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Gene Expression in Escherichia coli and Purification of Recombinant Type II Pullulanase from a Hyperthermophilic Archaeon, Pyrobaculum calidifontis.

Byline: Habib-ur-Rehman, Masood Ahmed Siddiqui, Abdul Qayyum, Arifa Bano and Naeem Rashid


Type II pullulanase also known as amylopullulanase hydrolyzes [alpha]-1,6 glycosidic bonds in pullulan and [alpha]-1,6 as well as [alpha]-1,4 glycosidic bonds in starch. Most of the previously reported type II pullulanases are metal ion dependent and hard to purify, to homogeneity, due to low level of expression or the proteolytic machinery of the host. Herein we report expression in Escherichia coli and purification of metal ion independent type II pullulanase from Pyrobaculum calidifontis. Thermophilic origin and metal ion independency of the enzyme reported in this study make it a potential candidate for starch hydrolyzing industry.

Key words

Hyperthermophile, Pyrobaculum calidifontis, Debranching enzyme, Type II pullulanase, Amylopullulanase.


Starch is one of the most abundant homopolysaccharide on our planet and is a polymer of [alpha]-D glucose residues (Kujawski et al., 2002). Starch is synthesized in most green plants as energy reserve, and is used as energy source for various microorganisms, higher plants and animals (Janecek, 1997). Starch is composed of two glucose polymers; one is amylose and second is amylopectin. Amylose is a linear polymer of approximately 6000 [alpha]-D-glucose residues which are linked through [alpha]-1,4 glycosidic bonds. Amylopectin is a branched polymer that in addition to [alpha]-1,4 glycosidic linkage also contains [alpha]-1,6 glycosidic linkage (Nigam and Singh, 1995).

Formerly starch processing industry was using mild acid for the hydrolysis of starch but nowadays, due to the discovery of more efficient thermostable starch hydrolyzing enzymes, the starch hydrolyzing industry has switched to the enzymes for starch hydrolysis to make beneficial products such as maltodextrin, dextrose, glucose and fructose syrups and sugar alcohols such as mannitol, sorbitol, erythritol etc.

The pullulanases (E.C. degrade pullulan, a linear [alpha]-glucan that contains repeating subunits of triose sugar (maltotriose) linked by [alpha]-1,6 glycosidic bond, into different types of saccharides. Pullulanases are also known as debranching enzymes due to their capability to hydrolyze [alpha]-1,6 glycosidic linkages in starch and other branched polysaccharides (Van der Maarel et al., 2002; Hii et al., 2012).

On the basis of reaction end products and substrate specificity five types of pullulanases have been reported till now, among them two are called pullulanases (pullulanase types I and pullulanase type II) while the other three are known as pullulan hydrolases (pullulan hydrolase types I, pullulan hydrolase type II and pullulan hydrolase type III). Type I pullulanase, hydrolyzes [alpha]-1,6 glycosidic bonds in pullulan, amylopectin and other branched chain polysaccharides but is unable to act on [alpha]-1,4 glycosidic bonds in substrates (Rudiger et al., 1995; Kim et al., 1996; Koch et al., 1997; Bertoldo et al., 1999; Ben-Messaoud et al., 2002; Han et al., 2013). The end product of reaction catalyzed by type I pullulanase is maltotriose (Wasko et al., 2011).

Type II pullulanase also known as amylopullulanase (APU) hydrolyzes [alpha]-1,6 glycosidic bonds in pullulan and starch but cannot hydrolyze [alpha]-1,4 linkage in pullulan and surprisingly can hydrolyze [alpha]-1,4 glycosidic linkage in starch (Melasniemi, 1988; Saha et al., 1988; Spreinat and Antranikian, 1990; Mathupala et al., 1993; Ramesh et al., 1994). The third and fourth types are pullulan hydrolase I (neopullulanase) and pullulan hydrolase II (isopullulanase) (Duffner et al., 2000; Niehaus et al., 1999; Matzke et al., 2000) hydrolyze [alpha]-1,6 glycosidic bonds in pullulan while fifth type is pullulan hydrolase III has the ability to degrade [alpha]-1,4 and [alpha]-1,6 glycosidic linkages in pullulan (Niehaus et al., 2000; Ahmed et al., 2014).

Nowadays the enzymes from hyperthermophiles are widely used in industries (Sabir et al., 2017) and especially are replacing the use of other amylolytic enzymes in starch industry. However, these enzymes are Ca2+ dependent for their activity and adding the Ca2+ in reaction may inhibits the activity of other enzymes involved in reaction processes or choke the heat exchangers. Previously we have cloned and expressed Pcal_1616 (apu) gene for the production of Ca2+ independent type II pullulanase from Pyrobaculum calidifontis strain VA1 (Siddiqui et al., 2014). P. calidifontis strain VA1 is an obligate heterotrophic and hyperthermophilic archaeon that was isolated from a terrestrial hot spring in Philippines (Amo et al., 2002). Unfortunately in our previous report the recombinant type II pullulanase could not be purified (Siddiqui et al., 2014). We report here the purification and activity assays of type II pullulanase from P. calidifontis strain VA1.


Chemicals, restriction enzymes and reagents

All the chemicals used in this study work were of molecular biology or analytical grade and were obtained from Sigma (St. Louis, Mo., USA). All restriction endonucleases, Phusion DNA polymerase, PCR cloning kit, (DNA) extraction and ligation kits, DNA markers and protein markers were obtained from New England Biolabs, Inc., Nebraska, USA.

Growth conditions for Pyrobaculum calidifontis

Routine cultivation of hyperthermophilic archaeon P. calidifontis was performed according to previously described method (Amo et al., 2002) in a medium containing 1% tryptone, 0.1% yeast extract and 0.3% sodium thiosulphate in distilled water at 90AdegC.

Host bacterial strains, plasmids and media

Plasmid pET101/D-TOPO (Invitrogen) was used as expression vector. One Shot TOP10 chemically competent E. coli F- mcrA I (mrr-hsdRMS-mcrBG) F80lacZIM15 IlacX74 recA1 araD139 I(ara-leu)7697 galU galK rpsL (Str R) endA1 nupG was used for cloning while E. coli BL21 Star(tm) (DE3) F- ompT hsdSB (rB-mB-) gal dcm rne 131 (DE3) was used for overexpression of the cloned gene. All E. coli strains were cultivated at 37AdegC.

Cloning of Pcal-apu gene

Based on the DNA sequence of Pcal_1616 and pET101 plasmid as described by Shuman (1991, 1994). The forward primers Apu-F1: 5'- CACCATGAAGATCCTCGCCCTCCTC-3', and a reverse Apu-R1: 5'- TATAGATTGTCGCGGCCTCC-3' primer for the amplification of apu gene were constituted. The gene amplification was performed by polymerase chain reaction (PCR) using Phusion DNA polymerase (New England BioLabs Inc.) as follows: 3 min at 98AdegC; 30 s at 95AdegC, 30 s at 53AdegC and 1.0 min at 74AdegC (30 cycles) using thermal cycler (Gene Amp PCR System 24000, Perkin Elmer, Foster, Calif). The resulting amplified PCR product of apu gene was ligated into TOPO pET101 vector and the resulting plasmid was named pET101-APU. One Shot TOP10 chemically competent E. coli cells were transformed using pET101-APU plasmid.

DNA sequencing

The gene fragment cloned in pET101 vector was sequenced using an automated DNA sequencer (Beckman Coulter 121 CEQ8000; Beckman Coulter Inc., Fullerton, CA).

Gene expression and purification of protein

For expression, E. coli BL21 StarTM (DE3) cells were transformed using pET101 plasmid harboring apu gene. Cells harboring recombinant vector pET101-APU were cultured at 37AdegC for 16 hours in Luria-Bertani broth (LB) medium containing (100 ug/mL) ampicillin antibiotic. This overnight grown preculture was added (1%) into freshly prepared LB medium containing (100 ug/mL) ampicillin and cells growth was continued at same temperature (37AdegC) with shaking at 100 rpm. The optical density (OD) was measured hourly at 660 nm and when it reached to 0.4 then the gene expression was induced with 0.5 mM isopropyl-[beta]-D-thiogalactopyranoside (IPTG). The incubation was continued at 37AdegC for further 4 h. The cells were obtained by centrifugation at 8,000 x g for 08 min and then washed with 50 mM Tris-HCl buffer (pH 8.0). The cell pellet obtained was resuspended in 50 mM Tris-HCl buffer. The cells were sonicated and centrifuged at 15,000 x g for 15 min to obtain the soluble and insoluble fractions.

The apu gene expression was confirmed by SDS-PAGE and enzyme activity assay. The soluble fraction was kept in shaking water bath at 85AdegC for 20 min and then again centrifuged at 15,000 x g for 15 min to separate soluble and insoluble fractions. The soluble fraction obtained after heat treatment was precipitated with the addition of 75% ammonium sulfate. The sample was centrifuged at 20,000 x g for 20 min, the pellet obtained after centrifugation was dissolved in 10 mL of 20 mM potassium phosphate buffer (pH 7.4) containing 0.5 M KCl and 40 mM imidazole and dialyzed against same buffer. The dialyzed sample was used for immobilized metal affinity chromatography (IMAC) by using His GraviTrap (Amersham Biosciences) column. The sample was applied, and the His tag protein was eluted from the His GraviTrap column with 20 mM potassium phosphate elution buffer (pH 7.4) containing 0.5 M KCl, and 0.5 M imidazole.

The elution buffer was exchanged with Tris-HCl (pH 8.0) utilizing Amicon Ultra-4 30K (Millipore) by centrifugation at 4,000 x g for 15 min, the collected fractions were stored at 4AdegC for initial examination of activity.

Enzyme assay

Type II pullulanase activity was determined according to previously reported method (Bernfeld, 1955) by measuring the amount of reducing sugar released during enzymatic hydrolysis of 1% pullulan or soluble starch in 125 uL 50mM acetate buffer (pH 5.6) with further addition of 125 uL diluted recombinant enzyme and incubation at 90AdegC for 15 min. A control without addition of enzyme was used. The amount of reducing sugar released was determined by the dinitrosalicyclic acid method (Miller, 1959). One unit of enzyme activity was defined as the amount of enzyme that released 1 umol of reducing sugars in one minute under standard assay conditions.

For the purpose of thermostability determination of APU in the presence or absence of Ca2+, the purified protein was incubated at 90AdegC for 5 h. Aliquots were withdrawn at every 30 min intervals of time and assayed for the activity at 90AdegC for 15 min. The activity of untreated enzyme at 90AdegC for 5 h was considered as 100%.

Activity staining

The amylopullulanase activity was confirmed on SDS-PAGE. The red pullulan 5 mg/mL was incorporated in polyacrylamide gel. After performing electrophoresis, the gel was incubated at 58AdegC for 4 h in 50 mM phosphate buffer (pH 7.0). After flooding with Lugol's iodine, yellowish white band against the red background on gel was appeared.

Effects of metal ions on amylopullulanase

The effects of various metal ions on the amylopullulanase enzyme activity were studied. The purified amylopullulanase (2.0 U/mL, final concentration) was incubated with 5 mM metal salts at 60AdegC for 15 min. Samples were then withdrawn, and activity of enzyme was measured at 90AdegC.


Gene cloning

The cells of hyperthermophilic archaeon strain P. calidifontis were cultivated as described in experimental section and chromosomal DNA was extracted. For the amplification of type II pullulanase gene (apu), sequence specific primers were designed according to the procedure described by Shuman (1991, 1994) by using topoisomerase I produced by Vaccinia virus. In this system to work additional bases (CACC) were added to forward primer to join the overhanging (GTGG) bases in the pET101 vector created by topoisomerase I. In the reverse primer stop codon was not added for the purpose to add 6xHis tag in PCR product. The polymerase chain reaction (PCR) was performed which resulted in the amplification of approximately 3.1 kbp DNA fragment (Fig. 1A).

The PCR product harboring apu gene was isolated from agarose gel (Fig. 1A), gene cleaned and ligated in pET101 vector to obtain pET101-APU and E. coli One Shot TOP10 competent cells were transformed with pET101-APU. In order to confirm the insertion of 3.1 kbp PCR amplified fragment, pET101-APU plasmid was digested with HindIII restriction enzyme. Two DNA bands were generated, a 1.9 kbp part of the inserted gene and the other 6.9 kb fragment containing 5.7 kbp vector and 1.2 kbp of the other part of the gene, indicating that the recombinant pET101-APU contained the apu gene (Fig. 1B). Complete nucleotide sequences of both strands of inserted apu gene were determined and no variation in both strands was observed. The result of gene sequence confirmed that it encodes type II pullulanase.

Heterologous expression of apu gene and purification of APU protein

For the expression of apu gene the pET101-APU plasmid was extracted. The E. coli competent BL21 Star(tm) (DE3) cells were transformed with pET101-APU and a number of colonies were appeared on selection plates. A colony was selected randomly for gene expression. Heterologous gene expression was achieved with 0.1 mM isopropyl-[beta]-D-galactopyranoside in 6 h. According to SDS-PAGE analysis approximately 111 kDa recombinant protein was produced (Fig. 2A).

Previously recombinant amylopullulanase from P. calidifontis could not be purified when apu gene was expressed in E. coli BL21 (DE3) cells culture (Siddiqui et al., 2014), similar to a number of reports on APU enzymes that could not be purified due to low gene expression or proteolytic effect (Duffner et al., 2000; Chen et al., 2001). In the current expression and purification system, the apu gene was cloned in pET101/D-TOPO plasmid that contains polyhistidine (6xHis) tag that facilitates the purification of recombinant fusion protein with metal chelating resin. The expression was also performed in competent BL21 Star(tm) (DE3) cells that contain rne131 mutation responsible for the enhancement of expression capabilities of competent BL21 Star(tm) (DE3) cells.

After gene expression, the soluble fraction was heat treated at 85AdegC for 20 min and then again soluble and insoluble fractions were separated by centrifugation. The heat treatment removed most of the heat-labile host protein. The soluble fraction obtained after heat treatment was precipitated with the addition of 75% ammonium sulfate. The sample was centrifuged at 20,000 x g for 20 min, the pellet obtained after centrifugation was dissolved in 10 mL of 20 mM potassium phosphate buffer (pH 7.4) containing 0.5 M KCl and 40 mM imidazole and dialyzed against same buffer. The soluble fraction was used for immobilized metal affinity chromatography by using His GraviTrap (Amersham Biosciences) column. The purified APU protein eluted from GraviTrap column was analyzed by SDS-PAGE (Fig. 2A). The purification steps and the amount of enzyme activity of APU are summarized in Table I.

Table I.- Purification of recombinant type II pullulanase.

Purification###TP###TA###SA###Yield Purification

steps###(mg)###(units) (units/mg) (%)###(fold)

Crude extract###1208###6548###5.4###100###1.0

Heat treatment###677###6210###9.2###94.8###1.7




Activity staining

The purified amylopullulanase activity was determined by activity staining on SDS-PAGE as described in materials and methods section. Lugol's iodine forms dark blue/black color with the interaction of amylose and amylopectin, the yellowish white band against the red background on gel appeared (Fig. 2B) indicating that the APU enzyme was active and converted the starch into product.

Effects of metal ions on amylopullulanase activity

The effect of various metal ions on the purified APU enzyme activity was studied. The metal ions such as Cu2+, Zn2+, Ni2+, Mn2+ and Co2+ have inhibitory effect on APU activity while Na+, K+, Mg2+ and Ca2+ did not effect on the APU enzyme activity (Table II).

Table II.- Effect of metal ions on type II pullulanase.

Metal###Concentration Specific activity Relative activity














The Ca2+ usually increases the activity and thermostability of Ca2+ dependent enzymes. In order to know the role of Ca2+ on APU, the enzyme was incubated with or without 0.5 mM Ca2+ for 5 h at 90AdegC and activity was determined as described in materials and methods section. In both cases no detectable difference was observed in the activity as well as thermostability of enzyme (Fig. 3).


We have purified type II pullulanase (APU) from a hyperthermophilic archaeon, P. calidifontis, by using gene cloning in pET101 cloning vector and purification of APU was achieved with immobilized metal affinity chromatography technique. As APU is Ca2+-independent thermostable enzyme therefore, purified APU is a good candidate to replace Ca2+-dependent enzymes being used in starch industry.


The research was funded by grant from the Higher Education Commission of Pakistan (No. 20-860/RandD).

Statement of conflict of interest

Authors have declared no conflict of interest.


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Author:Rehman, Habib-ur; Siddiqui, Masood Ahmed; Qayyum, Abdul; Bano, Arifa; Rashid, Naeem
Publication:Pakistan Journal of Zoology
Article Type:Report
Date:Aug 31, 2018
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