38

J. Jpn. Soc. Starch Sci., Vol. 34, No. 1, p. 38•`44 (1987)•l

Production of a Thermostable Pullulanase by a Thermus sp.

Nobuyuki NAKAMURA,* Nobuhiro SASHIHARA,** Hiromi NAGAYAMA and Koki HORIKOSHI***

Laboratory of Bacterial Metabolism, The Superbugs Project, Research Development Corporation of Japan (2-28-8, Honkomagome, Bunkyo-ku, Tokyo 113, Japan)

(Received November 29, 1986)

A moderate thermophile that produces a large amount of extracellular pullulanase was

isolated from soil. The isolate (AMD-33), that grew at 37 to 74•Ž with an optimum at 65•Ž, was identified as a Thermus sp. strain. Maximal enzyme production was attained after 3 days

shaking cultivation at 60•Ž on a medium composed of 1% pullulan, 2% gelatin, 0.1% K2HPO4, 0.03% MgSO4•E7H2O and 0.25% CaCO3. Pullulanase synthesis was enhanced by pullulan, soluble starch and dextrin as well as

maltose but not at all by glucose. The enzyme, which was most active at pH 5.5-5.7 and 70

, was stabilized by Ca2+, and the optimum temperature for activity shifted to 75•Ž in the

presence of 3mM CaCl2.

Pullulanase (pullulan 6-glucanohydrolase, EC grow during the processes and interfere with 3. 2. 1. 41) is a debranching enzyme which the saccharification. Accordingly, active and specifically cleaves the ƒ¿-1, 6-glucosidic linkages thermostable amylolytic enzymes will be very in pullulan and amylopectin. This enzyme is important for the starch processing industry. commercially produced using bacteria1•`5) and Although some mesophilic and thermophilic is generally used in combination with several bacteria which produce significant amounts of

amylases such as glucoamylases, ƒ¿-amylases, pullulanases have been reported, 8,11•`18) little is ƒÀ-amylases and maltooligosaccharides-forming known about the formation and biochemical enzymes for the production of glucose, maltose characteristics of pullulanases from Gram and some related starch conversion syrups, negative thermophiles except for the cell because it improves the saccharification rates associated enzyme from T. aquaticus.19) and yields.6•`9) Recently, branched cyclodextrins A pullulan-hydrolyzing and starch-liquefying

(CDs) have also been produced industrially, thermophile showing high extracellular pullu utilizing the condensation action of pullulanase lanase productivity was isolated from soil at a with CDs and maltooligosaccharides.10,11) hot spring and identified as Thermus sp. Starch saccharification processes are usually strain. The purpose of this paper is to report carried out at temperatures below 60•Ž because some taxonomical characteristics of the isolated most amylolytic enzymes are unstable above strain and the optimization of pullulanase 60•Ž. Below 60•Ž, various microorganisms synthesis by this strain (AMD-33). A few biochemical properties of the partially purified Present addresses: * Research Lab. of Nihon Sho enzyme are also reported. kuhin Kako Co., 30, Tajima, Fuji, Shizuoka 417, Japan; ** Technical Lab. of Q. P. Corp., 2-5, Senkawa, Chofu, Tokyo 182, Japan; *** Lab. of MATERIALS AND METHODS Applied Bacteriology, The Institute of Physical and Chemical Research, 2-1, Hirosawa, Wako, Materials. Pullulans for industrial and Saitama 351, Japan. analytical use were obtained from Hayashibara Production of Pullulanase by a Thermus sp. 39

Biochemical Lab. and Nakarai Chemical Co., acid.24) Blanks lacking the enzyme or substrate respectively. Mikacion brilliant red 5BS was a were run with each batch of assays. As a gift from Mitsubishi Kasei Kogyo Co. Mikacion standard, the pullulan and enzyme were re brilliant red-pullulan was prepared by the placed by 0.15mg of glucose and 0.05ml of method of Rinderknecht et al.20) with pullulan water, respectively. One unit of enzyme for industrial use. Kanamycin sulfate and activity was defined as the amount of enzyme tunicamycin were purchased from Wako Pure that produced 1ƒÊmol of reducing sugar equiv Chemicals Co. Penicillin G and streptomycin alent to maltotriose per min under the condi sulfate were from Meiji Seika Co. Actinomycin tions described above. Alpha-amylase and

D, ristocetin sulfate and cycloserine were ob glucosidase activities were determined by the tained from Makor Co., Nakarai Chemical Co. methods of Fuwa25) and Tabata et al.26) at 60 and Aldrich Chemicals Co., respectively. and pH 6.0 with amylose and p-nitropheny Other antibiotics were from Sigma Chemical ƒ¿-D-glucoside as substrates, respectively. Co. Epoxy-activated Sepharose 6B and DEAE Determination of bacterial growth. After Toyopearl 650M were from Pharmacia Fine mixing vigorously 2ml each of the culture Chemicals AB and Toyo Soda Co., respec broth and 0.1N HCl solution, the turbidity tively. was measured at 690nm with a Klett Nuclease P1 and standard deoxyribonucleic Summerson photoelectric colorimeter fitted with acids were purchased from Seikagaku Kogyo a No. 66 filter. Co. Other chemicals were of reagent grade Determination of the DNA base composi and available commercially. tion. Chromosomal DNA of the isolate was Isolation and identification of the microor prepared according to the method of Saito ganism. A small amount of soil (approximately and Miura.27) 0.1g), which was obtained from Arima hot The DNA composition (G+C content) was spring (Hyogo Prefecture), was suspended in determined by the HPLC method of Tamaoka water (1ml), and then the suspension (0.1ml) and Komagata28) with a pre-packed column of was spread on agar plates (15 ml medium/ HibarLichrosorb RP-18 (5ƒÊm, 4.0mmID•~250 plate, 9cm in diameter) containing 2w/v% mmL, E. Merck Darmstadt) under the follow agar, 1% pullulan for industrial use, 0.2% ing conditions: temperature, 30•Ž; mobile Mikacion brilliant red-pullulan, 0.5% poly phase, 10mM phosphate buffer (pH 7); flow pepton, 0.5% yeast extract, 0.1% K2HPO4 and rate, 1.0ml/min; and detector, UV (260 0.02% MgSO4•E7H2O in water. The pH of the nm). medium was adjusted to 7 with 1N NaOH Preparation of the pullulanase. The isolate solution before sterilization at 125•Ž for 20 was cultured in a 2l conical flask containing min. The plate cultures were incubated at 70 400ml of the enzyme production medium (1%

for 18hr. Strain No. AMD-33, with a large pullulan, 2% gelatin, 0.1% K2HPO4, 0.03% clear zone around the colony, was selected as MgSO4•E7H2O and 0.25% CaCO3) on a rotary a pullulanase producer. Microbiological proper shaker (200rpm) at 60•Ž. After 4 days ties of the isolated strain were investigated cultivation, the extracellular pullulanase was according to the methods described in "Bergey's purified partially by ammonium sulfate preci Manual of Systematic Bacteriology"21) and pitation and then DEAE-Toyopearl 650M ion "Laboratory Methods in Microbiology ."22) exchange chromatography followed by ƒÀCD Enzyme assays. Pullulanase activity was Sepharose 6B affinity chromatography to determined by the method of Wallenfels et remove a trace amount of ƒ¿-glucosidase activity al.23) with a slight modification at 60•Ž in 0.35 present in the culture supernatant. In this ml of a reaction mixture consisting of 0.1M work, even though the partially purified pul Na acetate buffer (pH 6.0), 2% pullulan for lulanase (213U/mg protein), which was analytical use and enzyme (0.05ml). Incuba dialyzed overnight at 4•Ž against 10mM Na tion was performed for 20min. Reducing sugar acetate buffer (pH 6.0), contained only about formed was determined with 3, 5-dinitrosalicylic 10% of the original ƒ¿-amylase activity (25.3 40 J. Jpn. Soc. Starch Sci., Vol. 34, No. 1 (1987)

U/ml culture supernatant), it was used as the Table 1. Effects of carbohydrates on pullulanase enzyme source because it lacked ƒ¿-glucosidase production. activity.

RESULTS

Characterization of the isolate

Strain AMD-33 grew at temperatures from

37 to 74•Ž, with an optimum at 65•Ž, on the isolation medium. The bacterium was strictly aerobic, non-motile, Gram-negative and non

sporulating rod-shaped cells (0.5-0.9 by 4-9 Table 2. Effects of nitrogen sources on pullulanase

m) with no flagella. The pH range for production. growth was 5.5 to 8.5, with an optimum near neutrality, on the isolation medium. The bacterium was able to grow on 3%, but not on 5% NaCl. Guanine plus cytosine comprised

55mol% of the deoxyribonucleic acid. The

isolate contained ornithine but lacked diamin

opimelic acid in the cell wall. The following

characteristics were also established: no growth

in anaerobic media with or without 1%

glucose; reduction of nitrate to nitrite; no indole or H2S formation; no milk coagulation As shown in Table 1, pullulanase secretion or peptonization; a negative Voges-Proskauer was enhanced by pullulan, soluble starch, reaction; positive catalase and oxidase reac dextrin and maltose.

tions; a negative urease test result; no growth A significant amount of enzyme was not in 0.1% Na laurylsulfate; and resistance to produced with glucose, fructose, mannose, lysozyme. The isolate could not grow on galactose, xylose, arabinose, sucrose, lactose, the isolation medium containing 10ƒÊg/ml of lactulose, cellobiose or trehalose. The enzyme

chloramphenicol, erythromycin, kanamycin, was also not synthesized with sorbitol, man

neomycin, streptomycin, tetracycline, penicillin nitol, maltitol, methyl ƒ¿-glucoside, glycerol,

G, cephalosporin C, ampicillin, ristocetin, methi Na citrate or Na acetate. Among the carbohy

cillin, novobiocin, actinomycin D, tunicamycin drates tested, pullulan and maltose were effec

or cycloserine, and was resistant to 100ƒÊg/ml tive carbon sources for the enzyme production.

of gramicidin D. Nitrogen sources. Table 2 shows the effects

of various organic nitrogen sources on the

Factors affecting pullulanase synthesis in the enzyme production. Strain AMD-33 was grown

Thermus sp. aerobically in the isolation medium containing Carbon sources. Some carbohydrates were 1% pullulan, 0.1% K2HPO4, 0.02% MgSO4 compared as carbon sources for the enzyme 7H2O and a nitrogen source under the same

production. Cultivation was performed 100ml conditions as described above. Among the conical flasks containing 20ml of the isolation organic nitrogen sources tested, defatted soy

medium without agar, in which pullulan was bean powder and gelatin were suitable nutrients replaced by various carbon sources, with for the enzyme production. A small amount shaking (200rpm) at 60•Ž on a rotary of enzyme was secreted with yeast extract,

shaker. Pullulanase activity in the culture polypepton, casein (milk), Casamino acid or supernatant after 4 days cultivation was deter meat extract. mined by the standard assay method described No enzyme was produced when the bacte above. rium was grown in the isolation medium Production of Pullulanase by a Thermus sp. 41

Fig. 1. Effects of the concentrations of pullulan (A) and gelatin (B) on growth and pullulanase production. Fig. 2. Effect of temperature on growth and The bacterial growth (•œ) and pullulanase pullulanase synthesis. activity (•›) were determined by the methods L-shaped tubes containing 5ml of the enzyme described in the text. production medium were incubated with rocking in a temperature gradient incubator for 4 days. Symbols: •œ, specific growth rate; •›, relative containing tryptone, corn steep liquor (C. S. L.), rice bran or peptone. No or only poor growth pullulanase activity after 4 days cultivation. was observed in the medium containing urea, glycine, ammonium citrate or an inorganic 33 was examined in a temperature gradient nitrogen compound such as NaNO3, NH4NO3, incubator. As shown in Fig. 2, the optimum NH4Cl, CH3COONH4, (NH4)2SO4 or (NH4)3 temperature for growth in the enzyme produc PO4. In this work, gelatin was used as a tion medium without CaCO3 was 65•Ž, whereas nitrogen source. that for pullulanase formation was 60•Ž. The

generation time of this bacterium was 27min Effects of the concentrations of pullulan and at 65•Ž and 38min at 60•Ž, respectively. gelatin The effects of the concentrations of pullulan Time course of pullulanase formation and gelatin on the enzyme production are The relationship of bacterial growth and shown in Fig. 1. Strain AMD-33 was grown enzyme formation was examined when strain aerobically for 4 days in the medium contain AMD-33 was cultured aerobically for a long ing 1% gelatin, 0.1% K2HPO4, 0.02% MgSO4•E time in the enzyme production medium. As 7H2O and pullulan, the carbohydrate content shown in Fig. 3, logarithmic growth continued being varied within the range of 0-3%. As for about 25hr without a lag time. During the shown in Fig. 1A, pullulanase secretion in stationary growth phase, the cell concentration creased with increasing pullulan concentration remained almost constant for about 15hr and in the medium and reached a maximum at 1%. then gradually decreased during the remainder Figure 1B shows the effect of the gelatin con of the experimental period. During the loga centration on the enzyme production. Pullu rithmic growth phase, the pH of the culture lanase synthesis was strongly stimulated by broth decreased from 7 to 6 and then increased increasing gelatin concentration, and the max during the stationary growth phase. The imum yield was obtained in the medium con formation of extracellular pullulanase increased taining 2% gelatin. gradually with bacterial growth and the maximum accumulation was achieved within Effect of temperature on growth and pullu 75hr. A significant amount of enzyme was lanase synthesis never present in the cells or on the cell sur The effect of the cultural temperature on faces during any phase of bacterial growth. growth and enzyme production by strain AMD 42 J. Jpn. Soc. Starch Sci., Vol. 34, No. 1 (1987)

Fig. 5. Effect of temperature on activity.

Fig. 3. Time course of pullulanase formation. Pullulanase activity was determined by the

AMD-33 was grown in aerobically in the standard assay method at various temperatures enzyme production medium at 60•Ž on a rotary in the presence (•œ) or absence (•›) of 3mM

shaker (200rpm). The bacterial growth and CaCl2.

extracellular pullulanase activity were deter mined by the methods described in the text.

Symbols: •¡, pH of the culture medium; •œ, bacterial growth; •›, pullulanase activity.

Fig. 6. Thermal stability.

Pullulanase was preincubated at pH 6.0 for 30min in the presence (•œ) or absence (•›) of

3mM CaCl2 at various temperatures. Residual Fig.4. Effect of pH on activity. activity was determined by the standard assay

Pullulanase activity was assayed at various method. pHs in Na acetate (pH 4-6) and Na phosphate

(pH 7) buffers. the presence or absence of Ca2+. The results

presented in Fig. 5 show that the pullulanase Properties of the pullulanase was most active at 70•Ž in the absence of Effect of pH an activity. The effect of pH Ca2+. When the reaction was performed in the on pullulanase activity was examined with the presence of Ca2+, the optimum temperature partially purified enzyme at various pHs. As for activity shifted to 75•Ž. shown in Fig. 4, the optimum pH range for The effect of temperature on the heat the enzyme activity was pH 5.5-5.7. stability of the pullulanase in the presence or Effect of temperature on activity and stabi absence of Ca2+ is shown in Fig. 6. Under lity. The relationship of temperature to pullu these conditions, the pullulanase activity was lanase activity and stability were examined in completely retained at 60•Ž in the absence of Production of Pullulanase by a Thermus sp. 43

Ca2+ for 30min. from thermophiles are entirely different from

The enzyme activity was abolished on treat those of the enzyme described here, i.e.,

ment at 75•Ž, and Ca2+ prevented this heat optimum pH and temperature for action and

inactivation of the pullulanase. In the presence thermal stability.

of 3mM CaCl2, the pullulanase was completely Although much works has been done on

stable at 65•Ž, and 50% activity was retained the characterization of intracellular and cell

after treatment at 75•Ž for 30min. associated enzymes of some Thermus sp.

strain,19,37•`41) little information is available on

DISCUSSION the secretion of enzymes out side of the cells

except for the protease production by T.

Although much information is available on caldophilus42) and T. aquaticus.43) The results

the synthesis and characterization of pullulan of this study suggest that the genus Thermus

ases from mesophilic microorganisms grown would be one of the better microbial producers

under neutral and alkaline pH conditions,8,12•`18) of extracellular thermostable enzymes useful in

little is known about the conditions influencing industrial fields. Purification of the enzyme is

the production of the enzyme by Gram-negative in progress in order to study its properties

thermophiles except for that by T. aquaticus19) further.

and a few Gram-positive thermophiles.29•`31)

The results of the present investigation REFERENCES

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