..... BiOlechllo!og\' Lellers 24: 1277-1280. 2002. 1277 " © 2002 Khllfer Academic Publishers. Prillled ill fhe Nefher!allds.

Development of a coupled enzyme assay for the measurement of alternanase activity**'***

Jeffrey A. Ahlgren & Gregory L. Cote" Fermentation Biotechnology Research Unit. National Center for Agricultural Utilization Research. Agricultural Research Service. U.S. Department ()fAgriculture. 1815 North University Street. Peoria. lllinois 61604. USA "Authorfor correspondence (Fax: 309-681-6427; E-mail: cotegl@ncalll:usda.gov)

Received 21 December 200 I: Revisions requested 8 January 2002: Revisions received 22 May 2002: Accepted 23 May 2002

Key words: alteman. altemanase. coupled enzyme assay. cyclic tetrasaccharide. panose

Abstract

Altemanase. an endoglucanase that hydrolyzes the bacterial exopolysaccharide alteman. will also hydrolyze the trisaccharide. panose. to produce glucose and a disaccharide that can be formed into a novel. cyclic tetrasaccharide. The glucose can then be selectively and quantitatively measured by enzyme-based reaction which forms the basis of a coupled enzyme assay to quantitate altemanase activity. By this method a preparation of altemanase purified by affinity chromatography on immobilized isomaltose had a maximum reaction rate (Vmax i of 0.75 /-w101 glu­ 1 cose min- and a K m of 34 mM for panose. Two competitive inhibitors of altemanase activity were also evaluated using this coupled enzyme assay: isomaltose had a Ki of 94 mM while the cyclic tetrasaccharide had a Ki of 66mM.

Introduction h ,'. • ',,, ,'-,.. , ,.","alt~~lfa[\.~v~p•. s.i~lli!lc;,antly underestimate altemanase " '-'. ",:.' . - ". , . . actIVIty due to the fact that about half of the to- Altemanase is an endoglucanase that: hydrolyzes' ral product is the ilOn-reducing cyclic tetrasaccharide. the a-D-Glcp-(I--..3i. a-D-Glcp-(I--..6) J.inkage se­ This method. while convenient for some qualitative quence of the bacterial exopolysaccharide alteman analyses. is not ideal for determining enzymatic pa­ (Biely et al. 1994. Cote & Ahlgren 200 I). The rameters such as maximum reaction velocity because main products of this enzymatic hydrolysis include the stoichiometry of the products is not consistent isomaltose. the trisaccharide a-D-Glcp-(I--..3)-a-D­ and reaction rates may be variable relative to the size Glcp-(I--..6)-a-D-Glc and the cyclic tetrasaccharide of the alteman. An alternate method involves Rema­ cyc/o {--.. 6)-a-D-Glcp-( 1--..3)-a-D-Glcp-( 1--..6)-a-D­ wI Brilliant Blue-conjugated alteman (RBB-alteman) Glcp-( 1--.. 3)-a-D-Glcp-( I--..} (Cote & Biely 1994). (Rinderknecht et al. 1967. Wyckoff et al. 1996). With The nature of the products of the hydrolysis of al­ this substrate it is possible to estimate the activity teman have complicated the development of a con­ of alteman by measuring the formation of ethanol­ venient assay method that can be used to quantita­ soluble oligosaccharide fragments after precipitation tively determine the specific activity of this enzyme. of the remaining unhydrolyzed RBB-alteman. One For instance. an assay based on measuring the in­ drawback to this method is that the RBB-alteman does crease in reducing sugar formed by the hydrolysis of not always have a consistent degree of bound dye per synthesis. which leads to inconsistencies between ''''The use of brand or trade names may be necessary to report fac­ tually on available data. The USDA neither guarantees nor warrants preparations. Furthermore. the release of soluble dyed the standard of the product. and the use of the name by USDA im­ fragments is not readily convertible to standard units plies no approval of the product to the exclusion of other that may of activity. We have also made radiolabeled alter­ also be suitable. nan by using the enzyme altemansucrase to polymer­ """'The US Government right to retain a non-exclusive royalty-free 4 license in and to any copyright is acknowledged. ize [14C]glucose derived from [1 C]sucrose (Cote & 1278

Robyt 1987). which after purification can serve as a 2 Panose Alternanase 2 Glucose + cyclo {-3j-D-Glcp-[1-6j-D-Glcp-[1-L substrate for alternanase. However this method ac­ Glucose +ATP Hexokinase Glucose-6-P + ADP tually measures the fraction of polysaccharide which Glucose-6-P + NAD G-6-P Dahydrogenase 6-PG + NADH remains insoluble in methanol by adhering to a piece Fig. I. Reaction scheme for the measurement ofalternanase activity of filter paper. and it is hard to calibrate this method using panose by coupled enzyme assay with hexokinase. Panose is U'-o-Glcp-( 1-6)-U'-o-Glcp-( 1-4)-U'-o-Glc. because small oligosaccharides have intermediate sol­ ubility in this method. Typically the results obtained from this procedure are derived from relatively small lvlaterials changes in large amounts of radioactivity. and also the correlation of radioactivity measured to the actual Panose. glucose. isomaltose. immobilized isomaltose number of bonds hydrolyzed is problematic. Alter­ affinity chromatography resin, and the hexokinase­ nanase will hydrolyze 4-nitrophenyl a-isomaltoside to based glucose determination kit were purchased from isomaltose and 4-nitrophenol. the latter being quan­ Sigma-Aldrich Chemical company (St. Louis, MO). titatable spectrophotometrically at 415 nm (Cote & The cyclic tetrasaccharide cyelo {--+6)-a-D-Glcp­ Ahlgren 200 I). But this substrate is not commercially (1--+ 3)-a-D-Glcp-( 1--+6)-a-D-Glcp-( 1--+3)-a-D-Glcp­ available. has low solubility (below the measured K II1 (I--+} (cGlec;) was prepared by hydrolyzing native for this compound). and the overall reaction is very alternan from Leuconostoc mesenteroides NRRL B­ slow. which taken together limit the usefulness of this 21297 with alternanase and purified as described (Cote compound for this application. & Biely 1994). All other chemicals were reagent grade The previous observation that the enzyme al­ or better. ternanase would hydrolyze 2 mol of the trisaccha­ ride. panose {a-D-Glcp-( I--+6)-a-D-Glcp-( I--+4)-a­ Enzyme assays D-Glc} to form 2 mol of glucose and the cyclic tetrasaccharide c.velo {--+6)-a-D-Glcp-( 1--+3)-a-D­ Mixtures of alternanase (5 fJ.g total protein) and Glcp-(l--+6)-a-D-Glcp-( 1--+ 3)-a-D-Glcp-(l--+ }(Cote panose (0-178 mM) contained in 100 l.iI in 0.5 ml & Ahlgren 200 I) led to the idea that an assay that capped plastic microfuge tubes were incubated at would specifically quantitate glucose could be used 45°C. A 10 Iii sample was removed immediately af­ to indirectly measure the activity of the enzyme alter­ ter the reactants were combined and thereafter every nanase in a coupled reaction scheme. This report de­ 5 min for 20 min. To each sample in a 2-ml capped scribes the use of a commercially available method to plastic microfuge tube in a wet ice bath. 1.5 ml of the quantitate glucose. based on the action of hexokinase reconstituted hexokinase reagent (as specified by the coupled with glucose-6-phosphate dehydrogenase in a manufacturer) was added and the mixture incubated a reaction that produces NADH. in a coupled enzyme minimum of an additional 5 min at ambient tempera­ assay to measure the enzymatic activity of alternanase. ture before measuring the absorbance of the solution at 340 nm. A series of incremental glucose concentra­ tions were used to calibrate the assay method under the Experimental same conditions used for the alternanase plus panose reactions. A blank reaction consisted of 10 Iii of the Enzymes 50 mM MOPS buffer alone. Control reactions contain­ ing an equivalent volume of alternanase alone as well Alternanase was purified from crude. cell-free concen­ as panose alone were also prepared: the absorbance trated supernatant from 3 day old cultures of Bacillus values obtained from these reactions were subtracted sp. NRRL B-2 I 195 using affinity chromatography from the absorbance values for the complete reaction on immobilized isomaltose (JA Ahlgren & GL Cote. mixtures in order to determine the reaction velocity. submitted). The preparation used in the reactions de­ scribed here contained 0.5 mg ml- I protein in 50 mM Protein concentration determinations MOPS. pH 7. containing I mM CaCh and 1.5 mM NaN3. The protein concentration of alternanase solutions was determined by the bicinchoninic acid protein assay method using bovine serum albumin as a reference (Pierce Chemical Company. Rockford, IL). 1279

Results and discussion 12

The coupled-enzyme assay reaction scheme (Figure I) 10 that measures the enzymatic activity of alternanase preparations is composed of two steps: in the first step the enzyme alternanase releases one mole of glu­ cose from each mole of panose. This is followed by a second step that selectively and quantitatively mea­ sures the glucose concentration in solution. Therefore a unit of alternanase activity is defined as the amount • Control (no Inhibitor) o + 50 mM Isomaltose of enzyme which produces a micromole of glucose T + 50 mM eGle, from panose per minute at 45°C. Two enzyme-based - Linear Regression methods were evaluated for the quantitation of glu­ -40 -20 20 40 60 80 100 120 140 cose. One is based on a hexokinase-coupled reaction and the second scheme uses a glucose oxidase-based 1/[8] (Panose, M)-1 reaction (data not shown). The advantage of using Fig. 2. EtTecl of panose concentration on the nlie of reaction of the hexokinase-based coupled assay over the glucose alternanase: (.1 control (no inhibitor): Ie> 1with 50 mM isomaltose: IT) with 50 m1',,[ cGlq. oxidase based assay is that the latter assay requires adhering to a 20-min incubation period for the glu­ cose determination phase of the method. because the l calculated. AVmax value of 0.75 flmol glucose min- absorbance value continues to change slowly over at 45 DC and a Kill value of 34 mM panose were time. The absorbance value from the glucose quanti­ derived for this preparation of alternanase. The ac­ tation phase of the hexokinase coupled assay reaches tual highest rate measured. 0.65 ~lmol glucose min-I a steady value after 5 min, which remains constant for when assayed with 178 mM panose for 20 min, con­ approx. I h, which greatly simplifies matters when sumed 73% of the available panose in the reaction. handling a large number of samples. Both methods so for routine assay work it would be preferable to otherwise yielded identical results. have a higher concentration of panose to maintain en­ Preliminary assays showed that it was not neces­ zyme saturation. A starting concentration of 0.3 M sary to inactivate the alternanase after the first step of panose would be reasonable, which in this case would the process as long as the reaction was chilled to 0 DC consume only 50% of available panose at a Vmax of or below after the desired incubation at 45°C; control 0.75 /.lmol glucose min- l in a 20-min single time reactions held for more than I h at O°C showed no point assay. increase in glucose levels over those processed im­ The coupled reaction method is also useful for mediately. The 10 ~d reaction volume was diluted to quantitating the effect of adding inhibitors of alter­ 1.5 ml for the second step of the reaction, conducted at nanase activity. such as isomaltose or cGlc4. both of room temperature, which diluted any residual panose which are reaction products. In the case of isomaltose, by more than ISO-fold so that it would not likely it would not be possible to quantitate these kinetic continue to be hydrolyzed by alternanase to any signif­ parameters based on a reducing sugar assay because icant extent during the glucose quantitation step. The it itself is a reducing sugar. By adding a constant analysis of the assays showed that 5 p.g purified al­ amount of inhibitor to a series of panose-alternanase ternanase per 100 VI reaction was suitable, as judged reactions identical to those used to measure the Vmax by yielding linear results. over a wide range of panose of the uninhibited reaction. the inhibitor constant (Ki) concentrations at 45 DC for at least 20 min. Plots were as well as the nature of the inhibition can be deter­ made of absorbance versus time, and the slope of mined. As seen in Figure 2. a double-reciprocal plot the line determined for each concentration of panose of the results of reactions with and without 50 mM tested. The slope of the line was then converted from isomaltose showed that the Vmax of the reaction did not change in absorbance units per minute to /.Lmol glu­ change (same intercept on the y-axis) but the appar­ cose formed per minute. As seen in Figure 2, a double ent Kill for the reaction was changed, indicating that reciprocal plot of the data yielded a straight line and the inhibition was competitive. The Ki value for iso­ the values for the maximum reaction velocity (Vmax! maltose can be derived from the x-axis intercept and and Michaelis-Menton constant (Kill) can be readily 1280

was determined to be 94 mM. A similar experiment References was performed with 50 mM cGlc4, which also showed Biely P. Cote GL. Burgess-Cassler A( 19941 Purification and prop­ competitive inhibition of alternanase, and had a K i value of 66 mM. erties ofalternanase. a novel endo-a-I.3-a-I.6-0-glucanase. £111: .I. Biochelll. 226: 633-639. Based on these results we conclude that the cou­ Cote GL. Ahlgren JA (20011 The hydrolytic and transferase action pled assay utilizing panose as the substrate and moni­ of alternanase on oligosacchararides. Carbohy{b: Res. 332: 373­ toring the formation of glucose provides a reliable and 379. Cote GL. Biely P (19941 Enzymatically produced cyclic a-l.3­ convenient method to quantitate alternanase activity. linked and a-1.6-linked oligosaccharides of o-glucose. £111: .I. As more research on alternanase is carried out. such Biochelll. 226: 641-648. as improved production processes including cloning Cote GL. Robyt JF (1982) Isolation and partial characterization of and potential overexpression to increase yields of the an extracellular glucansucrase from Leuconostoc lI1esellteroides NRRL B-1355 that synthesizes an alternating (I -61. (1-31-a-O­ enzyme, a method involving commercially available glucan. Carbohyd/: Res. 101: 57-74. components that provides consistent and reproducible Rinderknecht H. Wilding P. Haverback BJ (1967) A new method for results for the measurement of alternanase activity will determination of a-amylase. £xperielltia 23: 805. be very useful and appreciated. Wyckoff HA. Cote GL. Biely P (19961 Isolation and characteriza­ tion of microorganisms with alternan hydrolytic activity. CUrl: i'vlicrohiol. 32: 343-348.

Acknowledgements

The authors thank Drs Terence R. Whitehead and Lubomir Kremnicky for their insightful discussions on assay development and Mr James J. Nicholson for his technical assistance.

Suoollea bV U.S. DeOL of Agriculture National Center tor Agncultural UtilizatIOn Research, Peoria, lIfinois

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