Molecular cloning of the gene for β-d -xylosidase of Bacillus polymyxa and its expression in Escherichia coli

Molecular cloning of the gene for fl=D-xylosidase of Bacillus polymyxa and its expression in Escherichia coli Jasbir S. Sandhu MRC Dunn Nutrition Unit, University o f Cambridge, Milton Road, Cambridge CB4 1XJ, UK

and John F. Kennedy Research Laboratory for the Chemistry o f Bioactive Carbohydrates and Proteins, Department o f Chemistry, University o f Birmingham, Birmingham B15 2TT, UK

(Received 21 November 1985 ; revised 6 May 1986) Escherichia coli has been transformed with recombinant plasmids containing Bacillus polymyxa chromosomal DNA inserts. The resulting E. coli transformants were screened for [J-D-xylosidase activity using a spectrophotometric nitrophenyl glycoside-based assay. Two colonies gave positive results. The [J-D-xylosidase was found to have characteristics identical to the native protein, and was predominantly located in the E. coli intracellular space. The fl-D-xylosidase synthesized by the E. coli was found to hydrolyse (]~4)-fl-D-xylan oligosaccharides isolated from the mucilage Plantago ovata Forsk. These results suggest that it should be feasible to set up a large-scale degradation of (1-+4)-13-o-xylan using this enzyme. Keywords: DNA; t~-D-xylosidase;molecular cloning; Bacillus polyrnyxa xylosidase gene; Plantago ovata mucilage

Introduction

Materials and methods

Xylan, (l~4)-fl-D-xylan, is the main component of hemicellulose and is found in large amounts in agricultural wastes such as straw, hardwood and corn cobs. The functions, properties and applications of the xylanases, the enzymes which degrade xylans, have been reviewed. 1 Enzymatic hydrolysis of (l~4)-fl-n-xylan to n-xylose by Bacillus polymyxa is accomplished by sequential reactions; the first is the conversion of xylan to D-xylooligosaccharides by the extracellular en do-( 1-+4)-fl-D-xylanase (1,4-~-D-xylan xylanohydrolase, EC 3.2.1.8) followed by hydrolysis to D-xylose by the intracellular ~-D-xylosidase (see 1,4-~-D-xylan xylohydrolase, EC 3.2.1.37). Most microorganisms of industrial importance are able to metabolize n-xylose but not (l~4)-/3-D-xylan. Cloning of the DNA coding for B. polymyxa (1-~4)-/~-n-xylanase has already been reported by us. 2 To obtain /5-D-xylosidase on a large scale we decided to clone this gene from B. polymyxa, ligate it at the PstI site in pBR322 and transform Escherichia coil with this resulting plasmid. This paper describes the cloning of the /3-D-xylosidase gene from B. polymyxa into E. coli using the plasmid pBR322. The fl-o-xylosidase from E. coli was purified and found to be active against the various relevant substrates tested.

Bacterial strains, plasmids a n d m e d i a The strain ofB. polymyxa (NCIB 8158; ATC 842) was grown in penassay broth (Difco Laboratories) at 30°C with agitation; its growth on minimal media has already been described in detail? E. coli C600 was used as the cloning host and was grown in Luria Bertoni LB-X composition given for 1 litre: I0 g bacto tryptone, 5 g yeast extract, 5 g NaC1; adjusted to pH 7.0 (with 2 M NaOH) and grown at 37°C.

0141--0229/86/110677--04 $03.00 © 1986 Butterworth & Co. (Publishers) Ltd

E n z y m e s and reagents The plasmid pBR322 was purchased from Bethesda Research Laboratories (BRL, Cambridge, UK).T4 DNA ligase [polydeoxyribonucleotide synthetase-(ATP), poly(deoxyribonucleotide): poly(deoxyribonucleotide) ligase (AMPforming), EC 6.5.1.1], lysozyme (mucopeptide N-acetylmuramoylhydrolase, EC 3.2.1.17), restriction endonucleases (BamHI, endodeoxyribonuclease, EC 3.1.23.6) and calf intestinal alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] were also from BRL and were used under the conditions outlined by the supplier. 4-Nitrophenyl/3-D-xylopyranoside, D-xylose, ampicillin and tetracycline were obtained from Sigma (UK).

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Papers Spectrophotometric ~-O-xylosidase assay The activity of/3-D-xylosidase was measured with 4-nitrophenyl /3-D-xylopyranoside (1 mg m1-1 ) in 55 mM phosphate buffer, pH 7, as substrate. The reaction mixture consisting of 1 ml substrate solution and 1 ml of diluted enzyme was incubated at 37°C for 15 min. The reaction was terminated by the addition of 0.4 M Na2CO~ (2 ml) and the absorbance of 4-nitrophenol released was measured at 405 nm. One unit of enzyme activity was defined as the amount of enzyme capable of releasing 1 pmole of 4-nitrophenol in 1 min under the above reaction conditions.

Preparation o f cell ex tracts E. coil cells, grown in LB plus 0.1% w/v D-xylose with the appropriate antibiotics were isolated by centrifugation and washed with 50 mM phosphate buffer (pH 7) containing 1 mM EDTA. The cells were suspended in the same buffer and lysed by sonication at 20 MHz for 3 rain at 0°C. The extracts were then centrifuged at 10000 g for 10 rain and the supernatant was used for enzyme assay of /3-Dxylosidase, or for column chromatography.

Localization formants

o f ~-D-xylosidase in E. coli trans-

Cell extracts were prepared (at 6 and 24 h intervals) by the modified procedure of Tsukagoshi et al. 3 E. coli (harbouring recombinant plasmids coding for 13-D-xylosidase) were centrifuged (10 000 g for 10 min at 25°C). The resulting cell pellet was washed twice with 10 mM Tris-HC1 buffer (pH 7) containing 30 mM NaC1 and was osmotically shocked as described by Anraku and Heppel. 4 The periplasmic enzymes were recovered in the supernatant fraction after centrifugation. The shocked cells were suspended in 20 mM potassium phosphate buffer (pH 7.3). After washing with the same buffer solution they were sonicated at 20 MHz for 3 rain. The supernatant obtained by centrifugation at 10000 g for 10 min was further subjected to centrifugation at 20000 g for 1 h and the cytoplasmic and membrane fractions were recovered in the supernatant and pellet respectively. An alternative procedure to fractionate the bacterial cells was also used. This consisted of treatment of the bacterial cells for 25 rain at 37°C with lysozyme (2 mg m1-1) (Sigma, UK) in 10 mM Tris-HC1 buffer (pH 8) containing 20% w/v sucrose and 10 mM EDTA. The resulting spheroplasts were recovered in a pellet by centrifugation at 10000 g for 10 min and resuspended in 10 mM TrisHC1 buffer (pH 8) to cause the cells to burst. Cytoplasmic and membrane fractions were obtained as already outlined.

Prepara tion o f an tisera B. polymyxa was grown to a late log phase in LB. The bacterial cells were removed by centrifugation, and the resulting pellet was washed with 50 mM phosphate buffer (pH 7) containing 1 mM EDTA. The cells were suspended in the same buffer and sonicated as already outlined. The extract was centrifuged 10000 g for 10 min remove the debris. The supernatant was fractionated with ammonium sulphate (55-85% s~turation) followed by dialysis against 50 mM phosphate buffer (pH 7). The dialysate (3 ml, containing 200 mg protein) was applied to a column of Sephadex G-100 (1.5 cm x 100 cm) previously equilibrated with 50 mM phosphate buffer (pH 7). The column was eluted with the same phosphate buffer and 2 ml fractions were collected. The elution of protein was monitored by measuring absorbance at 280 nm and alternate fractions were

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assayed for /3-D-xylosidase activity as already outlined. A /3-D-xylosidase activity peak was detected in fractions 5 5 80 and these were pooled and concentrated by ultrafiltration (Amicon Diaflo membrane PM 10). The second stage of purification of the t3-D-xylosidase was then performed by chromatography on DEAE-Sephadex as outlined by us previously. 2 The fractions containing the 13-Dxylosidase activity were pooled and concentrated by ultrafiltration as outlined before. Anti-/3-D-xylosidase antibody synthesis was induced in New Zealand White rabbits by injection of purified /3-Dxylosidase (7.8 mg) emulsified in Freund's adjuvant. After three more injections of antigen over a period of 2.5 months, the rabbits were bled. The antibody was purified by ammonium sulphate precipitation, followed by DEAEcellulose chromatography. Final purification was achieved by anti-/3-D-xylosidase antibody affinity chromatography, s The anti-fl-D-xylosidase antibody was chromatographed on protein A immobilized on Sepharose CL-4B. 6

Immunoadsorption by E. coli

o f (~-D-xylosidase synthesized

Normal serum was bound to protein A-Sepharose CL-4B column. The column was washed with PBS (phosphate buffered saline: NaC1, 8 g 1-1 ; KH2HPO4"7H20, 0.2 g 1-1 , pH 7.4), then E. coil (pXYL 1) ~3-D-xylosidase (450 units) was loaded onto the column. The eluate was collected (1.5 ml fractions) and assayed spectrophotometrically for /3-Dxylosidase activity using 4-nitrophenyl ~-D-xylosidase as substrate. The column was then eluted with 1 M glycineHC1 buffer (pH 3.5) and re-equilibrated with PBS. The experiment was repeated using anti-t3-D-xylosidase serum instead of normal serum (see Figure 2).

Isolation o f DNA B. polymyxa DNA was prepared by the modified procedure of Dubnau and Davidoff-Abelson. 7 The phenol-treated DNA was extracted with ether, and ethanol precipitated twice. Finally the DNA was resuspended in 10 mM TrisHC1 (pH 7.5) containing 4 mM NaC1 and 0.1 mM EDTA and dialysed against the same buffer (three changes over 24 h). Plasmid DNA suitable for cloning was prepared as follows. Chloramphenicol (160 ~g m1-1) was added to an exponential phase culture of E. coil. The cells from the 400 ml culture were obtained by centrifugation, washed with 50 mM Tris-HC1 (pH 8) containing 1 mM EDTA and frozen at -70°C for 1 h. The frozen pellet was resuspended in 25 ml of a solution containing 25% w/v sucrose in 50 mM TrisHC1 (pH 8.0). Lysozyme (1 mg m1-1) was added followed by 250 mM EDTA (1 ml) and the suspension was placed on ice for 15 rain. Cells were lysed by the addition of 150 mM Tris-HC1 (pH 8.0) (13 ml) containing 0.3% w/v Triton X-100 and 200 mM EDTA. After incubation for 20 rain, RNA was removed by digestion at 37°C for 30 min with RNase (1 mg, boiled in 10 mM Tris-HC1 (pH 7.5) containing 1 mM EDTA (0.1 ml) for 20 rain to inactivate the DNase). The lysate was centrifuged at high speed (18 000 rev/min) for 1 h at 4°C. The supernatant was extracted twice with phenol, once with ether, and ethanol precipitated. The precipitated plasmid DNA was resuspended in 10 mM Tris-HC1 (pH 8.0) containing 1 mM EDTA and further purified by banding in CsCl-ethidium bromide density gradient. Mini plasmid preparation was performed according to the procedure of Birnboin~ and Doly. 8

Molecular cloning of gone for fl-D-xylosidase: ,I. S. Sandhu and J, F. Kennedy

Cloning procedures Plasmid pBR322 was cleaved with restriction endonuclease PstI and then treated with calf intestine alkaline phosphatase. Digestion was terminated by phenol and ether extraction. B. polymyxa chromosomal DNA was partially digested with PstI under the conditions described by the suppliers. The restriction digestion was terminated by phenol and ether extraction. The degree of DNA digestion was monitored by horizontal agarose gel electrophoresis. DNA molecules were separated on a 0.75% agarose gel in a running buffer consisting of 40 mM Tris-HC1 containing 30 mM sodium acetate and 1 mM EDTA (pH 8.0), the DNA was visualized by staining with ethidium bromide, according to Sharp etal. 9 A 2 ~g sample of linearized pBR322 DNA was mixed with 6 and 8 #g of PstI partiall3) digested B. polymyxa DNA. The ligation reaction was achieved with T4 DNA ligase overnight at 16°C in 50 mM Tris-HC1 (pH 8.0) containing 10 mM magnesium sulphate, 10 mM dithiothreitol and 1 mM ATP. Transformation of E. coli C600 was performed as described by Kushner. x° Transformants that were tetracycline resistant and ampicillin sensitive were selected and screened for /3-D-xylosidase-positive clones.

Digestion o f (l-~4)-/3-D-xylan oligosaccharides with /3-D-xylosidase (1-+4)-/3-D-Xylan oligosaccharides, particularly O- [~-Dxylopyranosyl-(l~4) ] -O-/3-D-xylopyranosyl-D-xylose (xylotriose), were prepared from the partially acid digested mucilage of Plantago ovata Forsk. Previous studies by us have shown that the mucilage is a complex polysaccharide possessing a (1-+4)-/3-D-xylan backbone, a1,12 The reaction mixture for the demonstration of /3-Dxylosidase activity contained 0.5 rrd of appropriately diluted enzyme solution and 1 ml of substrate D-xylooligosaccharides (4 mg m1-1) in 55 mM phosphate buffer (pH 7). The incubation was performed at 37°C for 2 h, after which the increase in reducing sugar produced was estimated by the Nelson-Somogyi x~'t4 procedure on the entire digest and the results were expressed in terms of D-xylose equivalent, i.e. the amount of D-xylose giving the same reducing sugar value. Blank values were obtained for the enzyme solution that had been inactivated by heating for 10 min at 100°C and for the non-contacted Dxylo-oligosaccharide substrate. One unit of/3-o-xylosidase activity is defined as the amount of enzyme required to release 1 /amole of o-xylose in 1 min from xylotriose at pH 7 and 37°C. Protein concentrations of the enzyme solution were determined by the Lowry is procedure.

Results The chromosomal DNA from B. polymyxa was isolated and partially digested with PstI. Fragments ranging in size from

Figure

1 Restriction map of the D N A fragment f r o m E. co/i ( p X Y L 1) coding f o r /3-D-xylosidase. Symbols; Pstl (e) H i n d l l l ( , ) Pvull (m) and Xbal (v). The scale at the b o t t o m is kb

4

T

"ID

C~

0

2

4

6

8

lwO

Fraction number

Figure 2

Immunoadsorption of E. coli ( p X Y L 1) fl-D-xylosidase by anti-fl-D-xylosidase sera and normal sera. (a) Normal serum was bound to a protein A-Sepharose CL-4B column. The column was washed with PBS and E. coli ( p X Y L 1) extract containing fl-Dxylosidase (450 units) loaded. The eluate was collected (1.5 ml fractions) and assayed for fi-D-xylosidase activity using the substrate 4-nitrophenyl /3-D-xylosidase (e). The column was then eluted with glycine-HCI (pH 3) and equilibrated with PBS. (b) The experiment was repeated using anti-fl-D-xylosidase serum instead of the normal serum (A)

4 to 8 kb were generated for cloning experiments. To increase the yields of recombinants, a 3 - 4 molar excess of partially digested chromosomal DNA was used during the ligation step. Chimeric plasmids containing PstI-digested DNA inserts transformed E. coli C600 cells with an efficiency of 3.2 x 10 a Tc(r)transformants per/ag of total DNA. About 10 s total transformants were obtained. After replica plating of the PstI-transformants on Ap-Tc plates, approximately 18% of the clones were Ap(s) indicating insertion of a Bacillus polymyxa DNA. The host cells E. coli C600 showed no detectable activity of fl-D-xylosidase even after the cells were lysed. The Ap(s) Tc(r) clones of E. coli C600 transformed with the newly constructed hybrid plasmids, consisting of pBR322 and the chromosomal DNA fragments of B. polymyxa, were tested for their fl-D-xylosidase productivity. Two clones among the 850 tested were found to produce the yellow pigment by incubation of the cell extract with 4-nitrophenyl fl-Dxylopyranoside for several hours. The plasmids in these clones were named pXYL 1 and pXYL 2. The two plasmids were isolated and digested with restriction enzymes to determine the size of the inserts (restriction map for the DNA insert in plasmid pXYL 1 is shown in Figure 1). Rabbit antiserum towards /~-D-xylosidase was prepared using the /3-D-xylosidase purified from B. polymyxa. This serum was bound to a Sepharose CL-4B-protein A column On passing the E. coli (pXYL 1) cell extracts containing active/3-9-xylosidase through this column it was found that the /3-D-xylosidase activity was removed (Figure 2). However, elution of the material bound by the column with glycine buffer was found to have activity towards the /3-o-xylosidase substrates. In contrast, normal serum bound

Enzyme Microb. Technol., 1986, vol. 8, November

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Papers Table 1 Level and localization of/~-D-xylosidase in E. coil ( p X Y L 1 ). Bacterial ceils were harvested at 6 and 24 h intervals. The levels of ~-D-xylosidase in extracellular, periplasmic and cytoplasmic fractions were determined as described in the text Time (h) 6 Extracellular Periplasm Cytoplasm

24

11

44

1

9

49

220

The values represent units of ~-D-xylosidase activity per mt of extract

to protein A-Sepharose CL-4B did not remove the t3-D-xylosidase activity from the E. coli (pXYL 1) extract. The results of the localization studies of 13-D-xylosidase are shown in Table 1. These data show that 13-D-xylosidase is predominantly found in the intracellular space of E. coli (pXYL 1), no 13-D-xylosidase was detected in the growth medium. The E. coli 13-D-xylosidase was found to be active not only against the 4-nitrophenyl t3-D-xylopyranoside substrate, but also against the (l~4)t3-D-xylan oligosaccharides isolated as above from the seed mucilage of Plan tago ovata Forsk.

not only against 4-nitrophenyl t3-D-xylopyranoside but also against (l~4)-t3-D-xylan oligosaccharides purified from the seed mucilage of Plantago ovata Forsk, confirming that the enzyme is active against (l+4)-13-D-xylan oligosaccharide. Confirmation that the /3-D-xylosidase synthesized by B. p o l y m y x a is identical to the 13-D-xylosidase produced by E. coli was obtained by immunoadsorption studies. These showed that rabbit anti-t3-D-xylosidase (from B. polymyxa) antiserum immobilized on a Sepharose column could eliminate 13-D-xylosidase activity from E. coli cell extracts. This demonstrates that the major epitopes present in the B. polymyxa ~-D-xylosidase are conserved in the ~3-D-xylosidase synthesized by E. coll. The fl-D-xylosidase localization studies show that the enzyme is mainly located within the E. coli celt. However we envisage that, for a large-scale economical purification procedure to be devised, it may be necessary to add a peptide sequence to the /~-D-xylosidase so that the active enzyme will be translocated into the periplasmic space of E. coll. This would also have the added advantage that if the 13-D-xylosidase was toxic to the E. coil at high levels of expression it could be removed from the intracellular space before the bacterial cells were debilitated. Dr D. Lonsdale is thanked for advice on the construction of genomic DNA libraries, and Miss L. Bowyer for her technical assistance.

Discussion

References

At present there is a great deal of interest in the use of microbes to degrade plant materials such as cellulose and hemicellulose. The aim of this project is to develop a commercially viable procedure to degrade the hemicellulose group of polysaccharides to their constituent monosaccharides. This could be achieved by cloning DNA coding for the microbial enzymes involved in the depolymerization of these polysaccharides by recombinant DNA techniques, and then to ligate these DNA sequences to a strong promoter in order to obtain high levels of expression of these proteins in E. coll. In accordance with this strategy we have cloned the DNA sequence coding for the enzyme (I~4)-13-Dxylanase 2 since (l~4)-13-D-xylan is a major component of the hemiceUulose group of polysaccharides. However, conversion of (l~4)-/3-D-xylan to D-xylose requires at least two enzymes. The (l~4)-t3-D-xylanase converts (1-+4)-13-Dxylan polymer to its oligosaccharide form, then 13-D-xylosidase is required to convert the xylan oligosaccharides to D-xylose. The screening method adopted to clone the DNA sequence coding for 13-D-xylosidase has the major advantage that it allows detection of only those E. coil cells that produce the /3-D-xylosidase in its active form. The enzyme activity of the ~3-D-xylosidase was established

1 Woodward, J. in Topics in Enzyme and Fermentation Biotechnology, (Wiseman, A., ed.) Ellis Horwood, Chichester, 1984, vol. 8, p. 9. 2 Sandhu, J. S. and Kennedy, J. F. Enzyme Microb. Technol. 1984, 6,271 3 Tsukagoshi, N., Ihara, H., Yamagata, H. and Udakae, S. Mol. Gen. Genet. 1984, 193, 58 4 Araku, Y. and Heppel, L. A. J. Biol Chem. 1967, 242, 2561 5 Livingstone, D. M.MethodsEnzymol. 1974, 34,723 6 Affinity Chromatography Principles and Methods Pharmacia Fine Chemicals, Uppsala, 1981, p. 47 7 Dubnau, D. and Davidoff-Abelson, R. J. Mol. Biol. 1971, 56, 209-221 8 Birnboim, H. C. and Doly, J. Nucleic Acids Res. 1979, 7, 1513 9 Sharp, P. A., Sugden, B. and Sambrook, J. Biochemistry 1973, 12, 3055 10 Kushner, S. A. in Genetic Engineering (Boyer, H. B. and Nicosia, S., eds) Elsevier, Amsterdam, 1978, p. 17 11 Kennedy, J. F., Sandhu, J. S. and Southgate, D. A. Carbohydr. Res. 1979, 75,265 12 Sandhu, J. S., Hudson, G. J. and Kennedy, J. F. Carbohydr. Res. 1981, 93,247 13 Somogyi, M. J. Biol. Chem. 1945, 160, 160 14 Southgate, D. A.Determination of Food Carbohydrates Applied Science, Barking, 1976 15 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. J. Biol. Chem. 1951, 193,265

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