SACCHAROSE PHOSPHORYLASE

Abstract

The invention relates to a saccharose phosphorylase that catalyzes the synthesis of glucose-1-phosphate and fructose from saccharose and phosphate, among other things. The saccharose phosphorylase according to the invention can be considered to be a mutation of the saccharose phosphorylase from Bifidobacterium adolescentis. In comparison to wild-type saccharose phosphorylase, the saccharose phosphorylase according to the invention is distinguished by improved activity, process stability, temperature stability, and lower product inhibition and is therefore particularly suitable for use in industrial processes.

Claims

1. A sucrose phosphorylase comprising an amino acid sequence which has an identity in relation to the amino acid sequence according to SEQ ID NO:1 of at least 80% and which, in comparison with SEQ ID NO:1, comprises an amino acid mutation in a sequence segment C) corresponding to positions 175 to 195 according to SEQ ID NO:1; and comprises an amino acid mutation in a sequence segment D) corresponding to positions 285 to 305 according to SEQ ID NO:1.

2. The sucrose phosphorylase as claimed in claim 1, wherein sequence segment C) corresponds to positions 180 to 190 according to SEQ ID NO:1; and sequence segment D) corresponds to positions 290 to 300 according to SEQ ID NO:1.

3. The sucrose phosphorylase as claimed in claim 2, wherein sequence segment C) corresponds to positions 184 to 186 according to SEQ ID NO:1; and sequence segment D) corresponds to positions 294 to 296 according to SEQ ID NO:1.

4. The sucrose phosphorylase as claimed in claim 3, wherein the amino acid mutations are H185 and position I295 or H185G and I295V.

5. (canceled)

6. The sucrose phosphorylase as claimed in any of claim 1, which, in addition to the amino acid mutations in the sequence segments C) and D), comprises at least one amino acid mutation in a sequence segment G) corresponding to positions: (i) 464 to 484 according to SEQ ID NO:1; (ii) corresponding to positions 469 to 481 according to SEQ ID NO:1; or (iii) corresponding to positions 473 to 477 according to SEQ ID NO:1.

7-8. (canceled)

9. The sucrose phosphorylase as claimed in claim 6, wherein the amino acid mutation is D474 or T476, or D474E or T476A.

10. (canceled)

11. The sucrose phosphorylase as claimed in claim 1, which, in addition to the amino acid mutations in the sequence segments C) and D) and optionally G), comprises an amino acid mutation in a sequence segment A) corresponding to positions: (i) 11 to 31 according to SEQ ID NO:1; (ii) 16 to 26 according to SEQ ID NO:1; or (iii) 20 to 22 according to SEQ ID NO:1.

12-13. (canceled)

14. The sucrose phosphorylase as claimed in claim 11, wherein the amino acid mutation is S21 or S21G.

15. (canceled)

16. The sucrose phosphorylase as claimed in claim 1, which, in addition to the amino acid mutations in the sequence segments C) and D) and optionally G), comprises an amino acid mutation in a sequence segment F) corresponding to positions: (i) 447 to 459 according to SEQ ID NO:1; or (ii) 447 to 456 according to SEQ ID NO:1.

17-18. (canceled)

19. The sucrose phosphorylase as claimed in claim 16, wherein the amino acid mutation is S451, or S451T.

20. (canceled)

21. The sucrose phosphorylase as claimed in claim 1, which does not comprise at least one amino acid mutation selected from the group consisting of Q331E, R393N, D445P, D446G, D446T, Q460E and E485H, preferably does not comprise all these amino acid mutations.

22. The sucrose phosphorylase as claimed in claim 1, which catalyzes the conversion of sucrose and phosphate to glucose 1-phosphate and fructose.

23. The sucrose phosphorylase as claimed in claim 1, which, in comparison with the sucrose phosphorylase according to SEQ ID NO:1, has (i) an increased activity with respect to the conversion of sucrose and phosphate to glucose 1-phosphate and fructose in the presence of equimolar amounts of glucose 1-phosphate and fructose; and/or (ii) a lower inhibition by fructose with respect to the conversion of sucrose and phosphate to glucose 1-phosphate and fructose in the presence of equimolar amounts of glucose 1-phosphate and fructose; and/or (iii) a higher temperature stability after incubation at 58 C. for 15 min.

24. The sucrose phosphorylase as claimed in claim 1, which has an identity in relation to the amino acid sequence according to SEQ ID NO:4 of at least 90%, or at least 95%.

25. (canceled)

26. The sucrose phosphorylase as claimed in claim 1, which has an identity in relation to the amino acid sequence according to SEQ ID NO:5 of at least 90%, or at least 95%.

27. (canceled)

28. The sucrose phosphorylase as claimed in claim 1, which has an identity in relation to the amino acid sequence according to SEQ ID NO:6 of at least 90%, or at least 95%.

29. (canceled)

30. The sucrose phosphorylase as claimed in claim 1, which has an identity in relation to the amino acid sequence according to SEQ ID NO:8 of at least 90%, or at least 95%.

31-32. (canceled)

33. A method for producing glucose 1-phosphate and fructose, comprising the conversion of sucrose and phosphate under enzymatic catalysis by the sucrose phosphorylase as claimed in claim 1.

34. A method for producing cellobiose and fructose from sucrose and glucose, comprising the steps of (a) synthesis of glucose 1-phosphate and fructose by conversion of sucrose and phosphate under enzymatic catalysis by the sucrose phosphorylase as claimed in any of claim 1; (b) synthesis of cellobiose and phosphate by reaction of the glucose 1-phosphate with glucose under enzymatic catalysis by a cellobiose phosphorylase.

Description

Example 1

Engineering of the Wild-type Sucrose Phosphorylase from Bifidobacterium Adolescentis

[0199] For the engineering of the wild type of the sucrose phosphorylase from Bifidobacterium adolescentis, the following engineering goals were defined: [0200] reduction in fructose inhibition [0201] increase in activity in general [0202] reduction in the Km value with regard to phosphate [0203] reduction in G1P inhibition [0204] maintenance of temperature stability

[0205] For the engineering, what was chosen was a semirational, iterative approach. In the first round of engineering, there was first of all, after analysis of all available data, the identification of potentially interesting positions and possible substitutions and the creation thereof as individual mutants. These were then screened in a multiparameter screening according to the desired engineering goals. Positive mutations from said first round were then combined in the following and the resultant variants were characterized.

[0206] All assays were carried out at 30 C. and pH 6.3. Commercial G1P (Sigma-Aldrich, article No. G7000) was exclusively used as G1P. The wild-type enzyme (with new codon optimization) was always co-assayed twice on each plate as comparative enzyme. All data were normalized to a comparative enzyme which was co-assayed on the same plate, making it possible to compensate for fluctuations in expression or measurements between different plates.

Overview of multiparameter screening:

TABLE-US-00002 Engineering goal Test conditions Detection Increase in 750 mM sucrose + G1P phosphorolysis 750 mM Pi activity Decrease in fructose 750 mM sucrose + G1P inhibition 750 mM Pi + 250 mM fructose Decrease in the 750 mM sucrose + G1P Pi Km value 20 mM Pi Maintenance of Pretreatment for G1P temperature 15 min/65 C.; stability reaction with 750 mM sucrose + 750 mM Pi

[0207] By means of MDM analysis, 169 positions and substitutions were selected for a first round of engineering. This corresponds to about 33% of all positions, but less than 2% of all possible individual mutants. Said individual mutants were created using the AGM method (automated generation of mutants). The sequence of the wild-type gene (with codon optimization) was used as template. The variants created were expressed and were screened according to the different engineering goals. The results were normalized to the wild-type co-assayed in duplicate on each plate. It was possible to identify improved variants for all the engineering goals.

[0208] In a second round of engineering, the 7 best mutations from the first round were combined with one another (128 variants). The complex bank was created using the mutagenesis method according to WO2009/146892. 376 clones of the complex bank were picked, expressed, and screened with regard to the engineering goals (2.9-fold oversampling, screening coverage 95%). It was possible to identify improved variants for all the engineering goals.

[0209] In a third round of engineering, the hits from the second round were additionally combined with further primary hits from the 1st round (105 variants). Said variants were created, expressed and screened using the AGM method (automated generation of mutants).

[0210] The results of the engineering with respect to phosphorolysis activity, fructose inhibition and also Km value with regard to phosphate (Pi) are depicted in comparison with the wild type (WT) in FIGS. 1A to 1C.

Example 2

Characterization of the 7 Best Variants

[0211] The 7 best variants from the second and third round of engineering were selected for a detailed, final characterization (SEQ ID NO:2 to 8):

TABLE-US-00003 SEQ ID A B C D E F G NO. Reference S21 H142 L151 H185 1295 N396 S451 D474 T476 1 WT pSE014 2 P1-C7 pPB115 A S E 3 P1-C10 pPB116 Y S E 4 P2-A3 pPB117 G G V E 5 P2-B7 pPB118 G V T E 6 P2-B9 pPB119 G V A 7 PB10- pPB111 S E 24-1 8 PB10- pPB113 G V E 24-3
a) Activity yield and soluble expression

[0212] The seven variants selected and the wild-type enzyme (WT) were expressed in two independent expression cultures in a shake flask, they were disrupted, and the activity yield per mL of expression culture was determined:

TABLE-US-00004 SEQ ID NO: Plasmid ID PU/mL culture s Factor WT 1 (=WT) pSE014 35.4 1.1 1.00 2 pPB115 35.6 2.2 1.00 3 pPB116 46.1 1.7 1.30 4 pPB117 47.1 3.0 1.33 5 pPB118 34.9 0.3 0.99 6 pPB119 40.7 1.9 1.15 7 pPB111 46.3 2.4 1.31 8 pPB113 41.2 2.5 1.16

[0213] For the assessment of soluble expression, the soluble fraction was normalized. No significant differences in the level of expression were observed between the different variants.

b) Melting curves

[0214] One of the engineering goals was to maintain the high temperature stability of the wild-type sucrose phosphorylase. To test the variants, the enzymes were incubated in a PCR cycler at temperatures between 40 and 72 C. for 15 min and the residual activity of the enzymes was subsequently determined. Five of the variants investigated exhibited the same temperature stability as the wild-type enzyme. The results are depicted in FIG. 2.

c) Fructose inhibition, G1P inhibition and phosphate Km value

[0215] For the assessment of possible changes in the variants with regard to fructose or G1P inhibition, the changes in the initial activities in the presence of rising fructose or G1P concentrations were measured in each case for the enzyme variants and for the wild-type enzyme. As substrate, 200 mM sucrose and phosphate were added in each case.

[0216] It became apparent that all the variants investigated had a distinctly lower inhibition in the presence of 250-750 mM fructose. Even though it was not possible to completely eliminate the fructose inhibition, the degree of improvement is notable. By contrast, in the case of the G1P inhibition, the improvements observed were not so distinct. However, the G1P inhibition of the wild type is also not so highly pronounced, like the fructose inhibition. In addition, the G1P inhibition takes effect only at G1P concentrationen above 100 mM.

[0217] Analogous to the experiments relating to enzyme inhibition, the initial activity in the presence of 10-250 mM phosphate was determined for all variants and the wild-type enzyme in order to capture changes in the Km value with regard to phosphate. None of the variants exhibited significant differences in the initial activity in comparison with the wild type at different phosphate concentrations. No variant exhibited a deterioration compared to the wild type.

[0218] The results with respect to fructose, phosphate and glucose 1-phosphate inhibition are depicted in FIGS. 3A to 3C.

[0219] Four selected variants were expressed in a shaking flask, harvested, resuspended in 50 mM Na MES buffer, pH 6.3, and disrupted using ultrasound. Cell fragments and insoluble constituents were removed by centrifugation. The supernatant was sterile-filtered and admixed 50/50 (v/v) with sterile glycerol. The phosphorolysis activities of the formulated enzymes were determined:

TABLE-US-00005 Phosphorolysis SEQ ID NO: Reference Volume [mL] activity [PU/mL] 8 pPB113 37.5 408.5 4 pPB117 45 460.2 5 pPB118 47 384.3 6 pPB119 43.5 444.1