# Patent

## Title
Modified glucansucrase and related methods

## Abstract

Disclosed is a genetically modified enzyme belonging to glycosyltransferases type of glucansucrase comprising at least one mutation at position 654 of said enzyme, wherein modified enzyme is capable to producing a glucan polymer.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This present application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Ser. No. 61/936,663, which was filed on Feb. 6, 2014, and is hereby incorporated by reference.


FIELD OF INVENTION
The present invention is related to a modified glycoside hydrolase enzyme comprising at least one mutation to the amino acid downstream from a transition state stabilizer site wherein the modified enzyme produces a glucan. The modified glucan can have a higher yield of 1,3-disubstituted α-D-glucopyranosyl units as compared to a glucan produced by a wild-type glucansucrase, a higher yield of 1,6-disubstituted α-D-glucopyranosyl units as compared to a glucan produced by a wild-type glucansucrase, lower water solubility as compared to a glucan produced by a wild-type glucansucrase, or higher water solubility as compared to a glucan produced by a wild-type glucansucrase.
BACKGROUND OF INVENTION
Glucansucrases are a type of glycosyltransferase (GTF) that belong to the glycoside hydrolase family 70 (GH70), as defined by the CAZy classification system (Cantarel B. L., et al., 2009, Nucleic Acids Res 37:D233-D238), and catalyze the transfer of D-glucopyranosyl units from sucrose to acceptor molecules to form α-glucan chains. Glucansucrases are capable of catalyzing the synthesis of several different α-glucosidic linkages that affect molecular mass, branching, and solubility of the polysaccharide. In general, α-glucans containing mostly α(1→6) linkages (e.g., dextran) are water-soluble, while those made primarily of α(1→3) linkages are water-insoluble. Sequences of α(1→6) linked glucose units tend to form a flexible chain which readily hydrates and dissolves in water, whereas sequences of α(1→3) linked glucose units tend to form extended ribbon-like helices which self-associate and are water-insoluble, similar to cellulose (Rees D A, et al., 1971, J Chem Soc 8:469-479; Yui et al., 2000, Biosci Biotechnol Biochem 64:52-60). The term “mutan” is often used to refer to the water-insoluble glucan produced by Streptococcus mutans and related bacteria (Guggenheim et al., 1970, Helv Odont Acta 14:89-108), and it has become well-established that “mutan” is a graft or block-type copolymer consisting of regions of dextran-like α(1→6) linkages sequences as well as sequences of α(1→3) linked regions. This block or graft copolymer structure is quite different from the highly water-soluble alternan, which has similar proportions of α(1→3) and α(1→6) linkages arranged in a regular, alternating fashion with no extended sequences of either linkage type (Côté, 2002, Chapter 13 in Biopolymers, Vol. 5. Polysaccharides I. Polysaccharides from Prokaryotes. E. J. Vandamme, S. DeBaets, A. Steinbiichel, Eds. Wiley-VCH, Weinheim, Germany. Pp. 323-350).
Whereas the utility of water-soluble dextran has been well-established (Leathers, 2002, Chapter 12 in Biopolymers, Vol. 5. Polysaccharides I. Polysaccharides from Prokaryotes. E. J. Vandamme, S. DeBaets, A. Steinbiichel, Eds. Wiley-VCH, Weiheim, Pp. 299-321), applications of the related water-insoluble glucans are much less developed. As such, there is a need to establish a range of related water-insoluble glucans with varying properties.
Three-dimensional structures and targeted modifications of glucansucrases have provided substantial information regarding the functionality of these enzymes (Vujicic-Zagar, et al., 2010, Proc Natl Acad Sci USA 107:21406-21411; Ito, et al., 2011, J Molec Biol 408:177-186), but the mechanisms that control the type of glycosidic linkage still remain unclear. Glucansucrases are often described based on amino acid alignments with other glucansucrase as having an N-terminal variable region, followed by a catalytic domain, and a C-terminal glucan-binding domain (Monchois, et al., 1999, J Bacteriol 181:2290-2292) however, structural analyses of Lactobacillus reuteri GTF180-AN and Streptococcus mutans GTF-SI show that these glucansucrase proteins that catalyze α(1→6)/α(1→3) linkages actually contain five domains (A, B, C; IV and V) that are formed through a U-shape configuration that involves two regions of the polypeptide for each domain, with the exception of domain C. The amino acid residues of the catalytic triad (aspartate-nucleophile; glutamate-acid/base; aspartate-transition state stabilizer) are located within a deep pocket of domain A, which has a (β/α)8 barrel structure (Vujicic-Zagar, id; Ito, id.).
It is thought that the amino acids following the transition state stabilizer determine the orientation of the acceptor molecules and therefore influence the type of glycosidic bond that is formed (Leemhuis et al., 2012, Biocatal Biotransform 30:366-376; Leemhuis et al., 2013, J Biotechnol 163:250-272). The fifth amino acid after the transition stabilizer is likely coupled with the +2 subsite that binds the acceptor molecule and is almost universally an aspartate or threonine among Streptococcus and a threonine with Leuconostoc species. In streptococci glucansucrase, aspartate is typically associated with insoluble glucan production, while threonine in this position usually results in synthesis of soluble glucan. Substituting D567T in S. mutans GtfB shifted production of soluble glucan from 0 to 24%. Conversely, T589D and T598E in GtfD went from 86 to 15% and 86 to 2%, respectively for each mutation (Shimamura, et al., 1994, J Bacteriol 176:4845-4850). Moreover, mutations of this equivalent position in S. downei GtfI influenced the structure of the glucan and size of oligosaccharide produced in studies using the catalytic core region in enzymatic assays (Monchois, et al., 2000, Appl Environ Microbiol 66:1923-1927).
Recently identified is a glucansucrase, DsrI, from the type strain of L. mesenteroides (NRRL B-1118) that produces a water-insoluble glucan containing approximately 44% α(1→3), 29% α(1→6), 15% terminal non-reducing end residues, and 9% branching through 1,3,6-trisubstituted α-D-glucopyranosyl units (Côté et al., 2012, Appl Microbiol Biotechnol 93:2387-2394). The linkage types apparently occur in discrete sequence domains, with linear segments of α(1→3) linkages interspersed with or grafted onto segments of α(1→6)-linked regions. There is no evidence for an alternan-like structure in the insoluble glucan. This enzyme contains a threonine in position 654 like most other Leuconostoc glucansucrases, but its product is unique due to the high percentage of α(1→3) linkages and water-insolubility. Given this property, there is a need to further scrutinize the amino acid at the 654 position to determine whether the catalytic properties of the enzyme and synthesize glucans with different structures can be modified.



BRIEF DESCRIPTION OF THE DRAWINGS
The present invention together with the above and other objects and advantages may best be understood from the following detailed description of the embodiment of the invention illustrated in the drawings, wherein:
FIG. 1 depicts an amino acid alignment of motif IV of GH70 enzymes. Sm GtfB, S. mutans GS-5 (GtfB (SEQ ID NO: 4), Accession AAA88588 and GtfD (SEQ ID NO: 7), AAA26895); Ss, S. salivarius ATCC 25975 (GtfJ (SEQ ID NO: 5), AAA26896); Sd, S. downei MFe28 (GtfI (SEQ ID NO: 6), AAC63063 and GtfS (SEQ ID NO: 8), AAA26898); Lm, L. mesenteroides B512F (DsrS (SEQ ID NO: 9), U81374 AAA53749) or NRRL B-1118 (DsrI (SEQ ID NO: 10), YP-_819212). Predominant glycosidic linkage in a-glucan polymer shown in second column; TS, transition stabilizing aspartate; boxed residues, putative amino acid associated with the +2 subsite that binds the acceptor molecule.
FIG. 2 depict yields of insoluble glucan from each mutant enzyme. Reactions consisted of 2 units glucansucrase (assayed in the absence of dextran) and 2.67 mmoles of sucrose in 8 mL of buffer. Reactions were mixed by rotation and inversion until all sucrose was consumed. Error bars indicate standard error of the mean. Maximum theoretical yield=432 mg.
FIG. 3 depicts endodextranase resistance of insoluble glucans produced by DSR-I from T654 substitution mutants. Bars represent relative amount of glucan remaining water-insoluble after treatment with Chaetomium sp. endodextranase. Error bars represent standard error of the mean.
FIG. 4 depicts anomeric peak height ratio of insoluble glucans produced by DSR-I from T654 substitution mutants. Ratios were calculated from height of 13C-NMR peaks representing carbon-1 in an ∀-(1→3) linkage (101.6 ppM) divided by peak height of carbon-1 in an ∀-(1→6) linkage (98.7 ppM). Error bars represent standard error of the mean.
FIG. 5 depicts graph from a methylation analysis for the ratio of 3-mono-O-substituted glucopyranosyl units to 6-mono-O-substituted glucopyranosyl units in insoluble glucans produced by DSR-I from T654 substitution mutants. Ratio was calculated by dividing molar proportion of 2,4,6-tri-O-methyl glucose derivative by molar proportion of 2,3,4-tri-O-methyl glucose derivative obtained in methylation analyses. Error bars represent standard error of the mean.
FIG. 6 depicts a graph for the activation or lack thereof by the addition of soluble dextran to radioassay mixtures. Mixtures contained 50 μL of enzyme solution, 50 μL of 0.3M 14C-U-sucrose, and 50 μL of either 2% (w/v) commercial dextran (Mw=2,000,000) or buffer. Error bars indicate standard errors of the mean ratios.
FIGS. 7A-C depict graphs of representative examples of radioassays of mutant enzymes in the absence or presence of dextran. Assays consisted of 50 μL of enzyme solution, 50 μL of 0.3M 14C-U-sucrose (52 cpm/μg), and 50 μL of either 2% (w/v) commercial dextran (Mw=2,000,000) or buffer. At indicated time points, 15 μL aliquots were analyzed for methanol-insoluble 14C-glucan. (FIG. 7A) T654S. (FIG. 7B) T654I. (FIG. 7C) T654Y. No error bars are included, as this figure is merely intended to show typical shapes of the different types of assay curves.



BRIEF DESCRIPTION OF THE SEQUENCES
The invention can be more fully understood from the following detailed description and the accompanying sequence descriptions, which form a part of this application.







SEQ ID NO: 1 is


DVSQNNGVVVATAVDQSNLDATTSDKSITTDDKAATTAATSTDDKATTTV


 


ATSTDDKDTTTAATSTDDKATTTVATSTDDKATTTAATSTDDKAATTAAT


 


STDDKAATTAATSTDDKAATTADTSTDDKAATTAATSTDDKATTTAATST


 


DDKTATTVGTSDNNNSATASDKDVSSSAQKSQTIDNNSKTADTTAALEAS


 


SKNLKTIDGKTYYYDDDDQVKKNFATVIDGKVLYFDKETGALADTNDYQF


 


LEGLTSENNTYTEHNASVGTSSDSYTNVDGYLTADSWYRPKDILVNGQNW


 


ESSKDDDLRPLLMTWWPDKATQVNYLNAMKYLDATETETVYTSDDSQDAL


 


NKAAQNIQVKIEEKISQEGQTQWLKDDISKFVDSQSNWNIASESKGTDHL


 


QGGALLYVNSDKTPDANSDYRLLNRTPTNQTGTPLYTTDPTQGGYDFLLA


 


NDVDNSNPVVQAEQLNWMYYLLNFGSITNNDADANFDSIRVDAVDNVDAD


 


LLQIAADYFKAAYGVDKSDAISNQHVSILEDWSDNDAEYVKDNGDNQLSM


 


DNKLRLSLKYSLTMPAVDQYGNKRSGLEPFLTNSLVDRTNDSTDNTAQPN


 


YSFVRAHDSEVQTVIAEIIKQRIDPDSDGLSPTMDQLTEAFKIYNADQLK


 


TDKEFTQYNIPSTYATILTNKDTVPRVYYGDMYTDDGQYMATKSLYYDAI


 


DTLLKSRIKYVSGGQTMSMKYMQGDSSMAADSYRGILTSVRYGNGAMTAT


 


DAGTNETRTQGIAVIESNNPDLKLSSTDQVVVDMGIAHKNQAYRPALLTT


 


KDGIDTYVSDSDVSQSLIRYTNSNGQLIFNSSDIVGTANPQVSGYLAVWV


 


PVGASDTQDARTESSTATTADGQTLHSNAALDSQVIYESFSNFQSTPTTE


 


AEYANVQIANNTDLYKSWGITNFEFPPQYRSSTDSSFLDSIIQNGYAFTD


 


RYDLGFNTPTKYGTVDQLRTAIKALHATGIKAMADWVPDQIYNLTGKEVV


 


AVQRVNNSGIYNQDSVINKTLYASQTVGGGEYQALYGGEFLDEIKKLYPS


 


LFEKNQISTGVPMDASEKIKEWSAKYFNGTNIQGRGAYYVLKDWATNEYF


 


KVSTSSNSSVFLPKQLTNEESNTGFISTDGGMTYYSTSGYQAKDTFIQDD


 


KSNWYYFDKNGYMTYGFQTVNDNNYYFLPNGIELQDAILEDSKGNVYYFN


 


QYGKQAVDGYYMLANKTWRYFDKNGVMANAGLTTVTVDGQEHIQYFDKNG


 


IQVKGTSVKDADGKLRYFDTDSGDMVTNRFGENTDGTWSYFGADGIAVTG


 


AQTISGQKLFFDADGQQIKGKEATDKKGKVHYYDANSGEMITNRFEKLSD


 


GSWAYFNKKGNIVTGAQVINGQHLFFESNGNQVKGREYTATDGKMRYYDA


 


DSGDMVTNRFERISDGSWAYFGANGVAVTGEQNINGQQLYFDANGHQVKG


 


AAVTQADGSQKYYDANSGEMIKS is the amino acid


sequence to wild type glucansucrase.


 


SEQ ID NO: 2


gcacatgatagtgaagtacaannngttattgctgaaattattaaac


is a primer construct.


 


SEQ ID NO: 3


gtttaataatttcagcaataacnnnttgtacttcactatcatgtgc


is a primer construct.






Definitions

As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.
As referred to herein, the wild-type (“WT”) glucansucrase cloned from Leuconostoc mesenteroides NRRL strain B-1118 (ATCC strain 8293).
As described herein, a single amino acid residue substitution can be indicated as follows: the original amino acid residue (expressed as a single-letter abbreviation), followed by the position of the original amino acid residue (i.e., a numerical expression), followed by the new amino acid residue (expressed as a single-letter abbreviation) to be inserted in place of the original amino acid residue. For example, “T654G” means that the original threonine (T) residue at position 654 is to be replaced by the new glycine (G) residue. For multiple substitutions (e.g., double-substitutions, triple-substitutions, and quadruple-substitutions), the various substitutions are separated by either a slash (/) or by a space.
As a practical matter, whether any particular amino acid sequence having at least 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence described in SEQ. ID. No. X can be determined conventionally using known computer programs to find the best segment of homology between two sequences. When using sequence alignment program to determine whether a particular sequence is, for instance, 96% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference peptide sequence and that gaps in homology of up to 4% of the total number of amino acids in the reference sequence are allowed.
Computer-assisted comparison of SEQ ID NO: 1 with previously reported sequences present in publicly available databases is useful for identifying functional glucansucrase nucleic acid and polypeptide sequences. It will be understood that protein-coding sequences, for example, may be compared as a whole, and that a high degree of sequence homology between two proteins at the amino acid level indicates that the two proteins also possess some degree of functional homology, such as, for example, among enzymes involved in glucansucrase function.
The present invention also relates to a method of recombinantly producing a mutant glucansucrase. This method involves transforming a host cell with at least one heterologous nucleic acid molecule of the present invention under conditions suitable for expression of the modified glucansucrase. The modified glucansucrase is then isolated. Suitable host cells for this method are as described herein (infra).
The present invention further relates to expression vectors wherein a nucleic acid which comprises an expression cassette and which is capable of replicating in a selected host cell or organism. An expression vector may be a plasmid, virus, retrovirus, bacteriophage, cosmid, artificial chromosome (bacterial or yeast), or nucleic acid sequence which is able to replicate in a host cell, characterized by a restriction endonuclease recognition site at which the sequence may be cut in a predetermined fashion for the insertion of a heterologous DNA sequence. An expression vector may include the promoter positioned upstream of the site at which the sequence is cut for the insertion of the heterologous DNA sequence, the recognition site being selected so that the promoter will be operatively associated with the heterologous DNA sequence. A heterologous DNA sequence is “operatively associated” with the promoter in a cell when RNA polymerase which binds the promoter sequence transcribes the coding sequence into mRNA which is then in turn translated into the protein encoded by the coding sequence.
Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding a glucansucrase and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y, and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.
“Amplification” as used herein refers to the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction (PCR) technologies well known in the art (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primers a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).
The invention is not limited by the host cell employed.
“Amino acid sequence” as used herein refers to an oligopeptide, peptide, polypeptide, or protein sequence, and fragment thereof, and to naturally occurring or synthetic molecules. Where “amino acid sequence” is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, amino acid sequence, and like terms, are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.
In the following description, the nomenclature used to define the proteins and peptides is that specified by Schroder and Lubke [“The Peptides,” Academic Press (1965)] wherein, in accordance with conventional representation, the N-terminal appears to the left and the C-terminal to the right. Where the amino acid residue has isomeric forms, it is the L-form of the amino acid that is represented herein unless otherwise expressly indicated.
A purified protein or polypeptide of the mutant glucansucrase of the present invention can be obtained by several methods. The purified protein or polypeptide of the modified glucansucrase of the present invention is preferably produced in pure form (preferably at least about 80%, more preferably 90%, pure) by conventional techniques well known in the art. Typically, the purified protein or polypeptide of the glucansucrase of the present invention is secreted into the growth medium of recombinant host cells. Alternatively, the purified protein or polypeptide of the glucansucrase of the present invention is produced but not secreted into growth medium. In such cases, to isolate the protein or polypeptide of the mutant glucansucrase, the host cell carrying a recombinant plasmid is propagated, lysed by sonication, heat, or chemical treatment, and the homogenate is centrifuged to remove cell debris. The supernatant is then subjected to sequential ammonium sulfate precipitation. The fraction containing the protein or polypeptide of the mutant glucansucrase of the present invention is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the proteins. If necessary, the protein fraction (containing the mutant phytase of the present invention) may be further purified by HPLC.
As used in the specification and claims, the term “dextranase” refers to an enzyme that catalyzes the endohydrolysis of α-(1→6) glucosidic linkages. Typically, commercial dextranase is obtained from extracellular purification of Penicillium sp. or Chaetomium erraticum cultures.
DETAILED DESCRIPTION OF THE INVENTION
Disclosed herein is a genetically modified enzyme belonging to glycosyltransferases type of glucansucrase comprising at least one mutation at position 654 of said enzyme, wherein modified enzyme is capable to producing a glucan polymer. In one embodiment of the invention, the modified enzyme is derived from Leuconostoc mesenteroides. In another embodiment of the invention, the glucan polyer is water-insoluble. In yet another embodiment of the invention the modified enzyme has an amino acid, threonine at position 654, which is replaced with an amino acid selected from the group consisting of glutamic acid, lysine, glutamine, cysteine, arginine, asparagine, isoleucine, serine, histidine, glycine, or aspartic acid, wherein the modified glucansucrase produces a glucan polymer having a higher yield of 1,3-disubstituted α-D-glucopyranosyl units as compared to glucan polymer produced by a wild-type glucansucrase. In yet another embodiment of the invention, the modified enzyme has an amino acid threonine at position 654, which is replaced with tyrosine, wherein the modified glucansucrase produces a glucan polymer having a higher yield of 1,6-disubstituted α-D-glucopyranosyl units as compared to a glucan produced by a wild-type glucansucrase.
FIG. 1 depicts an amino acid alignment of motif IV of several GH70 enzymes. In the figure “Sm GtfB” refers to the motif from S. mutans GtfB protein (SEQ ID NO: 4); “Sm GtfD” refers to the motif from S. mutans GtfD protein (SEQ ID NO: 7); “Ss GtfJ” refers to the motif from S. salivarius GtfJ protein (SEQ ID NO: 5), AAA26896); “Sd Gtf” refers to the motif from S. downei GtfI protein (SEQ ID NO: 6); “Sd GtfS” refers to the motif from S. downei GtfS protein (SEQ ID NO: 8); “Lm DsrS” refers to the motif from L. mesenteroides DsrS protein (SEQ ID NO: 9), and; “Lm DsrI” refers to the motif from L. mesenteroides DsrI protein (SEQ ID NO: 10). Predominant glycosidic linkage in α-glucan polymer produced is shown in the second column. “TS” indicates the transition stabilizing aspartate residue. Boxed residues, indicate putative amino acid residues associated with the +2 subsite that binds the acceptor molecule.
Disclosed herein is a glucansucrase comprising the amino acid sequence of SEQ ID NO: 11 with a substitution at position T654, the substitution being either glutamic acid, lysine, glutamine, cysteine, arginine, asparagine, isoleucine, serine, histidine, glycine, or aspartic acid wherein the modified glucansucrase produces a glucan polymer having a higher yield of 1,6-disubstituted .alpha.-D-glucopyranosyl units as compared to a glucan polymer produced by a wild-type glucansucrase.
Also disclosed herein is a glucansucrase comprising the amino acid sequence of SEQ ID NO: 11 with a substitution at position T654, the substitution being tyrosine wherein the modified glucansucrase produces a glucan polymer having a higher yield of 1,3-disubstituted α-D-glucopyranosyl units as compared to a glucan polymer produced by a wild-type glucansucrase.
Also disclosed herein is a method for producing a modified glucansucrase, the steps comprise of producing a modified glucansucrase, wherein the amino acid at position 654 is substituted by a method comprising the steps of: replacing the amino acid at position 654 with glutamic acid, lysine, glutamine, cysteine, arginine, asparagine, isoleucine, serine, histidine, glycine, or aspartic acid, expressing said modified glucansucrase; and using said modified glucansucrase to catalyze the synthesis of a glucan polymer from a carbohydrate source. In another embodiment of the method, the amino acid at position 654 is substituted by a method comprising the steps of: replacing the amino acid at position 654 with tyrosine, expressing said modified glucansucrase; and using said modified glucansucrase to catalyze the synthesis of a glucan polymer from a carbohydrate source.
Also disclosed herein is a modified glycoside hydrolase enzyme comprising at least one mutation to the amino acid downstream from a transition state stabilizer site wherein the modified enzyme produces a glucan polymer. In one embodiment of the invention, the modified enzyme of produces a glucan polymer having a higher yield of 1,3-disubstituted α-D-glucopyranosyl units as compared to a glucan polymer produced by a wild-type glucansucrase. In another embodiment of the invention, the modified enzyme produces a glucan polymer having a higher yield of 1,6-disubstituted α-D-glucopyranosyl units as compared to a glucan polymer produced by a wild-type glucansucrase. In yet another embodiment of the invention, the modified enzyme produces a glucan polymer having a lower water solubility as compared to a glucan polymer produced by a wild-type glucansucrase. In yet another embodiment of the invention, the modified enzyme produces a glucan polymer having a higher water solubility as compared to a glucan polymer produced by a wild-type glucansucrase. In yet another embodiment of the invention, the modified enzyme has a modified amino acid that is the fifth amino acid subsequent to transition state stabilizer.
The equivalent of mature full-length recombinant L. mesenteroides DsrI (i.e., only missing secretion signal peptide, which is normally removed during secretion in wt host) was produced using an E. coli expression system and purified by immobilized metal ion affinity chromatography as previously described and incorporated by reference herein (Côté et al, 2012, Appl Microbiol Biotechnol 93:2387-2394). Mutations for T654 substitutions in plasmid pSUMO-Dsr403 were introduced using a two-stage PCR protocol followed by DpnI digestion and transformation into E. coli (Wang et al., 1999, BioTechniques 26:680-682).
Oligonucleotides used for mutagenesis were Plus Strand 5′ gca cat gat agt gaa gta caa nnn gtt att get gaa att att aaa c 3′ (SEQ ID NO. 2) and Minus Strand 5′ g ttt aat aat ttc agc aat aac nnn ttg tac ttc act atc atg tgc 3′ (SEQ ID NO. 3), whereby nnn is coded for the modified amino acids described below.
Glucansucrase assays and polysaccharide structure determinations were carried out as before (Côté and Skory 2012). Enzyme assays were based on the incorporation of 14C-labelled glucose from 14C-(U)-labeled sucrose (PerkinElmer-NEN, Waltham, Mass.) into alcohol-insoluble polysaccharide (i.e., includes water soluble and insoluble fractions), according to a modification of the method described by Germaine et al., 1974, J Dent Res 53:1355-1360). This particular assay measures only glucan synthesis, unlike assays based on release of reducing sugars, which also measure competing reactions such as hydrolysis, leucrose formation, etc. In a typical example, 30 μL of 0.3M 14C-sucrose in acetate buffer was incubated with 60 μL of enzyme solution at 30° C. At timed intervals, 15 μL aliquots were withdrawn and absorbed onto 1.5 cm squares of Whatman 3MM chromatography/filter paper. The squares were immediately dropped into a beaker containing approximately 150-200 mL of stirred methanol. A metal screen was used to protect the paper squares from maceration by the stir bar. After washing with three changes of methanol for ten minutes per wash, they were dried under a heat lamp and counted for 14C content in Ecolume cocktails (MPBio, Solon, Ohio) using a Beckman-Coulter (Brea, Calif.) LSC-6500 liquid scintillation counter. An enzyme unit is defined here as the amount of enzyme activity that incorporates one μmole of glucose into glucan in one minute.
For product analysis, enzyme was incubated with 0.3 M sucrose and the reaction was monitored by thin-layer chromatography as previously described in Côté et al., 1982, Carbohydr Res 111:127-142 and incorporated by reference herein.
After all of the sucrose had been consumed, the insoluble product was collected, washed to remove remaining sugars and oligosaccharides, dried for mass yield determination, and analyzed by a combination of methylation analysis, 13C-NMR spectroscopy, and measurement of solubilization by endodextranase as previously described (Côté, 2012, Appl Microbiol Biotechnol 93:2387-2394). Water-insoluble glucans were soluble in either dimethyl sulfoxide-water mixtures (DMSO:H2O 9:1, vol) or 1M NaOH (aq.) (Lamberts et al., 1975, J Dent Res 54:857-866). 1M NaOH (aq.) with deuterated NaOD in D2O was selected for NMR analyses because most previous studies on streptococcal glucans have been done in that system. Yields of insoluble glucan from each mutant enzyme were determined under identical conditions for each mutant: reactions consisted of 2 units glucansucrase (assayed in the absence of dextran) and 2.67 mmoles of sucrose in 8 mL of buffer. Insoluble glucan was recovered by centrifugation, followed by three washes with 8 mL water, one wash in 50% ethanol, and one wash in absolute ethanol, followed by drying in vacuo at 50° C.
EXAMPLE 1
Product Formation
The yields of insoluble glucan produced by each mutant enzyme varied significantly, with most of the modified enzymes producing less insoluble glucan than the wild type enzyme under the tested conditions (FIG. 2). However, two modifications, T654G and T654Y, resulted in enzymes that produced more insoluble glucan than the wild type. The remaining mutants, with the exception of T654D, produced less than half of the amount obtained with the wild type enzyme. All of the yields were less than half of the theoretical yield, 432 mg, from sucrose, despite the fact that all of the sucrose was consumed. The remainder of the sucrose appeared to be diverted to the production of leucrose, palatinose and higher oligosaccharides, as large amounts of these products were observed on TLC. The higher oligosaccharides presumably arise from glucosylation of leucrose and palatinose, as they were chromatographically similar to those products from alternansucrase acceptor reactions with fructose (Côté et al., 2008, Biocatal Biotransform 26:161-168).
Interestingly, the six highest yields, with the exception of T654D, had amino acids in position 654 that are defined as neutral hydropathy (i.e., hydropathy index −0.4 to −3.2) according to the Kyte-Doolittle scale (Kyte & Doolittle, 1982, J Molec Biol 157:105-132). There did not appear to be any correlation between yield and charge, polarity, or amino acid size (e.g., molecular weight and volume). In fact, the two highest yields were obtained with the largest (Y; MW 181.2 Da) and smallest (G; MW 75.1 Da) amino acid substitutions.
α-Glucan Structure and Properties
The three methods used to measure the amount of 1,3-disubstituted α-D-glucopyranosyl units in the insoluble glucans were generally in agreement, although some small differences can be seen when comparing FIGS. 3, 4, and 5. When all three methods of measurement are taken together, the mutants may be divided into three groups. Those producing insoluble glucans with the highest proportion of 1,3-disubstituted α-D-glucopyranosyl units (α(1→3)/α(1→6) ratio>2) included Q, R, I, N, K, and H. The second group comprises those producing glucans with a α(1→3)/α(1→6) ratio<2 and includes C, S, G, E, D, and WT. The third group produced glucan with significantly lower α(1→3)/α(1→6) ratio and greater endodextranase susceptibility compared to WT and contains only one mutant, Y.
Contrary to the trends regarding yield and amino acid hydropathy, the highest percentage of α(1→3) linkages were associated with the most hydrophilic (Q, N, R, K, H; in order of hydrophilicity) and hydrophobic (I) residues. The exception was with the acidic residues D and E. The percentage of α(1→3) linkages for D was approximately the same as WT in all of the studies, but the α-glucan from E was significantly higher as indicated by NMR and endodextranase treatment. Glucan from these two acidic residues along with the most neutral amino acids (C, S, G, T, H, and Y) had the lowest percentage of α(1→3) linkages of all of those tested. The largest residue, Y, by both MW (181.2 Da) and volume 194 {acute over (Å)}3 produced glucan with the lowest percentage of α(1→3) linkages; however, the other aromatic residue, H, actually produced glucan with significantly more α(1→3) linkages.
The insoluble glucans produced by each mutant enzyme differed in appearance. Although some of the observed differences may be due to differing amounts and concentrations of glucan formed, some properties were consistently noted regardless of concentration. The glucans with lower levels of α(1→3) linkages, namely those from T654 Y, WT, G and D, were gel-like and adhered to the glass surfaces, whereas those with higher levels of α(1→3) linkages, namely T654 K, R, N, Q and I, were dispersed as fine particles, and did not adhere as strongly to the glass.
No general conclusion can be drawn regarding the type of amino acid substitution that will lead to lower levels of 1,3-disubstituted α-D-glucopyranosyl units, since only one example (tyrosine) was found. Furthermore, none of the tested amino acid substitutions yielded an enzyme with significantly higher specific activity.
When the mutant enzymes were assayed in the absence and presence of dextran, two different phenomena were observed. Some of the enzymes exhibited an increase in initial rate of glucan synthesis in the presence of added dextran, whereas others showed no such effect. FIG. 6 shows that mutants substituted at position 654 with I, K, R, N and Q were activated by dextran, but none of the other mutants were significantly affected. FIG. 7 shows three typical radioassay plots. In FIG. 7A, mutant T654S shows no effect of dextran on the initial rate. After approximately 15 minutes, small particles of gel were seen forming in the reaction mixture. These insoluble particles were not pipetted onto the filter paper squares, and thus not counted as newly synthesized 14C-glucan. This gives rise to a great deal of scatter and inconsistency in the assay plot. However, when dextran was present, no such particles were seen, and the solution remained uniform, thus enabling the consistent counting of increasing amounts of 14C-glucan product. A similar effect on product solubility was noted with mutant T654I, but in that case (as well as T654 mutants K, Q, R and N), the initial rate was significantly higher when dextran was present. Finally, with mutant T654Y, no activation was observed, and the product remained relatively soluble throughout the course of the reaction regardless of the presence or absence of dextran. The molecular weight of the dextran added apparently made no difference. With mutant T654I, for example, the assay curves were identical with dextrans of 2,000 kDa, 20 kDa, 10 kDa and 6 kDa.
The majority of the T654 substitutions yielded glucansucrase that produced an insoluble glucan with a percentage of 1,3-disubstituted α-D-glucopyranosyl units equal to or greater than the wild type enzyme (FIGS. 3, 4, 5); however, this was often at the expense of yield (FIG. 2) or specific activity. In general, most of the modified enzymes exhibited lower specific activity than the wild type. The only exception was tyrosine, which produced an enzyme whose product contains fewer 1,3-disubstituted α-D-glucopyranosyl units, but had a higher yield than the WT enzyme. Modified enzymes producing glucans with the highest amount of 1,3-disubstituted α-D-glucopyranosyl units included all of those with nitrogen in the side chains (Q, R, N, K). Histidine may also be included in this group, although the effect was less clear. It may be that the effect of the nitrogen in histidine was offset by the effect of the aromatic ring structure, rendering it similar to tyrosine in that regard. Modifications in which threonine 654 was substituted with acidic amino acids (D, E), alcohols or thiols (S or C) as well as G, seemed to be the most similar to the wild type in terms of 1,3-disubstituted α-D-glucopyranosyl units in their product glucans. In most cases, these latter enzymes also exhibited slightly higher specific activities, perhaps another indication of their greater similarity to the wild type enzyme.
EXAMPLE 2
Activation by Dextran
The addition of dextran to the radioassay solutions could markedly increase the initial rate of synthesis by some of the modified enzymes (FIG. 6). The molecular weight of various commercial (NRRL B-512F) dextrans seemed to have little effect on the degree of activation. Mutated enzymes that produced higher levels of α(1→3) linkages showed the greatest increase in activity, up to 5-fold for T654K. It was also noted that the addition of dextran to assay reactions of some of the mutants was effective in preventing or delaying the formation of insoluble product, resulting in linear assay curves for a longer period of time (FIG. 7). Enzymes that were not activated by dextran had overlapping curves (FIGS. 7A and 7C) until the solubility of the glucan was presumably exceeded. However, enzymes that were activated by dextran had distinctly different initial rate slopes (FIG. 7B). Other homopolysaccharides were also analyzed for their ability to similarly activate the mutant DSR-I enzymes. Soluble starch and pullulan had no measurable effect. Highly branched NRRL B-742 fraction S dextran (Côté & Robyt, 1983, Carbohydr Res 119: 141-156) and alternan from several NRRL strains activated the DSR-I from mutants K, R, N, Q and I nearly as well as did dextran. Upon information and belief, any glucan having unbranched sequences of α(1→6) linkages would activate the glucansucrase disclosed herein.
The effects of dextran addition as due to two different causes. The activation of initial rates appears to be an allosteric effect whereby the efficiency of glucan synthesis is improved by binding of dextran to the glucan-binding domain, although earlier researchers did not differentiate between such allosteric activation and a so-called “primer effect” of dextran on streptococcal glucansucrases (e.g., Germaine et al., 1977, Infect Immun 16:637-648; Fukushima et al., 1981, J Dent Res 60:1707-1712; Sato et al, 1982, Microbios 34:99-112; Hanada et al, 1986, FEMS Microbiol Lett 36:173-175). The solubility effect may be due to the formation of graft copolymers, whereby the dextran acts as an acceptor for the transfer of glucosyl and/or glucanosyl units to the dextran chain. This conclusion is based on previous reports that GTF-I from Streptococcus spp. carries out similar reactions.
To the extent that the term “includes” or “including” is employed in the detailed description or the claims, it is intended to be inclusive in a manner similar to the term “comprising” as that term is interpreted when employed as a transitional word in a claim. Furthermore, to the extent that the term “or” is employed in the detailed description or claims (e.g., A or B) it is intended to mean “A or B or both”. When the applicants intend to indicate “only A or B but not both” then the term “only A or B but not both” will be employed. Thus, use of the term “or” herein is the inclusive, and not the exclusive use. See, Bryan A. Garner, A Dictionary of Modern Legal Usage 624 (2d. Ed. 1995). Also, to the extent that the terms “in” or “into” are used in the specification or the claims, it is intended to additionally mean “on” or “onto.”
While the invention has been described with reference to details of the illustrated embodiment, these details are not intended to limit the scope of the invention as defined in the appended claims. The embodiment of the invention in which exclusive property or privilege is claimed is defined as follows:

## Claims

1. A modified glucansucrase enzyme comprising an amino acid sequence that is at least 96% identical to SEQ ID NO:11, wherein the threonine residue at position 654 is substituted with glutamic acid, lysine, glutamine, cysteine, arginine, asparagine, isoleucine, serine, histidine, glycine, or aspartic acid, and wherein the modified enzyme is capable of producing a glucan polymer.

2. The modified enzyme of claim 1, wherein the enzyme is obtained from Leuconostoc mesenteroides.

3. The modified enzyme of claim 1, wherein the glucan polymer is water-insoluble.

4. The modified enzyme of claim 1, wherein the threonine is replaced with tyrosine, and wherein the modified enzyme produces a glucan polymer having a higher yield of 1,6-disubstituted α-D-glucopyranosyl units as compared to a glucan produced by a wild-type glucansucrase.

5. The modified enzyme of claim 4, wherein the wild-type glucansucrase is from Leuconostoc mesenteroides NRRL B-1118.

6. The modified enzyme of claim 5, wherein the modified glucansucrase produces a glucan polymer that is more water insoluble as compared to a glucan polymer produced by a glucansucrase produced by Leuconostoc mesenteroides NRRL B-1118.

7. The modified glucansucrase of claim 1, wherein the threonine residue is substituted with tyrosine, and wherein the modified glucansucrase produces a glucan polymer having a higher yield of 1,3-disubstitutedα-D-glucopyranosyl units as compared to a glucan polymer produced by a wild-type glucansucrase.

8. The modified enzyme of claim 7, wherein the wild-type glucansucrase is from Leuconostoc mesenteroides NRRL B-1118.

9. A method for producing a glucan polymer with a modified glucansucrase, comprising the steps of expressing the modified glucansucrase of claim 1 in a recombinant host cell, and; using the modified glucansucrase to catalyze the synthesis of a glucan polymer from a carbohydrate source, thus producing the glucan polymer.

10. A method for producing a glucan polymer with a modified glucansucrase, comprising the steps of expressing the modified glucansucrase of claim 7 in a recombinant host cell, and; using the modified glucansucrase to catalyze the synthesis of a glucan polymer from a carbohydrate source, thus producing the glucan polymer.

11. A method for producing a water-insoluble gel wherein a glucan polymer is formed by the modified enzyme of claim 1, and a carbohydrate source.