,
`Carbohydrate Research, 36 (1974) 207-210
`© Elsevier Scientific Publishing Company, Amsterdam » Printed in Belgium
`
`Note
`
`A viscometric assay for pullulanase—type. debranching enzymes’
`
`DAVID G. HARDIE AND DAVID J. MANNERS
`
`Department of Brewing and Biological Sciences, H'en’o‘t— Watt University, Edinburgh (Great Britain)
`(Received February 21st, 1974; accepted for publication, March 1st, 1974)
`
`Bacterial pullulanases (E.C. 3.2.1.41) specifically hydrolyse the at-(l——>6)—
`D-glucosidic bonds which form the inter-chain linkages in certain 'ot—(l —->4)—linked
`D-glucose polymers such as amylopectinz. The limit dextrinases, which serve a similar
`function in higher plants, are now known to be qualitatively similar in specificity to
`these bacterial enzymess"; Both groups of enzymes maybe routinely assayed by
`following the release of reducing groups from the fungal polysaccharide pullulans,
`which consists of maltotriose » units connected through the terminal residues by
`oz-(1—+6)
`linkages to form a linear polymer‘. Pullulanases or limit dextrinases
`hydrolyse the oz-(1-+6) linkages to give maltotriose as the end product. As a substrate.
`pullulan has the great advantage that it is not attacked by alpha— or beta—amylases,
`and only very slowly by cc-D—glucosidases. Unfortunately, however,
`the oligo-
`saccharides that are released by pullulanase action form good substrates for 01-13-
`glucosidases. ‘Thus, if the reaction is monitored by using a reductometric method and
`oz-D-glucosidases are present, which is usual, for example, in many plant extracts, an
`erroneous estimate of debranching activity will be obtained.
`Viscometry has been "used to monitor the degradation of polysaccharides,
`especially /5’-D-g1ucans7, and it gives a very sensitive assay for enzymes which hydrolyse
`internal linkages in an endo-fashion. Since bacterial pullulanase has been reported to
`have an endo—rnechanism for the degradation of pullulans, and hydrolysis of the
`maltotriose released or exo-attack by oz-D-glucosidases would have a negligible effect
`on viscosity, the use of Viscometry for the assay of a pullulanase~type, debranching
`enzyme has been investigated.
`
`EXPERIMENTAL
`
`Limit dextrinase was purified from germinated ba.rIey“'9.' The preparation was
`free of amylases or oc—D-glucosidases, and had a specific activity of 3 units/mg of
`protein. One unit releases one pmole of apparent maltotriose per min at 30°, from
`pul1ulan5.
`»
`'
`Amyloglucosidase (8, gift from Dr. I. D. Fleming of Glaxo Research Ltd.) was
`
`‘Studies on debranching enzymes: ‘Part II. For Part I, see ref‘. 1.
`
`TATE & LYLE AMERICAS LLC
`
`EXHIBIT 1017
`
`

`
`208
`
`_
`
`g
`
`-
`
`L
`
`‘
`
`'
`
`_
`
`”
`
`'—
`
`'
`
`i
`
`UNOTE
`
`apurified preparation from Aspergillus niger, and had a specific activity of 20 inter-
`national units per mg.
`i
`Pullulan was a laboratory sample and its preparation had been described
`previously”. Assays of reducing power were performed by a modified Nelson-
`Somogyi procedure‘ K
`'
`'
`-
`,
`.
`Viscometry was carried out at 37°, and solutions, pipettes, and viscometer tubes
`were pre—warmed to this temperature. The reaction was started by mixing 1 ml of
`enzyme solution with 1.5 ml of 1% pullul-an in lO0mM sodium acetate buffer (pH 5.3).
`A portion (2 ml) of the mixture was rapidly pipetted into an 0stwald‘No.
`1 B.S.
`viscometer tube, and the flow time recorded at intervals of 2 min during the incubation.
`These conditions gave an initial flow-time of 50-60 sec.
`
`RESULTS
`
`Pullulan can beregarded as a homopolymer of repeating maltotriose units, so
`that the treatment of Bryce and Greenwood” can be used. They showed that for
`random degradation of a homopolyrner at a constant rate,. and assuming that the
`specific’ viscosity at any particular degree of degradation is proportional to the
`number-average degree of polymerisation, the reciprocal of specific viscosity should
`rise at a constant rate. When the viscometric assay was performed using several
`different concentrations of barley limit-dextrinase, this relationship was found to
`hold for each concentration tested (Fig. 1). For convenience, zero time was arbitrarily
`taken as the time when the first measurement of fiowtime was started.
`
`15
`
` .0.8
`
`0.4
`
`"D.
`L3
`5<1
`
`Do
`
`4
`
`a .
`Tlme(mln)
`
`12
`
`r
`
`16‘
`
`. Fig. 1. The,increase_'in reciprocal specific viscosity as a function of time at several difierent concen-
`trations of enzyme. For conditions, see Experimental section. Key V, E], Q, I, and V represent
`digests containing,.re_spectively, 1.62, 3.25, 6.5, 13.0,'a.nd 19.0 x 1O"3-units ofbarley limit-dextrinase.
`
`

`
`NOTE
`
`.
`
`- 209
`
`The rate of increase of reciprocal specific viscosity is a useful _measure of enzyme
`activity, since when the slopes of the lines in Fig.
`1 were plotted against enzyme
`concentration, a straight line was obtained (Fig. 2).
`’
`
`10
`
`
`
`5
`
`10
`Enzyme (units x1O‘3)
`
`15
`
`20
`
`O)
`
`toA
`
`
`
`a‘f(ln1;;,)(mln)“x102
`
`O
`
`Fig. 2. The rate of increase of reciprocal specific viscosity as a function of enzyme concentration.
`Data calculated from Fig. 1.
`
`The linear rise in reciprocal specific viscosity obtained is consistent‘ with a
`random, internal cleavage of pullulan by barley limit—dextrinase. Below 21 specific
`viscosity of ~0.5, the relationship became non-linear (results not reported). This could
`be due to a breakdown in the viscosity—average d.p. relationship at low d.p., or to a
`change in action pattern of the enzyme.
`
`TABLE I
`
`EFFECIS or AMYLOGLUCOSIDASE ON Ti-re REDUCIOMETRIC AND VISCOMETRIC Assxws or
`LIMIT D1‘-IX‘IRINASE°
`
`Enzyme
`
`Reductamerric
`assay
`(45600)
`
`Viscametric
`assay
`(A ram.‘ per min)
`
`0.0061
`0.171
`Limit dextrinase
`-
`0.01 1
`Amyloglucosidase
`
`
`0.398 »Limit dextrinase+amylog1ucosidase 0.0064
`
`
`
`“Digests contained 2.5 x 10”?’ units of barley limit-dextrinase or 5 X 10'“ units ofamyloglucosidase,
`or both. Incubations for the reductometric assay contained euzyme(s), and 5 mg of puilulan in 1 II_1l
`of 20mM citrate bufi‘er (pH 5.3); 100111 samples were assayed for reducing power. Results are
`expressed as the increase in extinction at 600 nm after incubation for 4 h at 30°. The viscometric
`assay is as described in the text.
`0
`'
`
`

`
`210
`
`.
`
`-
`
`~
`
`NOTE
`
`The method is also insensitive to the presence of cz-D-glucosidases.(Table I).
`A
`When a mixture of limit dextrinase and amyloglucosidase was assayed by the reducto~
`metric method, the value obtained was much more than the sum of the values using
`the two enzymes separately, indicating hydrolysis, by the arnyloglucosidase, of the
`products released by limit dextrinase. In contrast, the presence of amyloglucosidase
`had an insignificant efl‘ect on the viscornetric assay of pullulan. it may also be seen
`from Table I that viscometry is by far the more sensitive method. The increase in
`reducing power was measured over a period of 4 h, whereas a significant decrease in
`viscosity was obtained in only 2 min.
`In the absence of automated equipment, this method is more laborious than the
`conventional reductometric technique. It also has the disadvantage that absolute units
`cannot be obtained unless a purified debranching enzyme is available for calibration
`‘purposes. In view of its high specificity and sensitivity, however, it is to be re-
`commended for the assay of crude plant~extracts where the presence of other carbo-
`hydrases, particularly oz—D-glucosidases, is suspected.
`
`ACKNOWLEDGMENT
`
`We are indebted to the Eda, Lady Jardine Charitable Trust for the award of a
`research Studentship (to D.G.H.).
`
`REFERENCES
`
`1 D. I. MANNEns AND D. Ystmwuass, J. Inst. Brewing, 79 (1973) 377.
`2 E. Y. C. LEE AND W. J. VVHELAN, in P. BDYER (Ed.), The Enzymes, Vol. 5, Academic Press, London
`and New York, 3rd edition, 1972, p. 192.
`G. S. DRUMMOND, E. E. SMITH, AND W. J. WHELAN, FEBS Letters, 9 (1970) 136.
`G. DUNN, D. G. HARDIE, AND D. J. MANNERS, Bioclzem. 1., 133 (1970) 413.
`H. BENDER AND K. WALLENFELS, Methods Enzymol., 8 (1966) 555.
`B. J. CATLEY AND W. J. WHELAN, Arch. Biachem. BiopIxy.s'., 143 (1971) 138.
`D. I. MANNERS AND G. WILSON, Biachem. J., 135 (I973) 11.
`G. S. Dxrm£MoND, E. E. Smrm, AND W. J. WHELAN, FEBS Letters, 5 (1969) 85.
`D. G. HARDIE AND D. J. MANNERS, unpublished work.
`D. J. MANNERS AND D. YELLOWLEES, Staerke, 23 (1971) 228.
`J. F. Ronvr AND W. J. WnELAN, in J. A. RADLEY (Ed.), Starch and its Derivatives, Chapman and
`Hall, London, 4th edition, 1968, p. 432.
`12 W. A. J. BRYCE AND C. T. GREENWOOD, J. Po'Iym. Sci., 25 (1957) 480.
`
`t-a-4"‘O‘DO0*~lO\Ut-K89.)