IMMOBILIZATION OF BACTERIA AND PLANT CELLS

5. IMMOBILIZATION OF BACTERIA AND PLANT CELLS

It has already been described earlier that immobilization particularly refers to — ‘the imprison- ment of a biocatalyst in a distinct phase which necessarily permits only exchange, but is clearly sepa-

PHARMACEUTICAL BIOTECHNOLOGY

rated from the bulk phase wherein the substrate, effector, and inhibitor molecules are adequately dis- persed and monitored.’

5.1. Immobilization of Bacteria

The immobilization of bacteria (organism) may be explained by the help of fermentation of

E. coli. for the production of cytidine deaminase. It is well known that cytidine deaminase is extensively distributed among microoraganisms,

where its physiological role is confined to scavenge specifically exogenous and endogenous cytidine*. However, in the particular instance of enteric bacteria, for instance : E. coli. and Salmonella typhimurium,

the enzyme is appreciably inducible to high levels, thereby permitting these organisms to grow rapidly with cytodine as an exclusive source of nitrogen**. Therefore, it is worthwhile and also logical to look into E. coli as a source of large quantities of enzyme for actual usage in the production of ‘Lamivudine’

[Epivir (R) ; Zeffix (R) ], an antiviral drug.

It has been demonstrated that cytidine deaminase production by two variants of E. coli strains JM 103 and B, with and without cytidine are as follows ;

S.No. Strain of

(+) Cytidine (E. Coli)

0.01 0.13 (– ) = Without ; (+) = With ;

2 B 50 mL

Based on the above findings one would expect the actual production to be approximately 10 folds as great in the presence of cytidine. In order to obviate the need to add cytidine to the fermentation broth,

a constitutive mutant was virtually sought. Munch-Petersen et al.*** (1972) suggested that in E. coli both cytidine deaminase and uridine phosphorylase are coordinately expressed under the direct con- trol of the CytR repressor. It has been observed that the expression of both genes is duly induced by cytidine, but not by uridine. As a result, the specific mutants which may grow progressively upon uridine are likely to have a defective CytR gene ; and, therefore, will express both enzymes constitutively.

* Munch-Petersen A, (ed.) : Metabolism of Nucleotides, Nucleosides, and Nucleobasein Microorganisms, Academic Press, London : 95-148, 1983.

** Munch-Petersen A (ed.) Ibid, 203-258, 1983.

ENZYME IMMOBILIZATION

Nevertheless, the enzyme production by the constitutive mutant was just sufficient to support biotransformation process at a resonable substantial scale ; however, for production scale, one may even require a still better source and type of enzyme. Such a challenging and herculein task was duly accomplished by cloning the cytidine deaminase gene (cdd) onto a multicopy plasmid under the control of the λ P L promotor*. Subsequently, the ensuing plasmid p PLcdd E was strategically introduced

into E. coli TG 1 (Amersham) by transformation, thereby attributing resistance to tetracycline. Thus, the recombinant strain [E. coli TG1 {p PLcdd E}, 3804 E] afforded ultimately an extremely high level of cytidine deaminase production. Importantly, the specific activity was found to be raised comfortably upto 80 times than that accomplished with the constitutive mutant.

5.2. Immobilization of Plant Cells

Immobilization of plant cells has recently been developed as an alternative (substitute) method- ology to the age-old suspension cultures for the exclusive production of secondary metabolites. i.e., such metabolites which are known to be very necessary to plant life, many of them providing a defence mechanism against bacterial, viral, and fungal attack analogous to the immune system of animals.

Immobilized Plant Cells for Agriculture : In the recent past the enormous application of ‘mi- crobial inoculants’ have gained a tremendous momentum in the ever expanding domain of agriculture that could be anchored to the following four solid supporting facts, namely :

(a) Apparent noticeable increment in symbiotic or associative nitrogen fixation, (b) Biological control and management of soil-borne plant pathogens,

(c) Spectacular reduction in aflatoxin** contents, and (d) Biodegradation of xenobiotic*** compounds. It is, however, pertinent to emphasize at this juncture that in these bioactive processes, the critical

survival of microbes under biotic as well as abiotic prevalent stresses emanated in the soil poses a major limitation. It has also been amply advocated that immobilization of microbial cells via various

modes e.g., entrapment, encapsulation etc., in these specific instances has been proved to cater for adequate protection against these prevailing stresses.

Examples : The two typical examples are as given below : (a) Microbial cells entrapped in alginate : These cells did survive against the environmental

hazards of the soil ; whereas, the actual survival of ‘free cells’ (i.e., the untrapped ones) was minimised astronomically under the prevailing drying/welting cycles in soil parameters, and

(b) Algal Biofertilizers**** : In this particular instance, the algal biofertilizers the phenomenon of immobilization not only displayed a positive edge in efficacy but also afforded a definite advantage.

Tissue Culture : Tissue culture refers to the growth of tissue in vitro on artificial media exclu- sively for experimental research. It is indeed a stark reality that tissue culture has immensely facilitated the techniques of microbial genetics as applicable specifically to the higher plant cells. One may strate- gically induce genetic variability in a relatively large homogeneous population of plant cells by ad- equate exposure to either chemical or physical mutagens. Thus, it is now quite possible to prepare

* Mahmoudian M et al. Enzyme Microb Technol., 15 : 749-755, 1993. ** A toxin produced by some strains of Aspergillus flavus and A. parasiticus that causes cancer in laboratory

animals. It may present in unprocessed peanuts and other seeds contaminated with Aspergillus molds.

PHARMACEUTICAL BIOTECHNOLOGY

various suspensions of appropriate cell cultures of higher plants, and maintain them more or less exactly in the same state for as long as one may desire. As a result, each single cell may prove to be as good as

a potential bacterial cell for affording induction and followed by isolation of mutants and variants.

Nevertheless, the importance of cell culture depends upon the meticulous development of meth- odologies for enabling the isolation of a broad spectrum of cultured cell strains having apparent charac- teristic features that are entirely different from those of cells in the original cultures. Therefore, in order to isolate such obvious variant cell strains, established techniques in microbial studies may have to be enforced judiciously in cultured plant cell systems.

Application of Mutagens to Plant Cells : It is regarded to be an especially important methodol- ogy to enhance the ensuing frequency of variant strains in population of cells so that they may be easily identified and conveniently selected. The vigorous and constant search for ‘chemicals’ that are virtually effective upon a broad spectrum of plant cell is invariably considered to be an important aspect of plant somatic cell mutant isolation.

In order to ascertain whetehr or not a particular substance is mutagenic exclusively depends upon the expression of an easily observed characteristic feature, such as : resistance to a specific nucleic acid precursor analogue termed as 6-azauracil, present profusely different in parent cells and the subse- quent variants derived from them after due treatment with the agent. Interestingly, the parent cells growing in culture are highly sensitive to this compound, whereas the variants which are apparently resistance to it, may be observed explicitely ; and this difference forms the fundamental basis of its

‘assay’.

It has been established beyond any reasonable doubt that the ensuing ‘difference’ is caused due to a highly deficient enzyme present in the variant cells, which is known as uracilphosphoribosyl transferase, and this actually affords a ‘cidal action’ upon the cells. There are, in fact, two predominant strains of cells duly obtained from two altogether different species of plants, namely :

(a) Haploid* Datura innoxia, and (b) Diploid** Happolopappus gracilis

that are found to be exerting resistance to this particular analogue have been meticulously isolated*** and exhibited to lack the aforesaid enzyme i.e., uracilphosphoribosyltransferase.

Salient Features of Mutagens to Plant Cells : Following are some of the salient features of mutagens to plant cells :

(1) Higher plants e.g., Nicotiana tabacum, and ferns e.g., Todea barbara ; Osmunda cinnamomea helped in a big way for carrying out such studies.

(2) Several varieties of auxotrophic*** mutants that essentially requires amino acids and vita- mins have been skilfully raised in Todea barbara.

(3) Mutants that absolutely requiring amino acids, purines, and vitamins have been isolated with utmost success in Nicotiana tabacum.

* Possessing half the normal number of chromosomes found in somatic or body cells. ** Having two sets of chromosomes ; said of somatic cells, which contain twice the number of chromosomes

present in the egg or sperm. *** Requiring a growth factor that is quite different from that required by the parent organism.

ENZYME IMMOBILIZATION

(4) Mutants that offer resistance to Streptomycin — an antibiotic ; 8-Azaguanine and 5-Bromo-

2 ′′′′′ -deoxyuridine — base analogues have been isolated in higher plants as well.

(5) Auxotrophic and resistance mutants are of utmost importance in carrying out the analyses of genetic linkage, complementation, and recombination.

(6) Inhibitors* do play a major and pivotal role in the decephering of various metabolic path- ways in vivo. Thus, by using different metabolic inhibitors one may conveniently affect virtually complete blockade of the cellular metabolism at a specific site ; and, therefore, by critically examining the fall out of such blockages it becomes a lot easier to know exactly the nature of the pathway and also the factors responsible for controlling the entire process.

(7) Inhibitors also help to control several cardinal functionalities, for instance ; a specific step in DNA and RNA, protein anabolism (syntheses), and metabolic process of an organism.

(8) Mutants also throw sufficient in-depth knowledge with regard to appropriate selection of products emanated due to ‘fusion’ occurring between cells of variant genetic backgrounds (or antecedents).

(9) Mutants solve a host of fundamental biological intricated problems to a great extent. (10) Mutants recovered carefully from cultured plant cells are really of tremendous

biotechnological importance, and may serve as the basis of unfolding a good number of complex agricultural, industrial and nutritional problems speedily and logically.

Example : The wildfire disease in Tobacco is caused due to Pseudomonas tabaci. In fact, the tobacco cells that are specifically resistant to wildfire toxin have been recovered amongst a mutagenized haploid cell population. Therefore, disease resistant tobacco plants may be accomplished in two ways, namely :

(i) Direct selection for resistance to a pathogen in the culture itself, and (ii) Appropriate selection of toxin resistance as a generalized means for producing particularly

the disease resistant varieties.

* Substances that interfere with a biological process thereby restraining the natural activity of a particular function or metabolic activity of an organism.

PHARMACEUTICAL BIOTECHNOLOGY

RECOMMENDED READINGS

1. Bickerstaff GF : Enzymes in Industry and Medicine, New Studies in Biology, Edward Arnold, London, 1987.

2. Boyce COL (ed.) : Novo’s Handbook of Practical Biotechnology, Novo Industries AS, Copenhagen, 1986.

3. Bulow L and K Mosbach : Multienzyme System obtained by Gene Fusion, Trends in Biotechnology, 9 : 226-231, 1991.

4. Charbey W and H Herzog : Microbial Transformation of Steroids, 2nd. edn. Academic Press, New York, 1980.

5. Chibata I, and T Tosa, Transformation of Organic Compounds by Immobilized Microbial Cells, pp 1-27. In : Perlman D. (ed.), Advances in Applied Microbiology, Academic Press, New York, 1977.

6. Chibata I., Immobilized Enzymes, John Wiley & Sons, New York. 1978.

7. Cornish-Bowden A and R Eisenthal : Computer Simulation as a tool for studying metabo- lism and drug design. In : Technological and Medicinal Implications of Metabolic Control Analysis, Kluwer Dordrecht, The Netherlands, pp. 165-172.

8. Edginton SM : Taxol Out of the Woods., Biotechnology, 9, 933-938, 1991.

9. Flickinger MC., Anticancer Agents, pp. 231-273. In : Moo-Young M (ed.) : Comprehen- sive Biotechnology, III, Pergamon Press, Oxford, 1985.

10. Fogarty WM, and CT Kelly : Enzymatic Developments in the Production of Maltose and Glucose, pp. 149-163. In : Lafferty RM (ed.) : Enzyme Technology, Springer Verlag, Ber- lin, 1983.

11. Fry JC, and M Day (eds.) : Release of Genetically Engineered and Other Microorgan- isms, Cambridge University Press, Cambridge.

12. Greenshields R(ed.) : Industrialized Biotechnology International, Sterling Publications Ltd., Hond Kong, 1993.

13. Hacking AJ : Economic Aspects of Biotechnology, Cambridge Studies in Biotechnology

3, Cambridge University Press, Cambridge, 1986.

14. Hodgson J : Data-directed Drug Design, Bio/Technology, 9 : 19-21, 1991.

15. Jacobsson S, Jamison A, and Rothman H, The Biotechnology Challenge, Cambridge Uni- versity Press, Cambridge, 1986.

16. Kazlauskas RJ : Molecular Modelling and Biocatalysis : Explanations, Predictions, Limi- tations, and Opportunities., Curr. Opin. Chem. Biol., 4 : 81-88, 2000.

17. Kleyn PW, and ES Vesell : Genetic Variation as a Guide to Drug Development., Sci- ence, 281 : 1820-1821, 1998.

18. Marconi W, and F Morisi : Industrial Applications of Fiber-Entrapped Enzymes, pp. 219- 258. In : Wingard LBE, Katchalski-Katzir, and L Goldstein (eds.) : Applied Biochemistry and Bioengineering, Vol. 2. Academic Press, New York, 1979.

19. Mosbach K, and O Ramstrom : The Emerging Technique of Molecular Imprinting and its Future Impact on Biotechnology, Bio/Technology, 14, 163-170, 1996.

ENZYME IMMOBILIZATION

20. Office of Technology Asessment : Commercial Biotechnology : An International Analy- sis, US Congress, Washington, DC, 1984.

21. Petronia IP, and FH Arnold : Designed Evolution of Enzymatic Properties, Curr. Opin. Biotechnol., 11 : 325-330, 2000.

22. Powell W, and JR Hillman : Opportunities and Problems in Plant Biotechnology, Pro- ceedings of the Royal Society of Edinburgh, 99B, 1992.

23. Scientific American : Industrial Microbiology and the Adrent of Genetic Engineering, A Scientific American Book, WH Freeman & Co., New York, 1981.

24. Tischer W, and V Kasche : Immobilized Enzymes : Crystals or Carriers ? Trends in Biotechnology, 17 : 326-335, 1999.

25. Woodward J : Immobilized Cells and Enzymes, a Practical Approach, IRL Press, Oxford, 1985.

26. Yanchinski S : Setting Genes to Work : The Industrial Era of Biotechnology, Viking, New York, 1985.

PROBABLE QUESTIONS

1. (a) What do you mean by ‘Enzyme Immobilization’ ? Explain. Discuss briefly the salient features and carrier matrices with reference to the immobi- lization of enzymes.

(b) Give a brief description of the four types of methods of immobilization of enzymes.

2. How would you explain the mechanism of ‘Covalent Bonding’ in the enzyme immobiliza- tion ? Discuss the advantages of Covalent Bonding with reference to the support with functional groups, namely :

(a) OH— Group ; and (b) —COOH— Group. Give examples in support of your answer.

3. (a) Describe an elaborated approach of either ‘Entrapment’ or ‘Encapsulation’ invari- ably encountered in enzyme immobilization.

(b) Enumerate the various ‘Advantages’ and ‘Disadvantages’ of enzyme immobilization.

4. Give a brief account on the following aspects of enzyme immobilization :

(a) Enzyme Activity (b) Michaelis — Menten constant [K m ]

(c) Determination of K m .

5. Discuss Kinetics of ES-complex formation in a comprehensive manner.

6. Manner the various cardinal parameters that essentially govern the Enzymatic Reactions.

Discuss the following aspects in details : (a) pH Activity ; (b) Stability ; and (c) Optimum Temperature.

7. Enumerate the exhaustive profile of any two important enzymes : (a) Hyaluronidase

(b) Pencillinase

(c) Streptokinase

(d) Strepodornase.

PHARMACEUTICAL BIOTECHNOLOGY

8. What are ‘Amylases’ ? Discuss the following aspects explicitely : (i) Applications of Amylases. (ii) Production of Bacterial α -Amylases. (iii) Production of Fungal α -Amylases.

9. How would you classify the ‘Proteases’ ? Give a detailed account of any three important categories.

10. Write short notes on any four of the following topics : (a) Immobilization of Bacteria (b) Immobilization of Plant Cells

(c) Applications of Mutagens to Plant Cells (d) Salient features of Mutagens to Plant Cells

(e) Alteplase (Recombinant) (f) Development of New Proteolytic Enzymes.