# Patent

## Title
Increased poly (α 1, 3 glucan) yield using boric acid

## Abstract

A process for production of poly (α 1, 3 glucan) from a renewable feedstock, for applications in fibers, films, and pulps. The effect of addition of boric acid in increasing the yield of the desired end products, poly (α 1, 3 glucan) and fructose, and decreasing formation of the undesired by-product leucrose.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of priority to U.S. Provisional Application No. 61/577,274, filed on Dec. 19, 2011.
FIELD OF INVENTION
This invention relates to the field of production of a polysaccharide. Specifically, it relates to production of poly (α 1, 3 glucan) via an enzymatic reaction. More specifically, it relates to increasing the titer of poly (α 1, 3 glucan) formed during the enzymatic reaction.
BACKGROUND
Cellulose, a polysaccharide consisting of β (1, 4)-linked glucose, formed by natural processes, (Applied Fiber Science, F. Happey, Ed., Chapter 8, E. Atkins, Academic Press, New York, 1979) has become the preeminent fiber for use in manufactured textiles, films and resins. Cotton, an especially pure form of naturally occurring cellulose, is well-known for its beneficial attributes in textile applications.
Cellulosic fibers such as cotton and rayon increasingly present sustainability issues with respect to land use and environmental imprint. This may be a significant factor leading to increased level of research into textiles containing polyester fiber blends with cellulosic materials and more sustainable solutions for cellulosic-derived materials. It is highly desirable, therefore, to discover other glucose-based polysaccharides for application in films, fibers and resins that can be economically produced from renewable resources. In addition such polymers offer materials that are environmentally benign throughout their entire life cycle.
Poly (α 1, 3 glucan), a glucan polymer characterized by having α (1, 3) glycoside linkages, has been isolated by contacting an aqueous solution of sucrose with a glycosyltransferase (gtfJ) enzyme isolated from Streptococcus salivarius (Simpson et al., Microbiology, 141: 1451-1460, 1995). Poly (α 1, 3 glucan) refers to a polysaccharide composed of D-glucose monomers linked by glycosidic bonds. Films prepared from poly (α 1, 3 glucan) tolerate temperatures up to 150° C. and provide an advantage over polymers obtained from β (1, 4) linked polysaccharides (Ogawa et al., Fiber Differentiation Methods, 47: 353-362, 1980).
U.S. Pat. No. 7,000,000 disclosed preparation of a polysaccharide fiber comprising hexose units, wherein at least 50% of the hexose units within the polymer were linked via α (1, 3) glycoside linkages using the gtfJ enzyme. The gtfJ enzyme utilizes sucrose as a substrate in a polymerization reaction producing poly (α 1, 3 glucan) and fructose as end-products (Simpson et al., et al., Microbiology, 141: 1451-1460, 1995).
Production of low-cost poly (α 1, 3 glucan) derived from sucrose, for commercial applications, requires a high yield process producing minimal undesirable by-products. In addition to poly (α 1, 3 glucan), the other end product, fructose, is also a desirable product due to its application as a high value sweetener. However, fructose is also known to compete with glucose, acting as an acceptor in the gtf enzyme reaction thus hindering conversion of available glucose to poly (α 1, 3 glucan) and limiting the final titer of poly (α 1, 3 glucan) (Valdivia et al., (Ann. NY Acad. Sci. 542:390-394, 1988).
Robyt and Eklund (Bioorganic Chemistry, 11: 115-132, 1982) and Prat, D, et al., (Biotechnol. Letters, 9: 1-6, 1987) reported production of a by-product leucrose, a disaccharide of glucose and fructose with a (1, 5) linkages, as well as fructose, by the dextranase enzyme of Leuconostoc mesenteroides when sucrose was used as substrate. Dextranase enzymes (E.G. 2.4.1.2) belong to glycosyltransferases family of enzymes and catalyze α (1, 4) and α (1, 6) type glycoside linkages.
Boric acid is known to react with suitable diol containing compounds (e.g., carbohydrates) in aqueous solution, to produce borate esters (T. Acree, Adv. Chem.; Am. Chem. Soc.: Washington, D.C., pp 208-219, 1973). The suitability of a diol for reaction with boric acid is determined by oxygen-oxygen bond distance (2.49 A to 2.63 A) within the diol and oxygen-carbon-carbon-oxygen dihedral angle (<40°). Fructose, in the furanose form, is an excellent configuration for bond distance and dihedral angle compared to glucose or sucrose for reaction with boric acid. Thus, the equilibrium constant for ester formation with boric acid favors fructose over glucose or sucrose in solution (Pollak, V. and Mlynek, J.; Carbohydrate Research, 241: 279-283, 1993). The relatively tight association between fructose and borate can be used to sequester this carbohydrate in a solution containing other sugars. Sequestration of fructose prevents its use as an acceptor in the dextranase reaction resulting in reduced leucrose synthesis. Prat et al., (supra) and Valdivia et al., (supra) described altering the yield of end products in a dextranase reaction by adding sodium tetraborate under strict conditions including specific concentrations of sodium tetraborate (<110 mM) and at pH<7.0. In the presence of 60 mM sodium tetraborate and at pH 7.0, the dextranase enzyme used by Prat et al., (supra) showed no activity at all.
Interaction between boric acid, borate anions and tetraborate occurs with carbohydrates having a specific configuration (Pollak, and Mlynek, supra). It is not clear whether a similar interaction can occur between borate and poly (α 1, 3 glucan). Furthermore, it has been shown that tetraborate dramatically reduces the activity of Leuconostoc mesenteroides dextranase, which belongs to a family of enzymes that catalyze α (1, 4) and α (1, 6) type glycoside linkages. It is not known if similar effects can be observed with the general class of glycosyltransferases which produce a high percentage of α (1,3) glycosyl linkages.
Commercial production of poly (α 1, 3 glucan) and fructose from sucrose, using glycosyltransferases, requires development of methods to increase the yield of these products during the enzymatic reaction.
SUMMARY OF INVENTION
This invention is a process for production of poly (α 1, 3 glucan) from a renewable feedstock, for applications in fibers, films, and pulps. The effect of addition of boric acid in increasing the yield of the desired end products and decreasing undesired by-product leucrose formation is disclosed.
In one aspect, the disclosed invention is a reaction solution for the synthesis of poly (α 1, 3 glucan) comprising:

    
    
        a) at least one gtf enzyme;
        b) boric acid; and
        c) sucrose,

whereby poly (α 1, 3 glucan) is produced with a lower concentration of leucrose by-product than is produced in the absence of boric acid.

    
    


In another aspect, the disclosed invention is an improved process for producing (α 1, 3 glucan) having a reduced concentration of leucrose as by-product comprising the steps:

    
    
        a) providing a reaction solution comprising:
        
            i) at least one gtf enzyme;
            ii) boric acid; and
            iii) sucrose;

wherein sucrose is converted by the enzyme to poly (α 1, 3 glucan) and fructose and wherein the amount of the by-product leucrose formed in the conversion is less than 35% of the sucrose consumed.

        
        
    
    





DESCRIPTION OF FIGURES AND DNA SEQUENCES
FIG. 1 is a graph of gtfJ enzyme (SEQ ID NO:4) reaction solutions containing no boric acid (black diamonds); 300 mM boric acid (black squares) and 600 mM boric acid (black triangles). Sucrose concentration is shown on the X axis and time (hours) is shown on the Y axis.
FIG. 2 is a graph showing changes in leucrose concentration in the FIG. 1 reaction solutions containing no boric acid (black diamonds); 300 mM boric acid (black squares) and 600 mM boric acid (black triangles).



NUCLEOTIDE SEQUENCES
SEQ ID NO. 1—is the nucleotides sequence of the forward PCR primer of S. sobrinus gtfI gene (BAA02976)









GGGAATTCCCAGGTTGACGGTAAATATTATTACT






SEQ ID NO. 2—is the nucleotides sequence of the reverse PCR primer S. sobrinus gtfI gene, (BAA02976).










AGATCTAGTCTTAGTTCCAGCCACGGTACATACG






SEQ ID NO. 3—Amino acid sequence (amino acid 162 to 1597) for Streptococcus sobrinus gtfJ (Genbank accession number BAA02976).

SEQ ID NO. 4—is the amino acid sequence (amino acid 178 to 1518) for Streptococcus salivarius gtfJ (Genbank accession number Z11873).

SEQ ID NO. 5—is the coding sequence (base 466 to base 4773) for Streptococcus sobrinus gtfI, (Genbank accession number BAA02976).

SEQ ID NO. 6—is the coding sequence (base 532 to base 4557) for Streptococcus salivarius gtfJ, (Genbank accession number Z11873).

DETAILED DESCRIPTION OF INVENTION
Poly (α 1, 3 glucan) is a potentially low cost polymer which can be enzymatically produced from renewable resources such as sucrose using the gtfJ enzyme (e.g., SEQ ID NO:4) of Streptococcus salivarius. The present invention describes formation of by-products poly (α 1, 3 glucan), fructose and leucrose in gtf enzyme reactions and the effect of boric acid in increasing fructose formation and decreasing leucrose formation.
The term “glycosyltransferase (gtf) enzyme”, as used herein, refers to an enzyme excreted by oral streptococci, such as Streptococcus salivarius which utilizes the high free energy of the glycosidic bond of sucrose to synthesize poly (α 1, 3 glucan). A glycosidic bond can join two monosaccharides to form a disaccharide. The glycosidic bonds can be in the α or β configuration and can generate, for example, α (1, 2), α (1, 3), α (1, 4), α (1, 6), β (1, 2), β (1, 3), β (1, 4) or β (1, 6) linkages. The term “α (1,3) glycoside linkage”, as used herein, refers to a type of covalent bond that joins glucose molecules to each other through the ring carbons 1 and 3 on adjacent glucose rings.
The term “poly (α 1, 3 glucan)”, as used herein, refers to high molecular weight, linear polymers obtained from polysaccharide molecules resulting from linking glucose units via α (1,3) glycosidic linkages.
The present invention relates to a process for increasing the titer of the polysaccharide, poly (α 1, 3 glucan) and fructose and decreasing the titer of the undesired by-product, leucrose, in an enzymatic reaction solution using sucrose as the substrate and one or more gtf enzymes. The term “enzymatic reaction” refers to a reaction that is performed by the gtf enzyme. An “enzyme reaction solution” of the present invention generally refers to a reaction mixture comprising at least one gtf enzyme in a buffer solution comprising sucrose and possibly one or more primers to convert sucrose to poly (α 1, 3 glucan).
The glycosyltransferase enzyme used in the present invention can be any gtf enzyme. The gtf enzyme used can be from any streprococci. Suitable gtf enzymes can be, for example, the gtfJ of Streptococcus salivarius (e.g., SEQ ID NO:4), the gtfB and the gtfC from Streptococcus mutans, the gtfI of Streptococcus sobrinus (e.g., SEQ ID NO:3) and the gtfI from Streptococcus downei. Particularly, the Streptococcus species can be Streptococcus salivarius. More particularly, the gtf enzyme can be the gtfJ (E.C. 2.4.1.5) enzyme of Streptococcus salivarius (e.g., SEQ ID NO:4). Alternatively, the gtfI enzyme of Streptococcus sobrinus (e.g., SEQ ID NO:3) can be used.
In one embodiment, the enzyme reaction solution can comprise only one gtf enzyme as described herein. In another embodiment, the enzyme reaction solution can comprise a combination of more than one type of gtf enzyme.
For purposes of this invention, sufficient quantities of the gtfJ enzyme (e.g., SEQ ID NO:4) can be produced using a recombinant E. coli strain expressing the desired gtfJ enzyme (e.g., SEQ ID NO:4). Methods for designing the codon optimized genes and expression in E. coli are well known in the art. The E. coli strain (DH10B) expressing gtfJ enzyme (e.g., SEQ ID NO:4) was prepared as described in the commonly owned U.S. Pat. No. 7,000,000.
Methods for the growth of recombinant microorganisms are well known in the art. Recombinant microorganisms expressing the desired gtf enzyme to perform the instant reaction can be grown in any container, such as, for example: various types of flasks with and without indentations; any container that can be sterilized and sealed and temperature-controlled; or any type of fermenter. In one embodiment, production of the gtfJ enzyme (e.g., SEQ ID NO:4) for poly (α 1, 3 glucan) production in the present invention can be achieved by growing the recombinant E. coli DH10B, expressing the gtfJ enzyme (e.g., SEQ ID NO:4), in a fermenter.
The gtfJ enzyme of Streptococcus salivarius (e.g., SEQ ID NO:4), used as the catalyst for conversion of sucrose to poly (α 1, 3 glucan) in the current invention, is a primer-independent gtf enzyme. The primer-independent enzymes do not require the presence of a primer to perform the reaction. A primer-dependent gtf enzyme as referenced in the present application refers to a gtf enzyme that requires the presence of an initiating molecule in the enzyme reaction solution to act as a primer for the enzyme during poly (α 1, 3 glucan) synthesis. Thus a “primer”, as the term is used herein, refers to any molecule that can act as the initiator for the primer-dependent glycosyltransferases. For the purposes of the present invention, either or both a primer-independent enzyme, and/or a primer-dependent gtf enzyme can be used in the same enzyme reaction system during poly (α 1, 3 glucan) synthesis.
While gtfJ (e.g., SEQ ID NO:4) is a primer-independent enzyme, it also acts with primer. In the present invention, dextran, which is a complex, branched glucan was used as a primer for the gtfJ enzyme (e.g., SEQ ID NO:4). In addition to dextran other carbohydrate-based primers can be used in the gtf reaction of the current invention. In one embodiment, the primer can be from any low to med molecular weight (2,000-50,000 Dalton) glucose-based carbohydrate.
In another embodiment, the primer in the reaction solution can be hydrolyzed poly (α 1, 3 glucan). In another embodiment, the primer in the reaction solution can be from any low to med (340-50,000 Dalton) non-glucose-based carbohydrate. In another embodiment, the primer in the reaction solution can be from any combination of any low to med molecular weight glucose-based carbohydrate. In another embodiment, the primer is glucose.
The production of poly (α 1, 3 glucan), by the gtfJ enzyme of Streptococcus salivarius (e.g., SEQ ID NO:4), is inhibited by its end product, fructose. When fructose accumulates in the enzyme reaction solution it can inhibit poly (α 1, 3 glucan) production, presumably by competing for available glycosyl moieties and thus results in the formation of the undesirable by-product disaccharide, leucrose.
Boric acid can be used to sequester fructose in a solution containing other sugars. The sequestration of fructose prevents its use as an acceptor in the gtf reaction and therefore results in reduced leucrose synthesis. The term “sequestration of fructose”, as used herein, refers to formation of a tight association between fructose and boric acid thus preventing fructose from reacting with gtf to produce the undesirable by-product, leucrose.
In one embodiment, the yield of sucrose can be increased by decreasing the formation of leucrose to less than 75% of the amount of leucrose formed in the absence of boric acid. In another embodiment, the amount of leucrose formed in the presence of boric acid can be less than one-half the amount of leucrose formed in the absence of boric acid. In still another embodiment, the amount of leucrose formed in the presence of boric acid can be less than one-third the amount formed in the absence of boric acid. In still another embodiment, the amount of leucrose formed in the presence of boric acid can be less than one-tenth the amount formed in the absence of boric acid.
In one embodiment, the concentration of boric acid used in the gtfJ (e.g., SEQ ID NO:4) reaction can be from 100 millimolar (mM) to 600 mM. In another embodiment, the concentration of boric acid can be from 300 mM to 600 mM.
In another embodiment the concentration of poly (α 1, 3 glucan) in the enzymatic reaction solution is increased from 0.08 grams poly (α 1, 3 glucan) per gram of sucrose to 0.25 grams poly (α 1, 3 glucan) per gram of sucrose.
In one embodiment concentration of fructose in the enzymatic reaction solution is increased from 29% to 43% of sucrose converted.
Other materials that can be used in place of boric acid to sequester fructose can include, but may not be limited to: 2-Aminopyrimidine-5-boronic acid; Benzene-1,4-diboronic acid; Carboxyphenylboronic acid; Fluorene-2-boronic acid; Furan-2-boronic acid; Naphthalene-1-boronic acid; Nitrophenylboronic acid; n-Pentylboronic acid; Methylpropylboronic acid; Methoxyphenylboronic acid; and Phenylboronic acid, for example.
In the present invention, the effect of pH of the gtf enzyme reaction solution can be from 6.5 to 8.1. In one embodiment the pH of the gtf enzyme reaction solution is 7.75. In another embodiment, the pH of the gtf enzyme reaction solution is 8.1.
EXAMPLES
The invention is further described but not limited by the following specific embodiments thereof.
Materials
T10 dextran (D9260), Isopropyl β-D-1-thiogalactopyranoside (IPTG) (I6758) and boric acid (B1934) were obtained from Sigma, St. Louis, Mo.
Whatman-1 filter paper was obtained from Whatman Filters, Maidstone Kent, UK.
Solenoid driven micro-valve was from Bio-Chem Fluidics, Boonton, N.J.
Bellco spin flask was from Bellco, Vineland, N.J.
VWR Ag/AgCL pH probe was from VWR International, Radnor, Pa.
Eutech pH/ORP controller was from division of Thermo Fisher Scientific Inc., Waltham, Mass.
Luria broth (LB) medium was from Becton, Dickinson and Company, Franklin Lakes, N.J.
The bead beater was obtained from MP Biomedicals, Eschwege, Germany).
Eppendorf 5415D Centrifuge was from Eppendorf, Hamburg, Germany)
Protein concentration in samples were determined using the Coomassie Plus, Bradford Assay Kit (Thermo Scientific, Rockford, Ill.)
High pressure chromatography (HPLC) was performed using a 1200 series, Agilent, Santa Clara Calif.) instrument. The column used for analysis was an Aminex HPX-87C column, (Bio-Rad Laboratories, Hercules, Calif.) which was maintained at 85° C. using a flow rate of 0.6 milliliters per minute (mL/min) with water as the mobile phase. HPLC analysis was used to determine disappearance of sucrose and the accumulation of fructose, glucose and leucrose. Using this system the following retention times were observed for chemicals of interest: sucrose (8.29 minutes, min); leucrose (9.40 min); glucose (10.12 min) and fructose (12.89 min).
Example 1
Preparation of Crude Extracts of GtfJ (Seq Id No:4)
The gtfJ gene of S. salivarius is available in Genbank (Genbank accession number Z11873, SEQ ID NO. 4). To produce sufficient quantities of gtfJ (SEQ ID NO:4), a truncated gtfJ gene (SEQ ID NO. 6) was expressed in E. coli strain (DH10B) as described in the commonly owned U.S. Pat. No. 7,000,000. E. coli (DH10B), cells expressing the gtfJ enzyme (SEQ ID NO:4), were grown in the LB medium (10 grams per liter, g/L Tryptone; 5 g/L yeast extract; and 10 g/L NaCl). E. coli cells were inoculated to an initial optical density (OD at 600nm) of 0.025 and were allowed to grow at 37° C. in an incubator while shaking at 250 rpm. The cultures were then induced by addition of 1 mM IPTG when they reached an OD of 0.8-1.0. Induced cultures were left on the shaker and harvested 3 hours post induction. The cells were removed by centrifugation (25° C., 16,000 rpm) using an Eppendorf centrifuge and cell pellets were suspended in 0.01 volume of 5.0 mM phosphate buffer (pH 7.0) and cooled to 4° C. on ice. The cells were broken using a bead beater with 0.1 millimeters (mm) silica beads. The broken cells were centrifuged at 16,000 rpm at 4° C. to precipitate the unbroken cells and cell debris. The crude extract (containing soluble gtfJ enzyme, SEQ ID NO:4) thus obtained contained 2.9 milligram per milliliter (mg/mL) of protein as determined by the Bradford protein assay.
Example 2
Effect of Boric Acid Addition on the Amount of Product Formed by GtfJ (Seq Id No:4)
To determine the effect of boric acid addition on the yield of poly (α 1,3 glucan) enzyme reactions were performed in 50 mL reactors in the presence (test) or absence (control) of boric acid.
GtfJ (SEQ ID NO:4) reaction solutions contained sucrose (50-150 g/L); potassium phosphate buffer (10 mM); T10-dextran primer at a final concentration of 1 g/L; and total soluble enzyme (0.4-1.0 volume percent, v %). Reactions were performed at 25° C.-35° C. in the presence or absence of boric acid. Concentration of sucrose, leucrose, fructose and glucose were determined using HPLC. The yield of poly (α1, 3 glucan) was determined by measuring its dry weight after filtration and washing with 8 volumes of water and then drying for 48 hours at 35° C.
In one experiment, the reaction solution contained: sucrose (150 g/L); gtfJ enzyme (SEQ ID NO:4) (0.4 volume %) and T-10 dextran primer (1 g/L). The initial pH of the reaction solution was adjusted to 7.5 using phosphate buffer. The temperature was held at 30° C. and the reaction solution was left stationary for 51 hours. Boric acid (at either 300 mM or 600 mM concentrations) was added to the reaction solution and the pH was adjusted to 7.5 using sodium hydroxide, prior to gtfJ (SEQ ID NO:4) addition. The pH of the reaction solution was monitored throughout the test, but was not adjusted. The pH of reactions containing boric acid continued to drop from the initial adjusted pH, while the pH of the control reaction solution, without boric acid, did not change throughout the experiment.
Addition of boric acid into gtfJ (SEQ ID NO:4) reaction solution decreased the leucrose concentration by approximately 2 to 3-fold (Table 1). Additionally, the yield of poly (α1, 3 glucan) recovered in this reaction increased relative to the control reaction that did not contain any boric acid. The concentration of fructose was also proportionally higher in reactions containing boric acid.
As the reaction progressed, pH dropped and leucrose continued to accumulate suggesting that controlling the pH of the gtfJ (SEQ ID NO:4) reaction containing boric acid can potentially affect by-product synthesis.







TABLE 1







Effect of boric acid on gtfJ reaction product formation












300 mM
600 mM


Grams/liter
Control
Boric acid
Boric acid













Initial sucrose
153.28
155.34
152.88


End sucrose
16.16
6.68
8.96


Leucrose
58.85
29.95
21.06


Glucose
10.13
14.75
16.98


Fructose
45.15
64.19
69.32


% Sucrose used
89
96
94


Leucrose (% of sucrose)
43
20
15


Glucose (% of sucrose)
7
10
12


Fructose (% of sucrose)
33
43
48


Yield g poly (α 1, 3 glucan)/g
0.17
0.29
0.37


sucrose)









To study the effect of lower (i.e., less than 300 mM) concentrations of boric acid on leucrose formation in an experiment, either 100 mM or 200 mM of boric acid was used in the reaction solution. The reaction solutions contained sucrose (100 g/L), gtfJ (SEQ ID NO:4) (0.5 v %), T-10 dextran primer (1 g/L) and potassium phosphate buffer, (10 mM). The initial pH of the reaction was 7.0. The temperature of the reaction was held at 27° C. for 44 hours. Initial boric acid concentration in the reactions was 0 mM (control), 100 mM (pH adjusted with KOH or NaOH) or 200 mM (NaOH adjusted). The results listed in Table 2 indicate that presence of boric acid in the enzyme reaction solution reduced leucrose formation relative to the control reaction. Counter ions (potassium or sodium) used for initial pH adjustment did not affect leucrose formation.







TABLE 2







Effect of reduced concentration of boric acid on product formation 


during gtfJ reaction in the presence of counter ions













(Na)
(K)
(Na)




100 mM
100 mM
200 mM


Grams/Liter
Control
boric acid
boric acid 
boric acid














Initial sucrose
112.12
111.36
114.57
102.36


End sucrose
14.21
7.23
7.17
7.48


Leucrose
37.50
28.60
29.68
25.74


Glucose
6.56
8.86
8.98
9.11


Fructose
28.64
36.41
37.32
37.53


% Sucrose used
87
94
94
93


Leucrose (% of sucrose)
38
27
28
27


Glucose (% of sucrose)
7
9
8
10


Fructose (% of sucrose)
29
35
35
40









In summary, the data presented in this Example demonstrate that the addition of boric acid to gtfJ enzyme (SEQ ID NO:4) reaction solutions resulted in an increase in poly (α 1, 3 glucan) yield which corresponded to a decrease in leucrose accumulation.
Example 3
Effect of Boric Acid on Sucrose Uptake by GtfJ (Seq Id No:4)
To determine if the presence of boric acid affected sucrose conversion and hence poly(α1,3 glucan) formation by gtfJ (SEQ ID NO:4), reaction solutions were prepared containing sucrose (100 g/L), gtfJ (SEQ ID NO:4) (0.5 volume %) boric acid (either 300 mM or 600 mM) and potassium phosphate buffer, (10 mM); at an initial pH of 7.0. The control reaction was set up with similar ingredients but without any boric acid.
The reaction was monitored over time. Samples were taken at intervals and the concentration of soluble sugars was determined by HPLC analysis as described in Example 1. Results for sucrose use over time are shown in FIG. 1a. From the data, it is clear that the rate of sucrose use is higher for a reaction containing 300 mM boric acid relative to the control (no boric acid). Furthermore, the reaction with the highest boric acid concentration (600 mM) resulted in the highest sucrose use rate. Thus the presence of boric acid in above experiments resulted in an increased rate of conversion of sucrose to poly (α1, 3 glucan).
The concentration of leucrose continuously increased in an enzyme reaction solution containing 100 g/L sucrose, where the initial pH had been adjusted to 7.4 using potassium phosphate buffer (10 mM final concentration)(FIG. 1B). The data provides additional evidence that the presence of boric acid results in reduced synthesis of leucrose over time. Measuring pH of the control reaction solution during the experiment demonstrated only a slight drop in pH from 7.4 to 7.2 at the end of the experiment (77 hours). In contrast to the control, the pH drop was much more pronounced in both reactions containing boric acid. The pH in the 300 mM boric acid reaction solution decreased from 7.4 to 5.98 and the pH in the 600 mM reaction was at pH 5.60 at the end of the experiment.
Example 4
pH Controlled Enzyme Reactions Containing Boric Acid
To perform this experiment, a 150 ml capacity Bellco spin flask was used and the pH of the reaction solution was monitored using a VWR Ag/AgCL pH probe which was linked to a Eutech pH/ORP controller set to maintain pH at 7.5. Base was delivered to the reaction using a solenoid driven micro-valve from a reservoir containing 2.0 M sodium hydroxide. The gtfJ (SEQ ID NO:4) reaction solutions contained: sucrose (100 g/L); gtfJ (SEQ ID NO:4) (0.5 volume %), T-10 dextran primer (1 g/L) and boric acid at 300 mM in each reaction. Initial reaction pH was 7.5. The reactions were performed at 25° C. for 56 hours.
The data shown in Table 3 compares sucrose consumption and product synthesis in gtfJ enzyme (SEQ ID NO:4) reaction solutions, containing 300 mM boric acid, with or without pH control.







TABLE 3







Effect of pH control in substrate consumption and product formation









Grams/Liter
Control reaction
pH Adjusted reaction












Initial sucrose
105.51
105.79


End sucrose
66.77
35.13


Leucrose
7.74
3.10


Glucose
5.08
13.77


Fructose
12.76
30.38


% sucrose used
37
67


Leucrose (% of sucrose)
20
4


Glucose (% of sucrose)
13
19


Fructose (% of sucrose)
33
43









The results indicate that maintaining the pH at 7.5 during the gtfJ (SEQ ID NO:4) reaction was far more effective in reduction of leucrose formation in the presence of boric acid. At approximately reaction mid-point, the concentration of leucrose in the pH adjusted flask accounted for only 4% of the sucrose converted. The level of leucrose at the end of the reaction (94% of sucrose converted) increased to 5% of sucrose used in the pH controlled test. The concentration of Leucrose in the control reaction was 20% of converted sucrose at the same time-point and increased to 27% at completion of the experiment. In addition to the reduced by-product accumulation, the pH controlled reaction used twice as much sucrose relative to the control.

Since maintaining the pH of a reaction was shown to be an effective method for reducing leucrose accumulation the effect of controlling the pH of the reaction at higher pH than 7.5 was investigated. The reaction solutions contained: sucrose (100 g/L); gtfJ (SEQ ID NO:4) (0.5 v %); T-10 dextran primer (1 g/L) and boric acid (300 mM). One reaction was maintained at pH 7.75 and the other one was maintained at pH 8.1. The reaction solutions were maintained at 25° C. for 76 hours.
Table 4 shows the concentration of leucrose formed at the end of these reactions at 11% and 6% respectively. When the results obtained in these experiments are compared to those obtained in reactions with no pH control (e.g. Table 1, when leucrose accounts for 20% of sucrose used) it becomes apparent that leucrose formation in a gtfJ (SEQ ID NO:4) reaction can be more effectively reduced when pH of the reaction solution is effectively controlled.







TABLE 4







Effect of pH 7.75 and 8.1 on sucrose consumption and 


product formation in a gtfJ reaction solution













Grams/Liter
pH 7.75
pH 8.1

















Initial sucrose
114.40
114.36




End sucrose
4.55
3.55




Leucrose
12.16
6.61




Glucose
12.56
14.91




Fructose
46.34
48.16




% sucrose used
96
97




Leucrose (% of sucrose)
11
6




Glucose (% of sucrose)
11
13




Fructose (% of sucrose)
42
43










Example 5
Cloning and Expression of the Gtf Enzyme of Streptococcus Sobrinus (Seq Id No:3)
To examine the effect of boric acid on sucrose consumption and product formation in a gtf reaction performed by an alternative gtf enzyme, the gtfI gene from Streptococcus sobrinus (Genbank accession number BAA02976, SEQ ID NO. 3) was cloned using methods well known in the art.
A truncated version of the S. sobrinus 6715 gene coding for gtfI protein (SEQ ID NO:3) was isolated from (S. sobrinus ATCC 27351) (SEQ ID NO. 5) by PCR amplification using primers based on the gene sequence described in Genbank accession number BAA02976 and by Abo et al., (J. Bacteriol., 173: 998-996, 1991). PCR reactions were performed using a 5′-end primer (SEQ ID NO: 1) and a 3′-end primer (SEQ ID NO: 2).
In (SEQ ID NO:1) (5′GGGAATTCCCAGGTTGACGGTAAATATTATTACT), the coding sequence corresponded to bases 466 through 491 of the gtfI gene of S. sobrinus ATCC 27351, and additionally provided the sequence for an Eco RI restriction enzyme site that was used for cloning purposes.
In (SEQ ID NO: 2) (5′-AGATCTAGTCTTAGTTCCAGCCACGGTACATA) the coding sequence corresponded to the reverse compliment of bases 4749 through 4774 of S. sobrinus (ATCC 27351) gtfI gene. The reverse PCR primer also included the sequence for an XbaI site for cloning purposes. The PCR was performed by methods well-known in the art. All subsequent steps for preparation of E. coli expression vector for the gtfI gene (SEQ ID NO:5) were performed as recommended by the manufacturers of the products used.
The resulting PCR product was digested with Eco RI and Xba I restriction enzymes and then purified using a Promega PCR Clean-up kit (A9281, Promega, USA) as recommended by the manufacturer. The DNA fragment was ligated into an E coli protein expression vector (pET24a, Novagen, USA). The ligated reaction was transformed into the DE3 cell line (New England Biolabs, USA) and plated on solid LB medium (10 g/L, tryptone; 5 g/L yeast extract; 10 g/L NaCl; 14% agar; 100 μg/ml ampcillin) for selection of single colonies. Transformed E. coli cells were grown overnight in the LB medium. The cells were then diluted to an optical density (OD) of 0.025 (A600nm) then grown to a density of 1.0 OD in the LB medium. At this density, IPTG (final concentration, 1.0 mM) was added to the culture to induce the gtfI gene (SEQ ID NO:5) expression. Cultures were incubated at 37° C. for an additional 2 to 4 hours before cell harvest. Cell disruption, total soluble protein isolation and enzyme reactions were carried out as described in Example 1.
Enzyme reaction solutions for investigating the effect of addition of boric acid on substrate consumption and product formation by gtfI (SEQ ID NO:3) contained: sucrose (100 g/L); gtfI (SEQ ID NO:3) (0.4 volume %); T-10 dextran primer (1 g/L). The control experiment did not have any boric acid. The test experiment included 100 mM boric acid. The Initial pH of the reaction solution was 7.5 using potassium phosphate. The reaction pH was monitored, but not adjusted after addition of enzyme. The temperature was held at 30° C. for 44 hours.
The effect of boric acid on reduction of the by-products formed using VI enzyme (SEQ ID NO:3) is summarized in Table 5. The data indicate that presence of boric acid in the gtfI (SEQ ID NO:3) reaction resulted in a 3 fold decrease in leucrose formation.







TABLE 5







Streptococcus sobrinus gtfl reaction












0 mm
100 mM



Grams/Liter
Boric acid
Boric acid














Initial sucrose
99.83
98.84



End sucrose
8.79
5.08



Leucrose
50.73
14.69



Glucose
5.32
8.55



Fructose
28.25
48.20



% sucrose used
91
95



Leucrose (% of sucrose)
56
16



Glucose (% of sucrose)
6
9



Fructose (% of sucrose)
31
51









Example 6
pH Controlled Enzyme Reactions Containing Boric with Dextran or Glucose as Primer
To determine the effect of boric acid addition on the yield of poly (α 1, 3 glucan) enzyme reactions were performed in a 4 L jacketed glass resin kettle equipped with a glass agitator shaft with 3 glass turbine impellers was used. The pH of the reaction solution was monitored using a VWR Symphony gel epoxy flask combination electrode which was linked to a pH controller set (Cole-Parmer Digital Chemcadet pH meter/controller Model 5652-00) and maintained pH at 7.5. Base was delivered to the reaction using a Cole-Parmer, Masterflex, console drive pump, model #7521-40 pump from a reservoir containing 4.0 M sodium hydroxide. The gtfJ (SEQ ID NO:4) reaction solutions contained: sucrose (100 g/L); gtfJ (SEQ ID NO:4) (1.0 volume %), either T-10 dextran (1 g/L) or glucose (0.1 g/L) as a primer and boric acid at 300 mM in each reaction. Buffer system was 2.75 mM KCI, 2.75 mMK2SO4, and 0.75 mM potassium acetate. Initial pH of the reaction was 7.5 and the reactions were performed at 25° C. for 43-45 hours.
The gtf enzyme reactions were performed in the presence (test) or absence (control) of boric acid. The gtfJ (SEQ ID NO:4) control reaction solutions contained: sucrose (100 g/L); gtfJ (SEQ ID NO:4) (1.0 volume %), either T-10 dextran primer (1 g/L) or glucose (1.0 g/L) and 50 mM Potassium phosphate (KH2PO4) buffer pH was adjusted to 7.0 with 10% KOH solution. Reactions were incubated in an Innova 4200 incubator/shaker at 25° C. Concentration of products in the reaction solution was determined as described above.
The data shown in Table 6 compares sucrose consumption and product formation in gtfJ enzyme (SEQ ID NO:4) reaction solutions, with either glucose or dextran as a primer in pH-controlled experiments with or without borate.







TABLE 6







Effect of primer in substrate consumption and product formation














Borate—
Borate—





pH control 
pH control 



No borate— 
No borate—
at 7.5—
at 7.5—



dextran
glucose 
dextran 
glucose 


g/L
primed
primed
primed
primed














Initial sucrose
104.2
104.4
100.0
102.7


End sucrose
1.4
1.4
3.2
3.6


Leucrose
36.0
30.5
5.3
3.2


Glucose
5.1
5.0
12.8
11.9


Fructose
29.4
32.3
45.2
46.8


% sucrose used
99
99
97
97


Leucrose (% 
35
30
5
3


of sucrose)






Glucose (% of
5
5
13
12


sucrose)






Fructose (% 
29
31
47
47


of sucrose)






Yield of Glucan:
0.151
0.18
0.224
0.241


G poly (α 1, 






3 glucan)/g






sucrose









The results indicate that borate's effect on reducing leucrose and increasing poly (α 1, 3 glucan) formation is primer independent. The comparison with non-borate experiments also shows increased yield of poly (α 1, 3 glucan) with concomitant reduction in leucrose production.

## Claims

1. A reaction solution for the synthesis of poly (alpha 1,3 glucan) comprising:
a) at least one Streptococcus glucosyltransferase enzyme that synthesizes poly (alpha 1,3 glucan) from sucrose;
b) boric acid; and
c) sucrose,

whereby poly (alpha 1,3 glucan) is produced with a lower concentration of leucrose by-product than is produced in the absence of boric acid.

2. The reaction solution of claim 1 further comprising at least one primer.

3. The reaction solution of claim 2 wherein the primer is dextran.

4. The reaction solution of claim 2 wherein the primer is hydrolyzed poly (alpha 1,3 glucan).

5. The reaction solution of claim 1, wherein the Streptococcus glucosyltransferase enzyme is a Streptococcus salivarius glucosyltransferase enzyme or Streptococcus sobrinus glucosyltransferase enzyme.

6. The reaction solution of claim 5, wherein the Streptococcus salivarius glucosyltransferase enzyme comprises SEQ ID NO:4 and the Streptococcus sobrinus glucosyltransferase enzyme comprises SEQ ID NO:3.

7. The reaction solution of claim 5, wherein the reaction solution pH is maintained from 6.5 to 8.1.

8. The reaction solution of claim 7, wherein the concentration of boric acid in the reaction solution is from about 100 millimolar to about 600 millimolar.

9. The reaction solution of claim 8, wherein the concentration of boric acid in the reaction solution is from about 300 millimolar to about 600 millimolar.

10. The reaction solution of claim 2 wherein the enzyme of (a) is a primer dependent enzyme.

11. The reaction solution of claim 10 wherein the primer is glucose.

12. The reaction solution of claim 1 wherein the enzyme of (a) is a primer independent enzyme.

13. The reaction solution of claim 1 wherein more than one enzyme of (a) is present in the reaction solution.

14. The reaction solution of claim 13 wherein one glucosyltransferase enzyme is primer dependent and one glucosyltransferase enzyme is primer independent.

15. The reaction solution of claim 1 wherein the concentration of boric acid in the reaction solution is from about 100 millimolar to about 600 millimolar.

16. The reaction solution of claim 15, wherein the concentration of boric acid in the reaction solution is from about 300 millimolar to about 600 millimolar.

17. The reaction solution of claim 1 wherein the reaction solution pH is maintained from 6.5 to 8.1.

18. The reaction solution of claim 17, wherein the concentration of boric acid in the reaction solution is from about 300 millimolar to about 600 millimolar.

19. The reaction solution of claim 1 wherein the yield of poly (alpha 1,3 glucan) formed in the reaction solution improves from 0.08-0.1 g glucan/g sucrose to 0.25 g glucan/g sucrose.

20. A process for reducing the amount of byproduct leucrose formed during enzymatic synthesis of poly (alpha 1,3 glucan) comprising:
providing a reaction solution comprising:
i) at least one Streptococcus glucosyltransferase enzyme that synthesizes poly (alpha 1,3 glucan) from sucrose;
ii) boric acid; and
iii) sucrose;

wherein the sucrose is converted to poly (alpha 1,3 glucan) and fructose and wherein the amount of leucrose produced in the conversion is less than 35% of the sucrose consumed.

21. The process of claim 20 wherein the yield of leucrose formed decreases from 44% sucrose to 4% of sucrose converted.

22. The process of claim 20 wherein the reaction solution pH is maintained from 6.5 to 8.1.

23. The process of claim 20 wherein the yield of fructose increases from 29% sucrose to 43% of sucrose converted.

24. The process of claim 20, wherein the Streptococcus glucosyltransferase enzyme is a Streptococcus salivarius glucosyltransferase enzyme or Streptococcus sobrinus glucosyltransferase enzyme.