Hyperthermostable Endoglucanase from Pyrococcus horikoshii

An endoglucanase homolog from the hyperthermophilic archaeonPyrococcus horikoshii was expressed in Escherichia coli, andits enzymatic characteristics were examined. The expressed proteinwas a hyperthermostable endoglucanase which hydrolyzes celluloses,including Avicel and carboxymethyl cellulose, as well as ß-glucoseoligomers. This enzyme is the first endoglucanase belongingto glycosidase family 5 found from Pyrococcus species and isalso the first hyperthermostable endoglucanase to which cellulosesare the best substrates. This enzyme is expected to be usefulfor industrial hydrolysis of cellulose at high temperatures,particularly in biopolishing of cotton products.

INTRODUCTION

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Introduction

In the textile industry, cellulases have been used in largequantity for biopolishing of cotton products. This process isessential for removing fuzz and giving a soft touch and cleanappearance to the fabrics. The enzymes that are presently usedfor this purpose are mesophilic cellulases from fungi, and theiroptimum reaction temperatures are between 50 and 55°C. Ifthey are replaced by a hyperthermostable enzyme with an optimumtemperature close to 100°C, which will make it possibleto treat cotton products in steam, the processing will be muchmore simple, quick, and efficient than in the presently employedmethod. Desizing, the step to remove starch from the fabrics,is performed at temperatures at least 70°C, and higher temperaturesare preferred. Because amylases active at these temperaturesare available, this process is performed at temperatures higherthan 70°C. However, cellulase treatment, which usually followsdesizing, is performed at lower temperatures, since a cellulasethat is active and stable in this temperature range has notbeen available. If such a hyperthermostable cellulase is introduced,it will be possible to combine desizing and biopolishing ina single step.

For the purpose of producing such a hyperthermostable cellulase,we investigated the possibility of utilizing the genetic resourcesof hyperthermophilic archaea. Pyrococcus horikoshii OT3 is oneof those organisms with the optimum growth temperature above95°C (4). Based on its complete published genome sequence(8, 9), we have expressed some of the enzymes from this organismand characterized them, most of which have proven to be highlythermostable (1, 7). It is quite likely, therefore, that otheruseful enzymes that are active and stable at extremely hightemperatures remain to be found.

According to the classification of glycosidic hydrolases proposedat the CAZy website (http://afmb.cnrs-mrs.fr/~pedro/CAZy/db.html),the glycosidic hydrolases known to date have been classifiedinto more than 80 families. Among the three species belongingto the genus Pyrococcus, two endoglucanases showing regionsof homology with family 12 (2) and family 16 (5) have been foundin P. furiosus. It has been reported, however, that the hydrolyticactivity of the former enzyme toward celluloses was lower thanthat toward ß-glucose oligomers by at least 2 ordersof magnitude (2) and that the latter had no detectable activitytoward celluloses (5). There has been no report so far on theglucanases from Pyrococcus abyssi and P. horikoshii.

The genome sequence data of P. horikoshii (8, 9) suggest thatthe open reading frame PH1171 (1,377 nucleotides) is homologouswith genes from some other organisms encoding endoglucanasebelonging to family 5 (Fig. 1). There has been no report onhyperthermophilic endoglucanase belonging to this family. Therefore,we cloned this gene, expressed it in Escherichia coli, and characterizedthe expressed protein.

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FIG. 1. Comparison of the amino acid sequences of the endoglucanases from P. horikoshii (EGPh) and from A. cellulolyticus catalytic domain (EGAc) (43% identity). The first line shows the putative signal peptide sequence of EGPh. The sequences are aligned with dashes to indicate gaps. Asterisks (*) indicate that the amino acid is identical in both EGAc and EGPh sequences. The residues conserved in the family 5 endoglucanases are indicated by number signs (#). The active-site residues are shown in boldface. The C-terminal regions of both protein (residues 401 to 430 in EGPh and 379 to 521 in EGAc) were excluded from the alignment because of their low homology in these regions.

Preparation of recombinant protein.

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Preparation of recombinant...

P. horikoshii cells were a gift from Y. Kawarabayasi of theNational Institute of Technology and Evaluation (Tokyo, Japan).The cells were cultivated as reported previously (4), and theirgenomic DNA was prepared according to the method described previously(10).

The open reading frame PH1171 was amplified using the PCR withprimers having NdeI and BamHI restriction sites according tothe methods reported previously (7). The sequences of the primersused were 5'-TTTTGAATTCTTTCATATGGAGGGGAATACTATTCTTAAAATC-3'(upper primer, containing an NdeI site as underlined) and 5'-TTTTTCTAGATTTGGATCCTTTGGGCTACCTGGGAGCCCTTCTTAA-3'(lower primer, containing a BamHI site as underlined). Althoughthe 5' terminal sequence of PH1171 suggested the presence ofa signal peptide sequence (12) in the translated product, theprimers were designed to amplify the whole open reading frame.The amplified gene was digested with NdeI and BamHI and wasinserted into pET11a plasmid digested with the same restrictionenzymes. The nucleotide sequence of the inserted gene was determinedusing an LI-COR Model LIC-4200L(S) sequencer (Aloka, Mitaka,Tokyo, Japan) to verify identity with the anticipated sequence.The amplified gene was expressed using the pET11a vector systemin the host E. coli BL21(DE3) pLysS, according to the instructionsprovided by the manufacturer.

The expressed protein was purified by heating the crude extractat 85°C for 30 min, precipitation with 90% saturated ammoniumsulfate, ion-exchange chromatography using Hitrap Q (Pharmacia,Uppsala, Sweden), and gel filtration chromatography using HiLoad26/60 Superdex 200 (Pharmacia).

The detailed procedures for purification have been describedpreviously (7).

Characterization of recombinant protein.

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Characterization of recombinant...

The glucanase activity was assayed by measuring the reducingsugars according to the modified Somogyi-Nelson method (6).The substrates used were Avicel SF (microcrystalline cellulose;Asahi Kasei, Tokyo, Japan), carboxymethyl cellulose (CMC; Sigma),lichenan (from Cetraria islandica; Sigma), xylan (from birchwood; Sigma), curdlan (from Alcaligenes faecalis; Sigma), andxyloglucan (from tamarind; Megazyme International Ireland),and ß-glucose oligomers (from cellobiose to cellopentaose;Seikagaku Kogyo, Tokyo, Japan).

The hydrolytic activity toward various substrates was measuredat 85°C in 100 mM acetate buffer (pH 5.6). As listed inTable 1, this protein hydrolyzed celluloses such as CMC andAvicel SF. Lichenan, a ß-1,3 and -1,4 glucan, wasalso hydrolyzed. ß-Glucose oligomers (from cellobioseto cellopentaose) were hydrolyzed, although to much lesser extent.No activity was detected toward curdlan (ß-1,3 glucan),xylan (ß-1,4 glycan of xylose), and xyloglucan (mixedß-1,4 glycan of glucose and xylose). From these observationsit was concluded that this protein is a glucanase (hereinafterreferred to as EGPh) which specifically hydrolyzes ß-1,4glucosidic bonds. This was as expected from the amino acid sequencehomology of EGPh with the family 5 endoglucanases (8), exceptthat xyloglucan, which is usually hydrolyzed by endoglucanases,was not hydrolyzed by EGPh. In order to determine the mode ofhydrolysis of EGPh, the relationship between the viscosity andthe reducing power of the hydrolysis products by EGPh was determined.As shown in Fig. 2, the decrease in viscosity of the CMC solutionincubated with EGPh in relation to the increase in the reducingsugar showed a pattern characteristic of the endo-type cellulases(3). From these results, it was concluded that EGPh was an endo-typecellulase. The k_cat values for the substrates examined are listedin Table 1. The optimum pH was between 5.4 and 6.0. The totalyield of EGPh was 0.27 mg per liter of the transformant culture.The specific activity of the purified EGPh was 8.5 U/mg, where1 U is defined as the amount of enzyme producing reducing powerequivalent to 1 mg of glucose per min from CMC in the routineassay condition (0.5% CMC in 500 µl of 100 mM acetatebuffer, pH 5.6, 85°C).

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TABLE 1. Substrate specificity of EGPh^a

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FIG. 2. Mode of hydrolysis of EGPh. The viscosity values measured were plotted against the reducing sugar concentrations as measured by Somogyi-Nelson reaction using glucose as the standard. A solution of CMC (3.5% in 100 mM acetate buffer, pH 5.6, 50 ml) was subjected to hydrolysis by 4 U of EGPh/ml for 30 to 120 min at 90°C. After 30, 60, and 120 min of incubation, the viscosity of the solution was measured at 40°C, using Viscometer RE110L (Toki Sangyo, Tokyo, Japan). The initial value of viscosity was 18.92 mPa.

The sequence of the deduced translation product of this genewas aligned with that of the endoglucanase gene of Acidothermuscellulolyticus, a member of the family 5 endoglucanases, thecatalytic region of which has a known crystallographic structure(11). The alignment was performed using a Genetyx-Mac program(Software Development, Tokyo, Japan). As shown in Fig. 1, allthe seven residues characteristic of the family 5 endoglucanases,including the catalytic residues, are conserved in EGPh. Fromthe result of the sodium dodecyl sulfate-polyacrylamide gelelectrophoresis of the purified enzyme, the molecular size ofthe monomer of this enzyme was estimated to be approximately43 kDa. Since this was significantly smaller than the value(52 kDa) calculated on the basis of the sequence of the openreading frame, it was suspected that EGPh was an incompleteproduct of the gene. In order to make this clear, the N-terminalamino acid analysis was performed at the Custom Service Centerof Takara Shuzo (Kusatsu, Japan). The result indicated thatthe first 5 amino acids from the N terminus were ENTTY, suggestingthat the first 28 amino acid residues of the predicted translationproduct of the open reading frame were missing in EGPh. Thesequence of these 28 amino acid residues removed had all thecharacteristics unique to the signal peptide sequences, suchas a high ratio of the hydrophobic amino acid residues (12),implying that EGPh is possibly a protein destined for secretion.In order to see whether EGPh was also produced in the parentorganism, P. horikoshii was cultivated according to the methoddescribed previously (4) and glucanase activity was checkedfor both the extract of P. horikoshii cell mass obtained bysonication and the concentrated culture supernatant. The extractof the cell mass of this culture gave barely detectable activityof glucanase. However, no glucanase activity was detected fromthe concentrated culture supernatant, showing that this enzymeis not secreted in the medium. Thus, the use of E. coli transformantis a much better choice for production of EGPh than the useof the parent organism, P. horikoshii, considering the difficultyof cultivating P. horikoshii under strictly anaerobic conditionsat 95°C. The N-terminal sequence of EGPh purified from P.horikoshii cells was also determined, and it proved to be identicalto that of EGPh from E. coli transformant, reinforcing the possibilitythat the 28-residue segment removed from the protein is a signalsequence.

The temperature dependence of EGPh was examined by measuringthe hydrolytic activity in 100 mM acetate buffer (pH 5.6). Theassay was measured for 15 min. As shown in Fig. 3A, the optimumtemperature for the glucanase reaction was higher than 97°C.

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FIG. 3. (A) Effect of the temperature on the hydrolytic activity of EGPh on CMC. The hydrolytic activity was measured in 100 mM acetate buffer (pH 5.6). The assay was measured for 15 min. (B) Effect of heating on EGPh activity. EGPh solution was incubated at 97°C in 100 mM acetate buffer (pH 5.6). At the time shown, aliquots were taken out and the activity was measured in the same buffer at 85°C using CMC as the substrate.

Thermostability of EGPh was measured by incubating aliquotsof enzyme solution (0.1 mg/ml) at 97°C in 100 mM acetatebuffer (pH 5.6) for up to 3 h, followed by measurement of residualactivity. As shown in Fig. 3B, the residual activity after heatingfor 3 h at 97°C was 80% of that of EGPh that was not heated.

Because of its ability to hydrolyze celluloses at high temperaturesabove 90°C, as well as its thermostability, EGPh is expectedto be an excellent tool for industrial hydrolysis of cellulose,particularly for biopolishing of cotton products. The yieldof this enzyme from the E. coli transformant, however, is ratherlow for industrial application. In order to achieve more efficientproduction, a study is in progress for extracellular productionof this enzyme using Bacillus brevis as the host cell.

ACKNOWLEDGMENTS

We are grateful to Akio Shimomura of Rakuto-Kasei IndustrialCo. Ltd. for his helpful suggestions and instructions.

FOOTNOTES

* Corresponding author. Mailing address: Special Division for Human Life Technology, National Institute of Advanced Industrial Science and Technology (Kansai), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan. Phone: 81-727-51-9526. Fax: 81-727-51-9628. E-mail for Susumu Ando: andosusumu{at}hotmail.com. E-mail for Kazuhiko Ishikawa: kazu-ishikawa{at}aist.go.jp.

REFERENCES

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