(1974)
`Elsevier Scientilic Publishing Company, Amsterdam - Printed in Belgium
`207-210 0
`
`Note
`
`A viscometric
`
`assay
`
`for pullulanase-type,
`
`debranching
`
`enzymes*
`
`DAVID G. HARDIE
`
`DAWD J. MANNERS
`
`Herid-
`University,
`of
`(Received February 2lst, 1974; accepted for publication, March Ist, 1974)
`
`Britain)
`
`the a-(1+6)-
`Bacterial pullulanases (E.C. 3.2.1.41) specifically hydrolyse
`D-glucosidic bonds which form the inter-chain linkages in certain a-(1+4)-linked
`D-glucose polymers such as amylopectin’. The limit dextrinases, which serve a similar
`function in higher plants, are now known to be qualitatively similar in specificity to
`these bacterial enzymes
`Both groups of enzymes may be routinely assayed by
`following the release of reducing groups from the fungal polysaccharide pulMan5,
`which consists of maltotriose.
`connected through the terminal residues by
`a-(1+6)
`linkages to form a linear polymer6. Pullulanases or limit dextrinases
`hydrolyse the a-(1 46) 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 a-x@ucosidases. Unfortunately, however, the oligo-
`(r-D-
`glucosidases. Thus, if the reaction is monitored by using a reductometric method and
`or-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 P-D-glucans’, 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-mechanism for the degradation of pullulan’, and hydrolysis of the
`maltotriose released or exo-attack by a-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.
`
`Limit dextrinase was purified from germinated barley4sg. The preparation was
`free of amylases or a-D-glucosidases, and had a specsc activity of 3 units/mg of
`protein. One unit releases one pmole of apparent maltotriose per min at 3,0’, from
`pIllMa&
`Amyloglucosiclase (a gift from Dr. I. D. Fleming of Glaxo Research Ltd.) was
`
`*Studies on debranching enzymes: Part II. For Part I, see reT. 1.
`
`Carbohy&ate Research, 3 6
`AND
`Department
`Brewing and Biological Sciences,
`Watt
`Edinburgh (Great
`3s4
`.
`units
`saccharides that are released by pullulanase action form good substrates for
`

`

`NOTE
`
`and had a specific activity of 20 inter-
`
`a.puriIied preparation from
`national units per mg.
`Pullulan was a laboratory sample and its preparation had been described
`previouslylo. Assays of reducing power were performed by a modified Nelson-
`Somogyi procedure 1 !
`V&come&y 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% pullulan in 1OOmM sodium acetate buffer (pH 5.3)
`A portion (2 ml) of the mixture was rapidly pipetted into an Ostwald .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.
`
`Pullulan can be regarded 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 homopolymer at a constant rate, and assuming that the
`specifid 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 tist measurement of flow .time was started.
`
`Fig. 1. The~incr&ase~‘in reciprocal spbcific viscosity as a &~~ction of time. at s&~al dike&& concen-
`;kl, and V r.epresent
`trations of enzyme. For &nditions, see Experimental section. Key V, 17, e,
`digests~containing, respectively, 1.62, 3.25, 6.5, 13.0;and 19.0 x .10e3 units of.barley limit-dextrinake.
`
`268
`Aspergillus niger,
`_
`_
`

`

`NOTE
`
`-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).
`
`5
`
`Enzyme
`
`10
`(units x 10-9
`
`15
`
`I
`20
`
`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 a 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
`
`EFFECTS OF AMnOGLUCOSIDASE
`
`ON THE REDUCTOMETRIC
`
`Ati
`
`VISCOhlETRl
`
`C ASSAYS OF
`
`LIMIT DEXTRINASP
`
`Enzyme
`
`Limit dextrinase
`Amyloglucosidase
`Limit dextrinase + amyloglucosidase
`
`Reducrometric
`assay
`w%oo)
`
`0.171
`0.011
`
`Viscometric
`UssUy
`Cd IVlllp’ per min)
`
`0.0061
`-
`
`0.398 O.OO64
`
`ODigests contained 2.5 X 10m3 units of barley knit-dextrinase or 5 x LOS3 units of amyloglucosidase,
`or both. Incubations for the reductometric assay contained enzyme(s), and 5 mg of pullulan in 1 ml
`of 20mM citrate buffer (pH 5.3);
`IOO-~1 samples were assayed for reducing power. Results are
`expressed as the increase in extinction at 600 run after incubation for. 4 h at 30”. The viscometric
`assay is as described in the text.
`
`209
`

`

`210
`
`NOTE
`
`The method is also insensitive to the presence of a-D-glucosidases (Table I).
`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 amyloglucosidase, of the
`products released by limit dextrinase. In contrast, the presence of amyloglucosidase
`had an insignificant effect on the viscometric assay of pullulan. It may also be seen
`from Table I that viscometry
`is by far the more sensitive nielhod. The increase in
`reducing power was measured over a period of 4 h, whereas a s&Scant
`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 a-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
`
`D.
`
`D. J. MA?.&IS
`2 E. Y. C.
`in P. BOYER (Ed.),
`and New York, 3rd edition, 1972, p. 192.
`DRUSSMOND, E. E. SMITH, AND W. J. WHELAN,
`DUNN, D. G. H~RDIE, AND D. J. MANNERS,
`FEBSLefters, 9 (1970) 136. 4 G.
`
`YELL0 WLEES, J. Inst. Brewing, 79 (1973) 377.
`
`Vol. 5, Academic Press, London
`
`Biochem. J., 133 (1970) 413. 5 H. BENDERAND K.WALLENFELS,M~~~O~S ErrzymoZ.,8(1966)55.5. 6 f3.
`
`(1971) 138.
`
`J. CATLEY AND W. J.
`(1973) 11.
`WILSON,
`8 G. S. DRIJMMOND, E. E. SNIH, AND W. J. WHELAN,
`D. G.
`D. J. MANNERS, unpublished work.
`FEBS Letters, 5 (1969) 85. 9
`10 D. J. MANNERS AND D. YELLOWLEES,
`(1971) 228.
`11 J. F. ROSY-~ AND W. J. WHELAN, in J. A. RADLEY (Ed.),
`Hall, London, 4t!1 edition, 1968, p. 432.
`12 W. A. J.
`
`(1957)
`
`Chapman and
`
`1
`AND
`LEE AND W. J. WHELAN,
`The Enzymes,
`3 G. S.
`WHELAN, Arch. Biochem. Biophys, 143
`7 D. J. MANNERS AND G.
`Biochem. J., 135
`HARDIE AND
`Staerke, 23
`Starch and its Derivatioes,
`BRY~ AND C. T. GREENWOOD, J. P&m. Sci., 25
`480.
`