DE4425058C2 - Process for the preparation of a thermostable glucoamylase - Google Patents

Description

The invention relates to a method for producing a thermostable glucoamylase using an amylolytic, Starch as a carbon source of the yeast strain of the species Saccharomyces cerevisiae.

There has long been a high level of interest in the extraction of starch-degrading enzymes for industrial purposes or on the Construction of microorganisms containing such enzymes release extracellularly, in particular glucoamylase (1,4-α-D-glucan Glucanhydrolase, EC 3.2.1.3.), Which is the end product Monosaccharide forms glucose.

A number of microorganisms, especially fungi, are one Enzymatic starch degradation enabled. But also the use of yeast strains of the species Saccharomyces cerevisiae for the production of such enzyme preparations - and for example also from Glucoamylase - has been known for a long time (D Mot).

It has been shown that not every one of these products in the type and quantity desired for industrial use can be generated. Through different glycosylation, inaccurate splitting off of secretion sequences or missing or restricted transportation of the product within a Yeast cells and out of a yeast cell become namely Properties of the protein intended as a product of the Host strain itself negatively affected.

They are therefore outside of Saccharomyces cerevisiae whole range of (differently functioning) hosts Vector systems have been developed, all for one certain gene product good, for other, heterologous gene products are usually less suitable.

In EP 0 260 404 A2 and Derwent Abstract 92-22 32 41/27 and Derwent Abstract 87-17 36 94/25 is described, glucoamylase Genes from other donor strains in Saccharomyces cerevisiae (S.c.)  to transform. However, the amylases obtained do not have the properties you want based on what's essentially on different glycosylation and inaccurate splitting off of Selection sequences.

If you want one with the desired (natural) protein if possible identical protein (same primary, secondary and tertiary Structure, same characteristics) through genetic engineering of a yeast, then one test is complete in each case different host vector systems required because for example the degree and type of glycosylation or the Training of appropriate spatial structures on a host-specific basis are.

For this reason, the Arxula adeninivorans is also have been examined in more detail; especially some biochemical Properties such as insulation, cleaning and Characterization of a glucoamylase have already been described and molecular biological could be roughly the approximate size of the genome can be determined (DD patents 265 163 A1 and 268 254 A1).

DD patent 265 163 A1 describes a method for Obtaining a thermostable glucoamylase from the yeast Arxula adenine virus described. This makes this glucoamylase characterized in that the optimum of their pH between 4.0 and 5.5 lies, and in the counter type of the substrate it shows strength Optimal temperature of 70 ° C. Under these conditions it also has high thermal stability; after 15 min at 70 ° C there is still 80% of the initial activity. Higher pH values prevent inactivation of the glucoamylase. While at pH 4.5 in the presence of 5% starch after 15 minutes at 60 ° C only 50% of the activity is still present, takes place at pH 6.0 under otherwise identical conditions no inactivation takes place.

The disadvantage of this method is the isolation required of the enzyme preparation from the yeast Arxula adeninivorans, which  itself through the - inevitable - formation of pseudomycelium much worse than other yeasts biotechnologically worked up, especially since the knowledge of Arxula adeninivorans have been far less than others, for long times well-known, widely used and extensively examined Yeast species such as Saccharomyces cerevisiae.

The invention has therefore set itself the task such, which can be synthesized by the yeast type Arxula adeninivorans Glucoamylase instead in the biotechnologically light to synthesize manageable yeast Saccharomyces cerevisiae which should be constructed in such a way that the in this way produced amylolytic yeast strains starch as carbon Use source.

According to the invention the object is achieved in that Yeast strain from a donor strain of the Arxula adeninivorans species isolated glucoamylase gene is transformed, the Amino acid sequence with the secretion determining Partial sequence of the 16 (N-terminal) amino acids M-R-Q-F-L -A-L-A-A-A-S-I-A-V-A begins, with the glucoamylase gene using of a glucoamylase promoter expri is lubricated.

By expression of the glucoamylase gene in Saccharomyces cerevisiae, this yeast is able to be used as starch Carbon source to use. Using a yeast's own glucoamylase promoter is that in Saccharomyces cerevisiae synthesized glucoamylase both in terms of Amount as well as the biochemical properties with that from Arxula adeninivorans quite comparable. Obviously is responsible for the secretion of the enzyme preparation Signal sequence also effective in Saccharomyces cerevisiae.

In a preferred embodiment, the synthesized Glucoamylase using the centrifuged Culture fluid, unpurified, biochemically characterized become.  

Based on the drawing, the invention is in a Ausfü Example explained in more detail. Show it

Fig. 1 shows the cloning of the glucoamylase gene of Arxula adeninivorans,

Fig. 2 shows the gel electrophoretic analysis of a plasmid PGAA (this, the DNA was the plasmid with the restriction endonucleases PGAA (1) EcoRI / ApaI, (2) EcoRI / PstI and (3) Apa I / PstI digested and dissolved in a 0.8% agarose -Gen separated),

Fig. 3 shows the restriction map of the glucoamylase gene of Arxula adeninivorans,

Fig. 4 shows the gel electrophoretic separation of Chromo of Arxula adeninivorans somen Ls3 (the donor strain from the strain collection of the Institute for Plant Genetics and Crop Plant Research, Gatersleben) using the PFG and hybridization with the DNA fragment contained,

Fig. 5 PFG (1-7) and DNA hybridization (8-14) of the Chro somes of Arxula adeninivorans strains (1, 8) CSIR 1136, (2, 9) CSIR 1138, (3, 10) CSIR 1147, (4, 11) CSIR 1148, (5, 12) CSIR 1149, (6, 13) CBS 8244T and (7, 14) Ls3 (a DNA fragment with the glucoamylase gene as the radiolabelled probe was used for hybridization) ,

Fig. 6 is an agarose gel electrophoresis (1-3) and a Northern blot analysis (4-6) selected from (1, 4), (2, 5) free and (3, 6) ER-bound polysomes iso profiled RNS Arxula adeninivorans Ls3 (a DNA fragment with the glucoamylase gene was used as the radiolabelled probe for the Northern analysis),

Fig. 7/1 to Fig. 7/2 the nucleotide sequence of the glucoamylase gene and the corresponding amino acid sequence of the gluco amylase of Arxula adeninivorans Ls3,

Fig. 8/1 and

Fig. 8/3 a comparison of the amino acid sequence of the gluco amylase of Arxula adeninivorans Ls3 with the Glu koamylasen of Rhizopus oryzae, Aspergillus ni ger, Saccharomyces diastaticus and Saccharomycop sis fibuligera,

Fig. 9 shows the construction scheme of the plasmid pET-GAA,

Fig. 10, the separation by gel electrophoresis (1-4) and a Western blot analysis (5-8) of the intrazellulä ren proteins (1, 3, 5, 7) Escherichia coli HMS 174, and (2, 4, 6, 8) Escherichia coli HMS 174 / pET-GAA (gel electrophoretic separation was carried out on a 10% polyacrylamide gel, antibodies against the glucoamylase of the glucoamylase gene from Arxula adeninivorans were used for the Western blot analysis),

Fig. 11 shows the construction scheme of a plasmid Ycp50- GAA,

Fig. 12 shows the construction scheme of a plasmid pYGAP- GAA,

Fig. 13 growth and course of the secretory gluco amylase activity (Teilfig. 13b) of Saccharomyces ces cerevisiae SEY 6210 / pYGAP-GAA at a Kul minimal media yeast (Tanaka) tivierung in a glucose, maltose or starch as a carbon source ( Final concentration 1%) and

Fig. 14 shows the pH-dependence and the temperature-dependent ness of the secreted from Saccharomyces cerevisiae SEY 6210 / pYGAP-GAA glucoamylase.

1. Isolation of the Arxula Glucoamylase Gene adenine virus 1.2. Preparation of a phage cDNA library

To get a cDNA library from Arxula, first of all the total RNA from cells in a minimal yeast medium cultivated with maltose as a carbon source (Tanaka) which are isolated in a known manner (Elion). On finally, an mRNA clearing and cDNA synthesis takes place using a commercially available and relevant laboratory kit (mRNA Purification Kit Product No. 27-9258-O1 or cDNA Synthesis Kit Product No. 27-9260-O1 from Pharmacia). You can cDNA fragments up to a size of 4.5 Synthesize Kbp; they are used for ligation in the Phage vector Lambda gt11 (Pharmacia) used.

After the ligation and packaging of the DNA obtained therefrom using a commercially available laboratory kit (DNA Packaging Kit product no. 733 792 from Boehringer), recombinant phages can be isolated. In the gel electrophoretic analyzes connected in the laboratory experiment, it was found that the 4 × 10 ⁵ recombinant phage had incorporated cDNA fragments up to a size of 2 Kbp.

1.4. Creation of a phage library with chromosomal DNA by Arxula

Since in the expression of the glucoamylase gene from Arxula adeninivorans in different host systems the Te stung different transcription promoters, such. B. that of the glucoamylase promoter is also sought , a phage library with a chomosomal gene must be used at the same time DNS are made.

For this purpose, the chromosomal DNA of Arxula adeninivorans is isolated according to a method described in DD patent specification 298 821 A5 and partially cleaved with the restriction enzyme EcoRI; then 3-6 Kbp restrictase-generated DNA fragments are isolated. The DNA fragments obtained were then (as described under 1.2.) Built into the phage vector Lambda gt11. After ligation and packaging, 5 × 10 ⁵ recombinant phages with 3-6 Kbp DNA fragments of the chromosomal DNA of Arxula adeninivorans were isolated in the laboratory test.

At the same time, the one in DD patent 298 821 A5 wrote Charon 4A phage library with 10-15 Kbp Fragments of the chromosomal DNA of Arxula adeninivorans used.

1.5. Production of antibodies against glucamylase

For the immunization of rabbits are secretory Proteins of a donor strain Arxula adeninivorans Ls3 inserted puts. This donor strain is kept in a 1% for 3 days Cultivated maltose culture. Under these cultivation conditions the glucoamylase of the donor strain is induced and exported to the medium. It dominates compared to the other secretory proteins.

The culture medium is removed from the Cells separated and then by a 0.22 µm milli pore filter (Millipore) filtered. With the help of Affi Chromatography using hydroxyapatite Columns (equilibrated with 10 mM sodium phosphate buffer a pH of 6.8) the concentration and rei the secretory proteins. The Hydro Proteins of the cultivation medium bound to xylapatite columns um with 250 mM of the same sodium phosphate buffer eluted from the column, against distilled water below sterile conditions dialyzed and immunized three times used.

The antiserum obtained in this way is first removed by ammonium sul fat precipitation and subsequent dialysis against TBS (10 mM Tris HCl, pH 8.0; 150 mM NaCl) cleaned and for "Western Blot" - Experiments - see (b) - used. Then follows  the purification by immune adsorption, which makes the monospe specificity of the immune serum is reached - see (c) -.

(a) Localization of the corresponding glucoamylase Polypeptide band in SDS-polyacrylamide gel

After gel-electrophoretic separation of the secretory Proteins in a 7.5% polyacrylamide gel under native Conditions (Ornstein, Davis) this polyacrylamide gel cut horizontally into approx. 8 mm thick strips that Purpose of protein elution in 10 mM sodium phosphate buffer pH 7.0 can be incubated at 10 ° C for 15 hours. From the individual fractions eluted proteins are in be known way (Büttner 1987) on glucoamylase activity tests. The corresponding positive fractions are white ter cleaned (dialyzed against distilled water), con centered (lyophilization), suspended in Laemmli buffer and in SDS polyacrylamide gel electrophoresis in Ver immediately separated into the secretory proteins. Here was able to test an approximately 90 kDa polypeptide as a Glucoamylase band can be identified.

(b) "Western blot" experiments using the herge put immune serums

Those used as antigen for rabbit immunization Proteins are gel electrophoresis in a preparative SDS polyacrylamide gel separated and by electrical transfer immobilized on nitrocellulose (from Schleicher / Schüll). This blot is about 5 mm wide on both sides Strips cut off and blocked after 30 minutes 0.5% skim milk / TBS-T (TBS + 0.1% Tween 20) for 3 hours in primary anti purified by ammonium sulfate precipitation incubated serum. After washing the membrane strip three times For 10 minutes each in TBS-T, they are incubated in an anti-rabbit IgG alkaline phosphatase conjugate (Promega), which has been diluted 1: 7,500 in TBS-T. After repeated washing steps, as already described,  the alkaline phosphatase is mixed with the help of the substrate isolated.

The glucoamylase polypeptide band is made from the untreated Nitrocellulose membrane cut out and used as an antigen for used immunoadsorption.

(c) Serum purification by immunoadsorption

The nitrocellulose membrane strip on which the gluco Amylase polypeptide is immobilized for 30 minutes washed in TBS-T at room temperature with shaking and then blocked in blocking solution for the same time.

The subsequent 60-minute incubation of the Membrane strip in 1: 2 diluted by ammonium sulfate precipitation purified polyspecific antiserum that the glucoamylase antibodies. After a total of 5 washes The antibodies are incremented for 5 minutes in TBS-T with vigorous shaking of the nitrocellulose strip in Glycine buffer (0.1 M glycine, pH 2.5; 0.9% NaCl; 1% bovine rumalbumin) replaced. The buffer is immediately after detachment solution of the antibodies with saturated sodium phosphate solution neutralized. This purified monospecific antiserum is dialyzed overnight at 4 ° C against TBS and in aliquo stored at 20 ° C.

1.6. Immunological screening of the recombinant phages with cDNA from Arxula

Potential recombinant phages with appropriate glucose amylase insert are in a Y 1090 strain from Escherichia coli increased; this is brought to lysis in such a way that LB agar plates around 5,000-10,000 individual plaques occur. Those formed during the phage propagation cycle Proteins are made by simply contacting a nitrozel immobilized on this cellulose membrane (Sambrook). With Help of the monospecific antiserum against glucoamylase are glucoamylase-producing phages by means of a so ECL detection kits (from Amersham) detected.  

1.7 Screening of recombinant phages with chromosomal DNA from Arxula

The screening is carried out according to one in the DD patent 298 821 A5 described method. Modification is single Lich the hybridization temperature, which is 65 ° C. As radioactive labeled DNA probe is used by means of immunolo Chemical screenings isolated cDNA that contained part of the Glu contains Arxula adeninivorans koamylase gene.

With the help of the screenings described under 1.6 and 1.7 it is possible without difficulty to isolate the glucoamylase gene from Ar xula adeninivorans and to incorporate it into a plasmid pBlue script II KS (-) from Escherichia coli (Stratagene) ( FIG. 1) .

2. Characterization of the glucoamylase gene 2.1. Set up a restriction map

The DNA fragment, consisting of two EcoRI-DNA fragments with a total size of 21.3 Kbp (3.3 Kbp and 18 Kbp), on which the glucoamylase gene of Arxula adeninivorans is located, is characterized in more detail by means of restriction cleavages, to create a restriction map of the glucoamylase gene. Figs. 2 and 3 show Restriktionsspaltun gene and a graph of 5 kbp PstI-ApaI DNA fragment.

2.2. Localization of the glucoamylase gene in the genome of Arxula adeninivorans

With the help of the so-called "pulsed field" gel electrophoresis (PFG) it is possible to separate the four chromosomes of Arxula adeninivorans by gel electrophoresis (Kunze 1994, Smith), whose DNA can be transferred to nitrocellulose and radioactively labeled against it Hybridize DNA fragment with the glucoamylase gene (Bignell). The laboratory test selected 52 ° C as the hybridization temperature. It was found that the glucoamylase DNA fragment hybridized with chromosome 2 (molecular weight approx. 3.0 Mbp), which is in agreement with the results previously determined (Büttner 1990), which were achieved by genetic methods ( Fig . 4).

At the same time, gel-electrophoretically separated chromosomes of the wild arxula adeninivorans CSIR 1136, CSIR 1138, CSIR 1147, CSIR 1148, CSIR 1149 and CBS 8244 (Van der Walt) were also included in these investigations in addition to the donor strain Ls3. It could be shown that the glucoamylase gene is located on chromosome 2 in all 7 wild strains examined ( FIG. 5).

2.3. Determination of the transcript size of the glucoamylase gene

The size of the transcript can be determined using "Northern" hybridi be determined. For this (Stoltenburg) the Total RNA and the RNA from the free and from the endo plasmatic reticulum (ER) bound polysomes isolated and against the radiolabelled glucoamylase DNA probe hybridizes. In the subsequent autoradiography can be used for both the total RNA and the RNA ER-linked polysome hybridization signals detected become.

With the help of these hybridization signals it could be shown in the laboratory test that the glucoamylase gene transcribed a 2 kbp mRNA ( FIG. 6). Hybridization signals in the RNA isolated from the free polysomes could not be detected. Since it is known that the glucoamylase is a secretory enzyme which is transported via the endoplasmic reticulum and the Golgi apparatus, it must be translated on ER-linked polysomes, which has been confirmed by means of the "Northern" hybridizations described.

2.4. DNA and amino acid sequence of the glucoamylase gene

DNA sequencing can be carried out using an ALF DNA sequencer (from Pharmacia). The DNA sequence of both the glucoamylase gene and the associated transcription promoter can be found ( FIG. 7).

One "TATA" box each (pos. 47) can be placed in the promoter and identify the "CAAT" box (item -81). The real thing Gene spans from translation start (ATG) up to and including Stop codon 1875 bp. The adjoining terminator shows homologies to consensus sequences Saccharomyces cerevisiae were found (ATAATTATAT).

The amino acid sequence can be determined on the basis of this nucleic acid sequence ( FIG. 8). It is composed of 624 amino acids. When compared with the amino acid sequences of other glucoamylase, it could be demonstrated that the homology to the glucoamylase from Rhizopus oryzae 32.6%, to Saccharomycopsis fibuligera 23.1%, to Aspergillus niger 22.1% and to Saccharomyces diastaticus 15.4% amounts (Ashikari, Itoh, Boel, Yamashita).

One calculates with the computer program "P Signal" (PC Genes from IntelliGenetics, Inc.) used for secretion the amino acid sequence responsible for glucoamylase, see above shows that they are the first 16 N-terminal amino acids includes.

3. Expression of the glucoamylase gene in Escherichia coli

The glucoamylase gene can be expressed by the incorporation of the glucoamylase gene immediately behind the T7 transcription promoter of the plasmid pET-12 (from Novagen, Inc) and subsequent transformation in Escherichia coli. For this purpose, an EcoRV and a BamHI restriction site at the 3 'end of the gene must be synthesized by mutagenesis via a PCR reaction immediately before the start codon (ATG) ( FIG. 9). The plasmid obtained is transformed into the Escherichia coli strain HMS 174 (Novagen, Inc.) and the transformants obtained are characterized (Hanahan).

Glucoamylase activity is demonstrated after a already known method (Büttner 1987). In the laboratory test no glucoamylase was found intracellularly or extracellularly activity measured.

With the aid of polyacrylamide gel electrophoresis, the intra- and extracellular proteins can be separated by gel electrophoresis and then hybridized against antibodies to glucoamylase using a "Western blot" (Towbin) ( FIG. 10). It can be demonstrated that the Escherichia coli transformants synthesize an approximately 70 kDa protein which can be identified as glucoamylase according to the "Western blot" analysis. For example, the glucoamylase gene derived from Arxula adeninivorans can be expressed in Escherichia coli, but without showing glucoamylase activity.

This result can be achieved by incorporating the glucoamylase gene into other plasmids and subsequent transformation into Escherichia coli can be confirmed.

4. Expression of the glucoamylase gene in Saccharomyces cerevisiae 4.1. Expression using arxula glucoamylase promoter adenine virus

In the test of the expression of the glucoamylase gene using glucoamylase's own transcription promoter in the host system of Saccharomyces cerevisiae, the glucoamylase gene with promoter is incorporated into the Escherichia coli-Saccharomyces cerevisiae hybrid plasmid Ycp50 and this plasmid (Ycp50) the method described above (Ito, Dohmen) into a Saccharomyces cerevisiae strain SEY 6210 (α leu2-3 leu2-112 ura3-52 his3-Δ 200 trpl-Δ 901 lys2-801 suc2-Δ 9GAL - S. Emr / USA) ( Fig. 11). The transformants obtained in this way have neither intracellular nor extracellular glucoamylase activity.

4.2. Expression using Saccharomyces GAP promoter cerevisiae

The glucoamylase gene can also be brought to expression in Saccharomyces cerevisiae using a GAP promoter (from Ciba Geigy). For this purpose, the EcoRV-BamHI-DNA fragment described under point 3 with the glucoamylase gene is inserted into the plasmid pJDB2O7-GAP (Kunze 1991) immediately behind the GAP promoter and in front of the PHO5 terminator from Saccharomyces cerevisiae. The plasmid pYGAP-GAA obtained can be transformed into a Saccharomyces cerevisiae strain SEY 6210 and the transformants obtained can be characterized (Ito, Dohmen) ( FIG. 12).

Glucoamylase activity can be detected intracellularly and extracellularly in the Saccharomyces cerevisiae transformant SEY 6210 / pYGAP-GAA. The maximum concentrations of extracellular glucoamylase when using a minimal yeast medium with maltose as carbon source (Tanaka) could be measured in the laboratory test at the end of the log phase (55 hours) with 1558 pkat / ml ( Fig. 13). When using other carbon sources (glucose, starch), lower activities of secretory glucoamylase were detectable (glucose - 980 pkat / ml, starch - 510 pkat / ml). However, it must be taken into account that z. B. the Saccharomyces cerevisiae transformant can grow to starch as a zig carbon source, but here in the stationary growth phase the lowest cell titer it reaches ( Fig. 13).

Comparisons of intra- and extracellular glucoamylase Ab activities show that in Saccharomyces cerevisiae- Transformant SEY 6210 / pYGAP-GAA always more than 97% of the Glucoamylase activities are detectable extracellularly. These values are comparable to those for Arxula adenine virus Ls3 determined (Tab. 1).  

Tab. 1: Share of extra- and intracellular glucoamylase activities (%) of Saccharomyces cerevisiae SEY 6210 / pYGAP-GAA and Arxula adeninivorans Ls3 ¹⁾ .

4.3. Biochemical characterization of the in Saccharomyces cerevisiae synthesized glucoamylase

Analogously to the procedure described in DD patent specification 265 163 A1, the glucoamylase synthesized in the Saccharomyces cervisiae transformant SEY 6210 / pYGAP-GAA can be biochemically characterized ( FIG. 14). Here, however, is now working with unpurified enzyme preparations, ie with the centrifuged culture fluid. The pH optimum is 4.0. When using starch as a substrate (final concentration 1.25%) the temperature optimum is 50 ° C. Under these conditions the glucoamylase has a high thermal stability; after 15 minutes at 50 ° C, 95% of the initial activity is still present. These values are identical to those of the unpurified enzyme preparations of Arxula adeninivorans Ls3 (pH optimum 4.0; temperature optimum 50 ° C).

literature

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Claims (2)

1. A process for the preparation of a thermostable glucoamylase using a yeast strain of the species Sacharomyces cerevisiae, characterized in that a suitable gene for this glucoamylase is isolated from a donor strain of the Arxula adeninivorans type and is expressed in Sacharomyces cerevisiae so that the glucoamylase is expressed with the partial sequence determining the secretion starts from the 16 (N-terminal) amino acids MRQFLALAAAASIAVA, the glucoamylase gene being expressed by means of a glucoamylase promoter. 2. The method according to claim 1, characterized in that the synthesized glucoamylase using the centrifuged Culture liquid kit is characterized unpurified biochemically.