Enzymes from Genetically Modified Microorganisms - American

Chapter 17

Enzymes from Genetically Modified Microorganisms Sven Pedersen, Bent F. Jensen, and Steen T. Jørgensen

Downloaded by COLUMBIA UNIV on February 16, 2015 | http://pubs.acs.org Publication Date: October 5, 1995 | doi: 10.1021/bk-1995-0605.ch017

Novo Nordisk A/S, Novo Allé, DK-2880 Bagsvaerd, Denmark

Enzymes from genetically modified microorganisms play an increasing role in food technology. An example of such an enzyme with application possibilities both within the flavor and food area is cyclomaltodextrin glycosyltransferase (CGTase). A novel CGTase has been isolated from a strain of Thermoanaerobacter, a thermophilic anaerobe. The enzyme is extremely heat stable and has a temperature optimum of 90-95°C at pH 6.0. The gene encoding Thermoanaerobacter sp. CGTase has been transferred to a Bacillus host thus making possible large-scale production of the enzyme in commercially acceptable yields. This enzyme produces a mixture of α-, β-, and γ-cyclodextrins, not ideal from an industrial point of view, because β-CD's find an increasing industrial use. Possibilities of rationally designing mutants of CGTase, which mainly produce β-CD will be discussed. Safety aspects of the production of CGTase will be described. 1. Introduction Food enzymes produced by genetically modified microorganisms have been used commercially for a number of years. Examples are a maltogenic amylase for production of maltose syrups (1), a lipase from Mucor miehei for interesterification of fats (2), and acetolactate decarboxylase (ALDC) for maturation of beer (3). The properties and safety aspects of a novel CGTase from Thermoanaerobacter, an enzyme that has been introduced for production of cyclodextrins for technical applications and which will be introduced during 1995 as a food enzyme, will be described in this chapter. The necessary safety evaluations have been carried out for this enzyme, and the results are now being reported. A GRAS-petition will be filed for the enzyme in 1995, claiming that the CGTase, when produced according to current Good Manufact uring Practice, should be generally regarded as safe for use in the production of

0097-6156/95/0605-0196$12.00/0 © 1995 American Chemical Society

In Genetically Modified Foods; Engel, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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cyclodextrins. To illustrate the safety evaluation of the CGTase, the safety tests carried out for another enzyme, a maltogenic amylase (4), will be described. The safety tests carried out for the two enzymes, the maltogenic amylase and the CGTase, are the same. The possibilities of improving the properties of the enzymes by protein engineering will be discussed. 2. Cyclodextrins CGTase stands for cyclodextrin glycosyltransferase, describing the enzyme's primary function, to produce cyclodextrins from starch. Cyclodextrins are oligosaccharides with a closed ring structure, where the glucose units are joined together by a-1,4 linkages. Cyclodextrins containing 6, 7 or 8 glucose units are most common and are known as α, β and γ-cyclodextrins, respectively (Figure 1). The orientation of the molecule is such that the hydroxyl groups are on the outside of the ring structure and the interior of the cavity contains the C-H-groups and the glycosidic oxygens. Thus the cavity is hydrophobic while the external surfaces are hydrophilic. The dimensions of the ring are dependent on the number of glucose units (Figure 1). The hydrophobic interior is utilized in one of the main applications of cyclodextrins: flavors and spices form inclusion complexes with cyclodextrins and are protected against oxidation. Several spices, e.g., horseradish, are commercially available in Japan in this form. In addition to the production of cyclodextrins, CGTase catalyzes the transfer of glycosyl residues from a donor such as starch to a suitable acceptor. This is utilized commercially in glycosylation of the intense sweetener, stevioside (Figure 2). Stevioside is isolated from the leaves of the plant Stevia rebaudiana. The product is, however, bitter and its solubility is low. Glycosylation decreases bitterness and increases solubility. Examples of commercial applications of cyclodextrins are given in Table I. Table I. Applications of cyclodextrins Applications of cyclodextrins Stabilization of volatile substances - flavors, spices etc. Modification of physical properties - improve solubility of pharmaceuticals, eg., prostaglandin - reduce bitterness - mask unpleasant odors Selective adsorption - removal of cholesterol from egg, butter 3. Development of the Thermoanaerobacter CGTase Processing of starch at concentrations of industrial interest requires jet cooking as an initial step (5). Jet cooking is a continuous processing step, where the starch with an added amylase is liquefied in steam jet cookers or similar equipment operating at temperatures up to 105-110 °C. When a conventional α-amylase is used for liquefaction the reaction products are maltodextrins, which can act as acceptors in the cyclization reaction, thus reducing the cyclodextrin yield (6). Liquefaction with the CGTase itself became possible in 1985, when a CGTase from the thermophilic anaerobic genus, Ther-



GENETICALLY MODIFIED FOODS

α-Cyclodextrin

^-Cyclodextrin

y-Cyclodextrin

Figure 1. Structures of cyclodextrins

Stevioside

Figure 2. Stevioside



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moanaerobacter was isolated (Figure 3)(7,8). This enzyme is active and stable at high temperatures and low pH, and there are no traces of low molecular weight oligosaccharides produced in the initial stages of the reaction. The Thermoanaerobacter enzyme has its temperature optimum at about 90°C (Figure 4) and a broad pH-curve with a maximum at about pH 5.8 (Figure 5). A modification of the Pharmacia Phadebas ctamylase method has been chosen as analytical method, because it is both rapid and simple. Starch hydrolysis is accompanied by the release of a blue colour, which is measured spectrophotometrically at 620 nm. Anaerobic bacteria are poor enzyme producers and in order to produce the enzyme on an industrial scale gene transfer to a more suitable organism was chosen (7,8). The use of well known safe microorganisms with a long record of use in the food industry as host for cloning food enzymes may also be advantageous from a safety evaluation point of view. The gene coding for the CGTase was cloned into Escherichia coli as an intermediate host, and then into Bacillus subtilis. Figure 6 shows a simplified diagram of the basic steps. In Bacillus subtilis, CGTase is expressed extracellularly in quantities reaching approximately 40-fold higher than in the original Thermoanaerobacter strain (7,8). Characterization of the recombinant CGTase relative to the native CGTase with respect to molecular weight (SDS-PAGE), isoelectric point, thermostability, action pattern, liquefaction activity, and cyclodextrin production indicated no differences between the enzymes. The recombinant CGTase cross-reacted with antibody raised against the native CGTase. After the initial cloning and expression in B. subtilis, the gene was transferred into a Bacillus strain suitable for large scale production. The DNA-sequence of the CGTase gene, which has been transferred to B. subtilis and later to the Bacillus production strain has been determined. The DNA sequencing has been made on DNA extracted from E. coli. The N-terminal aminoacid-sequence of the native enzyme purified from Thermoanaerobacter has been determined. The cloned and sequenced DNA codes for this N-terminal aminoacid-sequence. 4. Safety aspects Manufacture and quality controls Microbial food enzymes are produced by pure culture fermentation of carefully selected strains of microorganisms grown on steam sterilized natural substances. Fermentation conditions and conditions during recovery of the enzymes should be carefully controlled to ensure Good Manufacturing Practice (GMP) throughout the course of enzyme production. The final products must meet the food grade quality criteria formulated by the FAO/WHO Joint Expert Committee on Food Additives (JECFA)(9) and Food Chemicals Codex (FCC)(10). These criteria are summarized in Table II, and include evaluation of raw materials, additives and processing aids, determination of chemical contaminants, microbial contamination, mycotoxins in fungal enzymes and antibiotic activity in the final product. Safety evaluation principles The safety in use of microbial food enzymes is often based on a combination of a history of safe use and a safety evaluation based upon scientific procedures. The scientific procedures often include both in vivo and in vitro toxicology studies or chemical analysis for suspected toxic compounds in enzyme preparations (11).


200

GENETICALLY MODIFIED FOODS

Starch slurry CGTase

ι 1


JET COOKING

1 CYCLIZATION

pH 5.0-6.0 15-30% DS 105°C 5min 25-50 NU/g DS

90°C 4-24 hours

Figure 3. CD-Production with Thermoanaerobacter CGTase

Figure 4. The effect of temperature on the activity of Thcrmoanaerohacter CG'I'ase



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PEDERSEN ET AL.

CHROMOSOMAL ONA FROM THERMOANAEROBACTER

Ο

P L A S M I

° o o ο ο

I PART IAL 0 IGESTI ON I WITH RESTRICTION PN7V] ENZYME

•

/

/ E

DIGESTION WITH RESTRICTION

On " G CENTRIFUGAL SEPARATION

°

m

Λ Ο

LIGATION

Ο

ο^

W

ο,

RECOMBINANT PLASMIO

I INSERTION OF PLASMID i INTO HOST RECOMBINANT PLASM 10

o & t -

CHROMOSOME

3. SUBTILIS

Figure 6. Basic steps in cloning of the CGTase gene from Thermoanaerobacter into Bacillus


201

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G E N E T I C A L L Y

M O D I F I E D

F O O D S

Table II. JEFCA and FCC specifications for enzymes


Parameter

FCC

JECFA

Raw materials

Free of harmful and undesirable substances

Same as JECFA

Additives and processing aids

Acceptable for food or insoluble and removed after use

Same as JECFA

Chemical contaminants

Lead Arsenic Heavy metals

Same as JECFA (ditto) (ditto)

Mycotoxins in fungal enzymes

Aflatoxin, Ochratoxin A Sterigmatocystin, T-2 toxin, Zearalenone must be absent

Ensure that products do not contain mycotoxins

Antibiotic activity

Negative

Not specified

Microbial contamination

Coliforms Salmonella E.coli Total viable count

Enzymes from Genetically Modified Microorganisms - American

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