Indian Journal of Biotechnology Vol 6, July 2007, pp 315-320

A unique thermostable lichenase from Thermotoga maritima MSB8 with divergent substrate specificity

Mohammed Abdul Sattar Khan 1*, Mohammed Akbar 2, Motomitsu Kitaoka 1 and Kiyoshi Hayashi 1 1Enzyme Laboratory, National Food Research Institute, Kannondai, Tsukuba, Ibaraki, 305-8642, Japan 2Section of Mass Spectrometry, Laboratory of Membrane Biochemistry and Biophysics National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, Maryland 20852, USA Received 9 March 2006; revised 7 March 2007; accepted 20 April 2007

A putative endoglucanase gene corresponding to locus TM 1752 (AAD36817.1, Q9X274) of Thermotoga maritima MSB8 was cloned and expressed in Escherichia coli . The contains a NEP ( Asn-Glu-Pro ) motif but lacks a PCD (proposed catalytic domain) block. This endoglucanase is grouped under family 5 of the glycosyl members of which predominantly hydrolyze β-1,4-linkages of carbohydrate polymers. The enzyme efficiently hydrolyzes the glycosidic bonds of mixed β-1,3-1,4 linkages in lichenan and barley β-glucan, but did not display any activity on crystalline cellulose or laminarin. However, negligible amount of was observed with CM-cellulose. This enzyme produced glucosyl β-1,3 glucosyl β-1,4 glucose as the major end product and glucosyl β-1,4 glucosyl β-1,3 glucose was not detected. The putative endoglucanase of T. maritima appears to be a unique endoglucanase being the first lichenase producing glucosyl β- 1,3 glucosyl β-1,4 glucose to be placed into family 5 of the glucosyl hydrolases.

Keywords: Thermotoga maritima, endoglucanase, lichenase, family 5, glucosyl β-1,3 glucosyl β-1,4 glucose, glucosyl β-1,4 glucosyl β-1,3 glucose IPC Code: Int. Cl. 8 C12N9/24

Introduction Almost all of the endoglucanases investigated from There is an increasing interest in the use of this species thus far have displayed hyperthermostable thermostable endoglucanases as biocatalysts in properties 1,6,7 . Endo-β-glucanases capable of carbohydrate technology and to date several such degrading β-glucans are classified as β-1,3- endoglucanases have been characterized. The endoglucanases (laminarinases, EC 3.2.1.39), β-1,4- discovery of new endoglucanases from endoglucanases (, EC 3.2.1.4) or β-1,3-1,4- hyperthermophilic organisms is facilitating an endoglucanases (lichenases, EC 3.2.1.73) 8. Based on assessment of their potential in industrial their amino acid sequence similarities, β-1,4- appplications. Recently, putative endoglucanase genes endoglucanases have been classified into at least 12 have been identified from a number of thermophilic different families: families 5 to 9, 12, 44, 45, 48, 51, bacteria such as Thermotoga maritima 1, 61 and 74 of the glucosyl hydrolases. in T. neapolitana 2 and Pyrococcus furiosus 3. T. maritima family 5 display predominantly activity by MSB8 is a fermentative marine hyperthermophilic randomly hydrolyzing β-1,4-linkages. Another eubacterium that can be grown at temperatures up to category of endoglucanases, namely β-1,3-1,4- 90 °C 4. This bacterium, known to be a treasure trove endoglucanases, are included in families 8, 11, 12, 16 of thermostable glucosyl hydrolases, contains a total and 17. They exhibit strict substrate specificity for the of eight endoglucanases belonging to different cleavage of β-1,4-linkages in the presence of β-1,3- glucosyl families ( http://www.tigr.org )5. glycosidic bonds to produce glucosyl β-1,4 glucosyl β- 1,3 glucose as the main product of hydrolysis 2,8-16 , but ______do not attack the β-1,4-linkages in CM-cellulose or *Author for correspondence: 16 Tel: 1-301-435-8011; Fax: 1-301-496-0599 the β-1,3-linkages in laminarin . In the present study, E-mail: [email protected] we report the characterization of a novel *Present address: endoglucanase, TM 1752 (AAD36817.1, Q9X274), Laboratory of Biochemistry, National Heart, Lung and Blood from T. maritima MSB8. This enzyme has been Institute, National Institutes of Health, Bethesda, MD 20892- β 8012, USA shown to specifically hydrolyze -1,3-1,4-mixed 316 INDIAN J BIOTECHNOL, JULY 2007

linkages to form glucosyl β-1,3 glucosyl β-1,4 Production and Purification of Recombinant Endoglucanase glucose as the major hydrolysis product in this The E. coli BL21-RIL codon plus transformed with investigation and represents the first lichenase pET-TM1752 was grown in 1L of Luria Bertani placed into family 5, which displays such a narrow medium supplemented with 50 µg/mL kanamycin at substrate specificity. 37 °C to give a final cell density of 0.6 at 600 nm. Protein production was induced by the addition of Materials and Methods isopropyl-β-D-thiogalactopyranoside (IPTG) to give a Bacterial Strains, Plasmids, Substrates and Chemicals final concentration of 1.0 m M, and cultivation was The Escherichia coli strain BL21RIL codon plus continued overnight at 25 °C. The cell pellet was and the plasmid vectors, pDrive and pET-28a, collected by centrifugation at 10,000 rpm for 3 min at (Novagen, Madison, USA) were used in this study. 4°C and then the pellet was washed with sterile water The genomic DNA of Thermotoga maritima MSB8 and resuspended in buffer A (50 m M sodium was kindly provided by Prof Dr Stetter, Lehrstuhl phosphate pH 8.0 and 300 m M NaCl containing fur Mikrobiologie, Universitaet Regensburg, 10 m M imidazole). Disruption of the cells was Germany. Carboxymethyl cellulose (CM-cellulose), accomplished by sonication (Branson sonifier 250D) β-glucan, laminarin, lichenan and other and the remaining cells and debris were removed by polysaccharides were purchased from Sigma, USA. centrifugation (10,000 rpm for 20 min at 4 °C). The All other chemicals of scientific grade were supernatant containing the crude cell extract was commercially obtained. mixed with 1 mL of NiNTA (QIAGEN, Hilden, Germany) agarose slurry and incubated under gentle Construction of Expression Vector shaking for 1 h to promote efficient binding of the To obtain the endoglucanase gene, the coding His-tag containing recombinant endoglucanase to the region of the enzyme was amplified from the genomic resin particles. The protein-bound resin was then DNA of T. maritima MSB8 by using PCR. packed into a column, the unbound protein washed off Amplification was achieved by using the with buffer A, and the bound endoglucanase was forward primer 5 ′-CATATG AATAACACCATT subsequently eluted with a linear gradient of CCAAGATG-3’ and the reverse primer 5 ′- imidazole (0-200 m M) in buffer A. The active AAGCTT TCAATATTTTCTCAATAGTTCCA-3′ fractions were combined and dialyzed overnight at (the Nde I and Hind III restriction sites are underlined). 4°C against 5 m M MES [2-(N-Morpholino) PCR was performed by using a GeneAmp PCR ethanesulfonic acid] buffer, pH 6.5. The purity of the System 9600 (Perkin-Elmer, Norwalk, CT, USA), enzyme was analyzed by sodium dodecyl sulfate- employing the following reaction conditions: 98 °C polyacrylamide gel electrophoresis (SDS-PAGE) 17 for 5 min, 98 °C for 30 sec, 60 °C for 30 sec and 68 °C using the method of Laemmli . A 10 kDa protein for 1 min, repeated for 25 cycles, and finally 68°C for ladder (Life Technologies, Gibco BRL, Rockville, 5 min. After amplification, the PCR product was USA) was used as a molecular weight marker. treated with Ex Ta q DNA polymerase at 72 °C for Assay of Endoglucanase Activity 10 min to create adenine overhangs. The PCR product In the standard assay, an aliquot of suitably diluted was autoligated in the pDrive vector, then the enzyme solution was incubated in a reaction volume recombinant plasmid was sequenced by the dideoxy of 200 µL mixture containing 50 m M MES buffer chain-termination procedure using a Big Dye (pH 6.6) and 1% lichenan for 20 min at 70 °C. The Terminator Cycle Sequencing Kit (Perkin-Elmer reactions were stopped by rapidly cooling the samples Applied Biosystems, Foster City, CA, USA) on a 310 on ice and the amount of reducing sugar released was Genetic Analyser (Perkin-Elmer). The pDrive plasmid estimated by using the Somogyi-Nelson method 18 . vector containing the endoglucanase gene and the One unit of endoglucanase activity against lichenan is pET-28a expression vector were subjected to defined as the amount of enzyme that catalyzed the restriction enzyme digestion with Nde I and Hin dIII liberation of 1 µmol of reducing sugar, estimated as and then the fragment and the pET vector were ligated glucose per min, under the above conditions. The at 16 °C for 2 h. The recombinant plasmid kinetic parameters Km and kcat were determined using (pET-TM1752) was then transformed into E. coli RIL the non-linear regression analysis program “Grafit” codon plus. by means of the double reciprocal plot method of KHAN et al : THERMOTOGA MARITIMA ENDOGLUCANASE IS A UNIQUE LICHENASE 317

19 Lineweaver and Burk using lichenan and β-glucan Preparation of Glucosyl β-1,3 glucosyl β-1,4 glucose and as the substrates at various concentrations in 50 m M Glucosyl β-1,4 glucosyl β-1,3 glucose MES buffer, pH 6.6. Glucosyl β-1,3 glucosyl β-1,4 glucose was synthesized by the reaction catalyzed by Analyses of Hydrolyzed Products by TLC and HPLC laminaribiose phosphorylase using cellobiose as the A one per cent solution of lichenan was hydrolyzed acceptor and β-D-glucose-1-phosphate as the by the endoglucanase at 70 °C for predetermined donor 21,22 . Glucosyl β-1,4 glucosyl β-1,3 glucose was lengths of time. 1 µL of a 10-fold concentrated aliquot prepared by the synthetic reaction of cellodextrin taken from each sample was spotted onto a silica gel phosphorylase with laminaribiose and β-D-glucose-1- plate (Merck Silica Gel 60F 254; E, Merck, Darmstadt, phosphate 23,24 . Germany). Thin layer chromatography was performed with chloroform-methanol-water (90: 65: 15, v/v) as Results the eluent 20 . The spots were detected after developing Multiple Alignment of Various Cellulases and Lichenases the plate twice in the same solvent system. Products in To investigate the diversity of endoglucanases the enzymatic hydrolyzate were analyzed by using an including TM 1752 (AAD36817.1, Q9X274) of HPLC system (DIONEX, USA) equipped with a T. maritima MSB8, multiple amino acid sequences of pulsed amperometric detector. Solvents A (100 m M a number of endoglucanases were aligned, as shown NaOH) and B (A containing 200 m M NaOAc) in a in Fig. 1. It is interesting to note that Cel5A linear gradient were used as the eluents (flow rate (TM1751), encoded by the upstream gene of 1 mL/min). Oligosaccharides were identified by the T. maritima , showed maximal amino acid sequence comparison of their retention times with those of identity (28%) over the entire polypeptide chain. As authentic saccharides. shown in Fig. 1A, this endoglucanase was aligned

Fig. 1—Multiple amino acid sequence alignment of β-1,4-endoglucanases (cellulases), β-1,3-1,4-endoglucanases (lichenases) with the endoglucanase of T. maritima (Tm-egl): A. Cellulases from T. maritima , Tm-egl [AAD36817.1]; T. maritima , Tm-cel [AAD36816.1]; Anaerocellum ther mophilum , At-cel [Z86104]; Bacillus amyloliquefaciens , Ba-cel [AF363635]; B. polymyxa , Bp-cel [M33791]; B. subtilis , Bs-cel [AF355629]; Clostridium thermocellum , Ct-cel [X03592]; Xanthomonas campestris , Xc-cel [M32700]; B. Lichenases from T. maritima , Tm-egl [AAD36817.1]; B. amyloliquefaciens , Ba-lic [M15674]; B. licheniformis , Bl-lic [X57279]; B. macerans , Bm- lic [X55959]; B. polymyxa , Bp-lic [X57094]; B. subtilis , Bs-lic [U60830]; C. thermocellum , Ct-lic [S70776]; Streptococcus bovis , Sb-lic [Z92911]. The amino acid residues that are completely conserved are indicated in bold type. Accession numbers are given in square brackets. INDIAN J BIOTECHNOL, JULY 2007 with a number of p-1,4-endoglucanses. A short conserved segment, NEP (Asn-Glu-Pro),was found in the core region of all the aligned P-1,4- endoglucanases and this is considered to be a salient feature of family 5 glucosyl hydrolases25.On the other hand, when the endoglucanase was aligned with bacterial lichenases it was devoid of a PCD (proposed catalytic domain) block, which is a prominent characteristic of most of the bacterial lichenasesZ6and this same block is also found in a few of the fungal li~henases~~.

Substrate Specificity of Endoglucanase Recombinant endoglucanase from the heterologous E. coli host was purified by nickel &oliotriaceticacid (NiNTA) column chromatography. A protein band corresponding to the theoretical molecular mass of 39,960 was observed by SDS-PAGE, as shown in Fig. 2. The endoglucanase showed pH and temperature optima of 6.6 WSbuffer) and 70°C, respectively. T. rnaritima generates an array of glucosyl hydrolases which act specifically upon a variety of linkages such as P-1,3, P-1,4, and P-1,3-1,4-mixed linkages. The ability of this enzyme to hydrolyze P-glucan, lichenan (P- 1,3- 1,4-mixed linkages), laminarin (only P- 1,3-linkages), and CM-cellulose (only P-1,4-linkages) was examined, as shown in Fig. 3. The endoglucanase showed Fig. 2--SDS-PAGE analysis of the purified enzyme. Proteins stained with coomassie brilliant blue R-250: Lane 1, molecular negligible activity towards CM-cellulose and no activity marker; lane 2, purified cloned enzyme. was observed against crystalline cellulose and larninarin, but the enzyme was highly active towards barley P- glucan and lichenan. The specific activity of the endoglucanase was 6.1 and 6.2 Ulmg for lichenan and P-glucan, respectively. It showed a low level of specific activity against CM-cellulose (0.23 U/mg) whereas only a tiny amount of activity was detected with oat spelt xylan, at 0.005 Ulmg. No hydrolysis was observed with avicel (P- 1,4), curdlan (P- 1,3), larninarin (k1,3), pustulan (k1,6), starch (a-1,4) or birchwood xylan (P-1,4), as shown in Table 1. These observations suggest that the substrate specificity of the endoglucanase is restricted to P-1,3-1,4-mixed linkages. It is interesting to find an endoglucanase that degrades P-l,3- 1,4-mixed Fig. 3--Comparison of lichenase activity against different linkages with such a narrow substrate specificity. The substrates. P-glucan (A), lichenan (o), CM-cellulose (a), K, and values of the endoglucanase toward lichenan laminarin (0). After 0, 10, 20, 30, 45 and 60 min of incubation at Lt 70°C, the amount of reducing sugar released frcm the substrates were 0.78 mgld and 7.0 sec-l, respectively and for measured by employing Somogyi-Nelson method [ 181. P-glucan they were 0.67 mg/ml and 6.2 sec-' , respectively as shown in Table 1. TLC and HPLC. When the degradation products were spotted on a TLC plate the main product moved just Assessment of Enzyme-catalyzed Products ahead of cellotriose, as shown in Fig. 4. By HPLC, The hydrolysis products of lichenan catalyzed by the main product was identified to be glucosyl P-1,3 the endoglucanase of T. maritima were analysed by glucosyl P-1,4 gluc--y (13.82 min) as it had the same KHAN et al : THERMOTOGA MARITIMA ENDOGLUCANASE IS A UNIQUE LICHENASE 319

Table 1—Substrate specificity of endoglucanase

Substrates Backbone Specific Km kcat kca t/K m . -1 -1 . -1. -1 linkages activity (mg mL ) (s ) (mg mL s ) (U/mg) Glucan Barley β-glucan β-1,3-1,4 6.2 0.78 7.0 8.9 Lichenan β -1,3-1,4 6.1 0.67 6.2 9.2 CM-cellulose β -1,4 only 0.23 Avicel β-1,4 only 0 Curdlan β-1,3 only 0 Laminarin β-1,3 only 0 Pustulan β-1,6 only 0 Starch α -1,4 only 0 Xylan

Oat spelt β-1,4 only 0.005 Fig. 4—TLC profile of end products generated by hydrolysis of arabinoxylan lichenan by the endoglucanase. A. Markers (glucose, cellobiose, Birchwood xylan 0 β-1,4 only cellotriose, cellotetraose and cellopentaose); B. Lichenan end retention time as that of the standard glucosyl β-1,3 products; C. Standard (glucosyl β-1,3 glucosyl β-1,4 glucose). glucosyl β-1,4 glucose (13.82 min), in contrast to that On the basis of the narrow substrate specificity, the of glucosyl β-1,4 glucosyl β-1,3 glucose (11.90 min). putative endoglucanase is considered to be a In addition to glucosyl β-1,3 glucosyl β-1,4 glucose, lichenase. Lichenases are distributed across families some other oligosaccharides were detected as minor 8, 11, 12, 16 and 17 of the glucosyl hydrolases. Two products with only negligible amounts of cellobiose enzymes showing both cellulase and lichenase or glucose being observed. activity have been recorded in family 5; β-1,4- endoglucanase (EngB) from Clostridium Discussion cellulovorans 30 and Cel5A (encoded by an upstream At least three distinct kinds of endoglucanases have gene of TM 1752) which was cloned in our laboratory been reported based on their modes of action towards from T. maritima 6. However, the enzymes that β-glucans. The first type is the cellulases, which possess only lichenase activity, as this enzyme does, cleave β-1,4-linkages. The second group includes are not found in family 5 of the glucosyl hydrolases. laminarinase, which attacks both the β-1,3-bonds in Based on the amino acid sequence, it can be laminarin and the β-1,4-bonds adjacent to β-1,3-bonds concluded that the endoglucanase of T. maritima in β-glucans. The third group is more specific in that MSB8 is the first lichenase without cellulase activity these enzymes are active on lichenan and cereal to be included in family 5. The analysis of the glucans but not on laminarin and CM-cellulose 16 . degradation of lichenan clearly demonstrated that this Substrates of β-glucan and lichenan are linear chains enzyme attacks only β-1,4-glycosidic bonds in the of approximately 400 to 1200 glucose residues vicinity of β-1,3-linkages to produce glucosyl β-1,3 composed of cellotriosyl and cellotetraosyl, separated glucosyl β-1,4 glucose instead of the more usual by β-1,3 linkages and their linkages are 25% and 35% glucosyl β-1,4 glucosyl β-1,3 glucose, and it does not β-1,3-glucosidic bonds, respectively 28 . Thus β-1,3- hydrolyze the isolated β-1,3 and β-1,4-linkages. Thus, 1,4-glucanases catalyze the hydrolysis of β-1,4- the characterization of this enzyme indicates that it is glucosyl linkages only where the glucosyl residue is a thermostable lichenase with unique substrate linked at the 3-O-substituted Glc in position-129 . The specificity. The application of such a unique endoglucanase TM 1752 (AAD36817.1, Q9X274) thermostable lichenase as an industrial biocatalyst was specific for lichenan and barley β-glucan, which presents an attractive proposition because of its consists of 1,3-1,4-β-mixed linkages. No hydrolysis potential importance in carbohydrate technology. was observed with avicel, curdlan, laminarin, pustulan, starch and birchwood xylan, and negligible Acknowledgement activities were detected with CM-cellulose and oat The Science and Technology Agency (STA) spelt xylan. The present experiments clearly fellowship of the JISTEC/JST availed by Mohammed demonstrate that this enzyme belongs to the third Abdul Sattar Khan is gratefully acknowledged. This group of endoglucanases. work was supported in part by a grant from the 320 INDIAN J BIOTECHNOL, JULY 2007

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