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Purification and Biochemical Characterization of Pullulanase Type I from Thermus Caldophilus GK-24

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Purification and Biochemical Characterization of Pullulanase Type I from Thermus Caldophilus GK-24 ELSEVIER FEMS Microbiology Letters 138 (1996) 147-152 Downloaded from https://academic.oup.com/femsle/article/138/2-3/147/540290 by guest on 30 September 2021 Purification and biochemical characterization of pullulanase type I from Thermus caldophilus GK-24 Cheorl-Ho Kim aq*, Oyekanmi Nashiru a, Jeong Heon Ko b ’ Department of Biochemistry and Molecular Biology, College of Oriental Medicine, Dong-Guk Unitiersit);, Sukjang-Dong #707, Kyung-Ju City, Kyung-Pook 780-714, South Korea b Korea Research Institute of Bioscience and Biotechnology, KIST, Taejon 305-600, South Korea Received 16 October 1995; revised 18 February 1996; accepted 26 February 1996 Abstract A thermostable pullulanase (pullulan 6-glucanohydrolase, EC 3.2.1.41) has been purified to homogeneity from Thermus culdophilus GK-24 by chromatographic methods, including gel-filtration and ion-exchange chromatography. The specific activity of the enzyme was increased 431-fold with a recovery of 13.2%. The purified enzyme was a monomer, M, = 6.5 kDa as estimated by SDS-PAGE and gel filtration. The p1 was 6.1. The enzyme was most active at pH 5.5. The activity was maximal at 75°C and stable up to 95°C for 30 min at pH 5.5. The enzyme was stable to incubation from pH 3.5 to pH 8.0 at 4°C for 24 h. The activity of the enzyme was stimulated by Mn*+ and Mg*+ ions. Ni*+, Ca2+, Co*’ ions and EDTA did not inhibit the enzyme activity. The enzyme hydrolyzed the (Y-1,6 linkages of amylopectin, glycogens, (Y,P-limited dextrin, and pullulan. The enzyme caused the complete hydrolysis of pullulan to maltotriose. The activity was inhibited by a-, /3-. or y-cyclodextrins. The N-terminal sequence [(Ala-Pro-Gln-(Asp or Tyr)-Asn-Leu-Leu-Xaa-ILe-Gly-Ala(Ser)] showed some similarity to those of bacterial pullulanases. Keywords: Thermus caldophilus GK-24; Pullulanase type I; Thermophilic bacteria 1. Introduction of microbial pullulanases have been purified and characterized from thermophilic bacteria by many Pullulanase (pullulan 6-glucanohydrolase, EC investigators [3- 131. However, most enzymes from 3.2.1.4 1) hydrolyzes the (Y-1,6-glucosidic linkages in thennophilic bacteria are type II pullulanases, which pullulan and produces maltotrlose as the end product hydrolyze the (Y-1,6-glucosidic linkages of pullulan, [l]. Since the discovery of Klebsiella pneumoniae as well as the (Y-1,4-linkages of other polysaccha- pullulanase by Bender and Wallenfels 121, a number rides. Pullulanases of type I, which exclusively hy- drolyze the (u-1,6 linkages of pullulan, are produced by K. pneumoniae [2], Bacteroides thetaiotaomicron * Corresponding author. Tel.: +82 (561) 770 2273; Fax: +82 95-1 [ 141, Bacillus sp. KSM-1876 151, T. aquaticus (561) 749 5117. [4], alkaliphilic Bacillus sp. S-l [16,17], Micrococ- 0378-1097/96/$12.00 Q 1996 Federation of European Microbiological Societies. All rights reserved PII SO378-1097(96)00089-4 L’USsp. Y- 1 [ 161, and B. ucidol,ullul~ticl.~ [ 181.Thus, was detected in the region of 0.17-0.25 M NaCl; the to our knowledge, little is known about thermostable active fractions were pooled and concentrated. Fur- pullulanases of type 1, except in the case of T. ther purification of the thermostable pullulanase pro- aquaticus [4]. In the present study, we report the tein was achieved by high performance liquid chro- purification and characterization of the thermostable matography on a DEAE Toyopearl 5PW column pullulanase type I from T. culdophilus GK-24. (10 X 100 mm; Tosoh Co., Tokyo. Japan) with a linear NaCl gradient (100 ml. 0.0-0.5 Ml. Enzyme Downloaded from https://academic.oup.com/femsle/article/138/2-3/147/540290 by guest on 30 September 2021 activity was detected in the region of 0.17-o. 19 M 2. Materials and methods NaCl. The collected active fractions (1 ml each) were concentrated and reapplied to a FPLC Mono-Q HR IO/ 10 column (IO X 100 mm; Pharmacia LKB, 2. I. Muterials und bacterial growth Uppsala, Sweden) and the tPUL was recovered as a single peak which eluted at 0.11-o. 13 M NaCl. Pullulan (m = 65 kDa, maltooligosaccharides, GE-. p-, and y-cyclodextrins (CDs) were from Hayashi- 2.3. Enzyme assay and enzyme churucterizution bara Co. Ltd. (Okayama, Japan). All other reagents were of the highest grade available. T. culdophilus Pullulanase activity was assayed by measuring the GK-24 cells (kindly supplied by Dr. S.T. Kwon and reducing sugar released from pullulan [2 1I. The reac- T. Ohta, Faculty of Agriculture, The University of tion mixture (1 .O ml), containing pullulan (1%. w/v). Tokyo, Tokyo, Japan) were grown in a medium (pH 50 mM Tris . HCI (pH 7.01, and enzyme. was incu- 7.2) containing 0.8% polypeptone, 0.4% yeast ex- bated at 73°C for 30 min. The reducing sugar was tract, and basal salts with 1% soluble starch as a measured by the dinitrosalicylic acid procedure [22]. carbon source [20]. The cultivation was carried out at I U of pullulanase activity was defined as the amount 75°C for 2 days in a 5liter stirred-tank fermentor of enzyme that produced 1 pmol of reducing sugar [ (Korea Fermentor Co. Ltd., Inchon, Korea) 19,201. as glucose per min. For effects of temperature and pH on the activity 2.2. Purification of the thermostuble tPUL .from T. and stability, 0.087 U of enzyme was incubated with caldophilus GK24 2% pullulan at various temperatures (20- 100°C) and pHs. Buffers used were 30 mM sodium acetate (pH After growth, cells (50 g) were resuspended in 3.0-6.0), 30 mM sodium phosphate (pH 6.0-8.01, 500 ml 50 mM Tris . HCl. pH 8.0 (buffer A) contain- 30 mM Tris . HCI (pH 8.0- 10.01, and 30 mM glycine ing 5 mM MgCl> and disrupted by a ultrasonic (pH 9.0-12.0). Protein was measured by the method homogenizer (Cole-parmer Co., USA), and followed of Lowry with bovine serum albumin as the stan- by centrifugation at 12 000 X g for 20 min. The dard. The absorbance at 280 nm was used to monitor crude enzyme was fractionated with 20-70s satura- protein in column elutes. The N-terminus of the tion of ammonium sulfate. The precipitates were protein was sequenced using a 470A gas phase pro- dissolved in 120 ml of buffer A and dialyzed against tein sequencer equipped with a 120A on-line the same buffer overnight. The dialysates were chro- phenylthiohydantoin analyzer (Applied Biosystems, matographed on a Sephacryl-S200 column (2.5 X 120 Warrington, Cheshire, UK). Native polyacrylamide cm) that had been equilibrated with buffer A. Frac- gel electrophoresis (PAGE) was done using 12% tions of 8 ml each were collected and enzyme activ- (w/v> gel, as described by Davis [23]. Sodium dode- ity was detected between fractions 17 and 24. The cyl sulfate (SDS)-PAGE was done essentially by the active enzyme fractions were combined and concen- method of Laemmli. Isoelectric point was deter- trated by ultrafiltration (Amicon PM30; Amicon Co. mined by isoelectric focusing (IEF) PAGE using Ltd., Danvers, MA, USA), dialyzed, and applied on precast PhastGels with a pH gradient from 3.5 to 9.0 a DEAE-Sephacel column (1.5 X 20 cm). The en- (Pharmacia). Oligosaccharides produced by enzy- zyme was eluted with a linear NaCl gradient of matic action were examined by thin layer chro- 0.0-0.4 M in 300 ml of buffer A. Enzyme activity matography, as described by Kim [24]. C-H. Kim et al./FEMS Microbiology Letters 138 (1996) 147-152 149 3. Results and discussion A 6 3 3.1. Purification and general properties of ther- 12 pH kDa mostable pullulanase from T. caldophilus GK24 T. caldophilus thermostable pullulanase was puri- fied 431-fold to homogeneity with a specific activity Downloaded from https://academic.oup.com/femsle/article/138/2-3/147/540290 by guest on 30 September 2021 of 86.2 U/mg protein and 13.2% recovery (Table 1). + 66 The purified enzyme migrated as a single protein band on SDS-PAGE, showing m = 65 kDa (Fig. + 4.3 1A). A calibrated column of Sepharose 4B gave a m = 63 kDa (data not shown). The value was similar 6.1~ + 29 to those of pullulanases reported to date. The molec- ular masses of pullulanases reported thus far are + 16.4 range between 70 and 140 kDa [2-6,13-15,17,18]. Some pullulanase-like enzymes, such as B. circulans amylase-pullulanase enzyme (220 kDa) /25], B. sub- Fig. 1. SDS-PAGE and IEF analysis of the purified thermostable tilis pullulanase-amylase complex (450 kDa) [28], pullulanase from i? caldophilus GK24. (A) SDS-PAGE. Arrow- Thermoanaerobacter B6A amylopullulanase (450 heads denote the position of tPUL. Lane 3, purified thermostable kDa) [7], and C. thermohydrosulfuricum cY-amylase- pullulanase (5 pg). Standard molecular weight markers (Bio-Rad): pullulanase (165 kDa) [8], have been regarded as myosin (200 kDa), phosphorylase b (97.4 kDa), bovine serum enzymes of high molecular masses. However, these albumin (68 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), and lysozyme (18.4 kDa). (B) PhaseGel IEF. Lane 1, enzymes are not true pullulanases in that they all purified thermostable pullulanase (5 pg); lane 2, the pI calibra- have dual activities of amylase and pullulanase. tion standards: trypsinogen (pl = 9.01, lentil lectin basic band Therefore, thermostable pullulanase from T. cal- (PI = 8.651, lentil lectin middle band (PI= 8.451, lentil lectin dophilus GK24 can be identified as a real pullu- acidic band ( p1 = 8.15).
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