MTG-HiS 6
• Deepwell microtiter plates (MTP, No. 0030502.310, Eppendorf, Hamburg) were incubated with 500 ⁇ l LB Amp medium (10 g / L tryptone / peptone, 10 g / L NaCl, 5 g / L yeast extract, pH 7.5, 100 ⁇ g / mL ampicillin) per well. Each well was inoculated from the agar plate using a sterile toothpick with cells from a single E. coli culture (preparation of the agar plate, see Example 5C.) The MTPs were sealed with two sterile adhesive sheets, first with BreathSeal (# 676050, Greiner Biochem.
• This MTP was in a shaking incubator (Multitron, Infors HT, Bottmingen, Switzerland) at 300 rpm, 28 0 C and 5 cm Scblinlradius incubated for 24 hours, then centrifuged at 4000 rpm for 20 minutes (centrifuge 5403, Eppendorf, Hamburg). The supernatant was discarded and the pellet stored at -80 0 C until cell disruption.
• a shaking incubator Multitron, Infors HT, Bottmingen, Switzerland
• lysis buffer 50 mM Tris / HCl, 2 mM MgCl 2 , pH 8.0, 1 mg / ml lysozyme (8259.2, Carl Roth GmbH & Co. KG) were added to the MTP with the frozen cell pellets (see Example 1) per well.
• 10 U / ml Benzonase No. 1,01695,0001, Merck KGaA, Darmstadt)
• the pellet was resuspended by pipetting up and down with a multichannel pipette.
• the MTP was then incubated at 650 rpm and 37 ° C. for 1 hour in a thermomixer (Eppendorf, Hamburg).
• Example 3 Activity test on FRAP-MTG-HiS 6 activity or its mutants in microtiter plate format
• the test for MTG activity was carried out by hydroxamate [FoIk and CoIe, 1966] and was adapted to MTP format.
• To 50 ⁇ l of supernatant containing the respective activated enzyme (see Example 2 or Example 4C) was added 90 ⁇ l of hydroxamate test solution (Final concentrations: 0.2 M Tris, 100 mM hydroxylamine, 10 mM reduced glutathione, 30 mM Z-GIn-Gly, pH 6.0).
• E. coli BL21 (DE3) pDJ1-3 was cultured in the bioreactor. These were the
• Preculture IM (LB Amp medium) were inoculated with preculture II such that a start
• Seed culture III inoculated The bioreactor cultivation was carried out in a 20 L Biostat C at a temperature of 28 0 C, a stirrer speed of 300 rpm and a gassing rate of 4.0 L / min.
• Antifoam was used silicone oil.
• the cells were harvested by centrifugation (4 0 C 1 6000 g, ZK 630,
• the biomass was resuspended in 0.9% NaCl solution, recentrifuged and as
• 5 ml LB amp medium was inoculated with E. coli BL21 (DE3) with the plasmids containing mutant FRAP MTG gene sequences (performing random mutagenesis see example 5) and incubated at 37 0 C and 200 rpm for about 6 hours incubated.
• E. coli BL21 DE3
• plasmids containing mutant FRAP MTG gene sequences performing random mutagenesis see example 5
• 3 ml of the preculture in each case a 500 ml Erlenmeyer flask with baffles with 100 ml of autoinduction medium (see below) was inoculated and incubated for about 20 hours at 28 ° C. and 120 rpm until the stationary growth phase had been reached.
• the cells were separated by centrifugation (20 min at 3000 g) and stored at -80 0 C until further use.
• the autoinducer medium was prepared as follows. To 93 ml LB medium, the following individually sterilized solutions were added: 200 ⁇ l ampicillin (50 mg / ml), 2 ml autoinduction solution 1 (final concentration 0.5 g / L glucose, 5 g / L glycerol, 2 g / L Lactose), 5 ml autoinduction solution 2 (final concentration 25 mM KH 2 PO 4 , 25 mM Na 2 HPO 4 ) and 100 ⁇ l autoinduction solution 3 (final concentration 2 mM MgSO 4 ).
• 200 ⁇ l ampicillin 50 mg / ml
• 2 ml autoinduction solution 1 final concentration 0.5 g / L glucose, 5 g / L glycerol, 2 g / L Lactose
• 5 ml autoinduction solution 2 final concentration 25 mM KH 2 PO 4 , 25 mM Na 2 HPO 4
• 100 ⁇ l autoinduction solution 3 final concentration 2 m
• the starting plasmid used was the previously described plasmid pDJ1-3 [Marx et al., 2007].
• the plasmid isolation was carried out according to the manufacturer's instructions by means of GeneJET Plasmid Miniprep Kit (No. K0503, Fermentas, St. Leon-Rot).
• the sequencing was carried out by the company MWG, Ebersberg.
• Mutagenesis of the FRAP-MTG gene was performed using GeneMorph II EZCIone Domain Mutagenesis Kit (Stratagene, Amsterdam, The Netherlands). 500 ng of target DNA (MTG gene in isolated pDJ1-3) were used for the error-prone PCR, the annealing temperature was 60 0 C and there were 25 cycles performed (Whatman Biometra thermocycler, Göttingen, Germany). The primers used were at the beginning and at the end of the MTG gene without pro-sequence (forward primer: 5'-GACTCCGACGACAGGGTCACC-3 ', reverse primer: 5 1 -
• Electrocompetent cells of E. coli BL21Gold (DE3) (Stratagene) were prepared as described [Marx et al., 2007].
• 1 ⁇ l of plasmid was transformed into 40 ⁇ l of competent cells. The transformation was repeated several times and the transformation mixture on a total of 30 agar plates with LB ampicillin medium plated. The plates were incubated at 37 0 C and then stored at 4 0 C. Approximately 30 plates each with about 200 clones were produced.
• Example 2 From the agar plates, individual cultures were picked by means of sterile toothpicks, thus inoculating medium in MTP and culturing was carried out as described in Example 1. Subsequently, the harvest, the cell disruption and the activation of the FRAP-MTG-HiS 6 or their mutants as described in Example 2.
• thermostable FRAP-MTG-HiS 6 50 L of the supernatant after activation at 55 0 C for 30 min were preincubated in the water bath. Subsequently, the standard activity test was carried out as described in Example 3.
• Example 7 Detailed characterization of Activa WM, MTG from Activa WM, FRAP MTG-HiS 6 and FRAP-MTG-HiS 6 mutants
• thermostable mutants From the positive clones from the screening (see Example 6), the plasmids were isolated and the gene of FRAP-MTG-HiS 6 amplified and sequenced (as in Example 5). Result: Mainly single nucleotide substitutions were found, which in some cases led to exchanges in the amino acids. The sequences found for the thermostable mutants are summarized in Tab.
• Activa WM (Ajinomoto Europe Sales GmbH, Hamburg, Germany) were dissolved in 1 ml of 50 mM Tris / HCl, 300 mM NaCl, 20 mM Imidazole pH 8.0.
• E. coli mutants of FRAP-MTG-HiS 6 were cultured under the same conditions as the strain with the unmodified plasmid [Marx et al., 2007].
• the respective Pro-MTG-HiS 6 was activated by TAMEP and purified by affinity chromatography as described in Example 4.
• the purified enzymes were pure by SDS-PAGE and used for characterization.
• the isolated enzymes (30 ⁇ l_ each) were PCR-vessel, and using a PCR thermal cycler (Whatman Biometra, Göttingen, Germany) pre-incubated at 60 0 C for 10 min. Subsequently, the activity was determined using the MTP version of the standard test for FoIk and CoIe [FoIk and CoIe, 1966] (see Example 7E).
• the inactivation curves of Activa WM, MTG from Activa WM, FRAP-MTG-HiS 6 and the selected mutants are shown in Figure 1. The results for all mutants are summarized in Tab. 6.
• thermostable variants which significantly reduced the decrease of
• the incubation was present even after half the initial activity could be increased from 1.7 minutes at the FRAP-MTG-HiS 6 to 4.6 minutes for the enzyme pCM203 (S2P) (ID SEQ N 0 4.).
• the residual activity after 10 minutes incubation at 60 0 C was significantly increased.
• the specific activity of the enzymes according to Example 7B was determined by the standard activity test according to FoIk and CoIe [FoIk and CoIe, 1966]).
• the protein concentration of the purified enzyme fractions was determined by absorbance at 280 nm.
• the results for all enzymes are summarized in Tab. 6.
• the specific activity of the parent enzyme was as high at 23 U / mg protein as that of the MTG from Streptomyces mobaraensis (22.6 U / mg protein [Ando et al., 1989].
• the commercial MTG preparation ( Activa WM), according to the manufacturer, has a specific activity of 80-140 U / g solids and contains 1% protein and 99% maltodextrin (corresponding to 8-14 U / mg protein) .
• the purified MTG from Activa WM had 7.6 U / mg a slightly higher specific activity than the commercial preparation.
• Some mutants have a decreased specific activity compared with the enzyme from Streptomyces mobaraensis (e.g., SEQ ID N 0 3.:. (CM201 Y24N) with 18 U / mg) as a mutant surprisingly a significantly higher activity (SEQ ID N °. 4: pCM203 (S2P) at 46.1 U / mg).
• Enzyme samples characterized according to Example 7D were diluted to approximately 5 U / mL. Subsequently, the activity was determined with the MTP version of the standard test for FoIk and CoIe [FoIk and CoIe, 1966] at different temperatures (10, 20, 30, 37, 40, 50, 60, 70 and 80 ° C.). Each 140 ⁇ L of substrate solution was preconditioned for 2 minutes at the respective temperature using a PCR thermocycler (Whatman Biometra, Göttingen, Germany) before the reaction was started by addition of 10 ⁇ L of the enzyme solution 150 ⁇ L Reagent A (see Example 3) Fig.
• thermostable mutants In thermostable mutants, a partially significantly increased activity is shown
• the FRAP-MTG-His 6 mutants were characterized according to Example 7.
• the results of the sequencing are summarized in Fig. 3.
• the amino acid changes found in thermostable mutants are highlighted.
• the mutations accumulate in certain areas of the primary structure. All mutations leading to improved properties lie in the left sidewall or at the bottom of the column, which according to Kashiwagi, 2002, forms the active center ("active site cleft" [Kashiwagi et al., 2002b] (see Fig. 4) ).
• this enzyme had previously been excluded from the 99%
• Activa WM (Ajinomoto Europe Sales GmbH, Hamburg, Germany) were dissolved in 4 ml of chromatography buffer (50 mM Tris, 0.3 M NaCl, 20 mM imidazole, pH 8)
• DMS dinitrosalicylic acid reagent
• the active fractions (0.5 mL each) in the range of the elution volume from 10.00 to 11.50 mL were pooled and used for the characterization according to Example 7.
• Fig. 1 Thermal stability of Activa WM (contains 99% maltodextrin), purified MTG from Activa WM, purified FRAP-MTG-HiS 6 and two selected (purified) FRAP-MTG-HiS 6 mutants (pCM201 (SEQ ID N ° 3) pCM203 (SEQ. ID N 0 4)). 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 min pre-incubation at 60 0 C. Thereafter, standard activity test after FoIk and Coie [FoIk and Coie 1966].
• Fig. 2 Specific activity at various temperatures of Activa WM, purified MTG from Activa WM, FRAP-MTG-His 6 (SEQ ID N 0. 2), the thermostable and active mutant pCM203 (SEQ ID N 0 4.) And the thermostable mutant pCM224 (SEQ ID N ° 8). 2 min pre-incubation of the substrate solution at the respective temperature, then 10 min reaction time at 10, 20, 30, 37, 40, 50, 60, 70 and 80 0 C. [FoIk and CoIe, 1966].
• FIG. 3 Primary sequence of FRAP-MTG-HiS 6 from Streptomyces mobaraensis. Marked are the altered amino acids found in thermostable mutants (highlighted in gray).
• Fig. 4 Crystal structure of the FRAP-MTG from Streptomyces mobaraensis (pdb 1iu4) [Kashiwagi et al., 2002b]. Spacefill visualization highlights the altered amino acids found in thermostable mutants and the active site Cys64.
• Fig. 5 Chromatogram of gel chromatography (Superdex 75) for the removal of maltodextrin from a solution of Activa WM. The conditions were described in Example 9. The standard activity test was carried out according to FoIk and CoIe [FoIk and CoIe, 1966], the concentration of maltodextrin was quantitatively determined by means of DNA reagent according to [Miller, 1959]. literature
• Motoki M. (1989). "Purification and characteristics of a novel transglutaminase derived from microorganisms.” Ag ⁇ c. Biol. Chem., 53, 2613-17. Ando, H., Uchio, R., Matsuura, A., Umeda, K., Motoki, M., Nonaka, M., Okiyama, A. and

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