thermophilic proteases: properties and potential applications

68

12 McGuire, J. C., Montgomery, J. P., Yang, H. H., Vollmer, P., Schruben, J. and Finkelman, M., patents pending, Genex Corp.

13 Schirch, L. (1982)Adv. Enzymol. 53, 83-112

14 Kallen, R. G. and Jencks, W. P. (1966)J. Biol. Chem. 241, 5851-5863

15 O'Connor, M. L. and Hanson, R. S. (1975).,7. Bacteriol. 124, 985-996

16 Alexander, N. and Greenberg, D. M. (1956) ,7. Biol. Chem. 220, 770--785

17 Blakely, R. L. (1960) Biochem. ft. 77, 459-465

18 Plamann, M. D., Stauffer, L. T., Urban- owski, M. S. and Stauffer, G. V. (1983) Nucleic Acids Research 11, 2065-2075

19 Anderson, D. M., Hsiao, H. Y. and Yang, H. H. (1984) Div. Microb. Biochem. Technol., Am. Chem. Soc., 188th ACS National Meeting, Phila- delphia, Abst. 70

20 Methods Enzymol. (1963) VI, 801-814 21 Knowles, W. S., Sabacky, M. J. and

Vineyard, B. D. (1972) Chemtech, 590-593

22 Anderson, D. M., Herrmann, K. M. and Somerville, R. L. (1983) US Patent No. 4,371,614

23 Snell, E. E. (1975)Adv. Enzymol. 42, 287-333

24 Sano Konosuke, M. and Koji Mitsugi, Y. (1975) US Patent No. 3,929,573

25 Hitoshi Enei, Z., Hidetsugu Nakazawa,

Trends in Biotechnology, Vol. 3, No. 3, 1985

K., Hiroshi Matsui, Y., Okumura, V. S. and Yamada, H. (1974) US Patent No. 3,808,101

26 Marechal, P. D., Calderon-Seguin, R., Vandecasteel, J. P. and Azerad, R. (1979) Eur. ft. Appl. Microbiol. 7, 33-34

27 Akio, M., Takahashi, Y., Yuasa, K. and Shibukawa, M. (1981) US Patent No. 4,360,594

28 Yoshiyuki, Asai, Y., Y., Masao Shimada, Y. and Kenji Soda, U. (1982) US Patent No. 4,335,209

29 Bang, W., Lang, S., Sahm, H. and Wagner, F. (1983) Biotechnol. Bioeng. 25, 1013-1025

30 Ulmer, K. M. (1983) Science 219, 666-671

Patents

Ajinomoto Co. Inc. (Enei, Z. H,, Nakazawa, K. H., Matsui, Y. H., Okumura, V. S. and Yamada, H, 1974) US 3808101, DT 2163964, FR 2119018, GB 1374454, J 74046917: L-tryptophan and its derivatives are produced by the action of tryptophanase produced by certain microorganisms (e.g. Escherichia, Aerobacter, Erwinia, Pseudomonas, Pro- teus, Salmonella) on indole and its derivatives and on pyruvic acid, oxaloacetic acid, fumaric acid, malic acid, maleic oxime, glyoxlyic acid or lactic acid. The tryptophase activity increases in the presence of a surface active agent. Broths containing microorganisms can be used as the source of the enzyme without further purification. Amino acids can be recovered continuously.

Ajinomoto Co. Inc. (Konosuke, M. S. and Mitsugi, Y. K, 1975) US 3929573, DT 2461188, FR 2256243, GB 1473244, J 50095484, J 76037353: tryptophanase from Proteus, Erwinia, Escherichia, Pseu- domonas or Aerobacter which catalyses the reaction of indole with serine to give L-tryptophan can also use fl-chloro- or/J- bromoalanine (instead of serine). This gives higher yields of L-tryptophan than other known methods. Cells can be used as an enzyme source in a batch system.

Mitsui Toatsu Chemicals Inc. (Asai, Y. Y., Shimada, Y. M. and Soda, U.K, 1982) US 4335209, CA 1128443, DE 3017861, FR 2456140, GB 2048266, J 55148095, J 56005098, J 56134992, J 82031438, NL 8002603; including a serine racemase from a Pseudomonos strain permits efficient use of cheap, chemically-prepared DL-serine as a substrate for tryptophan-

ase. The serine racemase has a low substrate specificity (it will act on 20 amino acids) but, importantly, it will not racemise tryptophan, nor is it inhibited by tryptophan. The enzyme can be used in the form of whole cells, dead cells, crude homogenates, or in an immobilized form, preferably in an acylamide lattice.

Purification Engineering Inc. (Wood, L. L. and Calton, G. J, 1984) US 4436813, AU 831212 I, EP 89165, J 58170480; The immobilization of E. coil is described. The immobilized cells are used to synthesize L- aspartic acid from ammonium fumarate in a batch or continuous fashion. Immobil- ization is in a fixed insoluble cross-linked polymer cured at below 40°C to mini- mize inactivation of aspartase. Immo- bilized cells can be virtually dried and still retain most of their original aspartase activity.

Thermophilic proteases: properties and potential applications

Don Cowan, Roy Daniel and Hugh Morgan

Proteases are arguably the m o s t i m p o r t a n t group o f industr ia l e n z y m e s and certa in ly f o r m a m a j o r port ion o f wor ld -wide e n z y m e sales. T h e r m o p h i l i c proteases , w i th the ir h igh speci f ic act ivi t ies and the ir super ior c h e m i c a l and phys ica l s tabi l i ty characteris t ics , w o u l d s e e m to be good candidates for current and future b io technolog ica l

appl icat ions .

Historically, proteases have played a very important role in the processing of natural products. For many centuries, protease-containing cultures, extracts and waste products have been used in

D. Cowan is now at the Department of Biochemistry, University College, Lon- don, UK, R. Daniel and H. Morgan are at the Thermophile Research Group, School of Science, University of Waikato, Hamil- ton, New Zealand.

such diverse applications as the tanning of hides and the production of condi- ments. Today, proteases are probably the most important of the classes of industrial enzymes, with world-wide sales (US $236 million in 1981) repre- senting about 60% of the total enzyme marketL Three enzymes (bovine and microbial rennins, used in cheese- making and Bacillus megaterium neutral protease, used in the detergent

© 1985, Elsevier Science Publishers B.V., Amsterdam 0166-9430/851502.00

industry) are responsible for nearly two-thirds of the protease market.

C o m m e r c i a l proteases These three are, of course, industrial

enzymes which are available in bulk and are relatively cheap. They are quite distinct from the speciality enzymes, which are usually highly purified, very expensive, and used on a much smaller scale (for example, in research work). The desirable properties for a commercial protease will depend primarily on whether the enzyme is intended for the industrial or specialist market. Within these categories, the requirements are extremely varied, depending entirely on the nature of the application. For example, a protease suitable for the detergent industry would ideally possess both high activity and stability in moderately alkaline conditions (pH 10-11), should be unaffected by oxidizing and chelating agents and should be available in bulk at low cost.

Trends in Biotechnology, VoL 3, No. 3, 1985

However, as a tool in protein sequenc- ing, the critical requirement for a protease is that it has a narrow and well- defined peptide bond specificity. Cost is of relatively little importance.

New proteases are constantly being isolated. Although almost all types of proteases have some potential application, one of the reasons that most are not even considered for commercial use is that any possible advantages are heavily outweighed by the necessary development costs. Even some which seem to have substantial advantages (e.g. the atkophilic proteases 2) have not yet found great commercial accep- tanceL It seems reasonable to predict that any new protease would be more likely to find application as a speciality enzyme than as an industrial enzyme.

In the following discussion, we will outline some of the properties of thermophilic proteases and attempt to identify areas of biotechnology where their novel characteristics could impart some process advantage.

Proteases from thermophilic organisms

Many proteases have been isolated and characterized from thermophilic organisms. The most detailed studies have been on thermolysin, a zinc metalloprotease isolated from Bacillus thermoproteolyticus, a strain of B. stearothermophilus 4. Other well- characterized thermophilic extracellular proteases include the B. stearothermophilus neutral proteases 5'6, thermo- mycolin (from Malbranchea pulchella var. sulfurea7), thermitase (from Thermoactinomyces vulgaris 8) and a protease from Streptomyces rectus 9. T h e distinction between thermo-

philes (optimum growth temperature, 55-65°C) and extreme thermophiles (optimum growth temperature above 6 5 ° C) is becoming less distinct with the isolation of new, high-temperature strains of B. stearothermophilus. To date, few proteases from extreme thermophiles have either been reported or studied in detail. Characterization studies are limited to the extracellular protease from Bacillus caldolyticus10 and four proteases from different strains of the genus Thermus'-~L

Protease production Protease-producing thermophiles are

routinely grown on undefined complex media4-1L In the laboratory, yeast extract and commercially available

Table I. Protease production by mesophiles and thermophiles

69

Microorganism Growth Protease Assay temp. activity temp. (°C) (PU/ml of (°C)

culture9

Ref.

Bacillus megaterium ATCC 14581

B. cereus ATCC 14579 B. polymyxa ATCC 842 B. subtilis SP 491 Pseudomonas

aeruginosa IFO 3454 Serratia

marcescens NCIB 10351 Aeromonas

proteolytica ATCC 15338 Aspergillus

oryzae ATCC 11493 Aspergillus

flavus ATCC 11498 B. stearo-

thermophilus 1503 Thermus

caldophilus GK 24 T. aquaticus T351 T. aquaticus YT1 Aq. I

Aq. II

30 4150 37 14 30 1280 37 14 30 4530 37 14 37 1110 37 14

37 4450 37 14

26 1750 37 14

26 19970 37 14

26 1190 37 14

26 1540 37 14

55 2 55 6

70 278 70 12 75 70 75 75 228 70 13 75 304 95 13

1 PU is defined as 0.5 #g tyrosine released per minute under the conditions of the assay

protein digests are normally used, while larger scale commercial producers substitute cheaper nutrient sources such as corn-steep liquor, fish meal and soybean extract ~4 which usually have high C:N ratios. While the thermophilic bacilli are rather non- specific in their nutrient requirements, Thermus species grow poorly in media containing high concentrations of carbohydrate/5.

Proteases are excreted at relatively low levels (Table 1) by most thermophilic bacteria. However, mesophilic organisms which excrete proteases appear to do so at activity levels at least one order of magnitude higher (Table 1). Data on the production yields of thermolysin, the only thermophilic protease currently used on an industrial scale (in the synthesis of aspartame) is not available. Strict comparisons between mesophilic and thermophilic protease activities are complicated by different assay temperatures and procedures. Nonetheless, it is apparent that substantial increases in protease production, whether brought about by genetic manipulation or nutrient optimization, would be necessary before these thermophilic proteases could become competitive as industrial enzymes. Production costs and yields would be less critical factors ifthermo-

philic proteases were to be considered only as speciality enzymes.

Little information is available on the mechanisms controlling protease production in extreme thermophiles. A study of the effects of amino acids, ammonium salts and sugars on the production of extracellular protease by Thermus aquaticus strain T351 showed that no significant induction or repression occurred (Cowan, D. A., unpublished results). However, a strong correlation was noted between growth rate and extracellular proteolytic activity. Akhough there are numerous examples of the induction and repression of the synthesis of proteases by mesophilic microorganisms (e.g. Ref. 3), no other data on similar effects in extremely thermophilic bacteria are currently available.

Fujii and coworkers 16 have recently cloned the structural gene for a B. stearothermophilus neutral protease into a plasmid. When the recombinant plasmid was expressed in B. stearothermophilus, protease production increased 15-fold. There have been no reports of attempts to increase protease production in cultures of extreme thermophiles by genetic manipulation. In our laboratory, attempts to select a highly productive, UV-induced mutant of T. aquaticus strain T351 have yielded a

70

Table 2. Thermostability of mesophilic and thermophilic proteases Source organism Growth Hag-life Incubation Ref

temp. (min) temp. (°C) (°c)

Bacillus licheniformis 37 10 70 34 B. subtilis 37 10 60 34 B. subtilis N' 37 12-18 50 36 B. stearothermophilus

NCIB 8924 55 15 74 35 B. stearothermophilus

NRRL 3880 55 15 87 35 Malbranehea pulchella var.

sulfurea 55 120 73 7 Streptomyces rectus var.

proteolyticus 50 30 82 9 B. thermoproteolyticus 55 60 80 4 Thermus aquaticus T351 75 1800 80 11 T. aquaticus YT1 75 180 80 * B. caldolyticus 72 >480 80 10 T. caldophilus 70 120 80 12 * H. Matsuzawa, personal communication

variant producing significantly more extraceUular protease than the wild type. It is quite possible that selection, mutation and genetic manipulation techniques will allow the isolation of thermophilic protease producers com- parable to mesophilic organisms in their enzyme production.

Protease recovery Predictions that expression of

thermophilic genes in mesophilic organisms would simplify protein purification procedures are supported by some experimental evidence 17. A heating step results in the denaturation of most of the relatively unstable mesophilic protein, while the thermophilic protein remains in solution. It is possible, however, that loss of proteolytic enzyme through its binding to precipi- tated (substrate) protein would make this technique less attractive for thermophilic proteases.

Extracellular proteases from Ther- mus caldophilus ~, T. aquaticus strain T351 u, M. pulchella 7, B. stearothermophilus 6 and three other Thermus strains isolated in our laboratory have been purified to near homogeneity with yields of 20-50%. These data do not appear to support the suggestion by Doig ~s that higher yields of purified enzymes should be obtained when using thermophilic microorganisms as a source. In view of the thermostability of these proteases (see later) and the fact that autolytic loss in concentrated solu- tions can be quite low 19, it is perhaps surprising that recovery levels are not higher. There is some evidence that

protein insolubility as a result of hydrophobic surface interactions is responsible for some losses during purification. Matsuzawa and coworkers 13 have found evidence for the formation of high-molecular-weight protease complexes in partially purified samples of Aqualysin II from T. aquaticus YT1. Such complexes could be caused by hydrophobic intermolecular interactions.

Structure and function All the thermophilic bacterial extra-

cellular proteases so far reported are serine- or neutral metaUo-proteases. Cysteine proteases appear to be restricted to the thermophilic fungi. The catalytic mechanism of a protease is an important consideration in commercial application. Cysteine proteases are susceptible to inhibition by many reagents, particularly metal ions, and lose activity by oxidation. Optimal activity is often only obtained in the


presence of reducing agents and such additions are often not industrially feasible. Neutral metalloproteases are rapidly inactivated by the removal of the catalytic metal ion and thus cannot be used in applications where chelating agents are present (e.g. in some detergents). The serine proteases, however, are not readily inhibited, although many are stabilized by metal ions, and are used in most established biotechnological applications.

Structural stability and its consequences

There is a good correlation between the growth temperature of a source organism and the stability of its extracellular proteases (Table 2). It follows, therefore, that a search for bacteria cap- able of existing at even higher temperatures should yield proteases of even greater thermostability.

The stabilization of proteins against thermal denaturation is generally conferred by small changes in the amino acid sequence 2°,21. Such changes can affect stability, without any obvious structural alteration, by giving rise to a relatively small number of additional intramolecular interactions. In proteases, specific binding of metal ions (particularly of calcium) further en- hances molecular stability. For example, the increased stability of caldolysin over thermolysin can be attrib- uted almost entirely to the binding of six calcium ions to the former ~9, as opposed to four calcium ions to the latter2L The abnormally high frequency of tyrosine in thermolysin 23 has also been implicated in its thermostability, although this proposed mechanism seems to be unique.

The stability of thermophilic proteases is not restricted to temperature but also includes resistance to denatur-

a % inactivation in 0.1% perborate at pH 9.5 for 30 minutes b Based only on laboratory-scale production NA, Data not available

Table 3. The suitability of proteases for use in detergent preparations Enzyme Papain Bacillus Bacillus Thermolysin Caldolysin Thermus properties neutral alkaline strain Tok3

protease protease protease Activity at low low high medium high high (pH 9-10.5)

Stability low low high high high high Effect of none inhibited none inh ib i t ed reduced none chelation stability

Effect of 100 NA 10 NA 0 0 oxidation ~

Cost low medium medium high v. high b v. high b


ing agents, detergents and organic solvents 1~,~2,23. Little evidence is available, however, to suggest that their stability at extremes of pH is any greater than that of their mesophilic counterparts. The thermophilic alkaline serine proteases are quite stable in mildly alkaline conditions (<pH 11.5) but lose activity rapidly below pH 4 (Refs 11, t2). In practical terms, hydrolyses can be per- formed satisfactorily at 75°C over extended periods at pH levels between 5 and 11.

Protein stability can be increased by various chemical modification procedures. Reports of increasing the thermostability of thermophilic proteases by immobilization 24'25 and metal ion substitution 19 have been published. Other methods of protein stabilization such as by covalent cross linking are well established 26,27.

Opportunities for commercial use The ability ofthermophilic proteases

to retain a significant level of activity after extended periods at high temperatures in aqueous solution (e.g. 4-5 h at 85 °C 11) must impart certain biotechnological advantages. The difficulty lies in identifying which existing enzymic processes could be satisfactorily per- formed at higher temperatures: most are currently carried out at less than 60°C 3. The multitude of industrial protein recovery or solubilization processes 3'~s are likely candidates. Advantages such as more efficient hydrolysis (see later), reduced mesophilic contamina- tion and reduced viscosity will be partly offset by the cost of maintaining the reactor temperature. In all these examples, the cost of the protease pre- paration is also likely to be an important consideration. Nevertheless, commercial processes usually have multiple requirements - for specificity etc. as well as stability. In the detergent industry, proteases must also be resistant to the effects of high pH, oxidizing agents and EDTA. Several thermophilic proteases can apparently fulfill these requirements (Table 3) but would need to be synthesized in much higher yields before they could become economically competitive. The same argument applies to many of the other existing commercial applications of proteases (Table 4). Enzyme cost aside, the func- tional properties of proteases from extreme thermophiles seem to be well suited to several applications.

Thermophilic proteases may be of

71

more immediate value in processes where the reaction environment pre- cludes using cheaper mesophilic enzymes. One example could be the use of thermophilic proteases in organic synthesis; the ability of proteases to synthesize peptide bonds (reviewed by Glass ~9) is enhanced in the presence of organic solvents but these solvents themselves may cause loss of enzyme activity. This loss can be minimized by using: (1) immiscible solvents, (2) immobilized (stabilized) proteases or (3) proteases of greater intrinsic stability.

the protein substrate at higher temperatures. This effect is also demonstrated by the increase in the rate of hydrolysis of casein by caldolysin with temperature; the temperature coefficient (Q10) for the reaction in the temperature range 20-90°C averages 2.1 with a maximum of 2.6, i.e. the rate approx- imately doubles with each 10°C rise in temperature over the whole range. The efficiency of hydrolysis of natural protein substrates by thermophilic proteases and their relative lack of specificity (these enzymes will also hydrolyse

Table 4. Some current commercial and industrial applications of mesophilic bacterial proteases a Industry Example of application Are existing thermophilic

proteases appropriate? b

Washing/cleaning Detergent preparations Yes Tanning Dehairing/bating Probably not Protein recovery/hydrolysis Soy protein Yes

Meat and fish hydrolysates Yes Gelatin hydrolysis Yes Tenderization Yes

Organic synthesis Aspartame synthesis Yes Beverage Clarifying wine and beer No Milling/baking Gluten hydrolysis Probably not Medicine Digestive aids, treatment of No

burns, ulcers, etc. Photography Recovery of silver from Yes

emulsions Data obtained from Ward 3 and Cowan 2a

b Enzyme cost is not included in this analysis. The primary criterion enzymic step be satisfactorily carried out at high temperatures?

for this assessment is: Can the

Immobilized enzyme preparations are expensive to produce, inevitably lose activity during synthesis and often have lower Vm~x values than the free enzyme. The stability of thermophilic proteases in organic solvents permits their use while minimizing activity losses. Indeed, several peptide syn- theses in miscible and immiscible organic/aqueous solvents by thermophilic proteases have been reported 3~32.

Temperature and activity At their in vivo temperatures, most

thermophilic enzymes have specific activities similar to or greater than com- parable mesophilic enzymes. However, the thermophilic proteases show very high specific activities on protein (as opposed to low-molecular-weight peptides) substrates at high temperatures. For example, the proteinase/amidase activity ratio ofcaldolysin (using casein and benzoyl-L-Phe-L-Val-L-Arg-p- nitroanilide as substrates) increases eight times from 40 to 70°C (Cowan, D. A., unpublished results). This reflects the enhanced susceptibility of

collagen, elastin and fibrin), are potential advantages for their industrial utili- zation.

Another useful property of these enzymes may be the 'thermal switching effect' in which a thermophilic reaction can be effectively stopped by cooling to ambient temperature. The caseinolytic activity of caldolysin at 20°C is about 1.3% of that at 75°C H. The meat- tenderizing industry provides an example for possible exploitation of this property.

Topical application of proteases to tenderize cuts of meat has never been a wholly successful technique. Limited diffusion often results in excessive proteolysis on the surface with little through the bulk of the tissue. Tech- niques involving injection and vascular diffusion ofa protease before slaughter are relatively successful 3 but are often opposed on humane grounds. Although some would argue that the injection of any foreign protein is in- humane, some difficulties might be overcome by the use of thermophilic proteases. Firstly, a relatively lower

72

Patents

Novo Terapeutisk Laboratorium A/S, (Andresen, O., Aunstrup, K. and Ottrup H., 1973) - US 3723250, BE 721730, CH 538539, DL 79270, DL 85605, GB 1243784, FP, 1_587801, GB 1243784: proteases were prepared from novel species of 6ocillus cultured on a medium of alkaline pH. The enzymes are particularly useful in detergent and dehairing compositions.

protein dose could be used because of the high specific activity of the enzymes. Secondly, protease activity during both vascular dispersal and sub- sequent post-slaughter storage would be limited by the ambient or cold temperatures, extending the shelf life of the tissue. Activity would rise only during the cooking and as cooking periods are well-defined, this could automatically limit the extent of proteolysis. The degree of ' tenderizing' could thus be accurately controlled by the dosage of the administered protease.

In other reaction systems the 'thermal switching effect' could pro- vide a convenient means of avoiding the use of inhibitors, heat treatment or expensive enzyme-removing procedures after the completion of the desired reaction.

Future prospects Both the study and biotechnological

applications of thermophilic proteases are still in their infancy. Relatively few proteases from extreme thermophiles have been isolated, let alone characterized in detail, and applications are largely hypothetical. Their future lies in the development of unique applications to match the unique properties of these enzymes. The discovery of proteases produced by the extremely ther-

mophilic archaebacteria (Cowan and Jasperse, unpublished results) could pave the way to the realistic use ofpro- teolysis at temperatures above 100°C.

A recent reporff 3 that both the stability and activity of enzymes are enhanced in anhydrous solvents sug- gests that a new range of process developments are iminent. The intrinsic stability of thermophilic enzymes, including proteases, may render them the most suitable candidates in these new technologies.

Acknowledgement The authors wish to thank P.

Reynolds for his assistance in the pre- paration of this manuscript.

References 1 Godfrey, T. and Reichelt, J. R. (1983) In

Industrial Enzymology (Godfrey, T. and Reichelt, J., eds), pp. 1-7, Nature Press, NY, USA

2 Andresen, O., Aunstrup, K. and Outtrup, H. (1973) US Patent 3723250; Publ. 730327 Publ., London, pp. 251-317

3 Ward, O. P. (1983)In MicrobialEnzymes and Biotechnology (Fogarty, W. M., ed.) pp. 251-317, Applied Science, London

4 Endo, S.(1962)HakkoKogakuZasshi40, 346-353 (in Japanese)

5 O'Brien, R. T. and Campbell, L. L. (1957) Arch. Biochem. Biophys. 70, 432-441

6 Sidler, W. and Zuber, H. (1980) European ft. AppL Microbiol. Biotechnol. 10, 197-209

7 Gancher, G. M and Stevenson, K. J. (1976) In Methods in Enzymology, (Lorand, L. ed.), Vol. 45, pp. 415-433, Academic Press

8 Hausdorf, G., Krfiger, K. and HShne, W. E. (1980) lnt. ft. Peptide Protein Res. 15, 420-429

9 Mizusawa, K. and Yoshida, F. (1973),7. Biol. Chem. 248, 4417-4423

10 Heinen, U. J. and Heinen, W. (1972) Arch. Mikrobiol. 82, 1-23

11 Cowan, D. A. and Daniel, R. M. (1982) Biochim. Biophys. Acta 705, 293-305

12 Taguchi, H., Hamaoki, M., Matsuzawa, H. and Ohm, T. (1983)ft. Biochem.


93, 7-13 13 Matsuzawa, H., Hamaoki, M. and Ohta,

T. (1983)Agric. Biol. Chem. 47, 25-28 14 Keay, L., Moseley, M. H., Anderson,

R. G., O'Connor, R. J. and Wildi, B. S. (1972) Biotechnol. Bioeng. Syrup. 3, 63-92, Wiley

15 Zeikus, J. G. (1979) Enzyme Microb. Tecknol. l, 243-251

16 Fujii, M., Takagi, M., Imanaka, T. and Aiba, S. (1983)J. BaeterioL 154, 831-837

17 Tanaka, T., Kawano, N. and Oshima, T. (1981)J. Biochem. 89, 677-682

18 Doig, A. R. (1974) In Enzyme Engineering (Pye, E. K. and Wingard, L. B., eds), Vol. 2, p. 17, Plenum Press

19 Khoo, T. C., Cowan, D. A., Daniel, R. M. and Morgan, H. W. (1984) Biochem. J. 221, 407-413

20 Griitter, M. G., Hawkes, R. B. and Matthews, B. W. (1979) Nature 277, 667-669

21 Hartley, B. S. and Payton, M. A. (1983) Biochem. Soc. Syrup. 48, 133-146

22 Roche, R. S. and Voordouw, G. (1978) Crit. Rev. Biochem. 5, 1-23

23 Ohta, Y. (1967)J. Biol. Chem. 242, 509-515

24 Cowan, D. A. and Daniel, R. M. (1982) Biotechnol. Bioeng. 24, 2053-2061

25 Kumakura, M. and Kaetsu, I. (1984) Enzyme Microb. Technol. 6, 23-26

26 Mozhaev, V. V. and Martinek, K. (1984) Enzyme Micro& TechnoL 6, 50-59

27 Maneepun, S. and Klibanov, A. M. (1982) Biotechnol. Bioeng. 24, 483-486

28 Cowan, W. D. (1983) In Industrial Enzymology (Godfrey, T. and Reichelt, J., eds), pp. 352-374, Nature Press, NY, USA

29 Glass, J. D. (1981) Enzyme Micro& Technol. 3, 2-8

30 Jakubke, H-D., Kuhl, P., K6nnecke, A., DSring, G., Walpuski, J., Wilsdorf, A. and Zapevalova, N. P. (1983) In Peptides 1982 (Bl~ha, K. and Malon, P., eds), pp. 43-54, Walter de Gruyter

31 K~nnecke, A., H~insler, M., Schel- lenberger, V. and Jakubke, H-D. (1983) Monatsh. Chem. 114, 433-444

32 Oka, T. and Morihara, K. (1980) J. Biochem. 88, 807-813

33 Biomass Digest (1984) 6, 1-2 34 Fullbrook, P. D. (1983) In Industrial

Enzymology (Godfrey, T. and Reichelt, J., eds), pp. 8-10, Nature Press, NY, USA

35 Sidler, W. and Zuber, H. (1977) Fur. ft. Appl. Microbiol. 4, 255-266

36 Fuke, I. (1963)ft. Biochem. 53, 304-313

thermophilic proteases: properties and potential applications

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