Cloning of the cellulase gene from Penicillium funiculosum and its expression in Escherichia coli

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...

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FEMS Microbtology Letters 66 (1990) 291-294 Pubhshed by Elsewer


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


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





' q5

TIME { ram)

Fig ] Decrease m speclhc vtscoslty I))sp ( 0 ) ] or CMC solu-

lion with a simultaneous mcreas~m flmd]tyl/v/sp (~)



+t'++, +r.+ Q


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





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