Gene. 96 (1990) 89-93 Elsevier

89

GENE 03824

Isolation and characterization of ievansucrase-encoding gene from B a c i l l u s amyloliquefaciens (Phagemid; recombinant DNA; sucrose regulation; signal sequence)

Leslie B. Tang, Reijer Lenstra, Torben V. Borehert and Vasantha Nagarajan Central Research and Development Division. E.L duPont de Nemours Company, Wilmington, DE 19880.0228 (U,S.A.) Received by R.E. Yasbin: 29 May 1990 Revised: 14 August 1990 Accepted: 17 August 1990

SUMMARY

The gene encoding levansucrase (LVS) from Bacillus amytoliquefaciens (sc~gB[BarnP]) wa~ isolated, sequenced and expressed in Bacillus subti~lis. Analysis of the nucleotide sequence of sacB[BamP] reveals extensive homology with that of the B. subtilis LVS-encodiag gene in the promoter and coding region. The sacB[BamP] gene cloned in a multicopy plasmid is induced by sucrose in B. subtilis.

INTRODUCTION

Sucrose metabolism in B. subtilis has been extensively studied by several laboratories (Lepesant etal., 1976; Steinmetz ¢tal., 1985; Shimotsu and Henner, 1986). Levansucrase (sucrose: 2,6-/kD-fructan-6-/~-D-fructosyltransferase.: EC 2.4.1.10) is secreted by B. subtilis upon addition of sucrose and seems to be regulated by multiple pathways (Shimotsu and Henner, 1986; Steinmetz et al., 1989). Several B. subtilis mutants that express LVS constitutively have been isolated and these mutations are located in three unlinked loci: (a)sacR which is a cis-regulatory region of sacB (structural gene for LVS) (Shimotsu and Henner, 1986); (b)sacS which codes for two genes, sacX and sac Y. Whereas sacX shares extensive homology to pts l ,

sacY is homologous to an Escherichia colt antiterminator protein BglG (Ayermich and Steinmetz, 1987; Debarbouilie et al., 1987; Steinmetz et al., 1988; Zukowski et al., 1988;) (c)ptsl which codes for phosphotransferase enzyme 1 (Crutz et al., 1990). In addition, the expression of sacB is also modulated by several unlinked genes such as degU and degQ that seem to affect most of the degradative enzymes in B. subtilis (Henner et al., 1988). The sacB gene from B. amyloliquefaciens was isolated to study the interaction &the various B. subtilis gene products such as sacS, degU) degR, degQ and ptsl with a heterologous LVS regulatory region. We report in this paper the isolation of the sacB[BamV] gene and its expression in B. subtilis.

RESULTS .'~ND DISCUSSION

Correspondence to: Dr. V. Nagarajan, E228/Rm 310C, Du Pont Co., P.O. Box 80228, Wilmington, DE 19880-0228 (U.S.~,.), Tel. (3027695-3984; Fax (302)695-9 ! 83. Abbreviations: aa, amino acid(s); Ap, ampicillin; B., Bacillus; bp, base pair(s); kb, kilobase(s) or 1000 bp; LVS, levansucrase; nt, nucleotide(s): oligo, oligodeoxyribonucleotide; ORF, open reading frame; R, resistance/resistant; RBS, ribosome-binding site; sacB, gene encoding LVS; sacB[BamP], gene encoding LVS from B. amyloliquefaciens; sacR[BamP], regulatory region of sacB [BamP]; tsp, transcription start point; [ ], denotes plasmid-carrier state. 0378-1119/90/$03.50 © 1990 Fl,~e~J~erScience Publishers B.V. (Biomedical Division)

(a) Isolation of the sacB[BamP] gene A partial EcoRl library of B. amyloliquefaciens ATCC23844 in ,~Zap (Stratagene) was screened with two oli8os, 5'-GACGTTGGACAGCTGGCCATTACAAAC and 5'-ATGAACGGCAAATGGTACCTG'FI'CACTGAC, which had been synthesized based on tile published B. subtilis sacB gene (Steinmetz et al., 1985). The two positive phage clones were converted to plasmids designated as

90

pBE300 and pBE301 using helper phage IRI. Comparison of EcoRl restriction analysis revealed that pBE300 contained a 1.5-kb insert and p~,E301 contained a 2.3-kb insert consisting of a 1.5-kb and 800-bp EcoRl fragments. The sacB oligo probe hybridized to the 1.5-kb fragment. The restriction map of pBE301 is shown in Fig. 1.

E

0

~

1 E

E

0

I

1 H

P

~

,.I H E

sacB[BamP] Fig. 1. R e s t r i c t i o n m a p o f p l a s m i d pBE301 containingsacB [BamP].T h i n line,vector;blackenedbox;s,::cB [Bame] ; z i g - z a g l i n e , B . amyloliquefaciens

DNA.B,BamHI;E, EcoRI, H,HindlII;M,Smal;P,PstI, gN,EcoRV;X, Xba.

(b) Nucleotide sequence determination Plasmid pBE301 was digested with various restriction enzymes and subcloned into both M13mpl8 and M13mpl9. Several independent and overlapping clones were sequenced resulting in a contigue of 2350 bp (Fig. 2A). Analysis of the nt sequence revealed the presence of a promoter sequence that can be recognised by B. subtilis RNA polymerase Ea A and a Gram + RBS (McLaughin etal., 1981; Moran etal., 1982). The nt sequence also suggested the presence of a stem-loop structure with a free energy of 24.5 kcal between the putative promoter and the RBS (Fig. 2B). Computer analysis of the nt sequence revealed the presence of a large ORF which coded for a mature protein containing 443 aa and a signal peptide coding for either 29 or 31 aa. However, only codon -29 had the correct spacing between the RBS and the start codon.

A GAATTCCTTCAGGAAAAGAACGATGGCTGTCTTATTAGCGGTTGCAGGcAcATTT&TTTTGGTcAcACAcGGGAATGTcGGCAGCCTGTcTATAT~CGGTCT •GCTGTTTTTTGGGGCATCAGCTCGGCATTTGCGCTGGcGTTTTA•AC•CTC•AGccG•ATCGG•TTTTGAAGAAATGGGGcTc•Gc•ATTATTGTcGGATGGGG•ATGCTGATGCGGAG CCGTTCTCAGCcTGATT~AGC~GC~TTGGAAGTTTGAAGGCCAATGGTCGTTGTCcG~ATATGc~GcGAT~GTGTTTATcATcATTTT~GGAA~GcTcATcGCTTTTTATTGCTATTTGG AAAGCCTGAAATATCTGAGTGccTcTGAAA•CAG••TccTcGccTGTG•AGAG•cG•TGTcAGcAGcTTTTTTAGCGGTGATcTGG•TGcATGTTcccTTcGGAATATcAGAATGG•TGG ~TACTTTA~TGATTTTAGccA~ATCGCTTTATTAT~TATcAAGAAAAAATAA~cTcTcTTTTTTA~AGAGGTTTTTcccTAGGccTGAAG~AcccTTTAGTcTcAATTA~ccATAAATT AAAAGGCcTTTTTTCGTTTTAcTAT~ATT~AAAAGAGGAAAATAGAccAGTTGTcAATA~AATcAGAGTcTAATAGAATGAGGTcGAAAAGTAAATcAcG~AGGATTGTTAcTGATAAAG CAG~CAAGA~CTAAAATGTGTTAAGGG~AAAGTGTATTcTTTGGcGTcATc~TTAcATATTTTGGGTcTTTTTTT~TGTAAcAAAc~TGccATCcATGAATTcGGGAGGAT~GAAACGG CAGATCGCAAAAAACAGTACATACAGAAGGAGACATGAAC ATG AAC ATC AAA AAA ATT GTA AAA CAA GCC ACA GTT CTG 901 me~ ash ~le lyS lys ile val lys gln ale ~hu val leu -29 ACT TTT ACG ACT GCA CTT CTG GCA GGA GGA GCG ACT CAA GCC TTC GCG AAA GAA AAT AAC ~h~ phe ~hr ~hr ale leu leu ala gly gly ale thr gln ale phe ale lys ~lu asn ash -1

+1

CAA gln

AAA lys

G~A a!a

TAC ~yr

AAA lys

GA~ glu

ACG thr

TAC tyr

GGC gly

GTC val

TCT set

CAT his

ATT ile

ACA ~h~

CGC arg

CAT his

GAT asp

ATG met

CTG leu

CAG gln

ATC ile

CCT pco

AAA iys

CAG gln

C~G gln

CAA gln

AAC ash

GAA glu

AAA lys

TAC ~yr

CAA gln

GTG val

CCT pro

CAA gln

TTC phe

GAT aap

CAA gln

TCA see

ACG th~

ATT ile

AAA lys

~AT a~n

ATT ile

GAG glu

TCT ~eE

AAA lye

GGA gly

CTT leu

GAT asp

GTG val

TGG ~=p

GAC asp

AGC see

TGG ~rp

CCG ~:o

CTG leu

CAA gln

AAC ash

GCT ala

GAC asp

GGA gly

ACA ~hr

GTA v~l

GCT ala

GCA ale 50 GAA glu

TAC ~yr

AAC asn

GGC gly

TAT tyr

CAC his

GTT val

GTG val

TTT phe

GCT ala

CTT leu

GCG ale

GGA gly

AGC set

CCG pro

AAA lys

GAC asp

GCT ale

GAT asp

GAC asp

ACA ~hr

TCA set

ATC ile

TAC tyr

ATG met

TTT phe

TAT tyr

CAA gln

AAG lys

GTC val

GGC gly

GAC a3p

AAC ash

TCA se~

ATC £1e

CAC asp

AGC set

TGG ~rp

AAA lys

AAC ash

GCG ale

GGC gly

CGT arg

GTC val

TTT phe

AAA lys

GAC asp

AGC se=

GAT asp

AAG lys

TTC phe

GAC asp

GCC ale

AAC ash

GAT asp

CCG p~o

ATC ile

CTG leu

AAA lys

GAT asp

CAG gln

ACG thr

CAA gln

GAA glu

TGG ~rp

TCC se=

GGT gly

TCT se=

GCA ale

~CC thr

TTT phe

ACA thr

TCT set

GAC asp

GGA gly

^~ lys

~TC ile

CGT arg

TTA leu

TTC phe

ACT thr

G~C asp

TAT tyr

TCC ser

GGT gly

AAA lys

CAT his

TAC tyr

GGC gly

AAA lys

CAA gln

AGC ser

CTG leu

ACA thr

ACA thr

GCG ale

CAG gln

GTA val

AAT asn

TAC ~yr 150 GTG val

TCA set

AAA lys

TCT set

GAT asp

GAC asp

ACA thr

CTC leu

AAA lys

ATC ile

AAC ash

GGA gly

GTG val

GAA glu

GAT asp

CAC his

AAA lys

ACG thr

ATT ile

TTT phe

GAC asp

GGA gly

GAC asp

GGA gly

AAA lys

ACA thr

TAT tyr

CAG gln

AAC a~n

GTT val

CAG gln 200

CAG gln

TTT phe

ATC ile

GAT asp

I02

342 582 822

1021

1141

1261

100

1381

1501

91 GAA glu

GGC Ely

AAT ash

TAT ty¢

ACA thr

TCC set

GGC Ely

GAC asp

~C asn

CAT his

ACG thr

CTG leu

AGA are

GAC aap

CCT pro

CAC his

TAC tyr

GTT val

GAA glu

GAC asp

AAA lys

GGC Ely

CAT his

AAA lys

TAC tyr

CTT leu

GTA val

TTC phe

GAA glu

GCC ala

AAC ash

ACG thr

GGA Ely

ACA tht

GAA glu

AAC asn

GGA Ely

TAC ryE

CAA gln

GGC gly

GAA glu

GAA glu

TCT see

TTA leu

TTT phe

AAA lys

GCG ala

TAC tyr

TAC rye

GGC Ely

GGC gly

GGC 91y

ACG thr

AAC asn

TTC phu

TTC phe

CGT a~

AAA lys

GAA glu

AGC ser

CAG gln

AAG lys

CTT leu

CAG gln

AAC asn 250 CAG gln

AGC see

GeT ala

AAA lys

AAA lya

CGC aEg

GAT asp

GCT ala

GAG glu

TTA leu

GCG ala

AAC ash

GGC Ely

GCC ala

CTC leu

GGT Ely

ATC ile

ATA ile

GAG glu

TTA leu

AAT asn

AAT ash

GAT asp

TAC rye

ACA the

TTG l~u

AAA lys

AAA lya

GTA val

ATG met

AAG lys

CCG pro

CTG leu

ATC ile

ACT thr

TeA set

AAC ash

ACG thr

GTA val

ACT the

GAT asp

GAA glu

ATe ile

GAG glu

CGC are

GCG ala

AAT ash

GTT val

TTC phe

AAA lys

ATG me~

AAC ash

GGC Ely

AAA lys

TGG trp

TAC rye

TTG leu

TTC phe

ACT the

GAT asp

TC& see

CGC age

GGT Ely

TeA see

AAA lys

ATG m~t

ACG the

ATC ile

GAT asp

GGT Ely

ATT ile

AAC aan

TeA sex

AAC asn

GAT asp

AT ~

TAC tyr

ATG met

CTT leu

GGT 91¥

TAT tyr

GTA val

TeA see

AAC ash

TCT set

TTA leu

Ace thr

GGC 91y

CCT pro

TAC tyr

AAG lya

CCG pro

CTG leu

AAC aan

AAA

ile ACA thr

GGG Ely

CTT leu

GTG val

CTG leu

CAA gin

ATG met

GGT Ely

CTT leu

G&T asp

CCA~ p¢o

ARC ash

GAT asp

GTG val

ACA the

TTC phe

ACT Chc

TAC rye

TCT set

CAC his

TTC phe

GCA ala

GTG val

CCG p~o

CAA gin

GCC ala

AAA 178

GGC Ely

AAC ash

AAT ash

GTG va~

GTT val

ATe £1e

ACA thr

AGC see

TAC tyr

ATG met

ACA thr

AAC asn

AGA arg

GGC g1¥

T~C ph~

TTC phe

G&G giu

GAT asp

AAA I¥s

AAG lys

GCA ala

ACA the

TTT phe

GGC Ely

CCA p~o

AGC a~r

TTC phe

TTA leu

&TG met

AAC ~sn

ATC ile

AAA lys

GGC 2221 91¥

AAT n3n

ARA lya

ACA the

TCC set

GTT yak

GTC yak

AAA lys

ARC asn

AGe see

ATC £1e

CTG leu

GAG

CAA

GGA

CAG

gin

Ely

gln

CTG leu

ACA thr

GTC val

AAC asn

TAA 2281

glu

30O

400

1621

1841

1961

208~

lya 2101

***

443

TAACAGCAAAAAGAAAATGCCGATACTTCATTGGCATTTTCTTTTATTTCTC~CAAGATGGTGAATTC 2350

B 710

720

730

u

740

u

-

AuO

AAGACCUAAAAUGUGU AAGGG

GUGU

UUCUGGGUUUUAUACA

UGCG

"

'

770

760

UUCCC -

-UAC

C U GUU

750

Fig. 2. The nt sequence ofsacB[BamP]gene and second,-,rystructureofsacR[BamP].(A) The sacBgene. FlasmidpBE301 (see Fig. 1) was digested with various restrictionenzymes and various fragmentswere subclonedinto Ml3mpl8 and M13mpl9 (Maniatis et al., 1982). Single-strandedDNA fromthe phageswereisolatedand the nt sequencewas determinedusinga Sequenasesequencingkit (United States BiochemicalCorp.,Cleveland,OH). Both strands have been sequencedusingoverlappingclones.The sequencehas beensubmittedto the EMBLdatalibraryunderaccessionnumberX52988. (B) Structureof sacR[BamP].The secondarystructureo[the putativeregulato,ryregion(nt sequence708-774) was generatedby computeranalysisusingFold programof sequence analysissoftwarepackage(Genetics ComputerGrou,p ofthe Universityof Wisconsin). Numbersreferto those in partA.

(c) Comparison of the nt sequence of sacB[BamP] with that of Bacillus subtilis sacB Shimotsu ~nd H ~ n e r (1986) have de~ermined the tsp of B. subtilis sacB. Comparison of the nt sequence from -55 to + 115 ( + 1 denotes the tsp for B. subtilis sacB) shows 90 % identity with extensive homology at both the promoter and regulatory region (Fig. "~). The co~ing region also showed 90% identity; 4% were conservative changes and 6% of the residues were different (data not shown). A small ORF has been described in the case of B. subtilis in the sacB region. However, this ORF is not translated in vivo

(Shimotsu and Henner, 1986). a similar ORF is absent in the case of sacR [BamP]. Henner et al. (1988) have identifled an upstream activating sequence for B. subtilis sacB as

the possible site of action for degU anddegQgeneproducts. Comparison of the upstream nt sequences from -56 to -263 showed only a 25 % homology. (d) Sucrose inducible expression of sacB[BamP] The sacB[BamP] gene from pBE301 was cloned into an E. coli-B, subtilis shuttle phagemid vector pB E20 (HindIII-

digested pTZ18RligatedwithHindIII-digestedpC194)re-

92 -263

1 ......

40 s a c B - B , subtili$

GATCCTTTTTAACCCATCA . . . . CATATACCTGCCGTTCACTAT I

IIIIII

I

I

I

I

I

I

I

II

III

II

399 C~G~CAGCA~CTTTTTTAGCGGTGATCTGGCTGCATGTTCCCTTCGGAAT 448

sacB[BarnP]

41 TATTTAGTG;~J%ATGAGATATTATGATATTTTCTGAATTGTGATTAAAAAG 90 i l II Ill I i II i l 449 ATCAG~TGGCTGGGT~CT~TACT~ATTTTAGCCACCATCGCTTTATTA.

497

91 GCAACTTTATGCCCATGCAACAG~,%CTATAAAAAATACAGAG~-TG;~ 140 I

I

II

~'

I

I II

l

498 .....TCTATC~.G~))$~,TAACCTCTCTTTTTTAGAGAGGTTTT~CCC 542 141AGAAACAGA~'AGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCA~TCCTT 190 I lllll Ill l II Illl 543 TAGGCCTGAAGCACCCTTTAGTCTCAATTACCCATAAATTAAAAGGCCTT 592 .-55

.

-35

191TTAT.GATTTTCTATCAAACAAAAGAGGAAAATAGACCAG~~TCCA II I I Ill llllll lllIIllllllllllllllll!lll II 593 ~ C G ~ C ~ C ~ C ~ G ~ G G ~ U ~ G ~ C C ~ C ~ G T C ~ T ~ G • -10___. +1 . . . 240 AACGAGAGTCT~~.TGAGGTCGAAAAGTAAATCGCGCGGGTTTG,T II lllIIllll}lIIIIlilllllllllllllIIlll Ill II lllll 643 AATCAGAGTCTAA~AGAAT~GAGGTCG_AAAAGTAAATCACGCAGGATTGTT -

239 6~2

289 692

sacR~

290 ACTGATAAAGCAGGC~AGA6CTAAAATGTGTAAAGGGCAAAGTGT~TACT 339 Illlllllllllllllllllllllllllllll

Illllllllllll~l

II

693 ACTGATAAAGCAGGC~.AGACCTAAAATGTGTTAAGGGCAAAGTGT~TTCT 742 340[TTGGCGTCACCC-CTTACATATTTT-AGGTCTT]TTTTTATTGTGCGTAACTA 389 ]llii!llll llllllllllllll llllllllllll I llIIl l 743 [TTGGCGTCATCCCTTACATATTTTGGGTCTT~TTTTCT .....GTAACAA 787 390 ACTTGCCATICTTCAAACAGGAGGGCTGG;%AGAAGCAGACCGC.TAACAC 437 II II I I I II II II I Ill Ill III II II ~

~CC~GCC~CC~G~CGG~G~CG~CGGC~C~C~C

a~

438 A G T A C A T ~ G G A G A ~ A T G A A C ~ C A T C A A ~ G T T T G C A ~ ]lllllll

! ~!l!!lll~llil;I

~l[~;f:llllllll

487 Ill

Illl

838 A~TACATAC~GI~O~GGAGA~;~YGAAC.[ATt~ACATC~O.AAAA~GTAAAA 886 •

RBS

488 CAAGCAACAGTAT 500 lllll I Ill 887 CAAGCCACAGTTC 899

f

met

Fig, 3. Comparison of the B. s.bti~-sacB sequence with sacB [BamP]. Nucleotide sequences of the promoter and upstream sequences of8. sub~iliz-sac8(top lines) and sacB [BamP] (bottom lines) are compared. + I denotes the tsp ~ r the B. subtilissacBtranscription, as determined by Shimotsu and Henner (1986).

suiting in pBE501, pBE504 is similar to pBE501 except it contains two additional restriction-enzyme recognition sequences in the signal peptide coding region and these changes do not alter the aa sequence of the signal peptide. B. subtilis strain BG4103[pBE504] was grown in medium B to an A6oo,m -- 0.5 and varying concentrations of sucrose (100/~M-100 mM) was added and 2 h lt°er the extracellular LVS activity was measured. The cell de~,sity of the various ,.ultures was comparable but the amount ,~f LVS activity varied with the amount, of sucrose added (Fig. 4). The uninduced culture had an activity of < 0. I0 units while a maximal activity of 2.'. units was observed when the initial sucrose concentration was 4 raM. However, with increasing sucrose concentration LVS activity decreased and at the maximal sucrose concentration tested (I00 mM) an activity of 0.80 units was observed. The concentration of sucrose needed to maximally induce B. subtilis sacB has been reported to be 30 mM sucrose (Lepesant et al.. 1976; Steinmetz eta!., 1989).

The sucrose induction of sacB[BamP] on a multicopy plasmid in B. subtilis suggests that B. subtilis-SacY probably can act as an antiterminator and interact with sacR[BamP]. Our results show that 4 mM sucrose was sufficient to maximally induce sacB[BamP] in B. subtilis. The presence of the multiple copies of the sacR[BamP] might titrate some of the factors and thus we expected to find a lower concentration of sucrose to induce the sacB[ RomP]. However, we did not expect to see a decrease in the amount of LVS accam, dqted when induced with high concentrations of s,lcrose. Whether this observed decrease in the accumulated LVS is due to sacR[BamP], gene dosage or growth medium is not clear. The regulation of sacB in B. subtilis seems to be similar to the regulation of the bgl operon of E. coli (Mahadevan et ~., 1937; Schnetz et al., 1987; Steinmetz et al., 1988). Amstar-Choder et al. (1989) have shown that BglG which is homologous to SacY, acts as an antiterminator when dephosphorylated. The phosphorylation of BglG is modu-

93

2.0

5 ~:

1.0

,z.,

0.1

I

I_

1

10

100

SUCROSE [mM]

Fig. 4. Expression of sacB [BamP] in B. subtilis. B. subtilis strain BG4103 (3sacB, trpC2) containing pBES04 was grown in medium B (per liter: tryptone 33 g/yeast extract 20 g/NaCi 7.4 g/Na,HPO 4 8 g/KH:PO4 4 g/casamino acids 20 g/glycerol 60/~M/MnCI: 0.06 mM/FeCI3 S0~ nM/NaOH to pH 7.57. LVS was partially purified from 10 ml culture supernatant by ethanol precipitation and resuspended in 500 #! of 50 mM K. phosphate buffer pH 6.0 containing 20% glycerol (Dedonder, 19667.50/~! ofthe partially purified LVS was preincubated in 50 mM K. phosphate buffer (850 pl) at 37 ° C for 5 rain. The reaction was initiated by the addition 100 #! of sucrose (400 raM). Samples were withdrawn at 0,15 and 30 min and LVS was inactivated by incubation at 70°C for 5 rain. The amount ofgiucose present at 0, ~5 and 30 rain was determined using Glucose Trindcr reagent from Sigma. O - ~ un~, of LVS activity is defined as 1/~g ofglucose released/min/ml of the culture supernatant.

lated by the protein kinase action of BglF. Regulation of sacB may be more complex than the E. coil bgl operon because Steinmetz et al. (I 989) observed a low level ofsacB induction in strains carrying a deletion in sac Y. In addition, ptsl mutants of B. subtili~' express sacB constitutively (Crutz et al., 1990). Thus, the availability of a second LVSencoding gene from another Bacillus species should aid in studies towards understanding the complex mechanism/s involved in the sucrose regulation of sacB in B. subtilis.

ACKNOWLEDGEM ENTS

We thank Dennis Henner for providing strain BG4103 and Georges Rapoport, Mark Zukowski, Ethel N. Jackson and Mark Payne for useful discussions and Helene Albertson for excellent technical assistance.

REFERENCES Amster-Choder, 0., Houman, F. and Wright, A.: Protein phosphorylation regulates transcription of the ~-glucoside utilization operon in E. coll. Cell 58 (1989) 847-855. Ayecmich, S. and Steinmetz, M: Cloning and preliminary characteriza-

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Isolation and characterization of levansucrase-encoding gene from Bacillus amyloliquefaciens.

The gene encoding levansucrase (LVS) from Bacillus amyloliquefaciens (sacB[BamP]) was isolated, sequenced and expressed in Bacillus subtilis. Analysis...
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