e xpres sed gene products which mi ght e xp lain their i ns t ab i lity in t h i s h o st ( Kane and Hart ley , 1 9 8 8 ) . In addition , protease that c le aved c e l lu lases i nt o sma l l e r peptides which wou ld f ac i li t at e the acti on of trans ferase but this expres s ion cou ld be lethal to the E . col i host . Further study o f t he action o f prot e o lytic proc e s s ing and glycosylation of c e l lu l a s e s i n R .
fl a ve fa c i ens i s needed .
Several strategies h ave been u sed t o overcome the problem of s e c retion o f foreign protein in E . col i . Overproduction has been reported with rec ombinant exoglucanas e of Ce o fimi in whic h the exoglucanase gene was fused to a s ynthetic ribos ome bind i n g s ite and plac ed under the c ontrol of the l e f tward promoter o f l ambda phage . The overproduced exoglucanase c an be i so l at ed e a s i ly i n an enriched f o rm a s inso luble aggregat e s and exoglucanase activity c an b e recovered b y s olubi l i z ation of the aggregat e s in 6 M urea or 5 M guanidine hydrochloride ( O ' Ne i l l et a l , 1 9 8 6 ) . Another experiment u s ing a high c opy number plasmid containing a l a c promote r to replace the promoter sequence of cenA c au sed an increas e of s ome 8 0 0 - fold in expres sion . Accumu l at io n of a prote i n to a high leve l i n the peripl a sm may destabi l i z e the outer membrane o f E . col i (
res u l t i n g in the l e akage o f perip l asmic protein to the medium ( Gatz and H i l len , 1 9 8 6 ; Abrahmsen et a l , 1 9 8 6 ) . I n addition , high l evel e xpres s ion o f Cl os tri di um thermocel l um c e l l u l a s e gene s c an b e achieved i n E . col i b y subc loning in a t empera ture-regu lated vect or which contains the l e f tward promoter of
l ambd a a nd u sing t hermal inactivation of the heat- sensitive l ambd a cI8 5 7 repre s sor . Overexpres s ion of the celA gene would resulted in a decrease i n c e l l viabi l ity c oncomitant with the accumU l at ion of endoglu c an a s e A in the membrane fraction .
( iv ) Cellulase c on s i st s of d i f ferent domains
CHAPTER 6 FINAL DISCUSSION AND CONCLUSION
which c ontains at least three domains ( ce l lu l o s e binding domain , c at a lyti c d omain and a c onnecting doma i n which is rich in pro l i n e , threon ine and s erine ; Ong et aI , 1 9 8 9 ) . In this report , the ORFs coding for e ndo- and exo-glucanase all had PTS rich r e gi o n s s eparate f rom the sequences of the other two domai n s . H owever , t h i s concept did not c ompletely f i t with the renA gene wher e the PTS rich region was c lo s e t o the s tart c odon ATG ( 2 1 bp d ownstream o f ATG ) . Al s o west et al ( 1 9 8 9 ) reported that the homologou s catalytic domains o f Ce o fimi
f ound i n fungi and Baci l l u s s p . apparentl y evolved by reshu f f ling o f catalyt i c doma i n s and s everal substrate-binding domai n s . When c omparing the binding domain of cenA and cex with ORF 1 and ORF2 in this s tudy , homologous regio n s of the N termin a l e nd o f ORF 1 and ORF2 were found . Al s o the catalytic doma i n s were f ound by comparing the c at alyt i c domains of cenA
and cex, which indicated the region between PTS rich region and
the binding regi on in ORF 1 and t he region near to the C t ermin a l e nd i n ORF 2 were the c atalytic domains ( summari z ed i n Fig . 6 - 1 ) . Won g e t a l ( 1 9 8 8 ) sugge sted that the function o f PT box o f celA and cex was to s ep arate the bind i ng and c at a lyt ic doma i n s . The c a s e in renA , a different structure of c e l lulase gene is shown in which the PTS rich region i s out s ide o f the c atalytic and bind i ng doma i n s . Further study of the function of PTS rich r egion is needed .
( v ) G+C content o f c e llu l a s e
The G+C c ontent of the nuc leotide in renA , rex and rbg were 7 1 . 7 % , 6 7 . 3 % and 6 6 . 6 % , respective l y , which i s higher than that of C . thermocel l um ( ce lA , c e l B , c e lC and celD , 3 9 . 4 % , 3 9 . 8 % , 3 5 . 5 % and 0 . 3 % , respective l y ; Beguin et a l , 1 9 8 5 ; Grepinet and Beguin , 1 9 8 6 ; S c hwar z et a I , 1 9 8 8 ; Joli f f et al , 1 9 8 6 ; H a l l e t a l , 1 9 8 8 ) and Baci l l us s p . ( strain 1 1 3 9 , 3 7 . 9 % ; pNK 1 , 3 9 . 3 % ; pNK 2 , 3 9 . 7 % ; Fukumori et a l , 1 9 8 6 ; MacKay et aI , 1 9 8 6 ; Nakamu r a et al , 1 9 8 7 ; Fukumori et a I , 1 9 8 9 ) , but c lose t o t h at o f Ce o fimi c enA and cex ( 7 4 . 3 % a nd 7 1 . 8 % i Wong et
CHAPTER 6 FINAL DISCUSSION AND CONCLUSION