Journal of Biotechttologv. 18 (1991) 0 1991 Elsevier Science Publishers ADONIS 016816569100088U

BIOTEC

243-254 B.V. 0168-1656/91/$03.50

243

00602

Expression of the Bacillus subtilis levanase gene in Escherichia coli and Saccharomyces cerevisiae Erich Wanker, Institut

fir

Biotechnologie, (Received

Kurt SchBrgendorfer

’ and Helmut

Arbeitsgruppe Genetik, Technische Universitiil and ’ Biochemie GmbH, Kundl, Austria 15 August

1990; revision

accepted

17 October

Schwab Graz,

Graz,

Austria

1990)

Summary

The gene coding for the inulin hydrolyzing enzyme levanase which was previously cloned from Bacillus subtilis was fused to the tat-promoter. Overexpression in Escherichia coli resulted in high amounts of intracellularly produced levanase (up to 20 U mgg’). After removal of the bacterial 5’ sequences, the levanase gene was also cloned into a yeast expression vector based on the PGK-promoter. Clones containing the intact levanase gene including the bacterial signal sequence gave rise to synthesis of active levanase by Succharomyces cereuisiae transformants. A considerable amount of levanase protein was found in the culture medium (around 0.5 U ml-‘) indicating efficient secretion of B. subtiiis levanase from yeast. Overexpression; Levanase; Escherichia coli; Saccharomyces cerevisiae; Inulin drolysis; tat-Promoter; PGK-promoter

hy-

Introduction Levanase is one of three enzymes of Bacillus subtilis involved in sucrose hydrolysis. The enzyme is secreted into the culture medium by B. subtilis (Kunst et al., 1977) and besides the hydrolysis of sucrose, it facilitates the hydrolysis of the Correspondence ro: H. Schwab, Institut fur Biotechnologie, Schlogelgasse 9, A-8010 Graz, Austria. ’ Present address: Biochemie GmbH, A-6250 Kundl, Austra. Abbreviations: bp, base pair(s); kb, kilobase pairs; kDa, kiloDalton( PAGE, polyacrylamide electrophoresis; RBS, ribosome binding site; SDS, sodium dodecyl sulfate; aa, ammo acids.

gel

244

polyfructans inulin and levan. Inulin is a reserve carbohydrate found in large quantities in numerous roots and nodules of various plants such as Jerusalem artichoke, dandelion, dahlia, and several other members of the family Composifae (Demeulle et al., 1981). Inulin has aroused wide interest as a raw material for biotechnological purposes (Vandamme and Derycke, 1983; Toran-Diaz et al., 1985). A 2.5 kb PsrI-EcoRI fragment of B. subtilis containing the levanase gene was previously cloned in Escherichia coli (Friehs et al., 1986). Expression of the levanase gene under the control of the natural B. subtilis promoter enabled E. cofi strains to grow on sucrose as the sole carbon source. However, expression in E. coli was found to be rather weak and the enzyme located mainly intracellularly. Only a small portion seemed to be secreted into the periplasm (Friehs et al., 1986). The entire 2.5 kb PstI-EcoRI levanase fragment containing the levanase gene was sequenced (Schbrgendorfer et al., 1987) and a polypeptide encoded within this fragment, with a molecular weight of 75 kDa, could be identified. The N-terminal end of the levanase polypeptide contains a signal sequence typical for secretory proteins of B. subtilis. The predicted amino acid sequence of levanase showed a particularly strong homology to yeast invertase (Martin et al., 1987; Schijrgendorfer et al., 1988). The ability of levanase to hydrolyze polyfructans is of substantial biotechnologicaI interest. Overexpression of the levanase gene to produce the enzyme in large quantities is one of the targets. A further objective is the introduction and expression of the levanase gene into various industrial micro-organisms resulting in strains which can utilize inulin as a carbon source. In this study we describe the overexpression of the B. subtilis levanase gene in E. coli and the expression of the gene in Saccharomyces cerevisiae laboratory strains. The results provide a good basis for further biotechnological utilization of the levanase gene.

Materials and Methods Strains and

plasmids

E. coli HBlOl (ATCC 33694) and E. cofi JMlOl (ATCC 33876) were used as hosts for recombinant plasmids and for Ml3 clones respectively. The S. cereuisiae strain DBY747a (uru3, trpl, feu2, his3, canR) (Botstein et al., 1979) was a gift from G. Kunze, Academy of Sciences, F.R.G. The S. cerevisiae strain W303a (ade2, ura3, trpl, feu2, canR) was obtained from S. Kohlwein, Institut fiir Biochemie, TU Graz. Plasmid pKF3 contains the levanase gene located on a 2.5 kb PstI-EcoRI B. subtifis DNA fragment (Friehs et al., 1986). pJF119HE and pJF119EH are tac-promoter based expression vectors (Fiirste et al, 1986) and were kindly provided by E. Lanka, MPI ftir Molekulare Genetik, Berlin, F.R.G. Plasmid pMA91 containing the S. cerevisiae PGK-promoter (Mellor et al., 1983) was a gift from S. and A. Kingsman, Department of Biochemistry, University of Oxford, U.K.

245

Growth media Bacterial cells were routinely grown at 37°C in LB medium (10 g 1-l bacto tryptone, 5 g 1-l bacto yeast extract, 5 g 1-l NaCl). For the selection of plasmids, the antibiotics tetracycline (15 mg I-‘) or ampicillin (100 mg 1-l) were added as required. To obtain selective growth of E. coli strains on sucrose, an M9 mineral salts medium (Miller, 1972), supplemented with essential amino acids (20 mg 1-l) and with thiamine (1 mg l-i), and containing sucrose (5 g 1-i) as the sole carbon source, was used. Sucrose was separately sterilized by filtration (0.2 pm membrane filters) to avoid hydrolysis by heat. Media were solidified by the addition of 1.5% agar. Yeast strains were usually grown at 30°C in YPD medium (1% Difco yeast extract, 2% Difco peptone and 3% glucose). For the selection of plasmid harbouring transformants, SD-Leu medium prepared according to Sherman et al. (1986) was used. To obtain selective growth on inulin, SD-Leu medium containing 5 g 1-l inulin as the sole carbon source was used. Inulin, free of low molecular weight hydrolysis products (Sigma) was dissolved in water at 85’C for 5 min and then separately sterilized by filtration. Media for yeast strains were solidified by the addition of 2% agar. Preparation of E. coli cell lysates E. coli cells were cultivated 37°C. Cells were harvested by in 1 ml citrate buffer (0.32 Labsonic 2000) on ice, with 4

in 300 ml M9-medium containing 5 g 1-l glucose at centrifugation, washed with 0.9% NaCl, resuspended M, pH 4.6) and disrupted by sonication (Braun, pulses of 30 s.

Preparation of protein fractions from yeast cultures Yeast cells were cultured in 50 ml YPD or SD-Leu selective medium containing 5 g I-’ glucose (T = 30°C 100 t-pm). Before harvesting the cells, the optical density of the cultures was determined (600 nm, Beckmann DU 50). Fractions representing different locations of synthesized proteins were prepared as follows. Preparation of L-fractions (ceil &sates) Yeast cells of 1 ml cell culture were centrifuged (3500 rpm, 5 min), washed and resuspended in 100 ~1 buffer (0.2 M Na,HPO,, 0.2 M sodium acetate, pH 5.0). 0.2 g glassbeads (0.2-0.3 mm) were added and this slurry was agitated on a vortex mixer at top speed for 1 min. After 1 min on ice, 200 ~1 buffer were added and the samples were vortexed for a further 30 s. Following centrifugation for 15 min in a microfuge (10,000 rpm), 200 ~1 of the supematant was removed and directly used for the determination of enzyme activity. Preparation of R-fractions (cell bound proteins) Yeast cells of 5 ml cell culture were centrifuged (3500 rpm, 5 min) and suspended in 0.5 ml R-buffer (0.3 M K,PO,, 8 mM L-cysteine, pH 7.2), according to Lam and

246

GrootWassink (1985). Following incubation for 30 min at 30°C, the cells were removed by centrifugation (15 min, 10,000 rpm). The supernatant was dialysed overnight at 0°C against 10 mM NaOAc (pH 5.0) before enzyme activities were determined. Preparation of S-fractions (secreted proteins) One ml cell culture was centrifuged (5 min, 3500 rpm) to remove yeast cells. 0.5 ml of the supematant was taken out, dialysed overnight at 0°C against 10 mM NaOAc (pH 5.0) and then used for the determination of enzyme activities. Determination

of levanase activity

Inulin degrading activities in E. coli lysates and different yeast determined as described by Friehs et al. (1986). Routinely, 200 ~1 fraction were mixed with 200 ~1 reaction buffer [0.2 M Na,HPO,, acetate, 100 g 1-l solid inulin (Sigma), pH 5.01. Following incubation 37’C the content of free fructose was determined enzymatically.

fractions were of the protein 0.2 M sodium for 30 min at

DNA manipulations and sequencing Standard methods as described by Maniatis et al. (1982) were used for recombinant DNA work. Specific DNA fragments were isolated from agarose gels by either binding to DEAE-membrane (NA 45, Schleicher & Schuell) and eluting with 1.5 M NaCl or by the “freeze-squeeze” method (Thuring et al., 1975). Rapid preparations of plasmid DNA from E. coli strains were carried out by using the alkaline lysis method of Bimboim and Doly (1979). Plasmid DNA for cloning experiments and Bal31 digestion was purified by CsCl gradient centrifugation in the presence of ethidium bromide. DNA sequencing was performed by the “Sequenase” method according to Tabor and Richardson (1987) using a commercial reagent kit (United States Biochemical). Restriction endonucleases and DNA modifying enzymes were purchased from Boehringer Mannheim, F.R.G., or Amersham, U.K., and used under the conditions recommended by the suppliers. E. coli and yeast transformations Transformation of E. coli cells was performed by the CaCl,-treatment method according to Cohen et al. (1972). The lithium acetate procedure of Ito et al. (1983) was used for the transformation of the yeast.

Results and Discussion Construction of levanase-tat-promoter

fusions

The 2.5 kb PstI-EcoRI B. subtilis levanase fragment was isolated from plasmid pKF3. Cloning of this fragment into EcoRI-PstI digested plasmids pJF119HE and

247

pJF119EH resulted in hybrid plasmids containing the levanase fragment in both orientations with respect to the strong inducible tat-promoter. In the plasmid pESI7HE the levanase gene is inserted in the proper orientation for transcription

Hindlll.Sphl.Pstl.Sall.Xbal.BamHI.Xmal.Kpnl.Sacl.EcoRl --

Pstl Pstl-EcoFtI

levanase

fragment I Ligation

Hindlll.Sphl.Pstl

------I

*r

pESl7HE 7.82

kb

I

Fig. 1. Construction of plasmids with the levanase gene fused to the rut-promoter. The 2.5 kb PstI-EcoRI levanase fragment was isolated from pKF3 and ligated into the PstI-EcoRI cleaved vectors pJF119HE or pJF119EH. The location of the tuc-promoter and its direction of transcription is indicated by the triangle. rrnB represents a part of the E. coli rrnB operon containing the gene for 5s RNA and its two transcriptional terminators. The p-lactamase gene bla for selection in E. co/i and the /acIQ gene responsible for regulation of the rat promoter are also marked.

248

from the strong &c-promoter, whereas in pESI7EH the gene is inserted in the opposite orientation. The cloning strategy for the plasmid pESI7HE is shown in Fig. 1.

E. coli HBlOl strains harbouring the plasmids pESI7HE and pESI7EH were tested for growth on M9-sucrose plates supplemented with 1 mM IPTG. After 2 d incubation at 37°C only clones harbouring the plasmid pESI7HE showed good growth, being much better compared to growth of strains harbouring pKF3 and indicating efficient expression of the levanase gene from the tat-promoter. With E. coli strains harbouring pESI7EH containing the levanase gene in the wrong orientation with respect to the induced &c-promoter, no growth at all could be detected. This suggests that enhanced transcription from the EcoRI end of the levanase gene by the tat-promoter might interfere with transcription of the gene from the original B. subtilis promoter. This hypothesis is supported by the fact that E. coli strains harbouring pESI7EH as well as pESI7HE grow poorly under repressed conditions (M9-sucrose plates without IPTG). Analysis

of levanase expression in E. coli HBIOI

E. coli HBlOl clones harbouring pJFl19HE, pESI7HE or pESI7EH were cultured in M9-glucose medium and cell lysates were analyzed by SDS-PAGE (Fig. 2). Levanase protein which has a size of 75 kDa (Schbrgendorfer et al., 1988) could be detected only in the lysate preparation of E. cofi HBlOl (pESI7HE) grown under induced conditions. The major proportion of the enzyme activity seems to be present in a soluble state in the cell lysates and we have no indications to suggest

s1234

Fig. 2. Analysis of proteins expressed in E. coli HBlOl clones. Crude cell extracts were prepared as described in Materials and Methods. Samples containing approximately 30 pg protein were analysed on a 12.5% SDS-PAGE. The gel was stained with Coomassie brilliant blue. The arrow indicates the levanase protein visible in lane 1. The lanes contained lysates of: 1, HBlOl (pESI7HE) induced with 1 mM IPTG; 2, HBlOl (pESI7HE) uninduced; 3, HBlOl (pJF119HE); 4, HBlOl; S, protein standard. The molecular mass (in kDa) of the proteins of the standard mixture is marked on the left side.

249

-

HBl 01 (pESl7HE) HBI 01 (pESl7HE) HBlOl(pJFl19HE)

+IPTG

I

I

I

0

1 INCUBATION

Fig. 3. Inulin degrading activities of E. co/i lysates were prepared and described in Materials and Methods. IPTG (pESI7HE) was diluted

2 TIME

cell the The with

(h)

lysates of E. coli HBlOl containing recombinant plasmids. The formation of free fructose from solid inulin was measured as lysate of E. coli which was obtained after induction with 1 mM reaction buffer 1: 500, all other lysates were diluted 1: 20.

the formation of inclusion bodies. The cell lysates were also tested for the ability to hydrolyze solid inulin in suspension. The results are shown in Fig. 3. Specific inulin degrading activities of about 20 U mg-’ protein were usually found under induced conditions. This value is approximately fifty times higher than values found in lysates where the gene was expressed from the original B. subtilis promoter (Schbrgendorfer et al., 1988). This inulin degrading activity is also relatively high in comparison with enzyme activity values usually obtained with enzyme preparations from “classical” inulinase producers. For example, in cell extracts of Debaromyces cantarellii, inulin degrading activities of about 0.75 U mg-’ were determined (Guiraud et al., 1982). Activities measured in the culture media of inulinase secreting organisms ranged from 50 mU mg-’ for Pichia polymorpha (Bajon et al., 1984) to 1 U mg-’ for Kluyveromyces fragiiis and Penicillium sp. (Vandamme and Derycke, 1983). Construction of recombinant yeast plasmids To remove the region gene, PstI cleaved DNA linear DNA-fragments BamHI-linkers according

containing the original bacterial promoter of the levanase of pKF3 was treated with Ba131 nuclease. The shortened were recircularized by addition of unphosphorylated to Seth (1984) and transformed in E. coli HBlOl (Fig. 4).

Fig. 4. Construction of recombinant yeast plasmids. Plasmid pKF3 was cleaved at the unique PsrI site and digested by exonuclease Be131 under controlled conditions. Following the addition of BumHI linkers, the DNA molecules were religated and transformed into E. co/i HBlOl. Proper deletion clones in the desired size range were selected and further characterized by DNA sequencing. For expression in yeast, the BornHI-EarnHI levanase fragments from the deletion clones pWE66 and pWE13 (Fig. 5) were isolated and ligated into the BglII site of the yeast expression vector pMA91 to obtain plasmids pMAEW66 and pMAEW13, respectively.

250

4

Pstl

4

Bal31

4 +

BarnHI-BamHI

levanase

BamHI-linkers Annealing

and transformation

in E. co/i HBlOl

fragment

ECORI

4 EcoRl \ ,Hindlll

Hindlll

Pstl

- Hindlll Sal1

Fig. 4.

251

A ATG 0

ATG

100 I

pKF3: PSfl

I I I I

200 1

300

pWE66: Barn/f/

400 I

,il I I I I I I I I I

WI I

(W c

*

pWE13:

c BamHl

pWE66::

Met ATG -

+ g/gatcqGCGAAGGAACAA

Lys Lys AAA AAG

ZbO ib!-W pWE13:

+ g/gatccgGCAAACTGG

Mel -ATG

Asn Asp AAT GAC

Fig. 5. Characterization of the deletion ends in plasmids pWE66 and pWE13. (A): the 5’ end of the levanase gene cloned in pKF3 and the positions of the deletion ends in pWE13 and pWE66 are schematically shown. (B): nucleotide sequence at the deletion ends. The nucleotides of the BumHI linker are printed in small letters. The first base of the original levanase sequence is indicated by the arrow. The first ATG codon in each insert is underlined.

Obtained levanase deletion clones were further characterized by restriction analysis and two sets of plasmids were selected according to the following criteria: (a) clones lacking the B. subtilis promoter but retaining the complete signal sequence, (b) clones lacking the entire 5’-region and the signal sequence. The nucleotide sequence at the deletion end point was determined for these clones and two recombinant plasmids were finally selected for overexpression of the levanase gene in yeast (Fig. 5). In pWE66 the levanase insert starts 12 bp upstream from the initiating ATG, retaining the signal sequence intact. In pWEl3 the levanase gene starts 9 bp upstream of the first in frame ATG following the signal sequence. The BamHI-BamHI levanase fragments of these recombinant plasmids were cloned into the Bg/II site of the yeast expression vector pMA91 (Mellor et al., 1983) as shown in Fig. 4. From the structure of the plasmids obtained, one may infer that translation initiation occurs at the first ATG codon present in each insert (underlined in Fig. 5B). The intact levanase preprotein including the complete signal peptide of 24 aa should be synthesized in the case of pMAEW66; an N-terminally truncated levanase protein lacking the signal sequence as well as the first 22 aa of the mature polypeptide is expected to be synthesized in the case of pMAEW13. Expression

of the PGK-leuanase

fusions

in yeast

Plasmids pMAEW66 and pMAEW13 and the insert-free vector pMA91 were transformed into S. cereuisiae laboratory strains W303a and DBY743a. Transformants were selected for the leucin marker on SD-Leu plates containing glucose (5 g

252

TABLE

1

Levanase

activity

in yeast

S. cereuisiae

strains

W303a(pMAEW66) DBY747a (pMAEW66) W303a (pMAEW13) W303a (pMA91)

transformants Levanase OQma 11.2 11.9 9.2 15.2

activity

[U ml-‘] L

R

S

0.111 0.282 nd nd

0.106 0.152 nd nd

0.487 0.472 nd nd

Yeast transformants were grown in 50 ml YPD medium at 30°C for 34 h (43 h for the DBY747a transformant). The levanase activity was measured in the culture fractions prepared as described in Materials and Methods. The given data for the different fractions are referred to 1 ml of the original culture. OD, optical density; nd. not detectable.

I-‘), and subsequently tested for the ability to grow on inulin as the sole carbon source (SD-Leu + inulin plates). All transformants harbouring the plasmid pMAEW66 showed good growth after 2 d incubation at 30°C, whereas transformants obtained with the plasmids pMA91 or pMAEW13 did not show significant growth. This suggests that yeast strains containing the intact levanase gene including the complete signal sequence are able to utilize inulin. Since in&n, as a high molecular weight substrate, cannot be transported into the cell, we assume that levanase is secreted at least into the periplasmic space, directed by the original signal sequence. To examine this hypothesis, yeast transformants were grown in different liquid media and inulin degrading activities were determined in the supernatant and the different cell fractions as described in Materials and Methods. The results (shown in Table 1) clearly indicate that the levanase enzyme is secreted into the culture medium to a considerable extent (S-fraction) in pMAEW66 transformants. In addition, parts of the synthesized enzyme are bound to the cell surface (R-fraction). Only a minor portion of the levanase activity can be found in cell lysates (L-fractions). With different yeast strains, there were no significant differences in expression and secretion levels. These data provide good evidence for the secretion of the levanase protein from yeast by the B. subtifis signal sequence. However, additional experiments are necessary to study the expression and secretion of levanase in yeast in more detail. Such experiments which are in progress will include replacement of the B. subtiiis signal sequence by homologous signal sequences (e.g. alpha-factor, invertase) as well as more detailed studies on the cellular localization of the levanase protein in yeast. Compared to other systems secreting inulinases, the enzyme levels measured in the supematants of cultured pMAEW66 transformants (0.47-0.49 U ml-‘) indicate a rather efficient secretion of levanase by S. cereuisiae. For example, with several thermophilic Bacillus species producing inulinase, enzyme levels between 0.073 to 0.282 U ml-’ were reported (Allais et al., 1987). Kluyueromyces fragilis is one of the most potent inulinase producers. Levels of up to 1.64 U ml-’ were reported (Negoro, 1978). With this organism however, significant amounts of enzyme are

253

usually bound to the cell surface or located in the periplasmic space (Workman and Day, 1984) and cost effective extraction methods are required for efficient recovery (Lam and GrootWassink, 1985). With yeast transformants harbouring the plasmid pMAEW13, inulin degrading activities could not be detected either in cell lysates (L-fraction) or in the culture supernatants (S-fraction). This suggests that deletion of the region coding for the signal sequence and further 22 aa at the N-terminal end of the levanase results in a situation where no active protein is synthesized. At the present state of our studies we do not have sufficient data available to determine whether expression of the deleted gene is affected or an expressed N-terminal truncated protein no longer exhibits levanase activity.

Conclusions The B. subtilis levanase gene which catalyzes the hydrolysis of polyfructans such as inulin and levan is of particular interest for biotechnological applications. Overexpression of the gene in suitable microbial hosts will be the basis for large scale production of the enzyme. Our studies demonstrated that overexpression in E. coli is a useful strategy and results in high levels of intracellularly produced levanase enzyme. However, secretion of levanase into the culture medium employing better suited host organisms would make enzyme production more efficient. We were able to demonstrate that the B. subtilis levanase gene can be expressed in S. cereuisiae hosts and that the levanase protein is secreted into the culture medium. This results in the ability of respective yeast transformants to grow on inulin as a substrate. Experiments to improve levanase secretion from yeast and to construct industrial yeast strains for ethanol production on the basis of inulin-producing crops are in progress.

Acknowledgements We would like to thank Dr. E. Lanka and Drs. S. and A. Kingsman for providing E. coli and yeast expression vectors as well as Dr. S. Kohlwein and Dr. G. Kunze for providing S. cerevisiae strains.

References Allais, J.-J., Hoyos-Lopez, G.. Kammoun, S. and Baratti, J.C. (1987) Isolation and characterization of thermophilic bacterial strains with inulinase activity. Appl. Environ. Microbial. 53, 942-945. Bajon, A.M., Guiraud, J.P. and Galzy. P. (1984) Isolation of an inulinase derepressed mutant of Pi&o polyn~orphofor the production of fructose. Biotechnol. Bioeng. 26, 128-133. Birnboim, H.C. and Daly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513-1523. Botstein, D., Falco, S.C., Stewart, S.E., Brennan, M., Scherer. S., Stinchomb, D.T., Struhl, K. and Davis,

254 R.W. (1979) Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene 8, 17-24. Cohen, S.N., Chang, A.C.Y. and Hsu, L. (1972) Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Esclrericltio co/i by R-factor DNA. Proc. Natl. Acad. Sci. U.S.A. 69, 2110-2114. Demeulle, S., Guiraud, J.P. and Galzy, P. (1981) Study of inulase from Debotyomyces phaffi Capriotti. Z. Allg. Mikrobiol. 21, 181-189. Friehs, K., Schbrgendorfer, K., Schwab, H. and Lafferty, R.M. (1986) Cloning and phenotypic expression in Escherichio co/i of a Bociflus subrilis gene fragment coding for sucrose hydrolysis. J. Biotechnol. 3, 333-341. Ftlrste, J.P., Pansegrau, W., Frank, R., Blocker, H., Scholz, P., Bagdasarian, M. and Lanka, E. (1986) Molecular cloning of the plasmid RP4 primase region in a multi-host range racP expression vector. Gene 48, 119-131. Guiraud, J.P., Bemit, C. and Galzy, P. (1982) Inulinase of Deboromyces cunrorellii. Foha Microbial. 27, 19-24. Ito, H., Fukuda, Y., Murata, F. and Kimura, A. (1983) Transformation of intact yeast cells treated with alkali cations. J. Bacterial. 153, 163-168. Kunst, F., Steinmetz, M., Lespesant, J.-A. and Dedonder, R. (1977) Presence of a third sucrose hydrolyzing enzyme in Bacillus subrilis: constitutive levanase synthesis by mutants of Bacilhu subrilis Marburg 168. Biochimie 59, 287-292. Lam, KS. and GrootWassink, J.W.D. (1985) Efficient, non-killing extraction of B-D-fructofuranosidase (an exo-inulase) from Kluyoeromyces fragilis at high cell density. Enzyme Microb. Technol. 7, 239-242. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Martin, I., Debarbouille, M., Ferrari, E., Kher, A. and Rapoport, G. (1987) Characterization of the levanase gene of Bocilluc subrilis which shows homology to yeast invertase. Mol. Gen. Genet. 128, 213-221. Mellor, J., Dobson, M.J., Roberts, N.A., Tuite, M.F., Emtage, M.F., White, S., Lowe, P.A., Patel, T., Kingsman, A.J. and Kingsman, S.M. (1983) Efficient synthesis of enzymatically active calf chymosin in Sacclraromyces cereoisioe. Gene 24, l-14. Miller, J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Negoro, H. (1978) Inulinase from Kluyueromyces /rogilis. J. Ferment. Technol. 56, 102-107. Seth, A. (1984) A new method for linker ligation. Gene Anal. Tech. 1, 99-103. Schorgendorfer, K., Schwab, H. and Lafferty, R.M. (1987) Nucleotide sequence of a cloned 2.5 kb PsrI-EcoRI Bacillus subrilis DNA fragment coding for levanase. Nucleic Acids Rcs. 22, 9606. Schorgendorfer, K., Schwab, H. and Lafferty, R.M. (1988) Molecular characterization of Bociks subrilis levanase and a C-terminal deleted derivative. J. Biotechnol. 7, 247-258. Sherman, F., Fink, G.R. and Hicks, J.B. (1986) Methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Tabor, S. and Richardson, C.C. (1987) DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 84, 4767-4771. Toran-Diaz, J., Jain, V.K., Allais, J.J. and Baratti, J. (1985) Effect of acid or enzymatic hydrolysis on ethanol production by Zymomonas mobilis growing on Jerusalem artichoke juice. Biotechnol. Lett. 7, 527-530. Thuring, R.W.J., Sanders, J.P.M. and Borst, P. (1975) A freeze-squeeze method for recovering large DNA from agarose gels. Anal. B&hem. 66, 213-220. Vandamme, E.J. and Derycke, D.G. (1983) Microbial inubnases: fermentation process, properties and applications. Adv. Appl. Microbial. 29, 139-176. Workman, W.E. and Day, D.F. (1984) The cell wall-associated inubnase of Kluyueromyces frugilis. Antonie van Leeuwenhoek; J. Microbial. Serol. 50, 349-353.

Expression of the Bacillus subtilis levanase gene in Escherichia coli and Saccharomyces cerevisiae.

The gene coding for the inulin hydrolyzing enzyme levanase which was previously cloned from Bacillus subtilis was fused to the tac-promoter. Overexpre...
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