Improved variants of starch phosphorylase active at high temperature

Description

[0001] Improved Variants of Starch Phosphorylase Active at High Temperature

[0002] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing, which has been submitted electronically in .xml format. The contents of the electronic sequence listing (CP0215W001.XML; Size: 1 2,641 bytes, and Date of Creation: November 10, 2025) is incorporated by reference herein in its entirety.

[0004] TECHNICAL FIELD

[0005] This disclosure relates generally to amino acid substitutions that improve the enzymatic activity of starch phosphorylase, which may be used in one pot reactions for producing sugars such as fructose or allulose from starch or dextrins derived from starch.

[0006] BACKGROUND

[0007] Starch phosphorylase is an essential enzyme that plays a crucial role in the metabolism of starch and glycogen in plants and bacteria, respectively. This enzyme catalyzes the transfer of phosphate groups to the non-reducing end of a glucose polymer, resulting in the formation of glucose- 1 -phosphate. This reaction is a crucial step in the synthesis and degradation of glycogen and starch, which are the major carbohydrate storage molecules in living organisms. The activity of starch phosphorylase is crucial for the proper functioning of many metabolic processes in plants and bacteria. Starch phosphorylase is industrially useful and a preferred enzyme among all glucan phosphorylases for phosphorolytic reactions for the production of glucose- 1 -phosphate and for the development of engineered varieties of glucans and starch. More recently, starch phosphorylase has been employed as the starting enzyme for conversion of starch (or starch derivatives like maltodextrin) to derivative sugars such as fructose, allose or allulose in a one pot reaction that begins by phosphoroly sis of starch or dextrin, then forming glucose- 1 -phosphate and converting the glucose- 1 -phosphate using a phosphoglucomutase and phosphoglucosisomerase and in some cases an epimerase and finally a phosphatase to dephosphorylate a 6 phosphorylated sugar residue that is ultimatety form the desired sugar. Other starch-hydrolyzing enzymes like pullulanase and glucanotransferase can be added to the enzymes cocktail.

[0008] However, the activity of starch phosphory lase can be affected by various factors such as pH. temperature, substrate concentration, and the presence of inhibitors. These factors can lead to reduced enzyme activity and therefore decrease the efficiency when used to produce starch derivatives in a commercial scale operation. There have been several studies aimed at improving the activity of starch phosphory lase enzyme. Some of the approaches used include directed evolution, site-directed mutagenesis, and rational design.

[0009] Directed evolution involves generating a library of randomly mutated enzymes and selecting those with improved activity. Site-directed mutagenesis involves altering specific amino acid residues in the enzyme to improve its activity. Rational design involves designing new enzy mes based on the know n structure and function of existing enzymes.

[0010] Research papers have reported success in improving the activity of starch phosphorylase enzy me using these methods. For example, one study reported a 16-fold increase in the activity of starch phosphorylase using directed evolution. Oyama. S., Toyama, H., Toyama, C., & Nishimoto, T. (2016). Directed evolution of a starch phosphorylase from Corynehacterium callunae with improved activity in the maltodextrin range - Applied microbiology and biotechnology, 100(4), 1723-1731. Another study used site-directed mutagenesis to improve the activity of the enzyme by 6-fold. Chang, J., Zheng, Y., Ge, X., Zhang, J., & Li, X. (2013). Enhancement of the activity) of maize starch phosphorylase by site-directed mutagenesis - International Journal of Biological Macromolecules, 60, 387-392.

[0011] Overall, these studies demonstrate the potential for improving the activity of starch phosphorylase enzy me, which could have important implications for various industrial and biotechnological applications. The desirable feature to improve is thermal tolerance, and specific activity of these enzymes. The optimal temperature and specific activity range for starch phosphorylase activity can vary depending on the source of the enzy me, as well as the experimental conditions. Thermostable starch phosphorylases are reported in the literature (Ubiparip Z. Et. al. Applied Microbiology and Biotechnology (2018) 702:8187-8202). The thermostable starch phosphorylases have lower specific activity < 66 U mg1of protein while the enzymes from Zea mays and Dioscorea cayenensis have low temperature tolerance while high specific activity (up to 659 U mg'1). However, in general, the optimal temperature for starch phosphory lase activity7is between 30°C and 40°C. Starch phosphory lase from different organisms may have slightly different temperature optima. For example, the optimal temperature for potato starch phosphorylase is around 30°C, while the optimal temperature for maize starch phosphory lase is around 37°C. In industrial-scale operations, it is desirable to use enzymes with high thermal stability7and high specific activity, operating at temperatures above 50°C.

[0012] It is therefore desirable to employ mutagenic techniques to generate improved starch phosphorylase enzymes operable at higher temperature ranges.

[0013] SUMMARY OF THE INVENTION

[0014] In one aspect, disclosed herein are improved starch phosphorylase enzyme comprising a set of three amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of: a) E199D, D806Q and H832Q; b) I268V, F349L and F349L; c) N67D, M90L and A272V; d) V776I, V829I and H832Q; e) E18K, L236I and E294S; f) D103N, A272V and H765R; g) L76I, P709K, and H382Q; h) N67D, D720N and D806Q; i) S62N, E293D and E294S; j) I126V, H755R and F811Y; k) D190N, T204A and D720N; 1) F349L, F811Y and D835N; m) M90L, E293D and F811Y; n) F668Y, P709K and D806Q; and o) L236I, M465L and A719G; wherein the improved phosphorylase enzyme exhibits at least one improved property7selected from having greater genetic expressibility in B. subtilis or having greater enzymatic activity7in phosphorylating starch or dextrins derived from starch than a starch phosphorylase enzy me comprising the amino acid sequence of SEQ ID NO: 1.

[0015] In another aspect disclosed here are improved starch phosphorylase enzymes comprising at least three amino acid substitutions in SEQ ID NO: 1 of N67D, M90L and A2HN , wherein the improved phosphorylase enzyme exhibits greater enzymatic activity in phosphorylating starch or dextrins derived from starch than a starch phosphory lase enzy me comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments the improved starch phosphory lase enzyme further comprises at least one additional amino acid substitution in SEQ ID NO: 1 selected from the group consisting of E18K, S62N, Y217I, L236I, V292I, E294S, F349L, E460N, H755R, and F811Y. In some embodiments, the improved starch phosphorylase further comprises at least two additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of E18K. S62N, Y217I, L236I. V292I. E294S. F349L, E460N, H755R, and F811Y. In still other embodiments, the improved starch phosphorylase enzyme further comprises at least three additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of E18K, S62N, Y217I, L236I, V292I, E294S, F349L, E460N, H755R, and F811Y.

[0016] In a further aspect, disclosed herein are improved starch phosphorylase enzymes comprising at least six amino acid substitutions in SEQ ID NO: 1 of S62N, N67D, M90L, A272V L236I and V292I wherein the improved phosphory lase enzyme exhibits greater enzymatic activity7in phosphory lating starch or dextrins derived from starch than a starch phosphorylase enzy me comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the improved starch phosphorylase comprises at least one additional amino acid substitution in SEQ ID NO: 1 selected from the group consisting of K175A, V217I, D227E, E293D, E295S, E460N, T478R, P709K, R726K, H755R and F811Y. In some embodiments, the improved starch phosphorylase comprises at least two additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of K175A, V217I, D227E, E293D. E295S, E460N, T478R, P709K, R726K, H755R and F81 1Y. In other embodiments, the improved starch phosphorylase comprises at least three additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of K175A, V217I, D227E, E293D, E295S, E460N. T478R, P709K, R726K, H755R and F811Y. In other embodiments, the improved starch phosphorylase comprises at least four additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of K175A, V217I, D227E, E293D, E295S, E460N, T478R, P709K, R726K, H755R and F811Y. In other embodiments, the improved starch phosphorylase comprises at least five additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of KI 75 A, V217I, D227E, E293D, E295S, E460N, T478R, P709K, R726K, H755R and F81 1Y. In still other embodiments, the improved starch phosphory lase comprises at least six additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of K175A, V217I, D227E, E293D, E295S. E460N, T478R, P709K. R726K, H755R and F811Y.

[0017] In still a further aspect, disclosed herein are improved starch phosphorylase enzymes comprising at least six amino acid substitutions in SEQ ID NO: 1 of S62N, N67D, L236I, V217I, A272V, and T478R wherein the improved phosphorylase enzyme exhibits greater enzymatic activity in phosphorylating starch or dextrins derived from starch than a starch phosphorylase enzyme comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the improved starch phosphorylase comprises at least one additional amino acid substitution in SEQ ID NO: 1 selected from the group consisting of M90L, D227E, Q463E, P709K, R276K, R435S, and H755R, In some embodiments, the improved starch phosphorylase comprises at least two additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of M90L, D227E, Q463E, P709K, R276K, R435S, and H755R. In some embodiments, the improved starch phosphory lase comprises at least three additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of M90L, D227E. Q463E, P709K, R276K, R435S, and H755R. In some embodiments, the improved starch phosphorylase comprises at least four additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of M90L, D227E, Q463E, P709K, R276K, R435S, and H755R. In some embodiments, the improved starch phosphorylase comprises at least five additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of M90L, D227E. Q463E, P709K, R276K, R435S, and H755R. In some embodiments, the improved starch phosphorylase comprises at least six additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of M90L, D227E, Q463E, P709K, R276K, R435S, and H755R. In still other embodiments, the improved starch phosphorylase comprises at least seven additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of M90L, D227E, Q463E, P709K, R276K, R435S, and H755R.

[0018] In yet another aspect, disclosed herein are improved starch phosphorylase enzymescomprising any of the set of amino acid substitutions in SEQ ID NO: 1 shown in Figure 4 wherein the improved phosphorylase enzy me exhibits at least one improved property selected from having greater genetic expressibility in B. subtilis or having greater enzymatic activity in phosphorylating starch or dextrins derived from starch than a starch phosphorylase enzy me comprising the amino acid sequence of SEQ ID NO: 1.

[0019] BRIEF DESCRIPTION OF THE FIGURES

[0020] Figure 1 is a chart that shows the starch phosphory lase activity measured as a result of screening 96 candidate starch phosphorylase genes identified by internal ID number at 50 °C (left bar) and 60 °C (right bar) for four hours in the presence of 150 mM phosphate and a maltodextrin sold by Archer Daniels Midland Company under the name Clintose™. Reactions were conducted for four hours. Phosphory lase activity is expressed as the amount of glucose-1 phosphate (G1P) produced per minute in the presence of the candidate phosphorylase enzyme in the presence of pullulanase and glucanotransferase to digest the maltodextrin releasing GIP. Numbers are the average of duplicate measurements.

[0021] Figure 2 is a chart showing the percent conversion of maltodextrin / starch that shows the relative specific activity of 12 starch phosphorylase enzymes of the present invention that are variants of a naturally occurring wild type enzyme (WT) having internal ID number 401436 (SEQ ID NO: 1) measured after 18 hours at 60°C.

[0022] Figure 3 shows embodiments of soluble phosphorylase levels of various starch phosphorylase enzymes of the present invention in polyacrylamide gel electrophoresis and densitometry'.

[0023] Figure 4 is a table showing specific amino acid substitutions made in the parent starch phosphorylase according to SEQ ID NO: 1 and subsequent improvements in starch phosphory lase activity over each immediate parent after 4 rounds of diversification and engineering.

[0024] DETAILED DESCRIPTION

[0025] This disclosure provides improved starch phosphorylase enzymes useful for phosphorolytic degradation any alpha glucan polymer such as starch, dextrins and maltotodextrins. The improvements were developed by synthesizing codon optimized nucleic acids encoding candidate starch phosphorylase genes or mutations thereof in a B. subtilis expression system, expressing the candidates in B. subtilis, forming crude extracts of the B. subtilis cells and measuring the lysine decarboxylase in the crude extracts using equal amounts of protein.

[0026] B. subtilis Expression of Variant Phosphorylase genes

[0027] Genes for glucan phosphorylases were designed for high expression in B. subtilis and cloned into the IPTG inducible expression vector pHT254. The resulting plasmid was used to transform B. subtilis by standard procedures. Transformants were selected by growth on LB agar plates containing appropriate antibiotics. Selected colonies were used to inoculate 0.5 ml of LB media with antibiotics and cultures were incubated overnight with shaking at 37°C. Overnight culture were used to inoculate 0.5 ml of fresh LB media containing antibiotics and the resulting culture was incubated with shaking at 37°C until an OD of 0.8 at 600 nm was reached. Cultures were cooled to 24°C, and induced with the addition of IPTG to 1 mM and incubation at 24°C. Cells were harvested and lysed. Lysate was treated at 70°C for 30 minutes and the remaining soluble fraction was recovered from supernatant after centrifugation. Soluble phosphorylase level was analyzed by polyacrylamide gel electrophoresis and densitometry and shown in Figure 3.

[0028] The candidate transformants were grown to mid-log phase and the lysed as described above. The soluble fraction of the lysates was quantified and evaluated for the formation of glucose- 1 -phosphate. Reaction was filtered through a PES membrane and the formation of glucose 1 -phosphate was measured using the colorimetric GIP assay kit from Millipore Sigma (Cat No MAK098)

[0029] Sequence Panning for Candidate alpha glucan Phosphorylase Genes

[0030] Several sequence libraries were initially probed to identify sequences that were known to be starch or alphaglucan phosphorylases based on actual evidence of starch phosphorylase activity, on notations in sequence databases indicating a predicted starch phosphorylase activity or because they share a high amino sequence identify to known starch phosphorylase enzy mes. Synthetic DNA constructs encoding the candidates with codons optimized for expression in B. subtilis were evaluated via the above B. subtilis expression system protocol. The assay conditions for the initial screening were 4-hour reactions with 20% w / v of a maltodextrin sold by Archer Daniels Midland Company under the tradename Clintose™ CR10 as a substrate, 0.5 mM pyridoxal phosphate (PLP), 2 mM MgCh, 1 0 mM NaPO4, at pH 7.0. After the reaction was stopped and liberated glucose- 1 -phosphate (G1P) that was measured colorimetric assay kits Sigma (Cat No MAK098). Figure 1 shows the activity of 96 initial candidate sequences screened for activity at 50°C and 60°C.

[0031] The candidate starch phosphorylase designed with Gene ID number 401436 (SEQ ID NO: 1) displaying one of the two enzymes exhibiting the highest levels of activity at 60°C was selected for improvement by substitution of 3 amino acid sequences at a time at various positions along the length of the protein over multiple rounds.

[0032] Generation of Variants of the SEQ ID NO: 1

[0033] Based on methods described in US 8,635,029, nucleotide changes encoding amino acid substitutions in SEQ ID NO: 1 were introduced into that gene, which was expressed and assayed using the B. subtilis expression system. In a first round of variant production, 96 sets of three amino acid substitutions distributed across the length of the protein were introduced into SEQ ID NO: 1. These round 1 variants and all subsequent variants were evaluated for starch phosphorylase activity by incubating equal protein amounts of B. subtilis crude extracts containing the variant proteins in reactions containing 20% w / v of Clintose™ CR10 as a substrate, 0.5 mM (PLP), 2 mM MgCb, 35 mM NaPO-i. at pH 7.0 for 24 hours at 65°C.

[0034] Unless indicated otherwise, the best performing variant SEQ ID NO: 1 from was selected and subjected to multiple rounds of diversification and machine learning (US 8,635,029) where substitutions having positive impacts on activity and stability are enriched.

[0035] Figure 4 is a table showing the specific amino acid substitutions introduced in sequential rounds of variant production. The substitutions are expressed in the form XNZ, where X is the one letter code for the original amino acid present in SEQ ID NO: 1, N is the amino acid position counting from the N terminus of the E.coli protein, and Z is the one letter code for the specific amino acid substitution made at that position. Those amino acid substitutions most preserved in multiple rounds of variant production are written across the top of the table. Those amino acid substitutions not preserved more than twice are listed in the penultimate column as “other substitutions.” The relative activity shown in the last column is the best measured activity relative to the immediate parent. Each reaction was done in triplicate and the relative activity reported is the average thereof.

[0036] Figure 4 shows that 16 round 1 and round 2 variants had improved activity ranging from 1.10 to 1.26 fold increased activity over the parent SEQ ID NO: 1. Those round 1 and round 2 variants had the following amino acid substitutions: a) E199D, D806Q and H832Q; b) I268V, F349L and F349L; c) N67D, M90L and A272V; d) V776I, V829I and H832Q; e) E18K, L236I and E294S; f) D103N; A272V and H765R; g) L76I, P709K. and H382Q; h) N67D; D720N and D806Q: i) S62N, E293D and E294S; j) I126V, H755R and F811Y; k) D190N, T204A and D720N; 1) F349L, F811Y and D835N; m) M90L, E293D and F811Y; n) F668Y, P709K and D806Q; and o) L236I, M465L and A719G.

[0037] Measured data indicated that some variants such as V264, V274, V246 and V285 may have shown higher measured activity because the variant protein was expressed better in B. subtilis than the parent, so Figure 4 also indicates whether the variant showed higher expression in B. subtilis. Although the variant designated V221 containing the N67D, M90L and A272V substitutions showed a rather small improvement over the original enzyme in measured activity', V221was used as the parent for round 3 variant production because the enzy me was not well expressed in B. subtilis yet demonstrated higher activity than the parent meaning the enzyme itself performs better than the parent although its expression in the B. subtilis host was worse. The substitutions in V264, V246 and V285, which seemed to show greater expressibility in B. subtilis were reintroduced in subsequent rounds of variant production.

[0038] The variants produced in round 3 retained at least two of the three substitutions of their immediate parent. Figure 4 shows that 14 round 3 variants had improved activity over their immediate parent. Almost all of these round 3 variants retained at least three amino acid substitutions of N67D, M90L and A272V and all demonstrated greater enzy matic activity' in phosphorylating maltodextrin than a starch phosphory lase enzyme comprising the amino acid sequence of SEQ ID NO: 1. In some variants the improved starch phosphorylase enzyme from round 3 further included at least one additional amino acid substitution in SEQ ID NO: 1 selected from the group consisting of E18K, S62N, Y217I, L236I, V292I, E294S, F349L, E460N, H755R, and F811Y. In some variants the improved starch phosphorylase further included at least two additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of E18K, S62N, Y217I, L236I. V292I. E294S. F349L E460N, H755R, and F811Y. In still other variants the improved starch phosphorylase enzyme further included at least three additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of E18K, S62N, Y217I, L236I, V292I, E294S, F349L, E460N, H755R, and F811Y.

[0039] Variant 309 showed the highest level of improvement over its immediate parent from round 2 so variant 309 was selected as the parent for a fourth of variant production retaining each at least six amino acid substitutions in from round 3 of S62N. N67D. M90L, A272V, L236I and V292I to make the round 4 variants by introducing three additional amino acid substitutions. Most variants with improved activity over their parent had at least one additional amino acid substitution in SEQ ID NO: 1 selected from the group consisting of K175A, V217I, D227E, E293D. E295S, E460N, T478R, P709K, R726K, H755R and F81 1Y. A large majority of round 4 variants had at least two additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of K I 75 A. V217I, D227E, E293D, E295S, E460N, T478R, P709K, R726K, H755R and F811Y. Many round 4 variants had at least three additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of K175A, V217L D227E, E293D, E295S, E460N, T478R, P709K, R726K, H755R and F811Y. Several of the improved round 4 variants had at least four additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of K175A, V217I, D227E, E293D, E295S. E460N, T478R, P709K, R726K, H755R and F811Y. Some of the round 4 variants had at least five additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of K175A, V217I, D227E, E293D, E295S, E460N, T478R, P709K, R726K, H755R and F811Y. More than a few improved round 4 variants had at least six additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of K175A, V217L D227E, E293D, E295S, E460N, T478R, P709K, R726K, H755R and F811Y.

[0040] Variant 439 from round 4 showed the highest level of improvement over its immediate parent from round 3 so variant 439 was selected as the parent for a fifth round of variant production retaining at least six of the nine amino acid substitutions from round 4 selected from the group consisting of S62N, N67D. M90L. KI 75 A, V217I, L236I, A272V . V292I and T478R. In many variants at least 7 of the foregoing substitutions were retained. In many variants at least 8 of the foregoing substitutions were retained in some embodiments. In several variants all of the foregoing substitutions were retained. The variants from round 5 that showed improved activity over their round 4 patent included at least one additional substitution selected from the group consisting ofD227E, Q463E, P709KR726K, R743S, andH755R. Many variants from round 5 that showed improved activity over the round 4 patent includes at least two of the foregoing additional amino acid substitutions and several variants included at least three of the forgoing additional substitutions.

[0041] The process of producing the variants shown in Figure 4 indicates that so long as any of the set key substitutions shown in any round of variant production are retained, it is possible to generate further variants having similar or improved activity starting with any of the set of variants show n in Figure 4.

[0042] Figure 2 shows the measured starch phosphorylase activity for the original starting parent phosphory lase according to SEQ ID NO: 1 (Wt) in comparison to the activity observed for representative variants from rounds 2 through 5. The measured activity is expressed as percent conversion of Maltodextrin to Fructose in 18 hours at 65°C, which was assayed by HPLC after digestion of the phosphory lated starch to monomers. As mentioned herein above, some variants displayed higher expressibility in the B. subtilis expression system used to produce the variants.

[0043] Figure 4 is a table showing specific amino acid substitutions made in the parent starch phosphorylase according to SEQ ID NO: 1 and subsequent improvements in starch phosphorylase activity over each immediate parent after 4 rounds of introducing 3 amino acid substitutions into the immediate parent. Asterisks indicate substitutions preserved in more than three variants. The sequence listing submitted herewith provides the amino acid sequence of representative variants shown in Figure 4.

Claims

CLAIMS1. An improved starch phosphorylase enzyme comprising a set of three amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of: a) E199D, D806Q and H832Q; b) I268V, F349L and F349L; c) N67D, M90L and A272V; d) V776I, V829I and H832Q: e) E18K, L236I and E294S f) D103N, A272V and H765R; g) L76I, P709K, and H382Q; h) N67D; D720N and D806Q; i) S62N E293D and E294S; j) I126V, H755R and F811Y; k) D190N, T204A and D720N; l) F349L, F811Y and D835N; m) M90L, E293D and F81 1Y; n) F668Y, P709K and D806Q; and o) L236I, M465L and A719G; wherein the improved phosphorylase enzyme exhibits at least one improved property selected from having greater genetic expressibility in B. subtilis and having greater enzy matic activity' in phosphorylating starch or dextrins derived from starch than a starch phosphorylase enzyme comprising the amino acid sequence of SEQ ID NO: 1.

2. An improved starch phosphorylase enzyme comprising at least three amino acid substitutions in SEQ ID NO: 1 of N67D, M90L and A2HN , wherein the improved phosphorylase enzy me exhibits greater enzymatic activity' in phosphorylating starch or dextrins derived from starch than a starch phosphorylase enzyme comprising the amino acid sequence of SEQ ID NO: 1.

3. The improved starch phosphory lase enzy me of claim 2, further comprising at least one additional amino acid substitution in SEQ ID NO: 1 selected from the groupconsisting of E18K, S62N, Y217I, L236I, V292I, E294S, F349L, E460N;H755R, and F811Y.

4. The improved starch phosphorylase enzyme of claim 2, further comprising at least two additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of E18K, S62N, Y217I, L236I, V292I, E294S, F349L, E460N. H755R, and F811Y.

5. The improved starch phosphorylase enz me of claim 2, further comprising at least three additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of E18K, S62N, Y2171, L236E V292I. E294S. F349L. E460N, H755R, and F811Y.

6. An improved starch phosphorylase enzyme comprising at least six amino acid substitutions in SEQ ID NO: 1 of S62N. N67D, M90L, A272V. L236I and V292I wherein the improved phosphor lase enzyme exhibits greater enzymatic activity in phosphorylating starch or dextrins derived from starch than a starch phosphory lase enzy me comprising the amino acid sequence of SEQ ID NO: 1.

7. The improved starch phosphorylase enzyme of claim 6, further comprising at least one additional amino acid substitution in SEQ ID NO: 1 selected from the group consisting of KI 75 A. V217E D227E, E293D, E295S, E460N, T478R, P709K, R726K. H755R and F811Y.

8. The improved starch phosphorylase enzyme of claim 6, further comprising at least two additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of K175A. V217E D227E, E293D, E295S, E460N. T478R, P709K, R726K, H755R and F811Y.

9. The improved starch phosphory lase enzy me of claim 6, further comprising at least three additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of KI 75 A, V217I, D227E, E293D, E295S, E460N, T478R, P709K, R726K, H755R and F811Y.

10. The improved starch phosphorylase enzyme of claim 6, further comprising at least four additional amino acid substitutions in SEQ ID NO: 1 selected from the groupconsisting of K175A. V217I, D227E, E293D, E295S, E460N, T478R, P709K, R726K. H755R and F811Y.I L The improved starch phosphorylase enzyme of claim 6, further comprising at least five additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of K175A. V217I. D227E, E293D, E295S, E460N. T478R, P709K, R726K, H755R and F811Y.

12. The improved starch phosphorylase enz me of claim 6, further comprising at least six additional amino acid substitution in SEQ ID NO: 1 selected from the group consisting of K175A. V2171. D227E, E293D, E295S. E460N. T478R, P709K, R726K, H755R and F811Y.

13. An improved starch phosphorylase enzyme comprising at least six amino acid substitutions in SEQ ID NO: 1 of S62N. N67D, L2361, V2171, A272V. and T478R wherein the improved phosphorylase enzyme exhibits greater enzymatic activity with starch or dextrins derived from starch than a starch phosphorylase enzy me comprising the amino acid sequence of SEQ ID NO: 1.

14. The improved starch phosphorylase enzy me of claim 13, further comprising at least one additional amino acid substitution in SEQ ID NO: 1 selected from the group consisting of M90L, D227E, Q463E, P709K, R276K. R435S, and H755R,15. The improved starch phosphorylase enzy me of claim 13, further comprising at least two additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of M90L, D227E, Q463E, P709K, R276K, R435S, and H755R.

16. The improved starch phosphorylase enzy me of claim 13, further comprising at least three additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of M90L, D227E, Q463E, P709K, R276K, R435S, and H755R.

17. The improved starch phosphorylase enzy me of claim 13, further comprising at least four additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of M90L, D227E, Q463E, P709K, R276K, R435S, and H755R.

18. The improved starch phosphory lase enzy me of claim 13, further comprising at least five additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of M90L, D227E, Q463E, P709K, R276K, R435S, and H755R.

19. The improved starch phosphory lase enzy me of claim 13, further comprising at least six additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of M90L, D227E, Q463E, P709K, R276K, R435S, and H755R.

20. The improved starch phosphory lase enzy me of claim 13, further comprising at least seven additional amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of M90L. D227E, Q463E, P709K, R276K. R435S, and H755R.

21. An improved starch phosphory lase enzy me that is a variant of SEQ ID NO: 1 comprising at least six amino acid substitutions in SEQ ID NO: 1 selected from the group consisting of S62N, N67D, M90L, KI 75 A. V217I. L236I, A272V. V292I and T478R wherein the improved phosphorylase enzyme exhibits greater enzymatic activity7in phosphory lating starch or dextrins derived from starch than a starch phosphorylase enzyme comprising the amino acid sequence of SEQ ID NO: 1.

22. The improved starch phosphorylase enzyme of claim 21 comprising at least seven of the amino acid substitutions.

23. The improved starch phosphorylase enzyme of claim 21 comprising at least eight of the amino acid substitutions.

24. The improved starch phosphorylase enzyme of claim 21 comprising all the amino acid substitutions.

25. The improved starch phosphory lase enzy me according any one of claims 21 - 23, further comprising at least one additional substitution selected from the group consisting of D227E, Q463E, P709K R726K, R743S, and H755R.

26. An improved starch phosphorylase enzyme comprising any of the set of amino acid substitutions in SEQ ID NO: 1 shown in Figure 4 wherein the improved phosphorylase enzyme exhibits at least one improved property selected from having greater genetic expressibility in B. subtilis and having greater enzymaticactivity in phosphorylating starch or dextrins derived from starch than a starch phosphorylase enzy me comprising the amino acid sequence of SEQ ID NO: 1.

27. The improved starch phosphorylase enzyme of claim 26 comprising an amino acid sequence according to any of SEQ ID NOs: 2-81.

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