The multidomain xylanase A of the hyperthermophilic ... - Springer Link

10 downloads 0 Views 360KB Size Report
xylan, /3-1,3-1,4-glucan and methylumbelliferyl cello- bioside (MUC) hydrolysing enzyme(s) (Dakhova et al. 1993). Here we report on the sequence of one open.
Appl Microbiol Biotechnol (1996) 45:245-247

^' Springer-Verlag 1996

SHORT CONTRIBUTION

V. Zverlov • K. Piotukh • O. Dakhova G. Velikodvorskaya • R. Borriss

The multidomain xylanase A of the hyperthermophilic bacterium Thermotoga neapolitana is extremely thermoresistant

Received: 19 May 1995/Received revision: 31 July 1995,/Accepted: 7 September 1995

Abstract The nucleotide sequence of the xynA gene, encoding extracellular xylanase A of Thermotoga neapolitana, was determined. The xynA gene was 3264 base pairs (bp) long and encoded a putative polypeptide of 1055 amino acids. Three different domains were identified by sequence comparison and functional analysis of proteins with N- and/or C-terminal deletions. The core domain displayed significant homology to members of the glycosyl hydrolase family 10. N- and C-terminal domains were dispensable for enzymatic activity and seemed to be responsible for thermostability and cellulose binding, respectively. The intact gene and its truncated variants were expressed in Escherichia coli and purified for biochemical characterization. The enzyme was shown to act as an endo-1,4-/3-xylanase, but minor activities against lichenan, barley glucan, methylumbelliferyl cellobioside and p-nitrophenyl xyloside were also detected. The specific activity and pH and temperature optima for hydrolysis of oat xylan were 111.3 U • mg ', 5.5 and 102 C, respectively. The endoxylanase was stable at 90'C and retained 50% activity when incubated for 2 h at 100°C. -

Introduction

Endoxylanase (1,4-f-D-xylan xylanohydrolase; E.C.3.2.1.8) and /3-xylosidase (1,4-/3-o-xylan xylohydrolase; E.C.3.2.1.37) are the major components of xylanolytic systems produced by many biodegradative

V. Zverlov • K. Piotukh • R. Borriss (® ) Humboldt Universitat, Institut fbr Biologic, Warschauer Str. 43, D-10243 Berlin, Germany. Fax: 49 30 5888807 V. Zverlov - 0. Dakhova - G. Vclikodvorskaya Institute of Molecular Genetics, Russian Academy of Sciences, Kurchatov Sq. 46, 123182 Moscow, Russia

microorganisms such as fungi and bacteria. On the basis of similarity in sequence comparison and hydrophobic cluster analysis, bacterial xylanases have been grouped into families 10 and 11 of glycosyl hydrolases (Henrissat 1991). The hyperthermophilic eubacterium Thermotoga neapolitana degrades a broad spectrum of carbohydrates including starch, cellulose and xylan. Recombinant clones from a genomic library encode different cellulose and hemicellulose degrading activities. Preliminary characterization of one of them, clone pTT 17, suggested that it carries a gene (or genes) encoding xylan, /3-1,3-1,4-glucan and methylumbelliferyl cellobioside (MUC) hydrolysing enzyme(s) (Dakhova et al. 1993). Here we report on the sequence of one open reading frame encoding a multidomain xylanase with wide substrate specificity. The temperature stability of the enzyme expressed in Escherichia coli exceeds that reported for other cloned thermostable xylanases.

Materials and methods E. coli DH5x [d(lctcZYA— argF), U169, endAl, recAl hsdRl7 (rk mk) deoR thi-I supE44 i. gvrA96 re/Al/F' (p80 dlacZ AM I5], vector plasmids pTZ18R, pTZ19R and pUC19 were used for subcloning procedures. Plasmid isolation, agarose gel electrophoresis, digestion of DNA with restriction enzymes, ligation and transformation of E. coli were all standard procedures (Sambrook et al. 1989). Subfragments prepared by restriction enzyme digestion from pTT 17 were cloned into vector plasmids and used for sequencing by the chain termination method (Sanger et al. 1977). The QlAexpress system (DIAGEN, Dusseldorf) was used as a tool for expression of different domains of xylanase A (XynA). DNA fragments cut at appropriate restriction sites and made blunt-ended were cloned into plasmids from the pQE series according to the protocol given by the supplier. E. coli DH5x cells transformed with recombinant plasmids encoding xylanase were grown in super broth (5 g NaCI, 12.5 g yeast extract, 25 g I ' of tryptone containing ampicillin (100 pg -ml - ') for 20 h. Xylanase was isolated from sonicated cell extracts as described previously (Olsen et al. 1991). Enzyme assays were performed at 80 C with purified protein samples in 50 mM acetate buffer, pH 5.5, supplemented with 5 mM CaCl2. using 0.5% of the -

246

terhalter et al. (1995) reported cloning and sequence analysis of a multidomain xylanase of Tt. maritima which displayed 88.7% identity with XynA from Ti. neapolitana. Examination of the primary structure of XynA showed significant similarity of its central domain `B" spanning residues 371-687 with enzymes of glycosyl hydrolase family 10 (Henrissat 1991). Surprisingly, besides the Tt. maritima enzyme, the highest similarity (58.5% identity) was found with the N-terminal catalytic domain of CelB of Caldocellum saccharolyticum (recently renamed Caldicellulosiruptor saccharolyticus, Saul et al. 1990). The C-terminal non-catalytic domain "C" has more than 80% identity with the cellulose-binding domain detected in Tt. maritima XynA. In order to characterize the function of the different domains, appropriately located restriction sites were used to delete some C-terminal regions of the gene (Fig. 1). The expressed truncated proteins AB" and AB'

appropriate polysaccharide as substrate. The appearance of reducing sugars was assayed by the dinitrosalicylic acid method (Miller 1959). One unit of enzyme activity is defined as the amount of enzyme producing 1 pmol reducing sugar (as glucose equivalents) per minute.

Results and discussion

The plasmid pTT17 with a 4.8-kb DNA from Ti. neapolitana chromosomal DNA encodes activities that degrade xylan, lichenan and MUC (Dakhova et al. 1993). The complete nucleotide sequence (the EMBL accession number of which is EMBL Z 46945, XYNA from Tt. neapolitana) was determined on both strands. The sequence revealed an open reading frame of 3264 bp corresponding to 1055 amino acid residues. The molecular mass of the enzyme deduced from the DNA sequence was 119 323 Da. This is in close agreement with the relative molecular mass of 116000 found by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Like other cellulases and xylanases the deduced primary translation product of xynA has three different domains A, B, and C which are preceded by a putative signal peptide 29 amino acids in length. Significant similarity covering the whole deduced amino acid sequence of 1055 residues was found with two representatives of glycosyl hydrolases XynX of Clostridium thermocellum (PROT SWISS entry P38853) and XynA from Thermoanaerobium saccharolyticum (Lee et al. 1993). Both enzymes share 27.4% identity and 27.9% similarity with the Thermotoga enzyme. Recently, Win1000

Fig. 1 Physical map of the xynA region of Ther•motoya neapolitana (top) and molecular architecture of xylanase A (XynA) protein and dimensions of truncated derivatives of XynA. The extent of remaining amino acid residues encoded by the subcloned specific restriction enzyme fragments is shown by filled bars. The domains identified in XynA are labelled as: SP signal peptide, A non-catalytic N-terminal domain `A' (position 30-367), B catalytic core domain `B' (position 371-687). C non-catalytic C-terminal domain `C' (696-1055), cl direct repeat Cl (position 720-851), c2 direct repeat C2 (position 895-1040). General properties of truncated proteins, namely enzymatic activity, stability and ability to bind to microcrystalline cellulose are indicated. Circles: 6 x His tags. Symbols: +, enzyme activity, thermoresistance, ability to bind cellulose; —, no enzyme activity, reduced thermoresistance, no ability to bind cellulose

bp Kpni

BamHl

BamHl

Ball Pstl EcoRV EcoRV

EcoRl

EcoRV

A 371 SP

A

alfst 600 687

B

Cl

C

1056

e2

0

0

1

AB I

AB"

I

AB

0

activity

stability

binding

ABC

1055

ABC - 6 x His

1055

+

+

+

6 x His - ABC - 6 x His

1055

+

+

+



n.d.

n.d

-

n.d

n.d

775

+

+

775

+

-

n.d.

+

-

+

572

668

248

6 x His - B

248

6 x His - BC - 6 x His

1055

247

containing either 572 or 668 N-terminal amino acids displayed no xylanase activity. Another protein, AB consisting of 775 N-terminal amino acids, was found to be fully active indicating that the catalytic domain B is intact in that protein. Sequences encoding six His tags (6 x His) adjacent to the N- and/or C-terminus were introduced on appropriate sites of the gene by taking advantage of the pQE system. Constructs containing either the B domain with residues 248-775 (6 x His—B), or the B and C domains with residues 248-1055 (6 x His—BC-6 x His) were found to be active, confirming again that the N- and the C-terminal parts of the multi domain enzyme are dispensable for enzymatic activity (Fig. 1). The characteristics of xylanase were determined in purified preparations obtained by ion exchange and molecular sieve chromatography of recombinant E. coli (pTT17) cell extracts. The specific xylanase activity of 111.3 u/mg of purified protein was relatively low. However, significant activity (2.3 u/mg and 4.0 u/mg) towards lichenan and barley glucan indicating /3-1,31,4-glucanase activity was also detected. Moreover, the enzyme displayed activity against MUC (2.8 u/mg) and p-nitrophenyl-j3- n-xyloside (0.47 u/mg) indicating cellobiohydrolase and xylosidase activity. The xylanase showed optimum activity at pH 5.5-6, with only marginal activity (less than 30%) below pH 4.5 or above 7.5. The temperature optimum was 102 C, with 50% activity at 70 C and only 20% activity at 50C. The enzyme was stable when incubated over a period of 200 min at 90 C. The half-life at 100C was 120 min. Different constructs containing domains ABC, AB, BC or B (see Fig. 1) were used to characterize the effect of different domains on thermostability of XynA. The results shown in Table 1 demonstrate the influence of the N-terminal A domain on the thermal behaviour of Thermotoga XynA. Deletion of the N-terminal A domain in the constructs 6 x His—BC-6 x His and 6 x His—B resulted in 86% loss of activity when incubated for 30 min at 80C. Deletion of the C-terminal C domain in XynA AB did not affect thermostability. The thermal stability of XynA was higher than that previously reported for xylanases cloned from any organism. Despite their highly related sequences, the xylananases cloned from Tt. neapolitana and Tt. maritima differ significantly in their thermostability. At 90C, the half-life of Tt. maritima xylanase was about 40 min (Winterhalter et al. 1995), which is significantly less than estimated for XynA of Tt. neapolitana (halflife of 200 min at 100°C). Because of its unique thermal behaviour, XynA might be an attractive candidate for some industrial applications, especially in the enzymatic treatment of paper pulp to remove xylan while preserving cellulose content. ,

Table 1 Thermostability and cellulose-binding ability of XynA and its truncated variants. (n.d. not determined) Enzyme

Thermostabilitya at: (% residual activity) 80 C

XynA ABC 89 XynA AB 88 6 x His—BC-6 x His 14 6 x His—B 14

90 C

100C

84 86 3 11

68 40 0.9 1.8

Activity bound on Avicel (%) n

27.2 2.3 58.8 n.d.

Cell extracts containing XynA or its deleted variants AB, BC and B were incubated for 30 min at the indicated temperatures in 50 mM acetate buffer, pH 5.5, supplemented with 5 mM CaCl 2 and residual activity was measured at 65 C for 10 min Binding to cellulose: cell extracts containing 1 unit xylanase activity were incubated with 10 mg Avicel PH 101, 1 mg bovine serum albumin, in I ml 50 mM acetate buffer pH 5.5. After a 1-h incubation at 37°C, the cellulose was separated and the residual activity in the supernatant was determined

Acknowledgements Dr. Wolfgang Liebl is thanked for communicating results prior to publishing. We are very grateful to Dr. Michael Yudkin for critical reading of the manuscript. The technical assistance of Carmen Hahstedt is gratefully acknowledged. This work and the stay of V.Z. in Berlin was supported by the Deutsche Forschungsgemeinschaft.

References Dakhova 0, Kurepina N, Zverlov V, Svetlichnyi V. Velikodvorskaya G (1993) Cloning and expression in Escherichia coli of Thermotoga neapolitana genes coding for enzymes of carbohydrate substrate degradation. Biochem Biophys Res Commun 194:1359-1364 Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280:309-316 Lee YE, Lowe SE, Henrissat B, Zeikus G (1993) Characterization of the active site and thermostability regions of endoxylanase from Thermoanaerohacteriurn saccharolyticum B6A-RI. J Bacteriol 175: 5890- 5898 Olsen 0, Borriss R, Simon 0, Thomsen KK (1991) Hybrid Bacillus (1-3.1-4)-/l-glucanases: engineering thermostable enzymes by construction of hybrid genes. Mol Gen Genet 225:177-185 Sambrook J. Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Sanger F. Nicklen S. Coulson AR (1977) DNA sequencing with chain termination inhibitors. Proc Natl Acad Sci USA 74:5463-5467 Saul DJ, Williams LC, Grayling RA, Chamley LW, Love DR, Bergquist PL (1990) celB, a gene coding for a bifunctional cellulase from the extreme thermophile "Culdocellum saccharolyticum". Appl Environ Microbiol 56:3117-3124 Winterhalter C, Heinrich P. Candussio A. Wich G, Liebl W (1995) Identification of a novel cellulose-binding domain within the multidomain 120 kDa xylanase XynA of the hyperthermophilic bacterium Thermotoga neapolitana. Mol Microbiol 15:431-444