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Cloning of the cellulase gene from Penicillium funiculosum and its expression in Escherichia coli
FEMS Microbtology Letters 66 (1990) 291-294 Pubhshed by Elsewer
291
FEMSLE 03833
Cloning of the cellulase gene from Penicillium funiculosum and its expression in Escherichia coli N.A. Sahasrabudhe a n d P.K. R a n j e k a r DwlslOn of Biochemical Sclentws, National ChemwalLaboratorL Poona. Inata Recewed 4 August 1989 Revlsmnrecelxed and accepted II September 1989 Key words: Cloning, Cellulase gene, Expression, Pemcdhum fumculosum 1. SUMMARY A gene of Pemcdlmm fumculosum encoding an endoglucanase was cloned and expressed m Eschenchta colt using the lacZ promoter of ~ector pUC 18. The gene product hydrolyzed carboxymethyl cellulose and showed strong cross reacttvity with P fumculosum anttcellulases
useful for producing genettcally engineered new orgamsms for effective sacchanficatton of cellulose/ hgnocellulose. This paper describes the clomng of an endoglucanase gene from P. fumculosum m E. colt, expressing earboxymethyl cellulase (CMCase) acttwty Thts ts the first report of gene cloning m P fumculosum
2, I N T R O D U C T I O N
3 MATERIALS A N D METHODS
Pertcdhum fumculosum (NCL strata) produces a powerful cellulase complex having fl-l,4 glueanase (1.5-2.0 U / m l ) and lugh fl-glucostdase (8-10 U / m l ) acUvity [1]. The homogeneous endoglucanase I preparauon of P fumculosum ts a very randomly acting enzyme and shows a broad substrate ~pectficity towards ,8-1,4, fl-l,3, fl-l,6, a-l,6 glucosidase hnkages [2] The presence of ,6'-1,4 glucan glucohydrolase acttxaty is its addluonal feature. It, therefore, appears that the cloning of a single homogeneous gone expressing endo I wdl he
3 1 DNA isolation Total genonuc D N A from P fumculosum, grown m a synthetic medium cont0anmg glucose, was prepared according to the method described previously [3]. Plasnud D N A was isolated from Escherwhta coh DH1 by alkah lysts method [4].
Correspondence w N A Sahasrabudhe. Divtston of Blochemacal Sciences. National Chermcal Laboratory. Poona 411008, lndta
3 2 DNA clonmg P fumculosum D N A (3 #g) and the pUC 18 D N A (1 /tg) were digested with Sinai, mixed and hgated Prior to hgatlon, the vector D N A was dephosphorylated. The hgated rmxture was used to transform E colt DH1 cells [5] and amptcdhn resistant transformants were selected on luna broth (LB) plates containing 50 ~ g / m l of ampicdhn. Clones expressing CMCase acttvsty were detected
0378-t097/89/$03 50 © 1989 Federation of European Mtcrobtologtcal Societies
292 by rephca platmg colonies onto earboxy methyl cellulose (CMC) plates m M9 medium contammg amptcdlm and were detected by congo red stainmg [6] The plates were incubated at 3 7 ° C for 16 h and after lys]s at 3 0 ° C for 48 h to allow CMCase diffusion. The msert m the plasnud D N A was obtained by Smal dtgestton and tts stze was determined by agarose gel electrophorests.
plates, prepared m M9 medmm supplemented with 50 t~g/ml amptcfllm, to search for CMCase postttve transformants. Of the 100 clones screened, only one colony showed a halo after lysls on the CMC plate and was destgnated as p U P F 1. Restnctton analysts of pUPF1 tsolated from E cob DH1 showed that the insert size was 1.8 kbp (Data not shown).
3.3. Enzyme producnon, endoglucanase assay and m:munodtffuswn For the endoglucanase assay and anmunodfffuslon, crude extracts were prepared by gently grindmg the overmght culture of E coh clones usmg glass beads (Stgma, ~°0 U) m cold PC buffer (50 m M K 2 H P O 4, 12.5 . .M citric act& pH 6 8). Endoglucanase actlwty was deterrmned by VlScometnc assay. Reaetton rmxture containing 1 ml of enzyme and 10 ml of 1% CMC made m PC buffer, pH 6.8 was incubated at 3 7 ° C for 10 ram. The samples were thermostated at 37 ° C and the vtscosRy was determined with an Ostwald type vtscometer [7] The reciprocal of the spectftc vtscosRy (flui&ty I/~sp) was calculated by the formula 1/rlsp = (loft - to) where t o ts the efflux time of buffer (50 s) and t is the efflux ttme of CMC solution. Enzymatte acuvtty was proporttonal to the increase m flmdRy [8]. For the tmmunodfffuston tests, crude extracts (prepared as above) were parttally purified by 5 rmn heating at 6 0 " C followed by ammomum sulfate prectpRatton at 0.9 saturation, dtalysls against PC buffer and concentratton to 1/25th of the inttlal crude extracts by lyopluhzatton. Antlbodtes were raised against the parttally punhed endo fl-l,4 glueanase of P fumeulosum and tmmunoassays were performed according to Ouchterlony [9].
4 2 Cellulase productwn E cob DH1 carrying p U P F I was ¢ultwated with 50 p.g/ml ampictlhn at 37 ° C overmght. The eellulase act]vlty m the extracellular and mtraeellular fractions was deternuned wscometncally. No CMCase activity was detected m the extracellular fraction. The spectflc acttvtty of the enzyme produced m E. cob is 2 0 as opposed to tts nattve state whmh is 20.0. Ftg. 1 shows the graph of l / r r s p vs time. A rapid reduction in the viscosity of 1% C M C in PC buffer revealed endo acuon of the enzyme. The lughly concentrated extracts of mtracellular enzymes were used for Ouchtedony double dlffuston test wtth anttserum to P fumculosum cenulases (Fig 2) The mtraeellular enzyme extract from the recombmant ~,ells gives a smgie preeipltm line which fused completely wtth that of cellulase from P fumculosum {21. Thts result shows
4. RESULTS 4 1 Clonmg of endoglueanase gene from P. fumculosum After the hgauon nuxture was transformed into £. cah DH1, 100 ampicillin resistant transformants per 40 ng of total D N A were obtained. The recombinant clones were rephca plated onto CMC
[
/.
051 X 3
....
6
9
12
' q5
TIME { ram)
Fig ] Decrease m speclhc vtscoslty I))sp ( 0 ) ] or CMC solu-
lion with a simultaneous mcreas~m flmd]tyl/v/sp (~)
293
tp
+t'++, +r.+ Q
A L
Furthermore, cross reacuvlty of the cloned enzyme with P fumculosum antlcellulases provides a strong evidence that the plasrmd p U P F I carries the structural gene for the intracellnlar endo ,8-1.4 glucanase geuc of P. fumculoswn A t t e m p t s are now being m a d e to enhance the celhilase production by the plasmld p U P F 1 m E
coll.
P
q
ACKNOWLEDGEMENTS
Fig. 2 Antigen-antibody reactton (C) +control (antigen Peniclll!um fumgulosum undo I). (P) undo I secreted by the clone pUPFI in E colt, (A} undo ] after ammomum sulfate prectpltaUon (cloned), (H) undo I after heat treatment (cloned) that E coh harbouring p U P F 1 produces cellulase wl'ach ts lmmunologically tdenttcal to the cellulase from P fumculosum
Authors wish to t h a n k Drs. Vtdya G u p t a for the perfect p l a n n i n g of experiments, D r A.H. Lachke and Mrs. A.K. Vyas for useful suggestions m the enzymologlcal work. W e also wish to t h a n k M r Yogesh M a w a l for assistance in carrying out tmmunologtcal w o r k
REFERENCES DISCUSSION Ours is the first report of cloning cellulase gene from Pemcdhum /umculosum T h e cellulase encoded by the gene wMch we have cloned ts mtrace:lular m & coil As /: cob is not cellulolytm, the enzyme was not ¢ontanunated by cellulases from E col: host. The mtracellular extracts, therefore, serve as an active enzyme source without purificatmn. Judging from the 58 k D a P fumculosum celhilase [2], the gene we have cloned must be m the range of 1 . 6 - 2 . 0 k b to encode the whole enzyme, and this is actually the case m the present work. In comparison, the fragment encoding the extracellular cellobmhydrolase of 56 k D a from Tnchoderma reesel is also 1.7 kb [10]. T h e mode of action and the substrate specfftetty define the enzyme as an undo ,8-1,4 glucanase.
[l] Deshpandc, V. Mlshra, C, Rao. M, Seeta, R, Snmvasan, M C and Jagannathan, V (1980) Reg. J Energy Heat Mass Transf 2(1), 23-29 [21 Sahasrabudhe N A, Lachk¢, A H and Ranjekar. P K (1987) Biotechnol Len 9, 881-886 [3] Sahasr~budhe. NA and Ranjekar, PK (1985) FEMS Mmrobtol Lett 30, 315-319 [4] Btrnbom, H C and Duty, T (1979) Nuclet¢ Actds Res 7, 1513-1523 [5] maffllatls, T, FrltSCh, E F and Sambrook, R (1982) Molecular clomng A laboratory manual, Cold Sprtog Harbor Laboratory, Cold Spring Harbor, New York [6] Teather. RM and Wood, PJ t1982) Appl Environ Mtcroblol 43, 777-780 [7] Cornet. P, Tromk. D. Mdlet, T and Aubert, J P (1988) FEMS Microblol Len 16, 137-141 [8] Schwartz, W H. Gradbmz, F and Staudenbau~r. W L (1986) Appl Envffon Mtcroblol 5,1293-1299 [9] Ouchtcrlony, O (1949)Acta Pathol Mtcrobtol Scand 26, 507-110 [10] Ten, T. Salovuon. I and Knowles. J (1983) Blotechaology 1,696-699
291
FEMSLE 03833
Cloning of the cellulase gene from Penicillium funiculosum and its expression in Escherichia coli N.A. Sahasrabudhe a n d P.K. R a n j e k a r DwlslOn of Biochemical Sclentws, National ChemwalLaboratorL Poona. Inata Recewed 4 August 1989 Revlsmnrecelxed and accepted II September 1989 Key words: Cloning, Cellulase gene, Expression, Pemcdhum fumculosum 1. SUMMARY A gene of Pemcdlmm fumculosum encoding an endoglucanase was cloned and expressed m Eschenchta colt using the lacZ promoter of ~ector pUC 18. The gene product hydrolyzed carboxymethyl cellulose and showed strong cross reacttvity with P fumculosum anttcellulases
useful for producing genettcally engineered new orgamsms for effective sacchanficatton of cellulose/ hgnocellulose. This paper describes the clomng of an endoglucanase gene from P. fumculosum m E. colt, expressing earboxymethyl cellulase (CMCase) acttwty Thts ts the first report of gene cloning m P fumculosum
2, I N T R O D U C T I O N
3 MATERIALS A N D METHODS
Pertcdhum fumculosum (NCL strata) produces a powerful cellulase complex having fl-l,4 glueanase (1.5-2.0 U / m l ) and lugh fl-glucostdase (8-10 U / m l ) acUvity [1]. The homogeneous endoglucanase I preparauon of P fumculosum ts a very randomly acting enzyme and shows a broad substrate ~pectficity towards ,8-1,4, fl-l,3, fl-l,6, a-l,6 glucosidase hnkages [2] The presence of ,6'-1,4 glucan glucohydrolase acttxaty is its addluonal feature. It, therefore, appears that the cloning of a single homogeneous gone expressing endo I wdl he
3 1 DNA isolation Total genonuc D N A from P fumculosum, grown m a synthetic medium cont0anmg glucose, was prepared according to the method described previously [3]. Plasnud D N A was isolated from Escherwhta coh DH1 by alkah lysts method [4].
Correspondence w N A Sahasrabudhe. Divtston of Blochemacal Sciences. National Chermcal Laboratory. Poona 411008, lndta
3 2 DNA clonmg P fumculosum D N A (3 #g) and the pUC 18 D N A (1 /tg) were digested with Sinai, mixed and hgated Prior to hgatlon, the vector D N A was dephosphorylated. The hgated rmxture was used to transform E colt DH1 cells [5] and amptcdhn resistant transformants were selected on luna broth (LB) plates containing 50 ~ g / m l of ampicdhn. Clones expressing CMCase acttvsty were detected
0378-t097/89/$03 50 © 1989 Federation of European Mtcrobtologtcal Societies
292 by rephca platmg colonies onto earboxy methyl cellulose (CMC) plates m M9 medium contammg amptcdlm and were detected by congo red stainmg [6] The plates were incubated at 3 7 ° C for 16 h and after lys]s at 3 0 ° C for 48 h to allow CMCase diffusion. The msert m the plasnud D N A was obtained by Smal dtgestton and tts stze was determined by agarose gel electrophorests.
plates, prepared m M9 medmm supplemented with 50 t~g/ml amptcfllm, to search for CMCase postttve transformants. Of the 100 clones screened, only one colony showed a halo after lysls on the CMC plate and was destgnated as p U P F 1. Restnctton analysts of pUPF1 tsolated from E cob DH1 showed that the insert size was 1.8 kbp (Data not shown).
3.3. Enzyme producnon, endoglucanase assay and m:munodtffuswn For the endoglucanase assay and anmunodfffuslon, crude extracts were prepared by gently grindmg the overmght culture of E coh clones usmg glass beads (Stgma, ~°0 U) m cold PC buffer (50 m M K 2 H P O 4, 12.5 . .M citric act& pH 6 8). Endoglucanase actlwty was deterrmned by VlScometnc assay. Reaetton rmxture containing 1 ml of enzyme and 10 ml of 1% CMC made m PC buffer, pH 6.8 was incubated at 3 7 ° C for 10 ram. The samples were thermostated at 37 ° C and the vtscosRy was determined with an Ostwald type vtscometer [7] The reciprocal of the spectftc vtscosRy (flui&ty I/~sp) was calculated by the formula 1/rlsp = (loft - to) where t o ts the efflux time of buffer (50 s) and t is the efflux ttme of CMC solution. Enzymatte acuvtty was proporttonal to the increase m flmdRy [8]. For the tmmunodfffuston tests, crude extracts (prepared as above) were parttally purified by 5 rmn heating at 6 0 " C followed by ammomum sulfate prectpRatton at 0.9 saturation, dtalysls against PC buffer and concentratton to 1/25th of the inttlal crude extracts by lyopluhzatton. Antlbodtes were raised against the parttally punhed endo fl-l,4 glueanase of P fumeulosum and tmmunoassays were performed according to Ouchterlony [9].
4 2 Cellulase productwn E cob DH1 carrying p U P F I was ¢ultwated with 50 p.g/ml ampictlhn at 37 ° C overmght. The eellulase act]vlty m the extracellular and mtraeellular fractions was deternuned wscometncally. No CMCase activity was detected m the extracellular fraction. The spectflc acttvtty of the enzyme produced m E. cob is 2 0 as opposed to tts nattve state whmh is 20.0. Ftg. 1 shows the graph of l / r r s p vs time. A rapid reduction in the viscosity of 1% C M C in PC buffer revealed endo acuon of the enzyme. The lughly concentrated extracts of mtracellular enzymes were used for Ouchtedony double dlffuston test wtth anttserum to P fumculosum cenulases (Fig 2) The mtraeellular enzyme extract from the recombmant ~,ells gives a smgie preeipltm line which fused completely wtth that of cellulase from P fumculosum {21. Thts result shows
4. RESULTS 4 1 Clonmg of endoglueanase gene from P. fumculosum After the hgauon nuxture was transformed into £. cah DH1, 100 ampicillin resistant transformants per 40 ng of total D N A were obtained. The recombinant clones were rephca plated onto CMC
[
/.
051 X 3
....
6
9
12
' q5
TIME { ram)
Fig ] Decrease m speclhc vtscoslty I))sp ( 0 ) ] or CMC solu-
lion with a simultaneous mcreas~m flmd]tyl/v/sp (~)
293
tp
+t'++, +r.+ Q
A L
Furthermore, cross reacuvlty of the cloned enzyme with P fumculosum antlcellulases provides a strong evidence that the plasrmd p U P F I carries the structural gene for the intracellnlar endo ,8-1.4 glucanase geuc of P. fumculoswn A t t e m p t s are now being m a d e to enhance the celhilase production by the plasmld p U P F 1 m E
coll.
P
q
ACKNOWLEDGEMENTS
Fig. 2 Antigen-antibody reactton (C) +control (antigen Peniclll!um fumgulosum undo I). (P) undo I secreted by the clone pUPFI in E colt, (A} undo ] after ammomum sulfate prectpltaUon (cloned), (H) undo I after heat treatment (cloned) that E coh harbouring p U P F 1 produces cellulase wl'ach ts lmmunologically tdenttcal to the cellulase from P fumculosum
Authors wish to t h a n k Drs. Vtdya G u p t a for the perfect p l a n n i n g of experiments, D r A.H. Lachke and Mrs. A.K. Vyas for useful suggestions m the enzymologlcal work. W e also wish to t h a n k M r Yogesh M a w a l for assistance in carrying out tmmunologtcal w o r k
REFERENCES DISCUSSION Ours is the first report of cloning cellulase gene from Pemcdhum /umculosum T h e cellulase encoded by the gene wMch we have cloned ts mtrace:lular m & coil As /: cob is not cellulolytm, the enzyme was not ¢ontanunated by cellulases from E col: host. The mtracellular extracts, therefore, serve as an active enzyme source without purificatmn. Judging from the 58 k D a P fumculosum celhilase [2], the gene we have cloned must be m the range of 1 . 6 - 2 . 0 k b to encode the whole enzyme, and this is actually the case m the present work. In comparison, the fragment encoding the extracellular cellobmhydrolase of 56 k D a from Tnchoderma reesel is also 1.7 kb [10]. T h e mode of action and the substrate specfftetty define the enzyme as an undo ,8-1,4 glucanase.
[l] Deshpandc, V. Mlshra, C, Rao. M, Seeta, R, Snmvasan, M C and Jagannathan, V (1980) Reg. J Energy Heat Mass Transf 2(1), 23-29 [21 Sahasrabudhe N A, Lachk¢, A H and Ranjekar. P K (1987) Biotechnol Len 9, 881-886 [3] Sahasr~budhe. NA and Ranjekar, PK (1985) FEMS Mmrobtol Lett 30, 315-319 [4] Btrnbom, H C and Duty, T (1979) Nuclet¢ Actds Res 7, 1513-1523 [5] maffllatls, T, FrltSCh, E F and Sambrook, R (1982) Molecular clomng A laboratory manual, Cold Sprtog Harbor Laboratory, Cold Spring Harbor, New York [6] Teather. RM and Wood, PJ t1982) Appl Environ Mtcroblol 43, 777-780 [7] Cornet. P, Tromk. D. Mdlet, T and Aubert, J P (1988) FEMS Microblol Len 16, 137-141 [8] Schwartz, W H. Gradbmz, F and Staudenbau~r. W L (1986) Appl Envffon Mtcroblol 5,1293-1299 [9] Ouchtcrlony, O (1949)Acta Pathol Mtcrobtol Scand 26, 507-110 [10] Ten, T. Salovuon. I and Knowles. J (1983) Blotechaology 1,696-699