Subtilisin - American Chemical Society

Chapter 7

Subtilisin Commercially Relevant Model for Large-Scale Enzyme Production

Downloaded by UNIV OF ARIZONA on September 29, 2015 | http://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0460.ch007

W. Douglas Crabb Department of Molecular Biology, Genencor International, Inc., 180 Kimball Way, South San Francisco, CA 94080 Subtilisins are a class of alkaline serine proteases produced by a variety of Bacillus species. The primary commercial use of subtilisins is as additives in detergent formulations to aid in the removal of proteinaceous stains. Until recently, commercial availability of subtilisins was limited to those produced by certain strains of either B.licheniformis or B.alcalophilus which had undergone years of traditional strain development to enhance overall productivity. The advent of new genetic engineering techniques has given industrial enzyme producers the opportunity to commercialize enzymes from new sources or site-specific modified enzymes designed for defined applications. Strategies for the cloning and expression of the Β.amyloliquefaciens subtilisin (ΒΡΝ') will be presented. The industrial enzyme market approaches approximately $600 million US dollars annually. The majority of these enzymes are produced by large scale fermentation from microbial or fungal sources. Typical uses for these enzymes include food processing (pectinases), biomass conversion (cellulases) and high fructose corn syrup production (amylases, glucoamylases). In general, the enzyme market can be considered a commodity market where the value of the enzyme lies in the processing advantages and subsequent reduced production cost recognized by the user. An example of this type of enzyme would be pectinase which increases the amount of free run liquid from fruit for wine or juice production. In some cases, however, the enzyme(s) is used to produce a more value-added product from a substrate. An example of this type of enzyme system would be glucoamylase/glucose isomerase for the production of high fructose corn syrups from corn starch. Most of the enzymes in commerce today were derived from natural isolates by the classical approaches of screening, strain selection/mutagenesis for overproduction and fermentation development. While these techniques have certainly proven useful in the past to identify and develop enzymes, there are limits to the effectiveness of this type of approach. There are two major difficulties facing the industrial microbiologist in developing future products. 0097-6156/91/0460-0082$06.00/0 © 1991 American Chemical Society

In Enzymes in Biomass Conversion; Leatham, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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First, the ability to identify a new enzyme relies on a labor intensive screening of large numbers of natural isolates. Though it has been possible to use plate screening methods to identify potential overproduces or to develop partially selective conditions for selecting overproduces, the practice still relies heavily upon shake flask analysis of large numbers of individual isolates. While advances in robotics has eliminated some of the tedium in screening isolates, it remains a "hands-on" field of endeavor. Second, when using a variety of natural organisms as enzyme production hosts, there must be an intensive strain and fermentation development program to increase enzyme yields. Routinely, information gathered during this process will be of little use in developing the next generation product, since a different host will most likely be employed. Additionally, the search for the new isolates is often limited, by regulatory necessity, to members of those genuses which have a history of safe production (Generally Recognized As Safe or GRAS). Why an rDNA Approach With the advent of recombinant technology, the enzyme producer now has a tool to aid in the development of new enzyme products and in the improvement of production costs for already commercialized enzymes. The search for new enzymes does not need to be limited to GRAS organisms, since it is possible to move the enzyme from its original host into a more suitable production organism. This may prove especially important in identifying enzymes from organisms which are pathogenic or not amenable to standard fermentation practices (such as extreme thermophiles). By expanding the sources of commercial enzymes, the potential exists to identify new and exciting applications. Recombinant DNA can be used to overcome a variety of potential problems in the industrial enzyme sector. A schematic outline of the types of problems encountered and the solutions by an rDNA program is shown in Figure 1. Basically, these problems can be divided into strict yield issues (making more of a given protein) and quality issues (making the product more useful). Because of the nature of the enzyme business, the highest return to the enzyme producer will usually come from increasing the value of the enzyme to the user. Historically, once a new enzyme was identified, it was often a laborious effort to identify the correct conditions for small scale fermentation in order to produce enough material for laboratory scale testing. This meant a major investment in resources and equipment simply to determine if the enzyme was suitable for further analysis. Pilot scale quantities required an even greater expenditure of effort. With a recombinant approach, it is possible to rather quickly clone and express an enzyme at high enough levels to enable applications scientists to test its utility. By moving this analysis earlier into the project, decisions with regard to which candidates should be pursued can be made more effectively in terms of resources spent. If the initial cloning and expression has shown the enzyme to be promising then a yield driven expression program can be instituted. Regardless of the host


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ENZYMES IN BIOMASS CONVERSION


PRODUCTS P R O R I F MR P r o d u c t i on costs

Aval labi I I t y VarlablI Ity

ι

* rDNA

More e f f i c i e n t expression host

F u n c t i onaI P r o b I ems

Not n a t u r a I I y a v a I I a b I e In quant Ity

* RDI

Clone and e x p r e s s In a l t e r n a t e host

ι

UTTONS E n g i n e e r an Improved protein

i

Clone and e x p r e s s In a l t e r n a t e host

ι FXAMPI F S

Amy I a s e s

C a l f Chymosin (renIn)

SubtI Ι I sin

Human g r o w t h hormone

Figure 1. rDNA solutions to commercial problems in the industrial enzyme sector.



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system chosen, the efforts will include placing the gene under control of a strong promoter and developing a host strain which will be compatible with a fermentation and recovery program. The main advantage of this approach is the ability to design generic host systems which are optimized for production from specific promoters. Figure 2 shows the effectiveness in utilizing recombinant DNA as compared to the classical methods for developing a series of products. As indicated in the figure, the generic host/expression system potentially shortens the time for achieving commercially viable yields of newly identified enzymes, primarily due to the elimination of a major strain improvement/fermentation development program on each new isolate. One of the most exciting potentials of the rDNA approach lies in the ability to design enzyme activity by site-directed mutagenesis of a naturally occuring enzyme. With this approach, one is freed from the constraints of natural isolate screening in order to identify an enzyme which has properties more suitable for commercial applications. Although enzyme design may never completely eliminate the need to screen culture collections, it will certainly increase the number of candidate enzymes available for commercial use. Already, functions such as thermostability (7) and oxidative stability (2) have been engineered into subtilisin by use of these techniques. Development of Subtilisin as a Model Enzyme In order to prove enzyme engineering feasibility, it was important to develop a model system. One of the prime considerations for any model would be the commercial potential of the model. Table I lists the major commercial enzymes and the market size in US dollars (3). The alkaline proteases (subtilisins) are clearly the major single class of enzymes in commercial use today, representing 25% of the total enzyme market of $600 million. The primary use of subtilisins is as additives in laundry detergents to aid in the removal of proteinaceous stains from cloth. Table I. The Major Commercially Available Enzymes and Estimated Market Size ENZYME

SOURCE

MARKET SIZE (millions US$)

Alkaline protease Neutral protease

Bacillus spp. Bacillus spp. Aspergillus orzae Bacillus spp. Aspergillus oryzae Aspergillus niger Streptomyces spp. Calf stomach Mucor spp.

150 70

Amylases Glucose isomerase Rennin

100 45 60



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CLASSICAL APPROACH ENZYME 1

ENZYME 2 Commercial


Commercial

Scale-up

Scale-up

LU

Identification

Identification

rDNA APPROACH ENZYME 1 Commercial

ENZYME 2 Commercial

û -1 LU

>-

Scale-up

Scale-up

Identification

TIME TO COMMERCIAL TARGETS Figure 2. Efficiency of an rDNA approach to enzyme commercialization. Areas in boxes refer to the typical times required for Identification, Scale-up and Commercialization. Efficiencies are recognized in Identification of enzymes (protein engineering) and shortened Scale-uptimes(generic hosts).



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Subtilisins are a class of related serine endo-proteases produced by members of the Bacillus genus. The B.amyloliquefaciens subtilisin (ΒΡΝ') is wellcharacterized with regard to its DNA sequence (4), protein sequence (5), X-ray crystal structure (6) and kinetic properties (7). With this wealth of information available, BPN' was chosen as the model enzyme for our recombinant approach. BPN' is a 275 amino acid protease with a serine in the active site. Since it is functional in an alkaline environment it has potential use in detergent applications. Our program was to change specific characteristics of BPN' to make it more effective in certain applications. Two main activities were targeted: pH range and oxidative stability (since bleaches are often components of detergents). Using X-ray structure data, amino acids which were believed to affect the desired characteristics were identified. Using either site-directed mutagenesis or "cassette" mutagenesis (8) different amino acid substitutions were made in the subtilisin structural gene (2,9). The BPN' variant genes were cloned onto expression vectors to produce sufficient quantities of the purified enzymes for analysis. The pH profiles of two variants compared to that of the native enzyme are shown in Figure 3. BPN' has a broad activity range from pH 7.5 to approximately pH 9.5. When the methionine which occurs at amino acid position number 222 in the native protein is replaced with a lysine (Lys-222), the pH profile shifts dramatically. The range of activity has been changed to a sharp region surrounding pH 9.5. When cysteine replaces the methionine at the same position, the optimal pH remains at approximately 8.5, but there is a small effect on the pH range with a slightly sharper dropoff in activity both above and below the optimum. An entirely different property of subtilisin was affected by substituting leucine at the 222 location. Native BPN' is extremely sensitive to the presence of oxidation agents, showing rapid inactivation when incubated in the presence of 0.3% H 0 (Figure 4). The Leu-222 variant, in contrast, was found to be totally stable under the same oxidation conditions. The data clearly show that single amino acid alterations can have dramatic effects upon the activity of the enzyme. Similarly, other changes have been shown to affect catalytic properties, substrate specificities and thermostability (7,2,9). 2

2

Development of a Host Production System From a commercial standpoint, it is not sufficient to merely identify those variations of the naturally occurring enzymes which have properties of interest. There must be a system devised which will allow production of the variants at commercially viable yields. Since, the subtilisins are produced by members of the Bacillus genus, Bacillus subtilis was chosen as the model host system. B.subtilis is the most characterized member of the gram positive group of microorganisms. Genes may be transferred among individuals by both transformation and transduction. In addition, there are numerous mutants available for genetic analysis. Most importantly, however, B.subtilis produces a number of extracellular enzymes, including an endogenous subtilisin.



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Figure 3. pH profiles of wild type and two variant subtilisins. Activity was assayed with synthetic substrates as described (2) at the indicated pH.


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120

ο. OH

0

•

1

•

2

1

4

1

1

1

1

6 8 TIME (min)

·

1

10

1

1

12

Figure 4. Oxidative stability of subtilisins. Purified wild type or variant subtilisin was mixed with 0.3% H 0 in 0.1M Tris buffer (pH 8.6). At thetimesindicated aliquots were removed and tested for remaining enzymatic activity (2). 2

2



90


Subtilisin expression in B.subtilis is controlled at several levels. Production begins during the transition stage between vegetative growth and sporulation. Though the exact mechanisms which activate expression are not clear, there are a number of known genes which will enhance levels of protease (10). Regulation appears to be primarily at the transcriptional level with both positive (enhancer) and negative (repressor) control elements (77). Figure 5 shows a typical production curve for subtilisin. Enzyme begins appearing after the cessation of logarithmic growth and increases as the culture progresses through the sporulation process. The relationship between sporulation and enzyme production remains unclear. Strains of B.subtilis which are lacking the subtilisin gene appear to sporulate normally (72). However, many of the mutations which affect spore production also affect the ability of the cells to produce the enzyme. Again, the control is at the level of messenger RNA production(7J). Table II is a partial list of known Bacillus genes which have an effect upon subtilisin production. Henner and coworkers (14) placed beta-galactosidase under the control of the subtilisin promoter. By using promoter deletions, they were able to identify regions of the promoter which are regulated by sacU, sacQ (present designation degU and degQ respectively, see Table Π) and hpr. Table II. Genetic Loci of Bacillus subtilis Which Regulate Subtilisin Production GENE

PROPOSED ROLE

spoOA spoOH degU degQ prtR senN abrB hpr sin

General sporulation control Sporulation specific sigma factor Transcriptional enhancer Transcriptional enhancer Transcriptional enhancer Transcriptional enhancer Repressor Repressor Repressor

The subtilisin gene and its upstream regulatory regions are shown in schematic diagram in Figure 6. The mature gene is preceeded by both a pre and pro sequence (29 and 106 amino acids respectively) which are removed during the secretion of the enzyme. Maturation of prosubtilisin to mature subtilisin appears to be due to a selfprocessing event (75). In order to develop a host strain for production of a variety of subtilisin enzymes, it was first necessary to delete the endogenous alkaline and neutral proteases (72,76). The strain was then constructed to contain the optimal combination of subtilisin regulatory genes which were compatible with the proposed fermentation and recovery process. Once this strain had been produced, the recombinant enzyme



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TIME

(hr)

Figure 5. Production of subtilisin. A culture of B.subtilis was grown in supplemented nutrient broth (13) and subtilisin production was followed. T-0 indicates the time of first departure from logarithmic growth. Subtilisin levels are indicated as arbitrary units/ml.


91


-400

hpr

''''''*'''''''' ^

-10

~ ~ abrB

sin

-35

PRE

PRO

MATURE

Figure 6. Regulation of the subtilisin promoter. Shaded areas refer to the promoter and upstream regions of the subtilisin gene. Numbers above the figure indicate base pairs upstream. Activators are shown above the figure, repressors below. Lines indicate possible regions of the DNA involved in binding the regulatory proteins.

sin

-200

degU degQ prtR senN


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under the control of the B.subtilis subtilisin promoter was cloned into the host organism. The strain was then moved into fermentation development for production optimization studies. After the initial fermentation process had been optimized, the production process was independent of the enzyme being produced. Therefore, production of alternative subtilisin variants (and other proteins as well) was no longer dependent upon individual strain improvement or fermentation development. When new enzymes were introduced, the time required to take them to commercial scale had been shortened considerably.


Summary Recombinant DNA technology has proven to be an effective means of producing enzymes for large scale commercial manufacture. The ability to genetically engineer preferred characteristics into a naturally occurring enzyme can help avoid extensive natural isolate searches for new enzymes. More importantly, it is now possible to tailor make enzymes for specific customers' needs. Since a protein engineering project includes production of a library of "variant" enzymes, the ability to identify new important activities will be limited solely by the ingenuity of applications screening methods. Production of laboratory and pilot scale quantities of a newly identified enzyme (either naturally occurring or engineered) can be facilitated by cloning and expressing the gene in an alternate host system. This host may be different from the host for final product of the commercial product (ie. E.coli vs Bacillus spp.). By developing a series of generic host production organisms, fermentation development time can be minimized when these host systems are used for production of multiple products. The investment of time and resources in developing the initial fermentation and recovery system can be recovered in subsequent programs in the form of more rapid commercial development timelines. Genetic engineering techniques have had a significant impact upon the research, development and commercialization phases of projects in the industrial enzyme business. The technology has proven successful and will play an even greater role in the future in expanding the present markets. Literature Cited 1. 2. 3. 4. 5. 6. 7.

Bryan, P.N.; Rollence, M.L.; Pantoliano, M.W.; Wood, J.; Finzel, B.C.; Gilliland, G.L.; Howard, A.J.; Poulose, T.L. Proteins 1986, 1, 326. Estell, D.; Graycar, T.; Wells, J. J. Biol. Chem. 1985, 260, 6518. Arbige, M.V.; Pitcher, W.H. Trends in Biotechnology. 1989, 7, 330. Wells, J.A.; Ferrari, E.; Henner, D.J.; Estell, D.A.; Chen, E.Y. Nucleic Acids Res. 1983, 11, 7911. Markland, F.S.; Smith, E.L. J. Biol. Chem. 1967, 242, 5198. Wright, C.S.; Alden, R.A.; Kraut, J. Nature 1969 221, 235. Markland, F.S.; Smith, E.L. In The Enzymes; Boyer, P.D., Ed.; Academic: New York, 1971; Vol. III, p561.


94 8. 9. 10. 11.


12. 13. 14. 15. 15.

E N Z Y M E S IN BIOMASS CONVERSION

Wells, J.A.; Vasser, M.K.; Powers, D.B. Gene 1985, 34, 315. Wells, J.A.; Estell, D.A. Trends Biol. Sci. 1988, 13, 291. Henner, D.J.; Yang, M.; Band, L.; Shimotsu, H.; Ruppen, M.; Ferrari, E. Proc. 5th Intl. Symp. on Genetics of Industrial Microorganisms 1986, p81. Valle, F.; Ferrari, E. In Regulation of Procaryotic Development Smith, I; Slepecky, R.; Setlow, P., Eds. American Society for Microbiology: Washington, D.C., 1989; p 131. Stahl, M.; Ferrari, E. J.Bacteriol.1984, 158, 411. Ferrari, E.; Henner, D.J.; Perego, M.; Hoch, J.A. J. Bacteriol. 1988, 170, 289. Henner, D.J.; Ferrari, E.; Perego, M.; Hoch, J.A. J. Bacteriol. 1988, 170, 296. Power, S.D.; Adams, R.M.; Wells, J.A. Proc. Natl. Acad. Sci. USA 1986, 83, 3096. Yang, M.Y.; Ferrari, E.; Henner, D.J. J. Bacteriol. 1984, 160, 15.

RECEIVED August 16, 1990


Subtilisin - American Chemical Society

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