Efficient production of active industrial enzymes in plants

Industrial Crops and Products, 10993) 241-250 0 1993 Elsevier Science Publishers B.V. All rights reserved. 0926-6690/93/$05.00

241

INDCRO PRO21

Efficient production of active industrial enzymes in plants Jan Pena, Albert J.J. van Ooyenb, Peter J.M. van den E1zena, Wim J. Quaxb and Andr6 Hoekema” “MOGENN. VI, Leiden, The Netherlands ‘Gist-brocades N. K, Research and Development, De&, The Netherlands (Accepted July 1992)

Abstract

Pen, J., van Ooyen, A.J.J., van den Elzen, P.J.M., Quax, W.J. and Hoekema, A. 1993. Industr. Crops Products 1: 241-250. An industrial bulk enzyme, Bacillus licheniformis a-amylase was produced in transgenic tobacco at a maximum level of about 0.3% of total soluble protein. The molecular weight of the enzyme produced in tobacco differed from the bacterial enzyme due to complex-type carbohydrate chains attached to the protein. Milled transgenic seeds containing a-amylase were successfully applied in the liquefaction of starch. The resulting hydrolysis products were virtually identical to those obtained from degradation with B. licheniformis a-amylase. No effect was observed of the presence of the enzyme on endogenous starch. Homogenization of transgenic leaves, followed by incubation at 95”C, resulted in degradation of the endogenous starch. Different approaches to the concept of production and application of industrial enzymes in plants are presented that show the efficacy of transgenic plants as a new source of active industrial enzymes. a-Amylase;

Industrial

enzymes; Transgenic plants; Tobacco

Introduction Genetically engineered plants may be a suitable alternative for the production of proteins. It has been demonstrated that foreign proteins can be successfully produced in plants (DeZoeten et al., 1989; During et al., 1990; Hiatt et al., 1989; Krebbers and VandeKerckhove, 1990; Sijmons et al., 1990; VandeKerckhove et al., 1989). Although the first examples have focused on the production of high-value pharmaceutical proteins, plants may be even more Correspondence: J. Pen, MOGEN N.V., Einsteinweg 97,2333 CB Leiden, The Netherlands.

suited as a competitive source of industrial bulk enzymes (Pen et al., 1992a). Enzymes are used in many industrial processes. Well-known examples are in the food and feed industry, where enzymes are added for a range of effects, for instance to obtain a desired product, to remove undesired components, to control texture, etc. In cases where the raw material is of plant origin, the enzymes used may be endogenous to the plant, as for the malt amylases, glucanases and proteases used in the brewery industry and the pectinases used in the fruitjuice industry (Peppler and Reed, 1987). However, for many purposes the use of heterologous enzymes is pre-

242

ferred because of their optimal characteristics, such as thermostability or suitable pH for the intended industrial process. To assess different possibilities for the production of industrial enzymes in plants and methods for their application, we have expressed a-amylase from Bacillus licheniformis in tobacco. Alpha-amylases from bacterial and fungal origins find wide application in a number of industries (Kennedy et al., 1988; Peppler and Reed, 1987; Schwardt, 1990; Whitaker, 1990). The a-amylase from B. licheniformis is the most commonly used enzyme in starch liquefaction, because of its extreme heat stability and its activity over a wide pH range (Peppler and Reed, 1987; Yuuki et al., 1985). Materials and Methods Transformation of tobacco with a-amylase expression vector Two binary expression vectors were constructed as described previously (Fig. 1; Pen et al., 1992a). Both contain the CaMV 35s promoter with a duplicated enhancer (Guilley et al., 1982), the AlMV RNA4 leader sequence (Brederode et al., 19801, the a-amylase gene fragment of Bacillus licheniformis encoding the mature enzyme and the terminator from the Agrobacterium tumefaciens nopalin synthase gene. Binary vectors pMOG229 and pMOG227 contain the signal peptide encoding sequences of B. licheniformis a-amylase and tobacco PR-S, respectively. Transgenic tobacco Wicotiana tabacum cv. Petit Havana SRl) plants were obtained by Agrobacterium-mediated transformation of leaf discs followed by selection on kanamycin and regeneration of shoots (Hoekema et al., 1983; Horsch et al., 1985). Expression analysis Loaf tissue (about 100 mg) was homogenized in 0.5 M glycine/HCl buffer pH 9.0 containing 10 mM CaClz. The homogenate was centrifuged for 10 min in a bench-top centrifuge (4”C, full speed), and the supernatant was collected. The soluble protein content was determined according to the method of Bradford (1976). Alpha-amylase activity was determined

spectrophotometrically as described by Saito (1973). Western blotting was performed using affinity purified antibodies (Kocken, 1989; Pen et al., 1992a). Staining for conjugate peroxidase activity was done with H202 and o-dianisidine or alternatively using the Enhanced Chemiluminescence (ECL) system (Amersham, U.K.; Pen et al., 1992a). Isolation of extracellular fluid Extracellular fluid was isolated as described before (Pen et al., 1992a). The fluid was assayed for marker peroxidase activity (Hendriks et al., 1985), a-amylase activity (Saito, 1973) and protein content (Bradford, 1976). Deglycosylation ofprotein extracts Endo+N-acetylglycosaminidase H (EndoH) of Streptomyces plicatus from a recombinant E. coli was used for the digestion of leaf protein extracts. Leaf extracts were incubated twice for 3 h at 37°C with each time 2 mU EndoH in a 0.05 M sodium acetate buffer pH 5.5, containing 0.2% SDS, 10 PM pepstatin A and 1 mM PMSF. After digestion the samples were dried in a speed-vat (Savant). Chemical deglycosylation was done with trifluoromethanesulfonic acid (TFMS) as described by Edge et al. (1981). Samples were finally lyophilized. Protein losses were found to be considerable during the deglycosylation by TFMS. Therefore, 50 times as much protein, based on the amount present before treatment, was loaded on the gel as compared with EndoH treated and untreated leaf protein extracts. For unknown reasons some protein loss was also observed upon Endo-H treatment. Qualitative &termination of leaf starch Starch was qualitatively determined in leaves collected half-way through the photoperiod by decolorizing the leaves overnight in 96% ethanol and subsequent staining for 30 min with 43.4 mM KI/5.7 mM Is in 0.2 M HCl (Caspar et al., 1986). Liquefaction of starch Potato or corn starch slurry were incubated with either milled transgenic seeds, an equal amount of non-transgenic seeds or the com-

243

mercial a-amylase from Bacillus licheniformis (Maxamyl@ WL7000, Gist-brocades, Delft, The Netherlands) or B. amyloliquefaciens (Dexlo’@ CL, Gist-brocades, Delft, The Netherlands). Alpha-amylase activity was determined by the Phadebas method according to the manufacturers instructions (Pharmacia diagnostics). The units are referred to as T.A.U. (thermostable a-amylase units). Transgenic seeds or microbial amylases were used to an equivalent amount of 4.4 T.A.U./g dry substance. Liquefaction was done at 95-100°C. Determination of the hydrolysis pattern by HPLC and calculation of the dextrose equivalents of the reaction products from the HPLC profile were done as described before (Pen et al., 1992a). Hydrolysis of leafstarch duringprocessing Top-leaves of transgenic tobacco plant-line MOG227.3, that expressed Bacillus licheniformis a-amylase and of control non-transgenie tobacco plants, grown for 19 days in the soil, were harvested half-way through the photoperiod. Leaf-material (approximately 40 mg) was homogenized in 2.5 pl/mg 0.5 M glycine/NaOH pH 9.0. The homogenates were subsequently incubated for 0,30 and 60 min at 95°C. The reaction was stopped by extracting the homogenate with 1 ml 96% ethanol (65”C), followed by centrifugation for 2 min at maximum speed in a bench-top centrifuge. The extraction procedure was repeated twice for the pellet. The pellet was washed once with 1 ml 0.5 M glycine/NaOH pH 9.0, each time followed by centrifugation as above, and subsequently suspended in 50 $0.5 M glycine/NaOH pH 9.0. Finally, 50 ul 1.5 N acetic acid and 50 ~1 2% KI/0.2% 12were added to stain for the presence of starch. As a control, non-transgenic leaf homogenates with various amounts of B. licheniformis a-amylase were used. All determinations were done in duplicate.

Results Expression analysis By Agrobacterium-mediated transformation with the binary vectors pMOG227 and pMOG229 (Fig. 11, 59 and 37 plants were ob-

tained, respectively. Leaves were assayed for a-amylase activity after the plants had been grown in soil for about three weeks. The maximum and average expression levels obtained were about 0.3% and 0.1% of soluble protein in leaves for both constructs, respectively, assuming the same specific activity for the Bacillus enzyme and the enzyme produced in tobacco. With Western immunoblotting, using polyclonal antibodies raised against the enzyme from B. licheniformis, it was demonstrated that the observed a-amylase activity is the result of the heterologous enzyme expressed in plants (Fig. 2). On Western blots the molecular weight was found to be around 64 000. At least two bands were found to be present. The molecular weight is clearly different from that of the Bacillus enzyme, which is 55,200 (Yuuki et al., 1985). Secretion of a-amylase The activity per mg soluble protein of a-amylase in the extracellular fluid isolated from the leaves of transgenic plants, was found to be approximately 30-fold higher than in the total leaf extract for both constructs (data not shown). The ratio of the activity in the extracellular fluid and in the total extract was similar for a-amylase and peroxidase, a marker enzyme for the extracellular fluid. From this it may be concluded that both signal peptides function in secretion of the a-amylase. The difference in amount of a-amylase in total extract and extracellular fluid of leaves was also demonstrated by Western blotting (Fig. 2). Glycosylation of a-amylase in tobacco An explanation for the difference in molecular weight between the Bacillus licheniformis aamylase and the enzyme as produced in tobacco is glycosylation of the enzyme. A total of six potential asparagine-linked sites are present in the primary structure (Yuuki et al., 1985). Deglycosylation of crude leaf extracts with Endo+&acetylglycosaminidase H (EndoH) did not result in any decrease of the molecular weight. However, deglycosylation with trifluoromethanesulfonic acid (TFMS) yielded a protein with a molecular weight identical to the Bacillus enzyme (Fig. 3). This

244

Hindlll \ RB

Alpha-amyl

Signal AIMV

peptide leader

3%

v BINARY ALPHA-AMYLASE

H

promoter/ enhancer

EXPRESSION

PLASMID

1.

LB

Encoded

signal

peptide

sequence

pMOG227

MNFLKSFPFYAFLCFGQYFVAVTHA

pMOG229

MKQQKRLYARLLTLLFALIFLLPHSAAAA

Fig. 1. Representation of the binary vectors containing the a-amylase genes. The amino acid sequences of the signal peptides encoded by the two constructs are shown below the plasmid drawing. Abbreviations: LB = left T-DNA border sequence; RB = right T-DNA border sequence; pnos= nopalin synthase (nos) promoter; tnas = nopalin synthase (nos) terminator; NPTII = neomycin phosphotransferase II gene, selectable kanamycin resistance marker; KmR = bacterial kanamycin resistance gene; oriRK = bacterial origin of replication; AlMV leader = leader sequence of Alfalfa Mosaic Virus RNAB.

showed that the difference in molecular weight was caused by glycosylation of the protein in tobacco and that the carbohydrate chains were of the complex type. The deglycosylation also showed that the existence of multiple forms of the enzyme can be explained by differential glycosylation of the protein (Fig. 3).

Phenotype of the transgenic plants The transgenic plants were visually indistinguishable from non-transgenic Nicotiana tabacum cv. Petit Havana SRl plants, i.e., no morphological differences were observed, and the growth-rate was identical. The presence of starch in leaves collected half-way through the

245

Amylase (w)

Total Extract A

67

B

C

2

51025A

Extracellular Fluid B

C

-

25.7-

Fig. 2. Immunoblot of soluble leaf protein from transgenic plants. Of tobacco leaf total extract, and extracellular fluid, 10 pg and 0.7 ug protein were applied to the gel, respectively. (A) Nicotiana tabacum cv. Petit Havana SRl control plant; (B) transgenic plant line MOG227.30; (C) Transgenic plant line MOG229.23. Alpha-amylase from Bacillus licheniformis was used as a control in the amounts indicated. Molecular weights (X 103) of marker proteins are indicated on the left.

photoperiod was qualitatively demonstrated by staining with KI/I2. Staining was similar in transgenic and non-transgenic plants (data not shown). This indicates that the enzyme has no access to the starch present in the leaves, as is expected from the extracellular location of aamylase and the chloroplastic location of starch. Direct application of transgenic seeds in starch liquefaction

a-amylase

Seeds of transgenic plant line 227.3 with an expression level of 0.2% of soluble protein in seeds, were milled and used directly in liquefaction of potato and corn starch. Non-trans-

genie seeds were used as a control. Commercial a-amylase preparations from Bacillus licheniformis or B. amyloliquefaciens were used for comparison. Transgenic seeds and bacterial aamylases to an equivalent of 4.4 T.A.U./g dry substance were added to the starch slurry. HPLC analysis of the degradation products obtained with the transgenic seeds showed that these were virtually identical to those obtained with B. licheniformis a-amylase, maltopentaose (DP5) being the predominant product. The products were significantly different from those obtained with B. amyloliquefaciens a-amylase, where maltohexaose (DP6) was the major degradation product (Fig. 4). No degra-

B. licheniformis

amylase

I III

1231

200

r

229.6

227.30

11111

2

had the same effect, while incubation of nontransgenic control leaf homogenate had no visible effect on the endogenous starch during the time of the experiment.

3123

97.4 67 43

25.7

Fig. 3. Deglycosylation of a-amylase produced in tobacco with EndoH and TFMS. Amount of protein, based on the amount determined before treatment, applied to the gel: Bacilluslicheniformisa-amylase 1: 5 ng; 2: 5 ng; 3: 10 ng; total leaf extracts of plant lines MOG229.6 and MOG 277.20: 1: 0.4 pg; 2: 0.4 pg: 3: 20 Kg. Treatment: 1. Non-treated; 2. treated with EndoH; 3. treated with TFMS. Molecular weights (x lOa> of marker proteins are indicated on the left. Following electrophoresis on a 10% polyacrylamide-SDS gel, samples were subjected to Western analysis using the ECL method. The film was exposed to the blot for 1 min.

dation was observed with non-transgenic SRl seeds. As shown in Table 1, the dextrose equivalents (DE), which were calculated from the HPLC values, were found to be similar to those obtained with the commercial preparations and within the commercially acceptable range (DE ~12, preferably 2 16; Reilly, 1985). Starch degradation was performed at 95-lOO”C, demonstrating the thermostability of the enzyme as produced in tobacco. Hydrolysis of leafstarch during processing Homogenization of transgenic leaf material containing Bacillus licheniformis a-amylase, followed by incubation at 95°C for 30 min hydrolyzed starch present in the leaves (Table 2). Addition of 2 T.A.U./mg B. licheniformk aamylase to non-transgenic control leaf homogenate for 30 min or of 0.2 T.A.U./mg for 60 min

Discussion The results presented here show that an active industrial enzyme can be expressed in plants at a considerably high level. From the determinations done so far it can be concluded that the biological activity of the enzyme does not differ from its natural bacterial counterpart. However, the protein is, in obvious contrast with the bacterial protein, glycosylated. The signal peptides of bacterial a-amylase and of the tobacco PR-S protein show a considerable structural difference (see Fig. 1). Nevertheless, they seem to function equally well in secretion of a-amylase. From the activity during starch liquefaction performed at 95-100°C it can be deduced that the enzyme produced in tobacco exhibits an extreme thermostability, similar to the Bacillus enzyme. The nature and quality of the hydrolysis products obtained from corn and potato starch with the transgenic seeds indicates that the enzyme as produced in tobacco is as suited for liquefaction of starch as the Bacillus licheniformk amylase. Different possibilities for the production of industrial enzymes in plants and the methods of application are discussed below. I. Production and purification from plants As a first possibility, plants can be used as a host to produce foreign proteins. The feasibility to produce an active industrial enzyme in plants at a considerably high level is shown in this study, and even higher expression levels may be obtained by screening for high expression (Pen et al., 199213).The success of producing industrial enzymes in plants will be determined by the feasibility of competing with traditional production methods, in general microbial fermentations. Starch potatoes or oilseed rape may be interesting target crops, because the enzymes may be produced as byproducts in addition to the primary product

247

a

Fig. 4. Comparison of oligosaccharide patterns obtained from starch hydrolysis. Corn (a) and potato @I) starch were hydrolyzed using milled transgenic seeds of plant line MOG227.3 (A), Bacillus licheniformis a-amylase or (B) Bacillw amyloliquef~iens a-amylase (0. Hydrolysis products were separated by HPLC. The degree of polymerization (DP) of glucose is indicated. The main difference between the products obtained with the seeds and Bacillus licheniformis amylase on one hand and Bacillus amyloliquefaciens on the other is the amount of DP5; this peak is indicated in black.

248 TABLE 1

TABLE 2

Dextrose equivalents (DE) obtained by hydrolysis of corn and potato starch

Degradation of endogenous starch in leaves during processing

DES

Transgenic tobacco seeds (MOG227.3) Non-transgenic

tobaccoseeds

Bacillus licheniformis amylase Bucillus amylaliquefaciens

amylase

Sample

Potato starch

Corn starch

16

13

0

0

Incubation time (min) 0

30

60

Control tobacco

+

+

+

Control tobacco

+

*

f

+

*

-

+

-

-

+

-

-

18

16

+ 0.02 T.A.U. B. licheniformis a-amylase/mg

15

18

Control tobacco + 0.2 T.A.U. B. licheniformis a-amylase/mg

(starch or oil), thus reducing the production costs. This approach may have the additional advantage of partial purification of the enzyme without added cost. Down-stream processing costs can be minimal in case of industrial enzymes, because the purity requirements are low compared with pharmaceutical proteins. These costs should generally be comparable with those after microbial fermentations. For industrial enzymes the economic production in plants (cheap biomass) may be combined with low down-stream processing costs.

II. Direct application A new concept is the direct use of engineered seed in an industrial process. The seed serves as a new way of formulation for the expressed enzyme. This approach is exemplified in this study by the direct application of transgenic seeds harboring a-amylase in liquefaction of corn and potato starch (Fig. 4 and Table 1). Manufacturing costs will be much lower compared with the first approach, because purification of the enzyme can be minimal and formulation is no longer required. A disadvantage may be the presence of the plant material in the industrial process. On the other hand, the presence may be compatible with or even be advantageous to the process. The plant material to be used will depend on the process but, in general, seeds may be the plant organ of choice because these create a stable environment for long term storage of the enzymes.

Control tobacco + 2 T.A.U. B. licheniformis a-amylase/mg Transgenic tobaccoline MOG227.3

The presence of starch, as demonstrated by IfiI-staining is indicated with +, intermediate staining with f, and absence with -.

II. Process-dependent conversion A further application of enzymes in crops is the expression and sequestered localization of an industrial enzyme in the crop where the substrate is also present. Alternatively, the enzyme may be expressed at the same location, but in an inactive form. The conversion will thus become process-dependent. During processing, enzyme and substrate are brought together or the enzyme is activated, resulting in conversion of the substrate. In this way the plant becomes equipped with the enzyme having the optimal characteristics for conversion of its endogenous substrate. This obviates the need for purification and formulation of the enzymes completely. Moreover, the production costs of the enzymes are reduced to the absolute minimum. As a consequence, in cases where this approach is applicable, the manufacturing costs will be extremely low. In vivo conversion of the substrate, by expression of the enzyme at the same location as the substrate will have the same advantages in terms of manufacturing costs, but often a clear dis-

249

advantage can be the effect of the enzymatic action on plant characteristics. The feasibility of this approach is exemplified in this study. Extracellular expression of a-amylase did not have an effect on endogenous starch, as expected from the separated location of enzyme and substrate. Moreover, the transgenic plants did not show any abberations in their phenotype. Homogenization of transgenic leaf material, followed by incubation at high temperature, resulted in conversion of the endogenous starch by the transgene product (Table 2). An industrial example of such an approach may be the expression of an extracellular a-amylase in potatoes or corn used for starch production. In potato the a-amylase expression levels as obtained in this study would be sufficient to hydrolyze the starch present in the tuber. Similar applications for a-amylase or for other enzymes may be found in other industrial processes, like for instance the application of a-amylase in the production of bio-ethanol and of a heat-stable (3-1,4-glucanase for the degradation of cell-wall material in the brewery industry. Another example is the clarification of juices by a-amylases or pectinases. Each of these approaches has its own benefits and limitations. The choice of application of one or the other method will be dependent on the nature of the intended industrial process. This study demonstrates the feasibility of these approaches and shows the competitiveness of production of industrial enzymes in plants. Acknowledgements We would like to thank Anja J. Luyt, Lucy Molendijk and Harry J.H. Hens for expert technical assistance and Drs Hunt and Chrispeels (University of California, La Jolla, USA) for sharing their deglycosylation protocols with us. References Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.

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