Method for producing serratia marcescens nuclease using a bacillus expression host

Abstract

A method for producing a nuclease of a gram negative bacterium or a nuclease preparation containing a nuclease of a gram negative bacterium including expression of the nuclease in a gram positive bacterium and subsequent secretion of the nuclease, as well as a nuclease or a nuclease preparation that can be obtained by this method.

Claims

1. A method for producing a Serratia marcescens nuclease comprising: culturing a Bacillus subtilis bacterium or a Bacillus amyloliquefaciens bacterium comprising a nucleic acid with a nucleotide sequence encoding the Serratia marcescens nuclease in a culture medium to express said nucleic acid to secrete the nuclease, wherein the Serratia marcescens nuclease is fused to a Bacillus subtilis or Bacillus amyloliquefaciens AmyE secretion signal, wherein the nucleic acid comprises a Bacillus subtilis or a Bacillus amyloliquefaciens maltose-inducible promoter and expression of the Serratia marcescens nuclease is controlled by the maltose-inducible promoter, and wherein the method achieves a yield of the secreted nuclease of at least 5,000 nuclease units per mL of culture medium.

2. The method according to claim 1, wherein said nucleic acid is integrated into the chromosome of the Bacillus subtilis bacterium or the Bacillus amyloliquefaciens bacterium.

3. The method according to claim 1, wherein the Bacillus subtilis bacterium or the Bacillus amyloliquefaciens bacterium is a low-protease Bacillus subtilis bacterium or Bacillus amyloliquefaciens bacterium.

4. The method according to claim 1, wherein the Serratia marcescens nuclease is fused to an affinity tag.

5. The method according to claim 1, wherein said nucleic acid is within an expression vector.

6. The method according to claim 1, excluding any purification step for removing endotoxins.

7. The method according to claim 1, wherein the bacterium is Bacillus subtilis and the secretion signal is Bacillus subtilis AmyE secretion signal.

8. The method according to claim 7, wherein said nucleic acid is integrated into the chromosome of the Bacillus subtilis bacterium.

9. The method according to claim 1, wherein the bacterium is Bacillus amyloliquefaciens and the secretion signal is Bacillus subtilis AmyE secretion signal.

10. The method according to claim 9, wherein said nucleic acid is integrated into the chromosome of the Bacillus amyloliquefaciens bacterium.

11. The method according to claim 1, wherein the bacterium is Bacillus amyloliquefaciens, the secretion signal is Bacillus subtilis AmyE secretion signal, and the nucleic acid comprises a Bacillus amyloliquefaciens maltose-inducible promoter.

12. The method of claim 1, wherein the Bacillus subtilis or the Bacillus amyloliquefaciens bacterium is cultured in a medium comprising 1% maltose.

13. The method of claim 1, wherein the Bacillus subtilis or the Bacillus amyloliquefaciens bacterium is cultured in a medium comprising 5% maltose.

14. The method of claim 1, wherein the Bacillus subtilis or the Bacillus amyloliquefaciens bacterium is cultured in a medium comprising 10% maltose.

15. A method for producing a Serratia marcescens nuclease comprising: culturing a Bacillus amyloliquefaciens bacterium comprising a nucleic acid with a nucleotide sequence encoding the Serratia marcescens nuclease in a culture medium to express said nucleic acid to secrete the nuclease, wherein the Serratia marcescens nuclease is fused to a Bacillus subtilis AmyE secretion signal, wherein the nucleic acid comprises a Bacillus amyloliquefaciens neutral protease (npr) promoter and expression of the Serratia marcescens nuclease is controlled by the npr promoter; and wherein the method achieves a yield of the secreted nuclease of at least 5,000 nuclease units per mL of culture medium.

16. The method of claim 15, wherein the Bacillus amyloliquefaciens bacterium is cultured in a medium comprising 1% or 10% maltose.

17. The method of claim 15, wherein the Bacillus amyloliquefaciens bacterium is cultured in a medium comprising 5% maltose.

18. The method according to claim 1, wherein the bacterium is Bacillus subtilis, the secretion signal is Bacillus subtilis AmyE secretion signal, and the nucleic acid comprises a Bacillus subtilis maltose-inducible promoter.

19. The method according to claim 1, wherein the amino acid sequence of the Serratia marcescens nuclease is SEQ ID NO: 3.

20. The method according to claim 7, wherein the amino acid sequence of the Serratia marcescens nuclease is SEQ ID NO: 3.

21. The method according to claim 9, wherein the amino acid sequence of the Serratia marcescens nuclease is SEQ ID NO: 3.

22. The method according to claim 11, wherein the amino acid sequence of the Serratia marcescens nuclease is SEQ ID NO: 3.

23. The method according to claim 15, wherein the amino acid sequence of the Serratia marcescens nuclease is SEQ ID NO: 3.

24. The method according to claim 18, wherein the amino acid sequence of the Serratia marcescens nuclease is SEQ ID NO: 3.

Description

EXAMPLES

(1) The following illustrative examples serve to explain the invention in more detail, but are not to be interpreted as restrictive.

Illustrative Embodiment 1

(2) Cloning of the Gene for the Serratia marcescens Nuclease into a Vacillus Expression Vector with a Bacillus subtilis Promoter

(3) Using two primers (SEQ-ID 173 and SEQ-ID 174) the gene of the Serratia marcescens nuclease including the signal peptide and a Bacillus subtilis ribosome binding site (SEQ-ID 175) from the synthetically created gene adapted to the codon usage of Bacillus subtilis is amplified by a PCR under the following conditions and the sequence (SEQ-ID 176) is obtained. On the amplified sequence the cut site for Pael is located upstream of the nuclease gene and that for Pstl downstream of the nuclease gene.

(4) 1.1 PCR:

(5) TABLE-US-00002 PCR batch: 20 μl 5 x Phusion polymerase buffer (Finnzymes) 2 μl dNTPs (per 10 mmol/liter) 100 pmol primer 1 (SEQ-ID 173) 100 pmol primer 2 (SEQ-ID 174) 1 μl original sequence (20 ng) 1 U Phusion polymerase (Finnzymes) to 100 μl dist. H.sub.2O Temperature profile of 1 min/98° C. the PCR: 1. 10 sec/98° C. (denaturing) 2. 20 sec/52° C. (addition) 30x 3. 2 min 20 sec/72° C. (elongation) 7 min/72° C.
The resulting PCR products are purified by the High Pure PCR Product Purification Kit (Roche, Diagnostics GmbH, Mannheim) according to the manufacturer's specification.
1.2 Restriction Digest:

(6) Any vector replicating in Bacillus species can be selected as expression vector. For preparation a promoter (SEQ-ID 171) with subsequent multiple cloning site (SEQ-ID 177) is inserted into this vector and any already present and interfering promoters and restriction cut sites removed, if necessary. Methods for producing such an empty expression vector are known to the person skilled in the art and are part of standard molecular biology.

(7) To clone the gene into an appropriately prepared expression vector, the PCR product and the vector are incubated with the restriction endonucleases Pael and Pstl (all MBI Fermentas, Vilnius, Lithuania) as follows:

(8) Restriction Digest Batches:

(9) TABLE-US-00003 PCR products: Vector: 2.4 μg PCR product 8 μg vector 5 μl 10x Tango (MBI) 4 μl 10x Tango (MBI) 30 U Pael 20 U Pael 10 U Pstl 10 U Pstl to 50 μl dist. H.sub.2O to 40 μl dist. H.sub.2O

(10) The restriction digest batches are incubated for 2 h at 37° C. 1 U SAP (MBI Fermentas, Vilnius, Lithuania) is then added to the “vector batch” for dephosphorylation and incubated for a further 30 min at 37° C. A further 1 U SAP (MBI Fermentas, Vilnius, Lithuania) is then added and the mixture incubated for 30 min at 37° C. once again. The enzymes are then inactivated for 20 min at 80° C., extracted with phenol and chloroform and the batch precipitated with PEG for further concentration. The cut PCR product is purified by Promega Wizard SV Gel and PCR Clean-Up System (Promega GmbH, Mannheim).

(11) 1.3 Ligation, Transformation of B. subtilis and Plasmid Reisolation

(12) The vector DNA and the PCR product are joined to one another by incubation with T4 DNA ligase as follows:

(13) TABLE-US-00004 Ligase batch: 50 fmol vector DNA 150 fmol PCR product 2 μl 10x Ligase Buffer (MBI) 1 μl T4 DNA ligase to 20 μl dist. H.sub.2O
The batches are incubated for 16 h at 16° C. and then the enzyme was inactivated by incubation for 10 minutes at 65° C. The batches are extracted with phenol and chloroform, precipitated with ethanol and taken up in 20 μl of deionised water. Before transforming 20 μl of 2×SMM are added to the batches. The batches are then used to transform Bacillus subtilis using the PEG protoblast method (Chang and Cohen, 1979). For isolation of the plasmids the High Pure Plasmid Isolation Kit (Roche, Diagnostics GmbH, Mannheim) is used according to manufacturers' specifications. The plasmids thus isolated are tested by sequencing the cloned gene with respect to its correct construction.

Illustrative Embodiment 2

(14) Cloning of the Gene for the Serratia marcescens Nuclease into a Bacillus Expression Vector with a Bacillus amyloliquefaciens Promoter

(15) Using two primers (SEQ-ID 178 and SEQ-ID 179) the gene of the Serratia marcescens nuclease adapted to the codon usage of Bacillus subtilis including the signal peptide and a Bacillus subtilis ribosome binding site is amplified by a PCR and with the plasmid constructed under 1. as template under the following conditions. At the same time, cut sites for Pael and Bpu11021 are contained on this PCR product (SEQ-ID 180).

(16) 2.1 PCR:

(17) TABLE-US-00005 PCR 20 μl 5 x Phusion polymerase batch: buffer (Finnzymes) 2 μl dNTPs (per 10 mmol/liter) 200 pmol primer 1 (SEQ-ID 178) 200 pmol primer 2 (SEQ-ID 179) 1 μl template vector from Example 1 (20 ng) 1 U Phusion polymerase (Finnzymes) to 100 μl dist. H.sub.2O Temperature 1 min/98° C. profile 1. 10 sec/98° C. (denaturing) of the PCR: 2. 20 sec/58° C. (addition) {close oversize parenthesis} 30x 3. 2 min/72° C. (elongation) 7 min/72° C.
The resulting PCR products are purified by the High Pure PCR Product Purification Kit (Roche, Diagnostics GmbH, Mannheim) according to the manufacturer's specification.
2.2 Restriction Digest:

(18) Any vector replicating in Bacillus species can be selected as expression vector. For preparation a promoter (SEQ-ID 172) with subsequent multiple cloning site (SEQ-ID 181) is inserted into this vector and any already present and interfering promoters and restriction cut sites removed, if necessary. Methods for producing such an empty expression vector are known to the person skilled in the art and are part of standard molecular biology.

(19) To clone the gene into an appropriately prepared expression vector, the PCR product and the vector are incubated with the restriction endonucleases Pael and Bpu11021 (all MBI Fermentas, Vilnius, Lithuania) as follows:

(20) Restriction Digest Batches:

(21) TABLE-US-00006 PCR products: Vector: 1.5 μg PCR product 4 μg vector 5.5 μl 10x Tango (MBI) 5 μl 10x Tango (MBI) 20 U Pael 20 U Pael 10 U Bpu11021 10 U Bpu11021 to 55 μl dist. H.sub.2O to 50 μl dist. H.sub.2O

(22) The restriction digest batches are incubated for 2 h at 37° C. 1 U CIAP (MBI Fermentas, Vilnius, Lithuania) is then added to the “vector batch” for dephosphorylation and 5 μl of CIAP buffer 10× (MBI Fermentas, Vilnius, Lithuania) added thereto and incubated for a further 30 min at 37° C. A further 1 U CIAP (MBI Fermentas, Vilnius, Lithuania) is then added and the mixture incubated for 30 min at 37° C. once again. 5 mM of EDTA pH 8 are then added and the enzymes are inactivated for 30 min at 80° C. The cut PCR product is purified by Promega Wizard SV Gel and PCR Clean-Up System (Promega GmbH, Mannheim).

(23) 2.3 Ligation, Transformation of B. subtilis and Plasmid Reisolation

(24) The vector DNA and the PCR product are joined to one another by incubation with T4 DNA ligase as follows:

(25) TABLE-US-00007 Ligase batch: 50 fmol vector DNA 150 fmol PCR product 6 μl 10x Ligase Buffer (MBI) 2 μl T4 DNA ligase to 60 μl dist. H.sub.2O

(26) The batches are incubated for 16 h at 16° C. and then the enzyme was inactivated by incubation for 10 minutes at 65° C. The batches are extracted with phenol and chloroform, precipitated with ethanol and taken up in 20 μl of deionised water. Before transforming 20 μl of 2×SMM are added to the batches. The batches are then used to transform Bacillus subtilis using the PEG protoblast method (Chang and Cohen, 1979). For isolation of the plasmids the High Pure Plasmid Isolation Kit (Roche, Diagnostics GmbH, Mannheim) is used according to manufacturers' specifications.

Illustrative Embodiment 3

(27) Cloning for Fusion of the Nuclease from Serratia marcescens with an AmyE Secretion Sequence

(28) Using two primers (SEQ-ID 174 and SEQ-ID 182) the gene of the Serratia marcescens nuclease adapted to the codon usage of Bacillus subtilis excluding the signal peptide is amplified by a PCR and with SEQ-ID 176 as template sequence under the following conditions.

(29) 3.1 PCR:

(30) 3.1.1. Nuclease PCR

(31) TABLE-US-00008 PCR 20 μl 5 x Phusion polymerase buffer batch: (Finnzymes) 2 μl dNTPs (per 10 mmol/liter) 50 pmol primer 1 (SEQ-ID 174) 50 pmol primer 2 (SEQ-ID 182) 2 μl template sequence (20 ng) 1 U Phusion polymerase (Finnzymes) to 100 μl dist. H.sub.2O Temperature 1 min/98° C. profile 1. 10 sec/98° C. (denaturing) of the PCR: 2. 20 sec/53° C. (addition) 3. 2 min 15 sec/72° C. {close oversize parenthesis} 30x (elongation) 7 min/72° C.
Using two primers (SEQ-ID 178 and SEQ-ID 183) the sequence encoding the secretion sequence of AmyE from Bacillus subtilis is amplified by PCR and the synthetically created sequence SEQ-ID 184 as template sequence under the following conditions.
3.1.2. AmyE Signal Sequence PCR

(32) TABLE-US-00009 PCR 20 μl 5 x Phusion polymerase buffer batch: (Finnzymes) 2 μl dNTPs (per 10 mmol/liter) 50 pmol primer 1 (SEQ-ID 178) 50 pmol primer 2 (SEQ-ID 183) 2 μl template sequence (20 ng) 1 U Phusion polymerase (Finnzymes) to 100 μl dist. H.sub.2O Temperature 1 min/98° C. profile 1. 10 sec/98° C. (denaturing) of the PCR: 2. 20 sec/56° C. (addition) {close oversize parenthesis} 30x 3. 30 sec/72° C. (elongation) 7 min/72° C.
The resulting PCR products are purified by the High Pure PCR Product Purification Kit (Roche, Diagnostics GmbH, Mannheim) according to the manufacturer's specification.
3.1.3. Fusion PCR AmyE Secretion Sequence with Nuclease from Serratia marcescens

(33) TABLE-US-00010 PCR 20 μl 5 x Phusion polymerase buffer batch: (Finnzymes) 2 μl dNTPs (per 10 mmol/liter) 5.5 μl PCR product 3.1.1 (600 fmol) 1 μl PCR product 3.1.2 (600 fmol) 1 U Phusion polymerase (Finnzymes) to 98 μl dist. H.sub.2O Temperature 1 min/98° C. profile 1. 10 sec/98° C. (denaturing) of the PCR: 2. 20 sec/72° C. (addition) {close oversize parenthesis} 7x 3. 2 min 15 sec/72° C. (elongation)
Directly after seven cycles elapsed 1 μl (100 pmol) in each case of primer 1 (SEQ-ID 178) and primer 2 (SEQ-ID 174) were added to the PCR batch and a further PCR was conducted with the following temperature profile:

(34) TABLE-US-00011 Temperature 1 min/98° C. profile of the PCR: 1. 10 sec/98° C. (denaturing) 2. 20 sec/53° C. (addition) {close oversize parenthesis} 30x 3. 2 min 15 sec/72° C. (elongation) 7 min/72° C.

(35) The resulting PCR products are purified by the High Pure PCR Product Purification Kit (Roche, Diagnostics GmbH, Mannheim) according to the manufacturer's specification and, as described under 1.2 and 1.3, were cloned into an expression vector that replicates in Bacillus species and carries a promoter with the following multiple cloning site (SEQ-ID 177).

Illustrative Embodiment 4

(36) Cloning for Fusion of the Nuclease from Serratia marcescens with an AmyE Secretion Sequence and an N Terminal Affinity Tag (His-tag) on the Secreted Protein

(37) Using two primers (SEQ-ID 185 and SEQ-ID 179) the gene of the Serratia marcescens nuclease adapted to the codon usage of Bacillus subtilis excluding the signal peptide is amplified by PCR and the plasmid constructed under 3, as template under the following conditions.

(38) 4.1 PCR:

(39) 4.1.1. His-Nuclease PCR

(40) TABLE-US-00012 PCR 20 μl 5 x Phusion polymerase buffer batch: (Finnzymes) 20 μl dNTPs (per 10 mmol/liter) 50 pmol primer 1 (SEQ-ID 185) 50 pmol primer 2 (SEQ-ID 179) 2 μl template sequence (20 ng) 1 U Phusion polymerase (Finnzymes) to 100 μl dist. H.sub.2O Temperature 1 min/98° C. profile 1. 10 sec/98° C. (denaturing) of the PCR: 2. 20 sec/57° C. (addition) 3. 2 min 10 sec/72° C. {close oversize parenthesis} 30x (elongation) 7 min/72° C.
The resulting PCR products are purified by the High Pure PCR Product Purification Kit (Roche, Diagnostics GmbH, Mannheim) according to the manufacturer's specification.
4.1.2. Fusion PCR AmyE Signal Sequence with His-Tag and Nuclease from Serratia marcescens

(41) TABLE-US-00013 PCR 20 μl 5 x Phusion polymerase buffer batch: (Finnzymes) 2 μl dNTPs (per 10 mmol/liter) 1.5 μl PCR product 4.1.1 (600 fmol) 1 μl PCR product 3.1.2 (600 fmol) 1 U Phusion polymerase (Finnzymes) to 98 μl dist. H.sub.2O Temperature 1 min/98° C. profile 1. 10 sec/98° C. (denaturing) of the PCR: 2. 20 sec/72° C. (addition) 3. 2 min 30 sec/72° C. {close oversize parenthesis} 10x (elongation)
Directly after ten cycles elapsed 1 μl (100 pmol) in each case of primer 1 (SEQ-ID 178) and primer 2 (SEQ-ID 179) were added to the PCR batch and a further PCR was conducted with the following temperature profile:

(42) TABLE-US-00014 Temperature 1 min/98° C. profile of the PCR: 1. 10 sec/98° C. (denaturing) 2. 20 sec/53° C. (addition) {close oversize parenthesis} 30x 3. 2 min 15 sec/72° C. (elongation) 7 min/72° C.
The resulting PCR products are purified by the High Pure PCR Product Purification Kit (Roche, Diagnostics GmbH, Mannheim) according to the manufacturer's specification and, as described under 1.2 and 1.3, were cloned into an expression vector that replicates in Bacillus species and carries a promoter with the following multiple cloning site (SEQ-ID 177).

Illustrative Embodiment 5

(43) Creation of the Expression Plasmids for Secretion of the Nuclease from Serratia marcescens

(44) The following plasmid constructs were constructed using molecular biological methods according to or in accordance with illustrative embodiments 1-4 and verified by sequencing: 1. Maltose-inducible promoter from B. subtilis+native nuclease (codon optimized), cf. illustrative embodiment 1 2. Maltose-inducible promoter from B. amyloliquefaciens+native nuclease including own signal sequence (codon optimized), cf. illustrative embodiment 2 3. Maltose-inducible promoter from B. subtilis+nuclease fused onto AmyE leader sequence (codon optimized), cf. illustrative embodiment 3 4. Maltose-inducible promoter from B. subtilis+native nuclease (codon optimized) including own signal sequence with C terminal His-tag (GGHHHHHHH) (SEQ ID NO. 186), analogous to illustrative embodiments 1 and 4 5. Maltose-inducible promoter from B. amyloliquefaciens+nuclease fused onto AmyE leader sequence (codon optimized), analogous to illustrative embodiments 2 and 3 6. Maltose-inducible promoter from B. subtilis+nuclease fused onto AmyE leader sequence (codon optimized) with N terminal His-tag (DHHHHHHGG) (SEQ ID NO. 187), cf. Illustrative embodiment 4 7. Maltose-inducible promoter from B. amyloliquefaciens+nuclease fused onto AmyE leader sequence (codon optimized) with N terminal His-tag (DHHHHHHGG) (SEQ ID NO. 187), analogous to illustrative embodiments 2 and 4 8. Growth phases induced npr promoter from B. amyloliquefaciens+nuclease fused onto AmyE leader sequence (codon optimized), analogous to illustrative embodiments 2 and 3

Illustrative Embodiment 6

(45) Comparative Expression Tests with the Expression Constructs Created Under 5.

(46) The plasmid constructs are tested for expression in B. subtilis wt168 trpC2 aprE nprE epr amyE bglC and also in B. amyloliquefaciens amy2 npr1 and amy 2 npr1 apr::ka. a) Different liquid media are used therein for the expression Medium 1: LB medium (10 g/l trypton, 5 g/l yeast extract, 10 g/l NaCl, pH 7.0) with 5 μg/ml erythromycin+0.1% glucose; For constructs with the maltose-inducible promoters the expression is induced by adding maltose (end concentration 1%) during transition to the stationary phase. Medium 2: 5% maltose+0.1% glucose+2% soy peptone+2.5% Solulys 095E+0.5% (NH.sub.4).sub.2SO.sub.4+0.1% KCl+0.05% Mg.sub.2SO.sub.4.7H.sub.2O Medium 3: 10% maltose+0.1% glucose+2% soy peptone+2.5% Solulys 095E+0.5% (NH.sub.4).sub.2SO.sub.4+0.1% KCl+0.05% Mg.sub.2SO.sub.4.7H.sub.2O Medium 4: 100 mM tris-HCl pH 7.5+10% maltose+0.1% glucose+2% soy peptone+2.5% Solulys 095E+0.5% (NH.sub.4).sub.2SO.sub.4+0.1% KCl+0.05% Mg.sub.2SO.sub.4.7H.sub.2O b) For the cultivation of precultures LB medium is used with 2% glucose and 5 μg/ml of erythromycin. c) For strain retention (glycerol stock) LB medium is used with 2% glucose and 5 μg/ml of erythromycin. The medium is inoculated with a single colony from the plate. The cultures are incubated in culture tubes overnight at 30° C. with agitation (200 rpm). The next morning 200 μl of 60% sterile glycerine is added to 600 μl of culture. The batches were mixed and frozen in liquid nitrogen. Storage took place at −80° C. d) For propagating the strains on plates LB medium is used with 5 μg/ml of erythromycin+2% glucose+1.5% agar. e) For detecting nuclease-positive colonies on the plate LB medium is used with 5 μg/ml of erythromycin+0.1% glucose+1% maltose+0.2% herring sperm DNA (AppliChem A2160)+0.2% RNA from yeast (Roche 109223) (for activity)+1.5% agar. Nuclease-positive colonies are made visible by flooding the plate with 1N HCl after 2 days of incubation. In this case, positive candidates are surrounded by a clear halo. f) For expression tests the cultures are incubated at 37° C. Liquid cultures are agitated horizontally at 150 rpm in this case. Precultures are incubated overnight at 30° C. g) Expression tests i. Medium 1 Bacillus subtilis or Bacillus amyloliquefaciens with plasmid constructs 1-7 Working from a preculture 100 ml of medium 1 are inoculated with 1 ml of preculture. The growth curve is determined and when the culture enters the stationary phase 2 ml of 50% maltose (end concentration 1%) are added to medium 2. The culture is incubated still with agitation overnight at 37° C. The next morning the nuclease activity is determined in the supernatant. ii. Medium 1 Bacillus subtilis or Bacillus amyloliquefaciens with plasmid construct 8 Working from a preculture 100 ml of medium 1 are inoculated with 1 ml of preculture. The culture is incubated for 24 hours with agitation at 37° C. The next morning the nuclease activity is determined in the supernatant. iii. Media 2, 3, 4 Bacillus subtilis or Bacillus amyloliquefaciens with plasmid constructs 1-8 Working from a preculture 100 ml of medium 1 are inoculated with 1 ml of preculture. The culture is incubated over a week still with agitation at 37° C. The nuclease activity is determined in the supernatant every day. h) Nuclease activity measurements
Chemicals DNA (salmon testes)—Sigma D1626.fwdarw.for assay RNA (yeast) Roche 109223.fwdarw.for plates DNA (salmon sperm) AppliChem A2160.fwdarw.for plates BSA (10 mg/ml) NEB B9001S
Buffer, Solutions 1 M tris-HCl pH 8.2; autoclave 10 mM MgCl.sub.2; autoclave assay buffer (always produce fresh)

(47) TABLE-US-00015 Example for 100 ml End concentration Stock solution tris HCl 50 mM 5 ml BSA 0.1 mg/ml 1 ml MgCl.sub.2 1 mM 1 ml Fill to 100 ml with sterile dist. water substrate buffer (1 mg/ml of DNA Sigma D1626 in assay buffer) 4% perchloric acid (slowly add 10 ml of 70% perchloric acid to 165 ml of water)

(48) 100 μl of substrate are mixed with 20 μl of solution containing DNase. If a dilution of the DNase is necessary, then the enzyme is diluted in assay buffer. The mixture must be diluted to different degrees, depending on activity and purity. The S. marescens nuclease is replaced by assay buffer as blank reading value.

(49) The solution is incubated for exactly 20 minutes at 37° C. and the reaction is then stopped by adding 100 μl of 4% perchloric acid. The solution is then immediately incubated on ice for 10 minutes to assure a complete precipitation of the unconverted DNA. The precipitated DNA is then centrifuged off (16 000×g; 10 min; 4° C.). 150 μl is removed from the supernatant and is measured diluted 1:5 with water. For this, the 150 μl of supernatant is mixed with 600 μl of water and the extinction is measured in a quartz cuvette photometer at 260 nm, which had previously been adjusted with water. In this case, a unit corresponds to the amount of enzyme that causes an absorption change at 260 nm of 1 in 60 minutes. i) S. marescens nuclease expression rates obtained (MU per liter of supernatant):

(50) TABLE-US-00016 Medium 1 2 3 4 B. subtilis aprE nprE epr amyE 0.4 bglC with plasmid construct 1 B. subtilis aprE nprE epr amyE 0.6 bglC with plasmid construct 2 B. subtilis aprE nprE epr amyE 2.5 bglC with plasmid construct 3 B. subtilis aprE nprE epr amyE 0.2 bglC with plasmid construct 4 B. subtilis aprE nprE epr amyE 3.7 0.7 bglC with plasmid construct 5 B. subtilis aprE nprE epr amyE 1.6 bglC with plasmid construct 6 B. subtilis aprE nprE epr amyE 1.0 0.3 bglC with plasmid construct 8 B. amyloliquefaciens amy2 npr1 0.9 with plasmid construct 1 B. amyloliquefaciens amy2 npr1 1.1 with plasmid construct 2 B. amyloliquefaciens amy2 npr1 0.5 with plasmid construct 3 B. amyloliquefaciens amy2 npr1 1.3 25.7 15.1 26.4 with plasmid construct 5 B. amyloliquefaciens amy2 npr1 1.0 12.1 with plasmid construct 8 B. amyloliquefaciens amy2 npr1 21.1 27.7 apr::kan with plasmid construct 5 B. amyloliquefaciens amy2 npr1 10.6 apr::kan with plasmid construct 6 B. amyloliquefaciens amy2 npr1 11.9 apr::kan with plasmid construct 7

Illustrative Embodiment 7

(51) Heterologous Expression of the Serratia marcescens Nuclease in Escherichia coli.

(52) Cloning and Expression

(53) Expression vectors were constructed by insertion of an open DNA fragment into the plasmid pBR327 (DSMZ, Braunschweig). The DNA fragment comprised the open reading frame of the Serratia marcescens nuclease including its native signal sequence under the control of the native Serratia marcescens nuclease promoter. Two variants were constructed, which differ in orientation of the DNA fragment with respect to the resistance gene of pBR327. The expression of the nuclease occurs in the E. coli strain MC1000 (from CGSC, New Haven USA) in the agitation vessel in LB medium with 0.2% glucose and 100 μg/ml of ampicillin at 37° C. for 72 h. The strain MC1000 with the empty plasmid pBR327 was used as control. Samples of the cultures were taken at different times. The cells were separated by centrifugation and the nuclease activity was determined in the supernatant. The determination of the expressed enzyme activities occurs as described under h) in illustrative embodiment 6. The following maximum expression yields were obtained:

(54) TABLE-US-00017 MU per liter of culture supernatant MC1000 pBR327 0.042 MC1000 pBR327 + nuclease variant 1 0.066 MC1000 pBR327 + nuclease variant 2 0.074

(55) In a second batch the Serratia marcescens nuclease was expressed in the fermenter. LB medium with 0.2% glucose and 100 μg/ml of ampicillin was used as medium. The growth temperature amounted to 35° C. The pH value was set at 8.4. Ventilation occurred at 500 rpm with 1 vvm of air. Fermentation occurred for 72 h. Samples of the cultures were taken at different times. The cells were separated by centrifugation and the nuclease activity was determined in the supernatant.

(56) The content of nuclease activity in the periplasm of the strains was also examined. For this, the separated pellet of the cells was taken up in 0.03 M tris-HCl pH 8 20% saccharose. 80 ml of buffer was used for 1 g of pellet EDTA (end concentration 1 mM) was then added to the suspension. The suspension was incubated for 10 minutes with agitation. The cells were then pelletised by centrifugation. The pellets were taken up in a sample volume of cold deionised water with a temperature of 4° C. and incubated at 4° C. for 10 minutes. The suspension was centrifuged and the nuclease activity measured in the supernatant.

(57) The determination of the expressed enzyme activities occurs as described under h) in illustrative embodiment 6. The following maximum expression yields were obtained:

(58) TABLE-US-00018 MU per liter MU per liter of culture of culture supernatant periplasm MC1000 pBR327 0.032 0.002 MC1000 pBR327 + nuclease variant 1 0.062 0.023 MC1000 pBR327 + nuclease variant 2 0.086 0.025

(59) The foregoing description and examples have been set forth merely to illustrate the invention and are not intended to be limiting. Since modifications of the described embodiments incorporating the spirit and substance of the invention may occur to persons skilled in the art, the invention should be construed broadly to include all variations within the scope of the appended claims and equivalents thereof.