DD272102B5 - Process for the preparation of thermostable Bacillus beta-1,3-1,4-glucanase - Google Patents

Description

For this 3 pages drawings

Field of application of the invention

The invention relates to a process for the preparation of thermostable Bacillus beta-i ^ -M-glucanase, wherein a suitable gene for this glucanase is isolated and expressed by means of a vector in a microorganism strain and further wherein a stable expression of this gene is ensured without the use of antibiotics.

Characteristic of the known state of the art

Glucanases are used in the production of various foods, foods and feeds, as well as adjuvants in biological research, where the hydrolytic cleavage of the beta-glycosidic linkages of beta-1,3-1,4-glucans is intended; especially in the brewing industry, the use of such plant glucan-hydrolyzing enzymes makes it possible to process larger quantities of raw fruits, for example barley instead of malt, without difficulties in filtering and refining the brews due to insufficiently degraded, viscosity-increasing glucan compounds to be feared.

It has long been known, the viscosity of the brewer's wort with the aid of beta-glucanases mesophilic Bacillus strains such as Bacillus amyloliquefaciens or Bacillus subtilis, reduce / 1 /. Also, the use of genetically engineered strains containing the beta-glucanase gene of Bacillus amyloliquefaciens in amplified form on a vector plasmid is already envisaged for improving the yield of mesophilic glucanase according to an earlier proposal of the same Applicant / 2 /.

The disadvantage of these previously known glucanases is their temperature sensitivity; they are effective only at an early stage of the mashing process, above 60 ⁰ C, but they are no longer active.

It is also known that Bacillus macerans produces a beta-glucanase which, in contrast, still exhibits its activity above the stated temperature / 3 /; but because of its low porosity such a microorganism is completely unsuitable for economic enzyme production.

Object of the invention

The invention aims to obtain a thermostable glucanase of the type described in more detail in economic industrial production.

-2- 272 102 Explanation of the nature of the invention

The invention is therefore based on the object to avoid the disadvantages described and to produce genetically engineered glucanase, which combines a thermostable behavior above 60 ° C with a productive manufacturing process. According to the invention, the object is achieved in that the gene is obtained from a donor strain of Bacillus macerans and brought to expression using a microorganism strain which is close to or belongs to the species Bacillus subtilis.

The stable expression of a gene that is not located in the environment of Bacillus subtifis is initially not expected in a microorganism of this type. As is known, the production of foreign protein is adversely affected by the instability of recombinant plasmid DNA in gram-positive bacteria and by the proteolytic degradation of the encoded gene product / 4 /. So far, only the production of gene products using manipulated bacilli appeared to be commercially viable, if the genes used were isolated from Bacillus subtilis itself or a closely related microorganism such as Bacillus amyloliquefaciens / 2 /. On the other hand, the use according to the invention of Bacillus macerans as donor strain with delivery of the desired yield is quite surprising, since Bacillus macerans must be clearly distinguished taxonomically from Bacillus subtilis and its related perimeter / 5 /.

It is particularly advantageous if the gene for the beta-glucanase is obtained from a donor strain E138 from Bacillus macerans. In a known manner, it is expedient that a vector plasmid, for example an Escherichia coli plasmid pBR 322/6 / or a streptococcal plasmid pDB 101/7 /, is used as the vector. Finally, it is particularly advantageous if the expression of the gene by means of an aqueous solution of Congo red, which is flooded with the lichenin-containing agar, is detected. The hydrolysis zones generated by recombinant, glucan-forming clones allow rapid identification of the clones sought. By this method isolated transformants of Escherichia coli containing Bacillus DNA in a size of 5000 base pairs.

By subcloning reaction fragments and carrying out unidirectional delegations with the enzyme exonuclease III, the foreign DNA surrounding the gene for the beta-glucanase can be largely removed in a known manner / 8 /. After transformation into Eecherichia coli, it can be shown that a DNA fragment of 900 base pairs still expresses beta-glucanase and consequently must contain the complete gene for the beta-glucanase. The level of expression of beta-glucanase is quite different for different vectors; strikingly obtained when using the Escherichia coli plasmid pBR322 and a DNA fragment with the gene for beta-glucanase an approximately 40 times higher enzyme activity, as it is achieved with the donor strain E138. By transformation of an Escherichia coli strain with a recombinant vector plasmid pBR 12/2 (see embodiment) is obtained about the same amount of beta-glucanase as from a production strain for mesophilic beta-glucanase of Bacillus amyloliquefaciens BE20 / 78/9 /. Purified enzyme preparations, which are isolated from the KuKurfiltrat of Bacillus macerans and from Escherichia coli cells with the vector plasmid pBR 12/2, have the same properties: at 65 ⁰ C and in the presence of calcium ions 50% of the enzyme activity is achieved after 40 minutes of incubation ,

Surprisingly, the expression of the gene for beta-glucanase from a donor strain of Bacillus macerans is particularly high in a strain of Bacillus subtilis and is about 30 times higher than in the donor strain E138 of Bacillus macerans itself. Therefore, for economical industrial production, microorganism strains particularly suitable, which are related to Bacillus subtilis or Bacillus amyloliquefaciens or these themselves, because they secrete the beta-glucanase in the culture medium and eliminate the inevitable difficulties with the digestion of the cells in practice.

Special concerns in the use of genetically engineered microorganism strains prepares their high instability. In order to obtain a recombinant vector plasmid in a cell, the addition of antibiotics to the culture medium is actually essential. It is already known that the streptococcal plasmid pDB101 is extremely stable in Bacillus subtilis and in similar microorganism strains; thus, it has been successfully used for the production of Bacillus amyloliquefaciens beta-glucanase in industrial microorganism strains / 2 /. However, the sequence of Bacillus subtilis beta-glucanase and Bacillus amyloliquefaciens beta-glucanase are largely homologous / 8 /, while the sequence of Bacillus macerans beta-glucanase is very distinct. It is therefore quite surprising that a DNA fragment with the gene of the beta-glucanase from Bacillus macerans also-without selection pressure by an antibiotic-is kept stable in the culture medium.

For a sufficient reduction of the viscosity of the braumaic addition of beta-glucanase produced according to the invention of less than 50% of the activity of mesophilic beta-glucanases is sufficient.

Ausfuhrungsbefspiele

In the following three embodiments, the invention will be explained in more detail with reference to the drawing. Show it

Fig. 1: a DNA fragment cloned from Bacillus macerans containing the gene for beta-glucanase (abbreviated: bgl), Fig. 2: the structure of the vector plasmids pBR 11/4 and pBR 12/2 and the construction of a plasmid pUC 19/1 (with the

Abbreviations Bl for BamHI, SHI for SflulllA, Pl for PstI, El for EcoRI and BII for BglII) and Fig. 3: the construction of a recombinant plasmid pBMG1 from the vector plasmid pDB 101 and the plasmid pUC 19/1 (with the abbreviations Hptt for HpaM , HpI for Hpal, HW for Hindlil and И for, inverted repetitive sequence '}.

BeispieM

Cloning of the gene for thermostable beta-glucanase

Chromoeomal DNA from Bacillus macerans E138 is partially digested with a Restrulctionsenzyme Sau3A. Twenty micrograms of this DNA are ligated with Б micrograms of the vector plasmid pBR 322 (linearized with BamH 1) and transformed into Escherichia coli DHS cells. There are about 20,000 ampicillin-resistant transformants; of these, about 20% are sensitive to tetracycline due to the inactivation of the resistance gene by inserted foreign DNA. The presence of the beta-glucanase gene is assessed by plating the tetracycline-sensitive transformants on lichenin agar. After overcoating with 0.1% solution of Congo red, colonies are found which have a hydrolysis farm. Part of the beta-glucanase-positive clones is isolated and the plasmid DNA is prepared. The DNA of the vector plasmids pBR 11/4 and pBR 12/2 is digested with various restriction enzymes, and the obtained DNA fragments are analyzed by agarose gel electrophoresis.

Both vector plasmids have a homologous region with the restriction sites Pst1, EcoR5 and Hpa1.

First, a subcloning of the EcoR 1 -BgI 2 fragment of pBR 12/2 is carried out in a vector plasmid pUC 19/10 / in which a resulting clone pUC 19/1 is obtained, which has a union-in hydrolyzing activity by a 2600 base pair DNA fragment is encoded. Further, when various parts of this DNA fragment are further subcloned, it is revealed that the gene for beta-glucanase is located on a 1800 base pair Pst 1-Bgl 12 portion of the DNA fragment.

A unidirectional deletion of the thus obtained vector plasmid pUC 19 / A is carried out in the following manner.

4 micrograms of plasmid DNA are digested with the restriction enzymes Kpn 1 and Sa11. In a total volume of 10 micrograms, the plasmid DNA is incubated with 1 microliter of exonuclease III at 30 ^° C. for up to 5 minutes. Each 1 minute intervals, 2 microliter aliquots are removed from the batch. Each sample is mixed at 70 ⁰ C with 3 microliters of water and then stored on ice. To remove the single-stranded DNA, 3 microliters of S 1 nuclease are added to each sample along with 1S microliter of S1 buffer and incubated for 10 minutes at room temperature. Subsequently, the reaction is stopped by addition of S microliters of 0.8 M Tris / HCl buffer pH 8. The deleted vector plasmids are recircularized and transformed into Escherichia coli. Except for beta-glucanase negative clones positive for it are found with a DNA insert of only 8SO base pairs (see Appendix 1). Thus, the gene for the beta-glucanase can be located within the associated DNA fragment of the vector plasmid pUC 19 / A.

The DNA fragment has restriction enzyme sites Pvull (Bp.34), Hinfl (Bp.92 and 146), Hpall (bp.130), Accl (bp.213 and 420), EcoRV (bp.376), Mbol (bp. 505), Haell (bp 701) and Dral (bp 789). Subcloning with various restriction fragments indicates that the genetic information for the beta-glucanase is located between base pairs 34 and 789, that is, 755 nucleotides are sufficient for the expression of Bacillus macerans beta-glucanase (Figure 1).

After SDS polyacrylamide gel electrophoresis of the purified beta-glucanase, its molecular weight is about 23 kD. Antibodies prepared by purified Bacillus amyloliquefaciens beta-glucanase show little or no cross-reaction with the Bacillus macerans enzyme, as well as Bacillus macerans beta-glucanase antibodies, with only very little reactivity with the Bacillus amyloliquefaciens enzyme are.

The temperature optimum of the beta-glucanase of Bacillus macerans is 60 to 65 ⁰ C, and thus about lOgrd above the mesophilic beta-glucanases of Bacillus subtilis or Bacillus amyloliquefaciens.

Example 2

Expression of the thermostable beta-glucanase gene in Escherichia coli

To check the rate of formation of beta-glucanase, various plasmids containing the Bacillus macerans beta-glucanase gene (Figure 2) can be transformed into Escherichia coli; the transformed strains are shaken for 24 hours in a glucose-peptone medium - culture volume: 50 ml at 37 ° C. and 220 min ^-1 . The activity of the beta-glucanase in the cell extracts prepared by ultrasonication is usually determined using lichenin as substrate ,

The result is recorded in Table 1. An enzyme unit is defined as that amount of enzyme which causes a release of reducing substance equivalent to one micromole of glucose.

Table 1: Comparison of the formation of beta-glucanase with various donor strains and vector plasmids

donor strain vector plasmid beta-glucanase in E / ml h Вас. macerans E138 _ 0.49 Вас. amylotiquefa- - 19.3 ciens BE 20/78 Escher, coli DH5 pBR322 0 Escher, coli DH5 pBRH / 4 28.7 Escher, coii DH 5 PBR12 / 2 20.0 Escher, coli DH5 PUC19 / 1 11.3

For comparison, the activity in the culture fluid of the donor strain E138 of Bacillus macerans and of an industrial use specific strain BE 20/78 of Bacillus amyloliquefaciens / 2 / is given, which is obtained under the same conditions. It can be seen that recombinant vector plasmid strains achieve up to 40-fold increased production of beta-glucanase compared to the donor strain. The quantitative level of activity produced by the transformed strains of Escherichia coli is on the order of magnitude of industrial bacillus production strains.

Example 3

Expression of the thermostable beta-glucanase gene in Bacillus subtilis

The vector plasmid used for expression in Bacillus is the streptococcal plasmid pDB101 (FIG. 3). 0.5 micrograms of plasmid DNA are cut with the restriction enzymes EcoRI and Hpall. A DNA fragment containing the Bacillus macerans beta-glucanase gene is obtained by digesting 0.5 micrograms of the pUC 19/1 plasmid with the restriction enzymes EcoRI and Hpal. The DNA fragment is ligated and the ligation mixture - the recombinant plasmid pBMG 1 - transformed into competent Bacillus subtilis cells prepared according to SPIZIZEN / 11 /. The host strain used is a glucanase-deficient strain BG 314/12 /. With 0.1 microgram of the ligation mixture, about 100 transformants are obtained, some of which are always capable of producing beta-glucanase. The isolates are used to prepare plasmid DNA and characterized by retransformation and restriction enzyme analysis. To check the stability of the recovered vector plasmids, strain BG 314, which has been transformed with one of the vector plasmids, is cultivated for 30 generations in erythromycin-free culture medium (glucose nutrient broth) without selection pressure. Subsequently, the culture is plated in a more suitable dilution on nutrient agar so that up to 200 colonies grow on a petri dish. Then it is overstamped on antibiotic-containing lichenin agar-5 micrograms / ml erythromycin and nutrient agar. The same number of colonies grow on both culture media, all forming on lichenin-agar lysis zones. After 30 generations of growth in antibiotic-free culture medium, 0.5 ml in 50 ml of glucose broth are inoculated and shaken at 37 ⁶ C for 24 h. It is found that the activity for beta-glucanase in the culture medium is about 3.0 U / ml · hr, whether or not erythromycin is added thereto. However, the activity is about 6 times higher than in the donor strain E 318 from Bacillus macerans.

literature

1 Borries, R., u. Schröder, K.-L, Zbl.Bakt. II. Dept., 136 (1981), pp. 324-329.

2 DD-PS 226012.

3 Borriss, R., u. Zemek, J., Zbl.Bakt. II. Dept., 136 <1981), pp. 63-69.

4 Borriss, R., Biotechnology Weinheim, 7a, pp. 35-62.

5 Bergey's Manual of Determinative Bacteriology, Baltimore 1974.

6 Bolivar, F., et al., Gene 2 (1977), pp. 95-113.

7 Behnke, D., u. Ferretti, J.F., Plasmid 4 (1980), pp. 130-138.

8 Hofemeister, J., et al., Gene 49 (1986), pp. 177-187.

9 Borriss, R., Bäumlein, H., u. Hofemeister, J., Appl. Microbiol. Biotechnol. 22 (1985), pp. 63-71.

10 Messing, J., Methods Enzymol. 101 (1983), p.20ff.

11 Anagnostopoulos, C / Spizizen, J., J. Bacteriol. 81 (1961), pp. 741-746.

12 Borris, R., et al., J. Gen. Microbiol. 132 (1986), pp. 431-442.

Attachment 1

-90 -80 -70 -АО -50 -40 -30 -20 -10 t * t * «« «t * A6C TCe 8AT ATA СТА TAA TTA CCC А66 TAA AAT ATT CCA АСА CCG Т66 CTC CAT AAC TTC GTT CAT ATT TAA AAT CAT TTT 66A GGT 6CA

1 10 20 30 40 50 60 70 80 t »t« «» t

TTA T6C ATT ATG AAA AAS AA6 TCC T6T TTT АСА CTG 6Т6 ACC АСА TTT 6C6 TTT TCT ТТБ ATT TTT TCT 6TA ABC GCT TTA BCB 6S6 ABT Het Lys Lys Lys Ser Cys Phe Thr Leu VaI Thr Thr Phe Ala Phe Ser Leu He Phe Ser VaI Ser Ala Leu AU Sly Ser

90 100 110 120 130 140 ISO 160 170

6TB TTC TG6 BAA CCA TTA AGT ACT TTT AAT CCG AGT AC6 GAA AAG 6CA GTG 6TA TCC AAT B6B 666 T6T TCA ATT 6CA CAT 6CT 6CC AAC VaI Phe Trp GIu Pro Leu Ser Tyr Phe Asn Pro Ser Thr GIu Lys Ala VaI VaI Ser Asn GIy Gly Cys Ser Ala His Ala Ala Al Asn

180 190 200 210 220 230 240 250 260

* * * Ι AAT 6TT AAT TTT ACG AAT 6AT 66A AAG CTC AA6 CTG 66C TTA AC6 ΑβΤ TCT 6CG TAC AAC AAA TTT 6AC T6C 6C6 6A6 TAC C6A TCA AC6 Asn VaI Asn Phe Thr Asn Asp Gily Lys Leu Lys Leu GIy Leu Thr Ser Ser Ala Tyr Asn Lys Phe Asp Cys Ala GIu Tyr Arg Ser Thr

270 280 290 300 310 320 330 340 350

* »II» I »It

AAC ATT TAC 66A TAC GGC CTG TAC 6A6 6TC A6T ATG AAG CCA GCC AAA AAT ACA 66A ATT GTC TCA TCC TTT TTC AC6 ACT АСА 66А CCT Asn lie Tyr GIy Tyr GIy Leu Tyr 6iu VaI Ser Het Lys Pro Ala Lys Asn Thr GIy He VaI Ser Ser Phe Phr Thr Tyr Thr GIy Pro

360 370 380 390 400 410 420 430 440

6CT CAT 66C ACA CAA T66 6AT 6AA ATA 6AT ATC 6AA TTT GSA AAA 6AC ACG AAA GTC CAG TTT AAC ACT ACC AAT 666 GTT Ala His GIy Thr Gin Trp Asp SIu He Asp He 6lu Phe Leu 6ly Lys Asp Thr Thr Lys VaI 61 η Phe Asn Tyr Tyr Thr Asn Giy VaI

450 460 470 480 490 500 510 520 530

("Tfl Il

66C 66T-CAT 6AA AA6 6TT ATC TCT CTT 66C TTT 6AT 6CA TCA AAG 66C TTC CAT ACC TAT 6CT TTC 6AT T66 CA6 CCA 666 TAT ATT AAA GIy GIy His GIu Lys VaI lie Ser Leu GIy Phe Asp Ala Ser Lys GIy Phe His Thr Tyr Ala Phe Asp Trp Gin Pro GIy Tyr He Lys

540 550 560 570 580 590 600 610 620

T6G TAT 6TA 6AC G6T 6TT TT6 AAA CAT ACC 6CC ACC 6C6 AAT ATT CCG A6T AC6 CCA 66C AAA ATT AT6 AT6 AAT STA T66 AAC 66A ACC Trp Tyr VaI Asp GIy VaI Leu Lys His Tbr Ala Thr Ala Asn lie Pro Ser Thr Pro GIy Lys lie Het Het Asn Leu Trp Asn GIy Thr

630 640 650 660 670 680 690 700 710

I III II »» I

66A 6T6 6AT 6AC T66 TTA G6T TCT ACT AAT 66A 6C6 AAT CC6 TTG TAC GCT 6AA ACT 6AC T66 6TA AAA ACT ACG A6C AAT TAA ACT 6AT GIy VaI Asp Asp Trp Leu GIy Ser Tyr Asn GIy Ala Asn Pro Leu Tyr Ala GIu Туг Asp Trp VaI Lys Туг Thr Ser Asn -

720 730 740 750 ι ι ι T6C ABC Τ66 BCA Τ6Α 6CT TTT TAB TCC ACT CCA 66C AT6 С

Claims (6)

claims: 1. A process for the preparation of thermostable Bacillus beta-1,3-1,4-glucanase, wherein - isolated a suitable gene for this glucanase and - Is expressed by a vector in a microorganism strain and wherein further - a stable expression of this gene is ensured without the use of antibiotics additives, characterized in that - the gene obtained from a Donar strain of Bacillus macerans and is expressed using a Bacillus subtilis or Bacillus amyloliquefaciens strain. 2. The method according to claim 1, characterized in that the gene for the beta-glucanase is obtained from a Donar strain E 138 of Bacillus macerans. 3. The method according to claim 1 and 2, characterized in that a vector plasmid is used as a vector. 4. The method according to claim 1 to 3, characterized in that the vector is an Escherichia coli plasmid pBR 322 is used. 5. The method according to claim 1 to 3, characterized in that a streptococcal plasmid pDB 101 is used as the vector. 6. The method according to claim 1 to 4, characterized in that the expression of the gene by means of an aqueous solution of Congo red, which is flooded with the lichenin-containing agar, is detected.