cellulase families and their genes

TIBTECH - SEPTEMBER 1987 [Vol. 5]

Cellulase families and their genes

Jonathan Knowles, P&ivi Lehtovaara and Tuula Teeri

Cellulases play a central role in the recycling of lignocellulose. Molecular biology is now helping to unravel the complex enzymology of cellulose hydrolysis and giving information that will be of use both for the improvement of existing processes and for the

development of completely new areas.

~t is frequently stated that lignocellulose is the most abundant renewable natural resource available. In nature, a large number of organisms, both :macro and micro, cooperate in the efficient recycling of lignocellulose. Thus, the natural biodegradation of lignocellulose is immensely complicated both from the ecological and enzymatic point of view.

A number of companies around the world now produce cellulases on an industrial scale and potential applications are to be found in any industrial or agricultural process where plant material is used. Cur- rently, however, the market for these enzymes is small. The substrates offered by industry and agriculture are considerably more complex and variable than the model substrates often used for the development of cellulolytic organisms. In addition the chemical complexity of lignocellulose makes its conversion to specific useful products difficult.

Lignocelluloses are complexes of three components present in varying proportions: cellulose, which consists of chemically simple but physically complex [~-1,4 linked glucose polymers; xylan which contains complex polymers of different pen- toses and hexoses, and lignin which is more or less a random polymerization of a limited number of different phenolic compounds. This is why large and complex batteries of different enzymes, many apparently acting synergistically, are required for natural recycling.

Fortunately, it is now possible to Jonathan Knowles, PMvi Lehtovaara and Tuu]a Teeri are at the Biotechnological Laboratory, VTT Tietotie 2, SF-02150 Espoo, Fin]and.

- Tab le I

use the powerful tools of molecular technology to explore in detail the complexity of lignocellulose degradation. Cellulases and their genes have been extensively studied in recent years, and it is already possible to understand something of their complexity 1'2. In this review, we discuss the different families of cellulases and indicate how knowledge concerning the molecular biology and enzymology of these systems is not only of fundamental interest but should have practical importance in the future.

Substrates of the cellulolytic enzymes and the detection methods most commonly used

Product measured CMC HEC [~-glucan

Soluble substrates

p-nitro- [3-ME-umbelli- phenyl-

Cello-oligo- feryl-oligo- oliogo- saccharides saccharides saccharides

Formation of + + + + reducing sugars (biochemical assay) Liberation of + chromophore (fluorescence, absorbance) Oligosaccharides + + + released (HPLC) Decrease in degree of polymerization

staining + + + viscosity + +

+ +

+ +

Insoluble substrates

Phos- Bacterial phoric micro- Amor., acid

Valonia Native crystalline Filter phous swollen Product measured cellulose Cotton cellulose Avicel paper cellulose cellulose

Formation of + reducing sugars (biochemical assay) Decrease in degree of polymerization

turbidity staining

Release of fibers light

microscope electron +

microscopy

+ + + + +

+ + + +

+

(~) 1987, ELsevier Publications, Cambridge 0166- 9430/87/$02.00


Cellulases Cellulolytic enzymes are generally

induced as multienzyme systems, composed of five or more different enzymes. Cellulolytic enzymes have been traditionally divided into three classes, endoglucanases (EC.3.2.1.4), exoglucanases or cellobiohydrolases (E.C.3.2.1.91) and [~-glucosidases (E.C.3.2.1.21).

In bacteria, these enzymes are often produced in small amounts (less than 0.1 g . l -1 ) 3 o r , in the case of C]ostridium thermocellum, form tight multienzyme complexes (cellu- losomes) which are difficult to dis- rupt without loss of activity of the individual components.

The fungal cellulases are available in large amounts (more than 20 g 1-1) 2 and do not seem to form physical complexes with each other, but they act in strong synergy. For this reason the purity of the enzymes preparations is of utmost importance for the elucidation of their enzymatic activities, since even traces of another type of activity can seriously bias the results.

Cellulases all cleave the same chemical bond, the ~-l,4-glycosidic bond, but there is variation in the microenvironment of these bonds in natural substrates. The same poly- saccharide molecule, which may contain up to 10000 glucose units, has both crystalline regions of highly ordered structure and inter- crystalline regions of more random structure. Moreover, in natural substrates, cellulose may be further distorted by association with lignin and hemicellulose. This may be the reason why cellulolytic organisms produce a family of different cellulases, each with different substrate and product specificities. Also, the specificities of many of the cellulases may well be much broader than the nomenclature and traditional classi- fication of cellulases would suggest.

The hydrolysis of the insoluble substrate, cellulose, is inconvenient to assay. Moreover, the insoluble substrate probably changes during hydrolysis (although this has not been studied in detail): if the loosely structured amorphous regions are hydrolysed first, this leads to a progressive increase in the crystal- linity of the substrate. For this

Table 2 S u m m a r y o f c loned cel lulase genes

Gene Organism Genes cloned sequence Refs

Bacteria Agrobacterium ATCC 21400 1 lS-glucosidase Bacillusamyloliquefaciens 1 [3-glucanase

Bacillus circulans

Bacillus subtilis e'f'9

alkalophlic strains Bacteroides succinogenes Cellumonas f imi

1 endo-l,3-1,4-13- glucanase 1 endo-[~-l,4- + glucanase 1 1%1,4-glucanase 1 13-1,4-glucanase + 313-1,4-glucanases all+ 1 endoglucanase 2 endoglucanases

1 cellobiohydrolase

Cellulomonas uda

Clostridium thermocellum

213-glucosidases 1 endoglucanase 7 endoglucanases

cenA (Eng)

cex (Ex9)

cel A (EGA) cel B (EGB) cel D (EGD)

i

4

j ,5 ,6

k

I

7

m

8

n

o

p ,9

10

11,12

aWakarchuk, W. W. etal. (1986) Mol. Gen. Genet. 205, 146. bBorriss, R. etal. (1985) Appl. Microbiol, Biotechnol. 22, 63. CHofemeister, J. et al. (1986) Gene 49, 177. dp-L. JSrgensen (pers. commun.), eHinchliffe, E. (1984) J. Gen. Microbiol. 130, 1285. fCantwell, B.A. et al. (1983) Gene 23, 211. gMurphy, N. et al. (1984) Nucleic Acids Res. 12, 5355. hMacKay, R. et al. (1986) Nucleic Acids Res. 14, 9159. iRobson, L. M. etal. (1986)J. Bacteriol 165, 612. JSashihara, N. etal. (1984) J. Bacteriol. 158, 503. kCrosby, B. etal. (1984) in: Proceedings of the Fifth Canadian Bioenergy R&D Seminar, Ottawa, Canada (Hasnain, S., ed.), p. 573, Elsevier Applied Science Publishers. ~Whittle, D. J. et al. (1982) Gene 17, 139. mGilkes, N. R. et al. (1984) J. Gen. Microbiol. 130, 1377. nNakamura, K. et al. (1986) J. Biotech-

reason, a wide range of different substrates have been used to study cellulases (Table I). The combination of such substrate complications with the synergistic and apparently over- lapping specificities of these enzymes, has meant that the specificities of many cellulases have not been unequivocally clarified.

Cloning of cellulase genes During the last five years, the

genes coding for a number of fungal and bacterial cellulolytic enzymes have been isolated (Table 2). Most of the bacterial cellulases cloned so far have been isolated by expression in Escherichia coli and subsequent screening for enzymatic activity towards carboxymethyl cellulose (CMC). Since there are no simple

enzymatic assays to detect activity on crystalline cellulose, genes coding for bacterial enzymes active on such substrates have proved more difficult to isolate. A different stra- tegy, based on the efficient induction of cellulolytic enzymes, has been used to isolate genes coding for both exo- and endoglucanases from fila- mentous fungi 14'15. Preliminary screening of gene libraries constructed in phage lambda vectors was performed by differential hybridization to two distinct cDNA probes: one synthesized using mRNA from mycelia grown on cellulose and thus enriched in cellulase sequences, and the other using mRNA from a culture grown on glucose. Among the clones hybridiz- ing only to the induced probe, the genes coding for several different


T a b l e 2

S u m m a r y o f c loned cel lulase genes

Gene Organism Genes cloned sequence Refs

Clostridium thermocellum 3 exoglucanases 2 ~-glucosidases 13 endoglucanases

Escherichia adecarboxylata 1 [3-glucanase Erwinia chrysantemi >1 endoglucanase

Microbispora bispora

Thermonospora YX

F u n g i

Aspergillus niger Candida pelliculosa Kluyveromyces fragilis Phanerochaete chrysosporium Schizophyllum commune Trichoderma reesei

1 endoglucanase 1 endoglucanase 5 endoglucanases >1 [3-glucosidases 1 endoglucanase

113-glucosidase 113-glucosidase 1 [3-glucosidase 1 cellobiohydrolase

1 endoglucanase 2 cellobiohydrolases

2 endoglucanases

q

15

r

s

t

u

v

w

x

Y

z

aa

b b

cbhl (CBHI) 15 cbh2 (CBHII) "=6,17 eg11 (EGI) 18,19 eg13(EGIII) co

(1984) J. Gen. Microbiol. 130, 1377. nNakamura, K. et al. (1986) J. Biotech- nol. 3, 239. °Nakamura, K. etal. (1986) J. Biotechnol. 3, 247. PMillet, J. etal. (1985) FEMS Microbiol. Lett. 29, 145. q Schwarz, W. et al. (1985) Biotechnol. Lett. 7, 859. rArmentrout, R. W. et al. (1981) Appl. Environ. Microbiol, 41, 1355. Svan Gijsegem, F. etal. (1985) EMBOJ. 4, 787. tBarras, F. etal. (1984) Mol. Gen. Genet. 197, 513. UKotoujansky, A. et al. (1985) EMBO J. 4, 781. VD. E. Eveleigh (pers. commun.). WCollmer, A. etal. (1983) Bio/-I-echnology 1,594. Xpenttit~i, M. E. et al. (1984) Mol. Gen. Genet. 194, 494. YKohchi, C. et al. (1985) Nucleic Acids Res. 13, 6273. ZRaynal, A. et al. (1984) Mol. Gen. Genet. 195, 108. aap. Simms (pers. commun.), bbv. Seligy (pers. commun.), ccSaloheimo et al. (pers. commun.)

exo- and endoglucanases were identified by hybrid selection of the corresponding mRNAs and subsequent immunoprecipitation of the proteins synthesized in vitro. An advantage of such an approach is that, in principle, all the genes efficiently induced during growth on a particular substrate, can be isolated.

Similarity in architecture between cellulases

Sequence analysis of the cloned genes has revealed a great variety in the structure of different cellulases. The primary structures of the enzymes characterized so far are very dissimilar even when the comparison is made between different cellulases derived from one organism, like the four endoglucanases of

C]ostridium thermoce]]um 9-12,2°, or three of the four T. reesei cellulases 14-19. However, despite the dissimilarity in amino acid sequences, many of the different cellulases show some interesting com- mon features in their protein architecture (Fig. 1).

In all the four Trichoderma reesei cellulolytic enzymes, a region of about 30 amino acids with 70% amino acid sequence conservation is found at either the C-terminal (EGI, CBHI) or the N-terminal (EGIII, CBHII) end of the deducted protein (Fig. 2). A strikingly similar arrange- ment is found in the Cel]ulomonas fimi cellulases where a 50% homologous region is present at the C- terminal (Exg) or N-terminal (Eng) end 7'8. The homologous terminal domain shared by all four Tricho-

derma cellulases is apparently different from the homologous terminal domain of the two Cellulomo- has cellulases, but both these sequences are joined to the rest of the protein by a sequence rich in hydroxyl amino acids and proline.

In the three sequenced Clostri- dium thermocellum cellulases, EG A, EG B and EG D, a direct repeat of 24 amino acids is located at each C- terminus 9-12'2°. In the case of EG B and less clearly in EG A, the repeated terminal sequence is pre- ceded by a region rich in proline and hydroxyl amino acids. In EG D this type of sequence cannot be detected.

Recent sequence data concerning a ~-l,4-glucanase of B. subti]is suggests that the gene codes for a longer polypeptide than the mature secre- ted enzyme. The N-terminal sequence of the mature protein agrees with the N-terminal sequence predicted from the gene and, therefore, it appears that approximately 150

- F i g . 1

Trichoderma reesei CBH I L

CBH II j

EG I L ~ . . . . . . . . .

EG III Ff4 I ]

Cellulomonas fimi ENG ~ . . . . . . . . ]

EXG r I L \ . ~

Clostridium thermocellum EG A I ~

EG B l

EG C I

Aspergillus niger G1 [

G 2 F

~ I I131

1 1I 11

,]

, , approx . 100 aa

Schematic structures of different cellulase and glucoamylase genes. Each enzyme consists of a catalytic domain probably linked via a flexible "hinge" region to a "taft' domain that is most likely responsible for disrupting the structure of the semi-crystalline substrate. (The drawing is only roughly to scale.) ~_ ~ hydrolytic domain; F J "h inge ' , '~ -~ ' ta i l " (T. r e e s e i ) ; ~ "tail" (C. fimi); ~====1 "tail" (C. thermocellum);[]]~[~[~ "tail" (A niger); G7 and G2 glucosidases.


amino acids are removed from the C- terminal of the precursor by an unknown mechanism 4. An alka- lophilic Baci l lus strain has two highly homologous cellulases coded by separate genes, but only one of them has a C-terminal repeat of 60 amino acids 5'6. This terminal sequence, like the adjoining sequence, is strikingly rich in prolines, and also contains hydroxylamino acids.

The analysis of the primary structure of cellulase genes thus focused attention on the terminal regions. There would appear to be at least two types of phenomena. In T. reese i and C. f im i cellulases, conserved structures are found at the termini of otherwise dissimilar enzymes. However, in the other cellulolytic bacteria studied so far, enzymes are found both with and without terminal extensions which often contain short direct repeats. The terminal homology shared by an enzyme family within a species, strongly suggests that the conserved region is functionally important and that its location at the end of the molecule also has functional significance.

Function of the terminal domains The T. reese i cellulase homo-

logous terminal domains, including the serine and threonine rich sequence, can be removed by limited proteolysis 21 (Tomme et al., unpub-

lished). The activity of these trunca- ted enzymes towards microcrystal- line cellulose is impaired, but towards small soluble substrates it is not affected. The hydrolytic active site, therefore, must be localized in the core protein, while the terminal domain seems to have a role in substrate binding or solubilization. However, when the homologous terminal region of the endo- and exoglucanase of C. f i m i is deleted, the enzymes still retain their activity on CMC 7. Similarly, the C-terminal repeated domain of B. subt i l i s endonuclease is not essential for its CMCase activity 6. Since CMC has a much more open, non-crystalline structure than native cellulose, these results do not conflict with the idea that the homologous region is required for hydrolysis of crystalline cellulose and not for other 'easier' substrates. It is of interest to note that fungal glucoamylases (glucoamylases hydrolyse terminal oc- 1,4-glycosidic linkages) have, at least superficially, a similar structure to cellulases: a C-terminal domain seems to be responsible for binding to raw starch and this is also linked to the hydrolytic domain by a region rich in serine and threonine (Fig. 1 ) 2 2 - 2 4 . We would like to propose that many enzymes hydrolysing solid carbohydrate substrates, such as cellulose and starch, will be

shown to have similar domain architecture.

The catalytic mechanism and molecular structure

Enzymatic cellulose hydrolysis is likely to occur by an acid catalysis mechanism, which is known to be used by some other enzymes splitting glycosidic bonds. The best example, where the structure and functioning of the active site is already under- stood in great detail is lysozyme. Lysozyme also cleaves [3-1,4- glycosidic bonds but hydrolyses substrates containing N-acetylmuramic acid and N-acetylglucosamine side chains. In different lysozymes, the tertiary structure of the active site has been conserved but the primary structures are variable. In each case, however, the cleavage of the O- glycosidic bond involves donation of a proton by the carboxyl group of the conserved active site Glu residue, to the bond to be cleaved, and stabiliza- tion of the carbonium ion intermedi- ate by the conserved Asp residue.

Some sequence homology has been found between the active sites of lysozymes and the sequences of fungal cellulases, containing puta- tive Glu and Asp active site residues ~7'25'26, but such homologies are too weak to allow any conclu- sions about their significance to be drawn. Computer-aided molecular

I Fig. 2 modelling can be used to study Hydrolytic domain whether the proposed active sites of

N-glycosylation cellulases could theoretically fold f ~ j J into the conformation of the lyso-

zyme active sites 27 but experiments are required to test this model.

, , . O - g l y c o s y l a t i o n The comparison of different cellulases has not revealed any conserved

• sequences which could be active site regions. On the contrary, some active site regions proposed on the basis of weak homology to lysozymes seem less likely in the light of such sequence comparisons. For example, there is strong homology between T. reese i EGIII (Saloheimo et al., unpublished) and S. c o m m u n e EGI throughout the sequence with the

Acti-veSite exception of the N-termini, which A hypothetical m o d e / o f a cellulolytic enzyme. This mode / i s based on the are totally different. It has been analysis o f amino acid s equence data and on low angle X-ray diffraction proposed that the S. c o m m u n e EGI studies 29. active site is at the N-terminal 25,26

but it is more likely that the active


- F ig . 3

I CELLULASES I I XYLANASES

r

L I G N I N A S E S

I I I

I

Process A Process B

Lignocellulose processing in the future. A wide range of different enzymes each modifying or hydrolysing lignocellulose in a different manner will be available separately in purified form through genetic engineering and~or efficient downstream purification. The required modification or hydrolysis will be optimized by varying the composition of the enzyme mix.

site would be located in a well conserved region. Thus, it may not be useful to attempt to draw close analogies between the cellulases and lysozymes. Comparison of all carbohydrate binding proteins with known three dimensional structure shows that there is a surprising amount of diversity in the tertiary structures of these proteins and, therefore, also in their carbohydrate binding sites 28. For instance, taka-amylase, the o~- amylase from Aspergi l lus niger which is of a similar size to many of the celluloses, has a tertiary structure completely different from that of lysozyme. (Lysozyme is the smal- lest of carbohydrate binding proteins and only one third of the size of a typical cellulase.) The catalytic Gin and Asp residues of taka-amylase are located in the middle of the protein chain ( n o t N-terminally as in lysozyme), and their spacing in the primary structure is much greater than in lysozyme. Therefore, amy- lases may provide a better model for

cellulases than do lysozymes.

Secondary structures Secondary structures predicted

from the primary structures of the four T. reesei cellulases suggest a predomination of [~-sheet structures. Partial proteolysis shows that the active site residues are not in the terminal homologous regions, which probably form a flexible tail region of the CBHI molecule. This hypothesis is supported by data of the molecular shape of T. reesei CBHI, obtained by small angle X-ray diffraction analysis, which suggest that CBHI is a tadpole-like molecule which has a flexible terminal tail 29. Rapid pro- gress in structural analysis can now be expected, since good quality crystals for X-ray diffraction have recently been obtained for two cellulases, EGD from C. thermocel lum 12 and CBHII from T. reesei (Alwyn Jones, pers. commun.). Our current hypothesis of what a cellulase looks

like is shown in Fig. 2. It contains three main structural elements, the hydrolytic domain, which hydrolyses the [~-l,4-glycosidic bond, the flexible 'hinge' region and the 'tail'. The 'tail', the region which is homologous within a cellulase family, probably helps to solubilize the cellulose chain from the crystal prior to hydrolysis.

The role of glycosylation in cellulase function

The presence of extensive glycosylation in most fungal and many bacterial cellulases would seem to imply a functional role for the carbohydrate (especially since the bacterial cellulases are some of the very few examples of glycosylated bacterial proteins). In the case of T. reesei cellobiohydrolases, a large part of the O-glycosylation has been shown to be localized to the conserved terminal regions. Most of the cloned bacterial cellulases and the CBHI of T. Teesei have been expressed, unglycosylated, in E. coli, and all are still active on soluble substrates. The hydrolytic domains of the T. reesei cellulases, lacking most of the O-glycosylation, were shown to be active on the cello- oligosaccharides 21 (Tomme et al., unpublished). Thus cellulases can hydrolyse glycosidic bonds without participation of their carbohydrate portions. On the other hand, the four T. reesei cellulases have also been produced in yeast, in an overglycosylated form 19'3°. The activity of overglycosylated enzymes towards various natural or synthetic substrates was the same as that of normally glycosylated enzymes. Purified, overglycosylated CBHII had one third lower specific activity towards [tglucan than the authentic enzyme, and its affinity towards crystalline cellulose was slightly impaired (Penttil~i et al., unpublished). Thus overglycosylation also seems to affect substrate binding but not the cleavage reaction.

Cellulase diversity Since bacterial cellulases have

been mostly studied by gene cloning and expression of CMCase activity, it is difficult to know if all the relevant enzymes have been identified.


However, a recent publication suggests that Clostr idium may produce as many as 21 different endoglucanases 15. The significance of this suggestion is not clear at present.

Fungal cellulolytic enzyme systems have been studied both bio- chemically and genetically and generally comprise at least two cellobiohydrolases and an ill- defined number of endoglucanases. It has been especially difficult to correlate the results obtained by different investigators on the various endoglucanases. Proteolytic processing in the culture filtrates has been proposed to explain in part the diversity of fungal endoglucanases, and recent results support this idea. Proteolytic removal of the terminal homologous regions could also be an important mechanism for the regula- tion and adjustment of the substrate specificities of cellulases during the hydrolysis of complex carbohy- drates. In the later stages of hydrolysis, enzymes with higher specific activities towards shorter and altered substrates can be obtained by removal of the terminal domain. In this way, the diversity of enzymes could be increased without increas- ing the number of genes. Removal of a part of the substrate binding domain may even be a general mechanism to generate exo-enzyme diversity during growth.

Evolution of cellulase genes Close evolutionary relationships

are obvious between some of the cellulases, which have evidently arisen by gene duplication. For example CBHI and EGI of T. reesei share 45% sequence identity 18. A gene similar but not identical to T. reesei CBHI has been isolated from P. chrysospor ium (P. Simms, pers. commun.), and T. reesei EGIII is clearly homologous to S. c o m m u n e EGI (Saloheimo et al., unpublished).

A number of cellulases contain conserved terminal regions attached to apparently unrelated core proteins. Shuffling of gene segments coding for functional domains and bordered by introns has been proposed to be the main mechanism of protein evolution 31. For instance, in the case of regulatory proteases of

the fibrinolytic and blood coagula- tion systems, macromolecule binding domains have been linked to the ends of different enzymes with varying specificities during evolution 32. It seems very likely that the species-specific terminal domains present in some bacterial and fungal cellulases (probably hav- ing a solubilizing or binding func tion) have also been acquired as the result of domain shuffling analogous to the assembly of the regulatory proteases.

The future The enzymatic hydrolysis of ligno-

cellulose will continue to present an exciting challenge to the molecular biologist and process engineer. The application of modern molecular technology to this interesting system will not only provide much important fundamental information concerning enzyme structure and function, but should also lead to a series of developments which will improve current industrial processes using lignocellulose raw materials and permit the development of new areas. One immediate research objective should be to prepare large amounts of single enzymes, either by the construction of novel micro- organisms or by development of sophisticated downstream processing for existing enzyme preparations.

It will also be important to improve the enzymes available, either by finding new enzymes and genes from nature or even by what has been called protein engineering such that the enzymes are better suited to potential process condi- tions. Process technologists can then use this selection of different specific enzymes to optimize the particular conversion required. This idea is illustrated schematically in Fig. 3.

Another exciting possibility is to use the cellulase genes to construct novel cellulolytic organisms. Good examples of this are the industrial brewing yeast strains producing the T. reesei EGI enzyme (Knowles et al., in press) which have been shown to have significant advantages in pilot scale trials since they remove ~- glucans from the beer and so improve filtration and clarity.

The aim of research into lignocellulose in the future will be to achieve modification and polymerization at least as much as the original aims of improving hydrolysis and solubilization.

Our understanding of the molecular biology and enzymology of lignocellulose degradation is advancing rapidly and already pro- viding information that is of use today. There is clearly reason to hope that by combining knowledge and skills from many different disci- plines, we shall be able to solve many of the challenges posed today by lignocellulose.

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Laboratory management of animal cell culture

processes Winfried Scheirer

Cell culture technology has been described in the past as more of an art than a science. With the imminent arrival of industrial cell culture, this no longer holds true. Without a wealth of industrial experience to call on, however, it is important that cell culture technologists adopt a logical and rigorous approach to process development; an approach that encompasses the biological aspects of the process (the cells and personnel) as much as the physical and

mechanical ones (facilities and equipment).

Within the last few years animal (and human) cell cultures have gained increased technical relevance. Pro- ducts of mammalian cell culture technology include monoclonal anti- bodies and many other complex" proteins like plasminogen activators. The successful and correct handling of animal cell cultures within development processes for industrial production must ensure:

• the protection of personnel and environment from direct and indirect adventitious risks;

• the protection of cultures from deleterious influences;

• the effectiveness and reproduci- bility of the process aimed at minimizing loss by accident;

• that regulatory requirements con-

Winfried Scheirer is at the Cell Culture Unit of Sandoz, Forschungsinstitut GmbH, Vienna, Austria.

cerning the facilities, the process and their documentation are met.

The first consideration necessary to meet these requirements is an appropriate facility.

The facility The facility should consist of

a suite of rooms accomodating each of the different aspects of the process1-3:

• laboratories for the cultivation of stock cell lines (at least one room for each simultaneously operated production cell line);

• fermentation rooms; • cleaning and sterilization de-

partment; • preparation and analytical labora-

tory; • incubator rooms; • cold room, freezing facilities; • air locks.

The sizing of all rooms must be designed with care to meet the required capacities. All rooms should be air-conditioned. The rooms where cultures are handled and where medium is prepared must provide the highest quality sterile aeration and be kept at positive pressure. The air-conditioning must be constructed to avoid contamination from room to room.

Connections between rooms should be designed for minimal air exchange and, in some cases, fitted with locks which can be opened by special permission only. The other route of room to room transmission of organisms is the plumbing. Each sink should, therefore, be piped directly to the disinfection unit to avoid back- contamination. An idealized layout with installations, air-condition data and equipment is shown in Fig. 1.

Several principles should be fol- lowed with all safety classes of cell culture laboratories. One is the isolation of laminar air flow cabinets from the through-traffic of colleagues to avoid turbulences from the environment.

Another principle is the separation or encapsulation of centrifuges because of the high risk of aerosol formation.

Fermentation equipment should be in a separate room because of heat generation, escape of steam or other 'wet' events.

Construction details like maxi- mum floor load, complicated piping and areas of high cooling capacities of the air condition must be con- sidered carefully.

Inside the rooms, it is important

© 1987, Elsevier Publications, Cambridge 0166- 9430/87/$02.00

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