Enzymic starch utilization and genetic engineering

TIBTECH- AUGUST 1988 [Vol. 6]

Subjects that were taboo only a few years ago - venture capital, tax benefits, and the potential of individual investment - are now being discussed, at least at the Round Table Conference. Hungary represents one of the most progressive of the Comecon countries and has one of

the strongest science bases. Biotechnology in Hungary may indicate the hope of the new technology within that group of nations.

Acknowledgements The authors wish to thank Dr M. Szab6-Pels6czi for economic infor-

Enzymic starch utilization and genetic engineering John F. Kennedy, Vivian M. Cabalda and Charles A. White Starch is widely used in the food and beverage, paper and textile industries. Genetic engineering will allow optimization of starch conversion technologies both by creating novel enzymes/microorganisms of the desired efficiency, stability and activity to fit any process, and by ensuring they are produced in commercial quantities. Plants could also be genetically engineered to produce starches of the desired amylopectin: amylose ratio. To ensure a continuous supply of starch, starch-producing plants could be genetically engineered to be disease- and insect-resistant, high yielding, and able to grow under any climatic and soil conditions. However, screening for novel microorganisms should not be neglected and should be used to complement genetic engineering. Starch is the principal food-reserve polysaccharide in the plant kingdom and is mostly found in the seeds of maize, wheat, barley, oats, rice and sorghum, in the tubers and roots of potato, arrowroot and cassava and in the pith of the sago palm._ Starch consists of a mixture of linear and branched homopolymers of D-glucose 1. Amylose is made up of linear chains of O~-D-glucopyranose units linked by (1-*4)-bonds and has a degree of polymerization of between 1 x 1 0 2 and 4 x 105. AmyloJohn F. Kennedy and Vivian M. Cabalda are at the Research Laboratory for the Chemistry of Bioactive Carbohydrates and Proteins, Department of Chemistry, PO Box 363, University of Birmingham, Birmingham, B15 2TT, UK. Charles A. White is at Chembiotech Ltd, Institute of Research and Development, University of Birmingham Research Park, Birmingham B15 25Q, UK.

pectin contains short linear chains (between 17 and 23 units long) of (1-*4)-linked C~-D-glucopyranose residues joined by (1--6) linkages to form a highly branched structure with a degree of polymerization of between 1 x 1 0 4 and 4 x 1 0 7. The degree of branching and the ratio of amylose: amylopectin varies with, for example, the source and age of the starch. Most starch is used in the food and beverage industries, mainly in the production of hydrolysates as sweeteners in drinks and confectionery and as thickneners in semisolid foods such as sauces, custards, pie-fillings, desserts. Approximately one-third of the total starch production is used for non-food purposes, mainly for sizing of paper and board and as adhesives in the paper, packaging and textile industries. Starch has a great potential as raw

( ~ 1988, Elsevier Publications, Cambridge 0167 - 9430/88/$02.00

mation on Hungary and its biotechnology efforts. We also thank Professor M. Simai of the Hungarian Academy of Sciences and Professor P.U. Kralov~nsky of the Office of Proteins and Biotechnology for the invitation to participate in the Round Table Conference. material for the chemical industry and as an energy source since glucose, the most important of the starch hydrolysis products, can be further converted to other products such as ethanol, hydrogen gas 2 and antibiotics by fermentation, fructose by enzymic isomerization, and sorbitol by 'chemical' hydrogenation. The use of starch as a raw material in the food, paper and textile industries has been widely developed and is well established. Its greater utilization will only be realized once it becomes a feedstock to the chemical industry. Starch will have to compete (especially in the chemical industry) both with other potential raw materials such as cellulose and with existing raw materials such as crude oil. Its competiveness depends on its continuous availability, the existence of a well developed technology by which starch is converted to the desired products and the relative economic positions of this welldeveloped conversion technology vis-a-vis the conversion technologies of other potential and existing raw materials. However, in this competition starch has several advantages: • it is easier to produce glucose from starch than from cellulose (even though both conversions are not yet fully optimized); • sources of crude oil are dwindling; • genetic engineering can be applied to improve the efficiency and economy of enzyme systems and fermentation processes for starch bioconversions; • genetic engineering can also ensure a steady, stable and abundant supply of starch by producing plants that are resistant to insects, disease and herbicides, tolerant of environmental stress, high yielding, and possibly nitrogen fixing.

TIBTECH- AUGUST1988 [Vol. 6]

ratable 1

Properties and industrial applications of hydrolysed starch products Type of syrup

DE a

Composition (%)

Properties

Application

Low DE maltodextrins

15-30

low osmolarity

clinical feed formulations; raw materials for enzymic saccharification; thickeners,

Maltose syrups

40-45

1-20 D-glucose 4-13 maltose 6-22 maltotriose 50-80 higher oligomers 16-20 D-glucose 41-44 maltose 36-43 higher oligomers 2-9 D-glucose 48-55 maltose

increased maltose

High maltose syrups

48-55

fillers, stabilizers, glues, pastes confectionary, soft drinks, brewing and fermentation, jams, jellies, ice cream, conserve, sauces hard confectionary, brewing and fermentation

high viscosity, reduced crystallization, moderately sweet

content

15-16 maltotriose

High DE syrups

56-68

25-35 D-glucose 40-48 maltose

confectionary, soft drinks,

increased moisture holding, increased sweetness, reduced content of higher sugars, reduced viscosity, higher

brewing and fermentation, jams, conserves, sauces

fermentability

Glucose syrups

Fructose syrups

96-98

98

95-98 D-glucose 1-2 maltose 0.5-2 isomaltose 48 D-glucose 52 D-fructose

soft drinks, caramel, baking, brewing and fermentation, raw material soft drinks, conserves, sauces,

commercial liquid 'dextrose' alternative industrial

sweeteners to sucrose

yoghurts, canned fruits

aDextroseequivalent(seeGlossary). Genetic engineering can improve

and introduce new characteristics into any living organism (animal, plant or microorganism) by transferring genes from one organism to another, changing undesirable genes and/or creating desirable genes and incorporating them in an organism 3. Of particular importance in starch utilization is its use in improving or creating (and producing more efficiently) novel proteins such as enzymes of stability and specificity suitable for any industrial application.

Improvement of starch-producing plants Since genetic engineering became possible, there have been predictions and speculations on its application to the creation of 'super' plants 4'5. Most genetic manipulation of plants has been performed with dicotyledons since transformation with Agrobacterium tumefaciens, the most common vector in plant engineering, is less successful in monocotyledons such as cereals. The direct uptake of plasmid DNA by cereal protoplasts is being used to transform cells of rice, wheat and maize 6, though transformation efficiency is low. Micro-

injection of DNA into the nuclei of cereal protoplasts may prove to be more successful for cereal plants. Cereals are also difficult to grow in the laboratory from tissue cultures, although rice plants have been efficiently regenerated from protoplasts 7. Similar methods for maize, wheat and other cereals are needed to advance genetic engineering in such plants. In potatoes, virus disease resistance has been conferred through genetic engineering. The gene for the protein coat from a virus has been inserted into the potato genome. Subsequent synthesis of the viral coat protein primes the cross-protection response of the plants (analogous to immune response of animals) rendering the plant more resistant to the infection 8. The methods used in the transformation of plants through incorporation of insect- and herbicideresistance genes 9 from bacteria and the observation that Gram-negative bacteria can transfer their genes directly to plant cells 1° could lead to the development of useful mechanisms for the manipulation of agronomically useful genes (those conferring resistance to bacteria, in-

sects, fungi and viruses or the ability to fix nitrogen) and their transfer from 'non-plant' sources into plants. For instance, potatoes are being produced that are resistant to a great variety of pests and diseases and nutritionally improved 11. Other agronomically desired improvements such as high yield, stress tolerance and photosynthetic efficiency are less well understood and are polygenic traits. The realization of these improvements is still a long w a y off. Even there, a better understanding of the control of photosynthesis and the suggestion that genes involved in the process of osmosis (osmogenes) play an important role in stress tolerance 12 opens up new possibilities for genetic manipulation. There is, therefore, a need for basic research into the biochemistry and physiology of cereal plants in order to extend applications of genetic engineering.

Improvements of starch-hydrolysing enzymes The major use of starch is in the food and brewery industries where most is hydrolysed to produce low molecular weight derivatives (Table 1).

TIBTECH- AUGUST 1988 [Vol. 6]

-- Fig. 1

o

*°

oDe OOoooooooo ° 0 0 0 O0

0

qHIO"0

gOo~~U ~D-glucosidase~ exo(1--~4 ) - a - D - g ~

l~-amyla~ pullulanase~Icyclomal~todextrin~isoamyl - ase D-glucotransferase" ~

Theenzymihydrol c ysiof s starch.0,Non-reducinD-gl g ucosylresidue;©,reducingD-glucosylresidueorD-glucose. The recent availability of starchdegrading enzymes such as (xamylases, amyloglucosidase and pullulanase on commercial scales has enabled the expansion of the enzymic hydrolysis of starch materials. Enzymic degradation gives higher yields of more specific hydrolysates with fewer by-products and lower energy consumption than traditional acid hydrolysis and is becoming preferred. Genetic engineering will enable greater quantities of starch-degrading enzymes to be produced at lower prices and may provide them with improved stability and specificity. There are many starch-hydrolysing enzymes in nature but only a few have commercial significance. Figure 1 shows the mechanism of actions of starch-degrading enzymes, both those presently used in industries and those with potential commercial significance. Other reviews provide more information on these starch-degrading enzymes 13.

Figure 2 shows some existing processes by which starch is hydrolysed. Optimization of these processes by the use of novel enzymes could lead to more economical bioconversion of starch to the desired hydrolysates. However, glucose is considered to be the most important of the starch hydrolysates since it can be further transformed to a range of products of m e d i u m to high value chemically (e.g. it is a chiral starting material for the synthesis of chiral non-carbohydrate products) 14, enzymically (e.g. in the production of fructose) 15, or through fermentation processes (e.g. in the biosynthesis of penicillin and enzymes) 16.

e~-Amylase and glucoamylase Thermostable

e~-amylase

from

Bacillus species is used for the liquefaction (thinning stage) in the enzymic production of starch-based products. They usually require C a 2+ for enzyme activity and conforma-

tional stability. However, such a requirement is deleterious since Ca 2+ denatures glucoamylase (used in the saccharifying stage) and destabilizes glucose isomerase used in the production of fructose syrups. This problem can be solved either by screening or by producing via genetic engineering thermostable e~-amylases that do not require Ca 2+ for stability. An enzyme has been found in Bacillus stearothermophilus that can retain at least 70% of its activity after 10 rain at pH 6.0 and 90°C in the absence of Ca 2+ (Ref. 17). Alternatively, screening and genetic engineering could be applied to the development of glucoamylases and glucose isomerases that are not denatured by Ca 2+. Such improvements are also appropriate for any of the enzymes used after the liquefaction stage in the production of other starch hydrolysates. Further optimization of the liquefaction/saccharification process might

TIBTECH-AUGUST1988[Vol.6] Fig. 2

Starch slurry I GELATION LIQUEFACTION

c~-amylase (bacterial)

pH 6.0--6.5, 105°C,5 min, or 95°C, 1--2 h SACCHARIFICATION

c~-amylase (fungal) and glucoamylase

Low DE I pH 5.0, 55°C, 42 h maltodextrins ~

~,,_1 "-I High DE syrup

\ ~

.

SACCHARIFICATION

~

oL-amylase(fung'al) or ~-amylase

SACCHARIFICATION ~ p H 5.5, 50--55°C, 40 h ,%.

glucoamylase or glucoamylase and pullulanase

pH 4.0--4.5, 60~C,48--72 h

"~. [ Malt°se I syrups

I Glucosesyrup I ION EXCHANGE ISOMERIZATION

D-glucose isomerase

pH 7.5--8.5, 60--65°C, 0.5--4.0 h

I Fructose [ syrup

REFINING

Very high fructose syrup

Starch processing using enzymes.

enable the whole conversion to take place under one constant condition (i.e. without temperature and pH adjustments). This would need a glucoamylase with the same thermostability and pH stability as the liquefying amylase. (A novel glucoamylase 18 from Clostridium thermoamylolyticum exhibits thermostability at pH 6-7.) Addition of pullulanase and/or eC-D-glucosidase (see below) of the same stability would further increase glucose yield. (A pullulanase from Thermus aquaticus YT-1 with an optimum ofpH 6.4 which is stable at 85-95°C has recently been reported, although its stability is Ca 2+ dependent19.) If such thermostable enzymes are found and/or created,

conversion of starch to glucose could occur in one step, shortening production time. However, this could be disadvantageous in that high temperature and therefore excessive energy would be used throughout the process. Another way to make the process more economical would be to do without the gelatinization (high temperature) stage and make use of highly raw-starch-degrading enzymes which are significantly active at lower temperatures. Ideally these should have the same pH optimum and temperature activity/stability as the glucoamylase. If a sterilization step is deemed necessary in addition to the lowering of temperature after-

wards to cut on energy cost, the brief use of a thermostable o~-amylase with a pH activity/stability profile similar to that of the glucoamylase and which is also appreciably active at lower temperatures would be another way of optimizing the starch hydrolysis process. This would allow the use of high temperature for sterilization, but decrease the ~time at high temperature and ~void the need to change the pH on cooling and addition of glucoamylase. These possibilities could also be applied to making high conversion syrups [DE 60-70 (see Glossary)], high maltose syrups and other hydrolysates involving e~-amylases, fungal amylases, cyclomaltodextrin D-glucotransferase and exo-acting glucanases (see below).

h-Amylase The industrial use of [3-amylase in the production of high-maltose syrups (80% maltose) has been restricted by limited availability of the enzyme from plant sources. However, extracellular [~-amylase can be obtained from microorganisms. Hyperproduction by a mutant strain of Bacillus cereus (220-fold increase in production over the wild-type strain) 2° or the possible expression of plant [3-amylase genes in microorganisms will make the enzyme more readily available. e~-D-Glucosidase C~-D-glucosidase has a very large potential in industry: it can be used (alone or in conjunction with glucoamylase) for the hydrolysis of maltodextrins and maltose obtained by prior enzymic hydrolysis to produce high-dextrose syrups or crystalline Dglucose (see Fig. 1). Used in combination with glucoamylase, O~-D-glucosidase might resolve the problemof the re-formation of glucosidic linkages that occurs w h e n high concentrations of dextrose are produced. (Re-formation of linkages is Glossary Dextrose equivalent (DE) - The extent of hydrolysis of starch to glucose, expressed as a percentage. A fully hydrolysed sample has a DE of 100.

TIBTECH-AUGUST 1988 [Vol. 6] - - Table 2

Bioproducts from starch Starch hydrolysates

catalysed by the e n z y m e - the reverse reaction of hydrolysis.) The combination could also provide more efficient production of high-glucose syrups. Once it becomes commercially available, o~-D-glucosidase could have widespread use in the production of high-dextrose syrups or crystalline D-glucose and might even usurp glucoamylase as the enzyme of choice. It hydrolyses oligosaccharides more rapidly than glucoamylase; the pH optima of bacterial enzymes fall in the range 5.0-7.0; the enzyme from Bacillus strains has a high temperature optimum (60-70°C); there is no evidence of denaturation in the presence of Ca 2+ or reverse reaction to re-form longer glucose.

Exo-acting OC-D-glucanases These enzymes can be used to hydrolyse starch to specific oligosaccharides (four to seven glucose residues). Such malto-oligosaccharides have been used as reagents for the determination of serum amylase activity and could be used as superior nutrient feeds for infants, the aged, patients with renal failure and those in a state of calorie deprivation.

Debranching enzymes (pullulanase and isoamylase) The use of debranching enzymes in conjunction with amylase is a method of rapidly increasing yields of hydrolysis products. Of the two enzymes, pullulanase has been more widely used because of its wider availability (isoamylase is only produced in Japan at present).

Cyclomaltodextrin D-glucotransferase This enzyme hydrolyses starch to a series of non-reducing cyclic oligosaccharides frequently referred to as cyclodextrins. Cyclodextrins are finding novel applications in the food industry and as chiral stationary phase for the separation of structural and optical isomers. This enzyme, again, is only available in commercial quantities in Japan. Exploitation in the starch industry oc-D-Glucosidase, exo-acting glucanases, cyclomaltodextrin D-glucotransferase and isoamylase can be made more available to the starch

maltodextrins glucose syrups fructose syrups maltulose cyclodextrins amyloses maltose

oligoglucosylfructosides Organic acids, solvents and alcohols acetic acid citric acid thuconic acid lactic acid

fumaric acid maltobionic acid butyric acid propionic acid ketogluconic acid staconic acid kojic acid ethanol

isopropanol butanol

2,3-butanediol glycerol

mannitol arabinitol acetone acetaldehyde ethylene

1,2 propanediol malic acid Amino acids

L-glutamic acid L-methionine L-lysine L-phenylalanine aspartic acid

Nucleotides and nucleosides

guanyl acid inosinic acid xanthyl acid

industry using genetic engineering to produce microorganisms that hyperproduce and secrete these enzymes. The mutant hyperproducing strain of Bacillus cereus2° (see ~-Amylase) could be exploited to synthesize any desired enzyme. Screening21 both for hyperproducing microorganisms and for enzymes of the desired stability, activity and specificity will also be important 3. Creation, through protein engineering, of novel starch-degrading enzymes requires an understanding of the structural basis of their activity, specificity and stability. One approach is to compare the genetic characteristics 22 and amino acid

Biopolymers

polyalkonates xanthan dextran pullulan alginate scleroglucans curdlan Erwinia polysaccharide polyhydroxybutyric acid glycane polysaccharide PS7 Beyerinckia polysaccharide Fats and lipids single-cell oil Proteins single-cell protein Enzymes

amylases proteases

Vitamins

vitamin B12 folic acid

Antibiotics penicillin

cephalosporin Hormones gibberelin Alkaloids

muscarine ergot alkaloids

Food enhancers

monosodium glutamate Alternative fuel

hydrogen gas methane Genes

sequences 23 of similar enzymes; for example, thermolabile and thermostable oc-amylases, acid- and alkalitolerant oc-amylases, liquefying and saccharifying oc-amylases, endo- and exo-acting amylases, and ec-amylases of differing abilities to digest raw starch. Another approach is to study the tertiary structures using protein crystallography, molecular simulations, site-directed mutagenesis 24 and studies on electrostatic potentials and effects in the active sites 25. Ultimately, all the genes encoding any enzyme system might be engineered into one hyperproducing bacterium. This could reduce enzyme production costs since only one kind

TIBTECH- AUGUST 1988 [Vol. 6]

of microorganism would need to be cultured. Each of the enzymes could be engineered so that the hydrolysis of any particular type of starch could be tailored to a given syrup type. The microorganisms used should not produce any toxic byproducts (to be acceptable to the Food and Drug Administration) and should also be capable of secreting the enzymes 3. The efficiency of secretion could also be improved through genetic engineering.

Tailor-made starches Genetic engineering of plants could be used in producing tailor-made starches. Amylose: amylopectin ratios could be predetermined to suit the application of the starch. The polymer could be made more susceptible to enzymic action (another way of optimizing starch conversion) and/or chemical modifications. One can also envisage that starches could be endowed with properties that could make them substitutes for other polymers: cellulose,, for instance, might be simulated ~y modified 100% amylose starches. The creation of tailor-made starches would only be possible with a greater understanding of starch biosynthesis. More ambitiously, microorganisms could be genetically engineered to produce novel starches and derivatives. In addition genetic manipulation of cereals could be used: to alter the type and/or amount of protein produced to improve its breadmaking quality 26 and nutritional value; to lessen the amounts of fatty substances which can cause rancidity on storage (or to reduce the 'off' flavours which have been attributed to lipid content); and to make starch more susceptible to gelatinization.

Starch-fermenting microorganisms In fermentation, starch can be used as a substrate for bioconversion to a range of chemicals and foodstuffs organic acids, solvents, antibiotics, vitamins, enzymes, amino acids and flavour enhancers. Hydrolysis of starch to D-glucose is an integral part of most fermentation processes since glucose is usually the central intermediate. But most microorganisms used lack starch hydro-

lysing enzymes. However, genes for starch-hydrolysing enzymes can be inserted into fermenting microorganisms to enable them t o use starch directly. This has been done in Clostridium a c e t o b u t y l i c u m for the production of butanol 27 and in yeasts for the production of ethanol 28. If this approach can be extended, production costs on fermentation processes may be reduced. (Wheat or maize starch costs £240 tonne -1 of glucose monohydrate producible; glucose monohydrate powder costs up to £400 tonne -1 to produce.) Replacing glucose with starch as feedstock could also minimize catabolite (glucose) repression and could yield greater amounts of the desired fermentation products. Table 2 lists some products that could be produced from starch biologically29'3°. Starch utilization will also be increased indirectly by other genetically engineered improvements to fermentative microorganisms. Increasing their productive efficiency, their stability to toxic products (e.g. solvents and organic acids), their independence from oxygen, and their pH/temperature stability and activity could lead to fermentation becoming more economical than petrochemical methods for fine chemical production. The chemical industry is increasingly using enzymes in catalysis. As the efficiency, specificity and activity of enzymes and the regeneration of cofactors are improved, the utilization of starch by the microorganisms used to produce the enzymes will increase further.

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nology 4, 1087-1090 8 Vaeck, M, Reynaerts, A., Hofte, H. et el. (1987) Nature 328, 33-37 9 Della-Cioppa, G., Bauer, S. C., Taylor, M. L. et el. (1987) Bio/Technology 5, 579-584 10 Buchanan-Wollaston, V., Passiatore, J.E. and Cannon, F. (1987) Nature 328, 172-175 11 Jaynes, J. and Dodds, J. (1987) N e w Sci. 115, 62-64 12 Dawnton, W. J. S. (1983) CRC Crit. Rev. Plant Sci. 1, 183-197 13 Kennedy, J. F., Cabral, J. M. S., SaCorreira, I. and White, C.A. (1987) in Starch: Properties and Potential (Galliard, T., ed.), pp. 122-130, John Wiley & Sons 14 Kieboom, A. P. G. and van Bekkhum, H. (1985) in Starch Conversion Technology (van Beynum, G. M. A. and Reels, J.A., eds), pp. 294-304, Marcel Dekker 15 Luenser, S. (1987) Brew. Dig. 62, 36-39 16 Suzuki, S. and Karube, I. (1979) in Immobilized Microbial Cells (Ventkatsubramanian, K., ed.), pp. 59-72, American Chemical Society, Washington DC 17 CPC International Inc. (1979) UK Patent Application 1539694 18 Katocin, D. M., Wood, N. S. and Yang, S.S. (1985) UK Patent Application 2145094 A 19 Plant, A. R., Morgan, H. W. and Daniel, R.M. (1987) Enz. Microb. Techno]. 8,668-672 20 Nanmori, T., Numata, Y. and Shinke, R. (1987) Appl. Environ. Microbiol. 53, 768-771 21 Cheetham, P. S. J. (1987) Enz. Microb. Technol. 9, 194-213 22 McConnell, D. J., Cantwell, B. A., Devine, K. M. et el. (1986) Ann. N Y Acad. Sci. 469, 1-17 23 Nakajima, R., Imanaka, T. and Aide, S. (1986) Appl. Microbiol. Biotechnol: 23,355-360 24 Knowles, J. R. (1987) Science 236, 1252-1258 25 Gilson, M. R. and Honig, B. H. (1987) Nature 330, 84-86 26 Flavell, R. and Payne, P. (1987) in Biotechnology in Agriculture (Hardcastle, J., ed.), pp. 14-15, Agriculture and Food Research Council, London 27 Blaschek, H. P. (1986) Food Technol. (Chicago) 40, 84-87 28 Hammond, J. (1986) Brew. Rev. 16, 15-18 29 Wiseman, A., ed. (1977 onwards) Topics in Enzyme and Fermentation Biotechnology, (Vols 1-7), Ellis Horwood 30 Busche, R. M. (1985) Biotechnol. Prog. 1,165-180