A Novel Bacillus Pullulanase-Its Properties and ...

200

(J.Jpn.Soc.Starch Sci., Vol.30, No.2, p.200•`211 (1983)]

A Novel Bacillus Pullulanase-Its Properties and

Application in the Glucose Syrups Industry

B.E. NORMAN

NOVO INDUSTRI A/S

(Novo Alle, DK-2880 Bagsvaerd, Denmark)

Introduction

The majority of starches used in the manu-

facture of glucose syrups contain 75-85% am

ylopectin.1) Amylopectin is a highly branched

polysaccharide consisting of linear chains of

1, 4-ƒ¿-linked D-glucose residues, joined together

by 1, 6-ƒ¿-glucosidic linkages. The branch points

occur on average every 20-25 D-glucose units,

so that amylopectin contains 4-5% of 1, 6-ƒ¿-

glucosidic linkages.2-4)

The 1, 6-ƒ¿-glucosidic linkages act as a kind of

barrier to the action of exo-acting, saccharif ying

amylases such as glucoamylases or maltogenic

/3-amylases. Endo-acting a-amylases are able to

by-pass the branch points,5' but in general are

not capable cf hydrolyzing the 1, 6-a-glucosidic

linkage. Recent work by Kobayashi and co-

workers has shown that at least one a-amylase

(from Thermoactinomyces vulgaris) can hydro-

lyze 1, 6-ƒ¿-glucosidic linkages, in addition to

1, 4-a-glucosidic linkages.6)

Glucoamylases can slowly hydrolyze 1, 6-ƒ¿-

glucosidic linkages in amylopectin and partially

hydrolyzed amylopectin,7) but the action of mal

togenic exo-amylases ceases as a branch point

is approached.8) It is therefore obvious that the

efficiency of the saccharification reaction could

be improved by incorporating a specific amylo

pectin debranchina enzyme in the system. Debranching enzyme such as isoamylase [EC

3.2.1.68, glycogen 6-glucanohydrolase] and

pullulanase [EC 3.2.1.41, pullulan 6-glucanohy-

drolase] have been known for many years,9)

but their use in the glucose syrups industry is

far from widespread. Pullulanases from Kleb

siella pneumoniae,10,11) Streptomyces sp.,12) and

Bacillus cereus var, mycoides13) and isoamylases

from Pseudomonas amyloderamosa14,15' and Cy-

tophaga sp.16' are not sufficiently thermostable

to be used at 60•Ž. Moreover the Pseudomonas

isoamylase was the only debranching enzyme

sufficiently acidophilic to be used at a pH of

around 4. 5.15,17,18)

After an extensive screening programme, our

research laboratories succeeded in isolating a

species of Bacillus which produced a thermo

stable, acidophilic pullulanase which was free

from glucoamylase, ƒÀ-amylase and a-amylase

side activities.

Some of the properties of this enzyme will

now be described.

Properties of Bacillus sp, pullulanase Determination of activity. Pullulanase activity

is determined by incubating a reaction mixture

consisting of 1 ml 4% w/v pullulan (Sigma) in

0.1 M acetate buffer, pH 5.0, with 1 ml of a

suitably diluted enzyme solution for 30 min at

60•Ž. The reaction is stopped by the addition

of 3 ml 0.5 M bicarbonate/carbonate buffer (pH

10.0) . The reducing sugars formed are mea

sured according to the Somogyi-Nelson method

using D-glucose as the standard.19,20)

One pullulanase unit (PNU) is defined as the

Footnotes: Amyloglucosidase activity, 1 AG unit is

defined as the amount of enzyme which hydrolyzes

1 timol of maltose per minute at 25•Ž, pH 4.3

(NOVO Analytical Method AF 22); ƒÀ-amylase

activity, 1 ƒÀ-amylase unit is defined as the amount

of enzyme which under standard conditions produces

1 pmol of reducing sugar per minute at 60•Ž, pH

5.0(1% soluble starch, 30 min reaction time).

201A Novel Bacillus Pullulanase

amount of enzyme required to produce 1 pmol

of reducing sugar per minute under the above

standard conditions.

The effect of temperature on activity and

stability. The effect of temperature on pullula-

nase activity at pH 5.0 (for Bacillus sp. pullu-

lanase) and pH 6.0 (for K. pneumoniae pullula-

nase) is illustrated in Fig.1. The optimum

temperature under the conditions of the analysis

for Bacillus sp. pullulanase is 60•Ž and for K.

pneumoniae pullulanase, 55•Ž.

Figure 2 illustrates the effect cf temperature

on enzyme stability. The data were obtained

by incubating the enzyme in a 29% w/w D-

glucose solution for 3 days and measuring the

residual debranching activity using an amylopec-

tin substrate. Because the pullulanase is heat

stable, it can be safely used at 60•Ž during

saccharification with either Aspergillus niger

glucoamylase or soybean ƒÀ-amylase.

The effect of pH on enzyme activity. Figure

3 illustrates the effect of pH on pullulanase

activity at 60•Ž (Bacillus sp. pullulanase) and

55•Ž (K. pneumoniae pullulanase) in the presence

of acetate buffer at different pH values. Under

the conditions of the analysis the optimum pH's

for pullulanase activity are 5.0 (Bacillus sp.)

and 6.5 (K. pneumoniae).

The optima temperature and pH of the pullu-

lanase (60•Ž and pH 5.0) are close to both

those of A. niger glucoamylase (60•Ž and pH

4.5) and soybean ƒÀ-amylase (60°C and pH 5.5).

This means that it can be used in combination

with either of these saccharif ying enzymes under

near optimum conditions. While K. pneumoniae

pullulanase is fully compatible with soybean ƒÀ-

amylase, it is most unsuitable for use with

A. niger glucoamylase because this pullulanase

is rapidly inactivated at pH's below 5.0.

Action on pullulan. Pullulan is a linear D-

glucose polymer produced by the fungus Aure-

obasidium pullulans.21) It consists essentially of

maltotriosyl units joined by 1, 6-a-linkages.

Several workers have reported the presence of

maltotetraose groups in pullulan,22-24) and we

have observed in our laboratories that prolonged

hydrolysis of "Sigma" pullulan results in the

formation of 2-3% DP4.

The viscosity of a pullulan solution is rapidly

reduced by the action of Bacillus sp. pullula

nase. The initial products of hydrolysis are

maltotriose and a whole range of 1, 6-ƒ¿-linked

Fig.1. The effect of temperature on enzyme activity.

Substrate: 2% pullulan(Sigma), 0.05 M acetate buffer, 30 min reaction.

Fig.2. The effect of pH and temperature on

pullulanase stability.

Fig. 3. The effect of pH on enzyme activity.

Substrate: 2% pullulan (Sigma), 0.05 M acetate buffer, 30 min reaction.

202 J Jpn. Soc.. Starch Sci., Vol. 30, No. 2 (1983)

(a)

(b)

Fig.4. Gel-chromatograms showing the action of

crude Bacillus sp. (a) and K. pneumoniae

(b) pullulanase on pullulan (Sigma), pH

4.5 (a) and pH 5.5 (b).

0.05 M acetate buffer, 50•Ž, 10 PNU/g DS (or

equivalent). Bio-Gel P 2 column, 100•~1.5 cm,

65•Ž, 18 ml/hr flow (H2O), RI detector.

Table 1. Amino acid composition of Bacillus sp.

pullulanase.

oligomers of maltotriose (63-ƒ¿-maltotriosyl-malto-

triose, 63-ƒ¿- (63-ƒ¿-maltotriosyl-maltotriosyl) -malto-

triose, etc.). Prolonged hydrolysis results in

more than 90% conversion to maltotriose (Fig.

4a). The action pattern of this enzyme on

pullulan is similar to that observed for K.

pneumoniae pullulanase (Fig. 4 b) and resembles

random endo-attack.10)

The action of Bacillus sp. pullulanase on low

molecular weight branched oligosaccharides has

not yet been investigated.

Activators and inhibitors. Heavy metal ions

such as Cot, Nit, Fe3+, Mn2+, Zn2t, Age,

Hg2+, and Cu2+ at concentrations greater than

1 mM inhibit pullulanase activity.

Cat, which reported by Ohba and Ueda10)

to enhance K. pneurnoniae pullulanase activity,

has no activating effect on Bacillus sp, pullu

lanase.

p-Chloromercuribenzoate (p-CMB) inhibits

Bacillus sp. pullulanase indicating that a sulf

hydryl group is involved in the configuration of

the active enzyme. Activity can be restored by

the addition of cysteine or ƒÀ-mercapto-ethanol.

In this respect the enzyme resembles the B.

cerceus var. mycoides pullulanase which is also

reported by Takasaki13) to be inactivated by

p-chloromercuribenzoate and reactivated by cys

teine.

ƒ¿-Cyclodextrin has been shown to inhibit

Bacillus sp. pullulanase.

General. The molecular weight of Bacillus

sp. pullulanase as determined by SDS-polyacryl-

amide gel electrophoresis is about 100,000

Daltons. The amino-acid spectrum of the

enzyme is given in Table 1. Calculation of the

molecular weight from these data gives a value

of 101,200. The specific activity is estimated to

be 300 PNU per mg protein.


The isoelectric point of the enzyme was

determined by isoelectric focusing using LKB

Ampholine-PAG plates, and found to be 5.0. Bacillus sp. pullulanase is immunologically

distinct from K. pneumoniae, B. megaterium

and B. cereus var. mycoides pullulanases. Using

serum obtained from rabbits immunized with

purified Bacillus sp. pullulanase, no cross reactivity with the other pullulanases was observed.

High dextrose syrups

High dextrose syrups are normally used for the production of crystalline dextrose or as the starting material for high fructose syrups (HFS) . For both applications the aim is to produce a syrup with the highest possible D-glucose level (normally 94-96%), economically. It is well known that increasing D-glucose levels can be obtained by decreasing the substrate concentration during saccharification,25) but this in turn will increase evaporation costs and require a larger saccharification tank volume as well as increasing the risk of microbial infection.

If we use a debranching enzyme, such as

pullulanase, and glucoamylase simultaneously

during saccharification, the glucoamylase re

quirement is reduced. In such a dual enzyme

saccharification system, the pullulanase spe

cifically hydrolyzes the branch points in the

amylopectin residues so that the glucoamylase

has only to hydrolyze the linear 1, 4-ƒ¿-glucosidic

linkages. Less glucoamylase activity is required,

therefore less enzyme catalyzed polymerization

of D-glucose to isomaltose takes place. This

results in increaed D-glucose levels.

In a normal dextrose syrup manufacturing

process an aqueous starch slurry is first liquefied

and partially hydrolyzed to a DE of about 5-15

by the action of thermostable bacterial ƒ¿-amylase.

The liquefied starch is then saccharified by the

action of a fungal glucoamylase. During sacchar

ification the partially hydrolyzed amylose and

amylopectin molecules are depolymerized by

the action of glucoamylase which removes D-

glucose units in a stepwise manner from the

non-reducing chain-ends. In addition to hydro-

lyzing the 1, 4-ƒ¿-glucosidic linkages in the linear

oligosaccharides, the glucoamylase is also able

to hydrolyze the 1, 6-ƒ¿-glucosidic linkages at

the branch points in the amylopectin residues,

although at a much slower rate, so that almost

complete conversion can be obtained.

The changes in the carbohydrate spectrum taking place during a normal saccharification

process are illustrated by the gelchromatograms shown in Fig.5. It can be seen that the high molecular weight fraction (DP) gradually disappears as the hydrolysis progresses; the panose which is initially formed is gradually hydrolyzed; the isomaltose level increases due to the D-glucose polymerizing action of the enzyme. There is also an accumulation of trace amounts of oligosaccharides (DP, and DPs) which dis-appear on prolonged hydrolysis.

The next series of gel-chromatograms (Fig.6) illustrate the change in carbohydrate spectra in a dual enzyme saccharification process using

pullulanase and only half the amount of glucoamylase. The high molecular weight fraction has been almost completely depolymerized after 24 hr. After 72 hr it can be seen that there

Fig.5. Gel-chromatograms showing the carbohydrate spectra of enzyme liquefied corn starch (DE 6) saccharified with A. niger

glucoamylase alone.

31% DS, initial pH 4. 5, 60•Ž, 0.225 AG/g DS.

204 J. Jpn. Soc. Starch Sc., Vol. 30, No. 2 (1983)

Fig.6. Gel-chromatograms showing the carbohydrate spectra of enzyme liquefied corn starch (DE 6) saccharified with A. niger

glucoamylase and Bacillus sp. pullulanase.

31% DS, initial pH 4.9, 60t, 0.113 AG/g DS

(glucoamylase), 2.4 PNU/g DS (pullulanase).

Table 2. Glucoamylase/pullulanase dosage required to give 96% D-glucose in 96 hr at 30% DS.

Substrate: DE 7 enzyme liquefied corn starch, 60•Ž,

pH 4.8-4.2.

Table 3. Glucoamylase/pullulanase dosage required to give 94.5% D-glucose in 72 hr at 38%

DS.

Substrate: DE 7 enzyme liquefied corn starch, 60•Ž,

pH 4.8-4.2.

has been no accumulation of DP7 and DP8, and also that the isomaltose level is lower than in

Fig. 5.

Higher D-glucose levels have been obtained because oligosaccharides above DP3 are almost

entirely absent, and less isomaltose has been formed because less glucoamylase has been

used.

The saving of glucoamylase and the possibil

ity of increasing the maximum D-glucose level are not the only advantages that can be obtain

ed. Saccharification may also be carried out

at a higher substrate concentration, and the reaction time may also be reduced significantly.

These points will now be considered in more

detail.

Reduction in glucoamylase. Low dosages of

pullulanase can be used to replace a significant

portion of the glucoamylase used for saccharification. Two examples of this are given in

Tables 2 and 3. Table 2 gives the enzyme dos-ages required to produce 96% D-glucose in 96 hr from a 30% DS, DE 7 enzyme liquefied corn starch substrate. Table 3 gives the enzyme dosages required to produce 94.5% D-glucose in 72 hr from the same substrate at 38% DS.

Decreasing the glucoamylase dosage reduces reversion or "back-polymerization," and therefore the risk of "overconversion" is minimized. Another important consideration is that although the D-glucose levels might be the same, the carbohydrate spectra will be different. As the

glucoamylase level is lowered, the isomaltose level will be lower and the DP3/DP4+ fraction will be higher, for a given D-glucose level.

The reduction in isomaltose may be of impor.

tance in the manufacture of crystalline dextrose or in a fructose enrichment process where the

raffinate stream is to be "re-saccharified." Increased D-glucose. Figure 7 illustrates the effect of pullulanase activity on the increase in maximum D-glucose for a DE 7 enzyme liquefied corn starch substrate at 30% DS. In the standard saccharification, without pullulanase, the maximum D-glucose that could be obtained under


Fig.7. The effect of pullulanase activity on

maximum D-glucose.

Fig.8. The effect of pullulanase dosage on

D-glucose formation after 48 and 72 hr

saccharification.

Fig.9. The effect of substrate concentration on

maximum D-glucose.

Reaction time: 60-100 hr.

Fig.10. The effect of pullulanase dosage on D-

glucose at high substrate concentrations.

the given conditions was 96.0%. Using half the

glucoamylase dosage and a pullulanase dosage corresponding to 0.25 PNU/g it was possible to increase the maximum D-glucose by 0.5%. At very high pullulanase dosages the maximum could be increased by 1.5%.

Figure 8 illustrates the effect of pullulanase dosage on % D-glucose after 48 and 72 hr sac-charification. In these experiments a DE 11 enzyme liquefied corn starch substrate at 38% DS was used. For a standard saccharification

process 94.1% and 94.5% D-glucose could be obtained after 48 and 72 hr, respectively. Using half the amount of glucoamylase and a pullulanase dosage of 1.2 PNU/g DS, 95% D-glucose could be obtained after 48 hr and 96% D-glucose after 72 hr. By using pullulanase and a substantially lower

glucoamylase dosage it is possible to significantly increase the D-glucose level. For dextrose

production this will mean an increase in the crystallization yield, for 42% fructose syrup

production there will be a potential saving in isomerization costs, and for 55% fructose syrups, obtained by fractionation and blending, the

polysaccharide content in the final product can be reduced.

Substrate concentration. Figure 9 illustrates the effect of substrate concentration on maximum D-glucose. When a debranching enzyme and

glucoamylase are used together, saccharification may be carried out at higher substrate concentrations than in the normal process. For example 96% D-glucose can be obtained with the given substrate at about 32% DS in the standard saccharification. With half the glucoamylase activity and a pullulanase dosage of 1 PNU/g DS, 96% D-glucose can be obtained at 37% DS.

206 J Jpn. Soc. Starch Sd., Vol. 30, No. 2 (1983)

Figure 10 illustrates the effect of different

pullulanase dosages on D-glucose when saccharif ying at high substrate concentrations. With a pullulanase dosage of 0.5 PNU/g DS it was

possible to obtain 95% D-glucose at 40% DS in 72 hr under the given conditions. By saecharif ying at a higher substrate con

centration, substantial savings can be made in evaporation costs.

Reaction time. Typical saccharification reaction times vary from 48-96 hr. The capacity of a starch syrups plant can be increased by reducing the saccharification time, but in the single enzyme saccharification process this can only be achieved by increasing the glucoamylase dosage. If the dosage is doubled, then the saccharification time can be reduced to about 30 hr. Un-fortunately, the reversion reaction (isomaltose formation from D-glucose) is very significant, and it is therefore difficult to stop the saccharification at, or close to, maximum D-glucose,26) especially when using the more thermostable

glucoamylases from A. tiger. By maintaining the normal glucoamylase dosages and using a debranching enzyme, the reaction time can be reduced without the problems of over-conversion. In Fig. 11 it can be seen that without the debranching enzyme, maximum D-glucose is obtained after about 80 hr. When the pullulanase is added at a level of 0.4 PNU/g DS, the same D-glucose level can be obtained after about 30 hr. Practical considerations. Addition of de-branching enzymes: As already mentioned,

glucoamylases act on amylopectin and partially

hydroyzed amylopectin by rapidly catalyzing

the stepwise removal of D-glucose units from

the non-reducing ends of the linear chains. If,

in our dual enzyme saccharification system, the

glucoamylase is in excess or is added prior to

the debranching enzyme, it is highly probable

that some of the side chains will contain single

D-glucose units27) (Fig.12). In this case, the

pullulanase is unable to hydrolyze the 1, 6-ƒ¿-

glucosidic linkage containing the single glucose

unit.28,

On the other hand, if the liquefied starch is first debranched with pullulanase, there is a

potential risk of retrogradation. (In the case of isoamylase, the risk is very real.) 27) However, experiments in our laboratories have indicated that the time of addition of the pullulanase is not absolutely critical, but for convenience, simultaneous addition of the two enzymes is recommended. Saccharifcation temperature: In order to

minimize the risk of microbial contamination

during the 48-96 hr reaction, saccharification is

normally conducted at 60•Ž. At temperatures

above 60•Ž glucoamylases from A. niger are

rapidly inactivated.

The curves shown in Fig.13 clearly demon

strate that Bacillus sp. pullulanase is sufficiently

heat stable to be used at 60•Ž, and in this

respect it is superior to other known debranching

enzymes.

Saccharifcation pH: From the pH/activity curve in Fig.3 it can be seen that the pullulan-


reaction time.

Fig.12. Susceptibility of various branched oligo

saccharides to K. pneumoniae pullulanase

attack.

After Kainuma et al.28)


Fig.13. The effect of temperature on saccharifi

cation.

Table 4. The effect of pH on saccharification.

Substrate: DE 7 enzyme liquefied corn starch, 30%

DS, 60•Ž. Enzyme dosage: 1 PNU/g DS(pullulanase),

0.113 AG/g DS (glucoamylase).

ase is less active at lower pH's. The optimum pH

for combined saccharification with glucoamylase has been found to be 4.5, and at pH's below 4.2 the debranching enzyme is deactivated. The

pH operating recommendations for the glucoamylase/pullulanase system are therefore 4.5-5.0. The data presented in Table 4 serve to illustrate this point. In cases where large pH drops are encountered during saccharification,

pH control may be necessary. Residual ƒ¿-amylase activity: Laboratory ex

periments have shown that the presence of ƒ¿-

amylase activity originating from the liquefaction

stage has a negative effect on maximum D-

glucose when pullulanase and glucoamylase are

used in combination. This yield loss can be

seen as an elevated PD3 (panose) level (Table

5).

The problem is greatest with the thermostable

ƒ¿-amylases from B . lichenif ormis which are still

active at pH 4.6 (60ƒ¿). In order to minimize

this effect it is therefore recommended that the

pH of the liquefied starch is adjusted to below

4.5 at 90-95•Ž before cooling down to 60•Ž

(saccharification temperature). This will effec-

tively inactivate residual a-amylase activity. If

necessary fine pH adjustment to 4.5 can be

made after the addition of the glucoamylase

and pullulanase.

Starting DE: The data presented here has

been obtained using liquefied starch substrates

Table 5. The effect of residual ƒ¿-amylase activity on carbohydrate composition .

Substrate: DE 7 enzyme liquefied (B.licheniformis) corn starch, 30% DS, 60t, pH

4.8-4.3, 0.113 AG/g DS (glucoamylase), 1 PNU/g DS (pullulanase). Initial ƒ¿-amylase

dosage: 1 kg Termamyl 60 L/ton DS (NOVO INDUSTRI A/S, Denmark).

208 J. Jpn. Soc. Starch Sc., Vol.30, No.2 (1983)

with DE's of 6, 7 and 11. No effect of start DE on

carbohydrate composition or maximum D-glucose

could be seen. With higher starting DE's the

risk of maltulose precursor formation increases.26)

There is also a risk that "ƒ¿-limit dextrins" with

single glucose residues in the side chain will

be formed, which would not be susceptible to

pullulanase attack. A starting DE within the

range 8-14 is therefore recommended.

High maltose syrups

High maltose syrups are characterized by their

mild sweetness, low viscosity in solution, low

hygroscopicity and good heat stability.29,30) They can replace conventional acid glucose syrups in

many formulations where acid glucose and su

crose are employed, the high maltose syrup

replacing both the acid glucose and a portion of the sucrose.

In recent years there has been an increasing interest for pure maltose in the pharmaceutical

industry. Maltose may be used instead of D-

glucose for intravenous feeding, the advantage being that it can be administered at higher

concentrations without elevating blood glucose levels.31,32) Pure maltose may also be used as a

starting material for the production of maltitol

and crystalline maltitol.

A number of methods have been described in the literature for obtaining pure maltose from

extra-high maltose syrups. These include chro

matographic separation, solvent precipitation,

membrane separation and crystallization.33) Conventional high maltose syrups. When

starch or liquefied starch is hydrolyzed with a

maltogenic exo-amylase such as a soybean ƒÀ-

amylase, the amylopectin fraction and the

amylopectin residues containing 1, 6-ƒ¿-glucosidic

branch points are only partially degraded. Malt

ose units are removed in a stepwise manner

from the outer chains until a branch point is

approached. Hydrolysis then ceases and the

residual material-the n-limit dextrins-is resist-

ant to further attack by the enzyme.1) The

maximum amount of maltose that can be ob-

tained under these conditions is about 60%.

Extra-highh maltose syrups. If a debranching

enzyme is used together with an ƒ¿-amylase free

j9-amylase, considerably higher maltose levels

can be obtained. The maximum amount of

maltose formed is limited by two factors. Firstly, D-glucose polymers with an uneven chain length (DP5, DP7, DP9, etc.) which are formed during liquefaction will give rise to maltose

plus maltotriose. The maltotriose level in a high maltose syrup is very dependent on the DE after starch liquefaction, therefore the starting material should have as low a DE as is

practically possible.30,34) Secondly, at high substrate concentrations and

high enzyme dosages, pullulanases will catalyze

the condensation of low molecular weight oli

gosaccharides such as maltose and maltotriose

to form branched tetra-, penta- and hexa

saccharides.35) The condensation of maltose to

DP4 (maltosyl-maltose) by Bacillus sp. pullu

lanase is illustrated in Fig.14. The DP4 com

ponent can be hydrolyzed by low levels of

glucoamylase to glucose and panose, suggesting

that it is 62-ƒ¿-maltosyl maltose.

Fig.14. The 'condensation' of maltose by Bacillus

sp. pullulanase.


maltose formation.


Table 6. The effect of pullulanase dosage on carbohydrate composition.

For conditions: see Fig.15.

Fig.16. The effect of substrate concentration and

ƒÀ-amylase dosage on maltose formation.

However, using a combination of Bacillus sp.

pullulanase and soybean ƒÀ-amylase, it is possible

to obtain about 80% maltose using a 30% DS,

DE 5 enzyme liquefied corn starch substrate,

at 60•Ž (Fig.15).

From the carbohydrate spectrum given in

Table 6 it can be seen that the DP4 fraction increases with increasing pullulanase dosage.

Higher maltose levels can be obtained by reducing the substrate concentration during saccharification. This is illustrated in Fig. 16. The other approach that has already been mentioned is to use a lower DE (0-3) substrate, but this increases the risk of yield losses due to retro

gradation. In the field of high maltose syrups there is

considerable room for process development. The availability of a new pullulanase, which

is more thermostable than previously described debranching enzyme, should provide some of

the incentive that is necessary.

Conclusion The discovery of a novel Bacillus pullulanase

which is both thermostable and acidophilic and

therefore fully compatible with A. niger gluco

amylase and soybean ƒÀ-amylase is an important

step forward. The saccharification process is

more efficient resulting in higher D-glucose of

maltose yields. Moreover, saccharification can

be carried out at 60°C so that the risk of

microbial contamination is minimized.

Acknowledgement. Figures and tables are used with the kind permission of NOVO INDUSTRI A/S, Copenhagen.

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　グルコース単位あるいはマルトース単位で水解する糖

化酵素と,アミロペクチン枝切り酵素,たとえばプルラ

ナーゼを使って,澱粉からグルコースあるいはマルトー

スを製造する工程について述べる.本方法は,澱粉をよ

り効率的に分解するものであるが,枝切り酵素の使用に

制限があるため,広く活用されていない.

　新プルラナーゼ[EC3.2.1.41]は最近デンマークの

ノボ社がBacillusの一菌株より分離したもので,熱安

定性が高く,好酸性で,至適温度60℃,至適pHは4.5～

5.0である.したがって,Aspergillus nigerグルコ

アミラーゼとともに使用すれば,澱粉のグルコースへの

分解効率を高め,あるいは,たとえば大豆から得た β-ア

ミラーゼとともに使用すれば,ハイマルトースシラップ

を製造できる.ここではBacillussp,プルラナーゼの

性状をいくつか述べ,グルコースあるいはマルトース製

造への応用例を挙げる.

【質問】神戸女子大原田

1. Dose your organism produce extracellulary

pullulanase?

2. If it is possible, could you let me know

the yield of pullulanase in the culture medium?•y“š•z

1. The pullulanase is recovered from the

fermentation medium without extraction. We

have not established whether the enzyme is

produced extracellularly of whether it is pro

duced intracellularly and simply leaks out of

the cells into the fermentation medium.

2. I am sorry, but we are not willing to

divulge the yield of pullulanase in our culture

medium.

【質問】食品総研貝沼 I would like to give a comment on the des

ignation of oligosaccharide. You mentioned the

formation of "G4" from maltose by the pullu-

lanase action on G2. I prefer to use "B4" instead of G4, as this is a branched tetrasaccharide.

【答】

I agree. The tetrasaccharide formed by the

catalytic action of pullulanase on maltose is a

branched tetrasaccharide and should, therefore,

be referred to as "B4" and not "G4." Another

alternative would be to use the designation "DP 4 ."

【質問】大阪市大理山本 1. I would like to hear about the effect of

your pullulanase on starch previously dextrinized in regard to the increase of reducing sugars or

the structure of branched dextrins.

2. Is there any difference in the effect of

pullulanase depending on the time of addition


of the enzyme to the reaction mixture with

glucoamylse ?

【答】

1. When a maltodextrin (with a DE of about 20) which gives a yellow colour reaction with iodine, is treated with our pullulanase an increase in reducing sugars is observed. By

gel-chromatography it can be seen that there is an increase in the level of low molecular weight oligosaccharides (DP 2-9). After pullulanase treatment the dextrinized starch gives a red/violet iodine reaction. 2. Experiments in our labs have shown that for dextrose syrup production the pullulanase can be added just before, together with, or just after the glucoamylase. However, if addition

of pullulanase is delayed, there is a risk that

the glucoamylase will degrade the branched

oligosaccharides to such an extent that they do not meet the minimum substrate requirements

for pullulanase.

With isoamylase the point of addition is more

critical. If added before the glucoamylase there

is a chance of retrogradation, and if added later, the problem of minimum substrate requirement

is even greater.

【質問】東北大農松田

　このプルラナーゼはグリコーゲンに作用しますか?

【答】

Our pullulanase has very little action on

glycogen and in that respect is similar to the pullulanase from K. pneumoniae.

A Novel Bacillus Pullulanase-Its Properties and ...

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