DD272102A1 - PROCESS FOR PREPARING THERMOSTATIC BACILLUS BETA-1,3-1,4-GLUCANASE - Google Patents

Abstract

The invention relates to a process for the preparation of thermostable Bacillus beta-1,3-1,4-glucanase which can be used in the brewing industry for processing large Rohfruchtmengen to hydrolyze the viscosity of the brewing erhoehende glucan compounds; however, this glucanase must retain its efficacy even above 60C. For this purpose, without antibiotics for the expression of a suitable gene in a microorganism of the species Bacillus subtilis or the like. Provided. The gene is thereby obtained from a donor strain of Bacillus macerans and transferred by vector plasmid. The yield has been found to be suitable for industrial use, especially for the brewing industry. Fig. 3

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-IS-M-glucanoso, 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 that additives of antibiotics are needed.

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 crude fruit quantities, for example of barley instead of malt, without difficulties in filtering and refining the brewing owing to insufficiently degraded, viscosity-increasing glucan. Connections to be feared.

It has long been known to reduce the viscosity of brewing wort with the aid of beta-glucanases of mesophilic Bacillus strains, such as Bacillus amyloliquefaciens or Bacillus subtilis / 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temperature sensitivity; they are effective only at an early stage of the mashing process, above 60 ⁰ C, but they are no longer active.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 has the aim of obtaining a thermostable glucanase of the type described in more detail in economic industrial manufacture.

-ί 272 102 Explanation of the essence 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 ⁰ 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 subtilis is not expected in a microorganism of this kind at first. It is known that the production of foreign protein is adversely affected by the instability of recombinant plasmid DNA in gram-positive bacteria and by the protoolytic degradation of the encoded gene product / 4 /. To date, only the production of gene products by means of manipulated bacilli has appeared to be commercially viable, if the gones used were isolated from Bacillus subtilis itself or from a closely related microorganism such as Bacillus amyloliquefaciens / 2 /. On the other hand, the use according to the invention of Bacillus macerans as a donor strain with delivery of the desired yield is quite surprising, since Bacillus macerans must be clearly differentiated from Bacillus subtilis and its related perimeter by taxonomy / 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 restriction 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 a transformation into Escherichia 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 pBR 322 and a DNA fragment with the gene for beta-glucanase about 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 example), the same amount of beta-glucanase is obtained as from a production strain for mesophilic beta-glucanase of Bacillus amyloliquefaciens BE 20/78/9 /. Purified enzyme preparations isolated from the culture filtrate of Bacillus macerans and from Escherichia coli cells with the vector plasmid pBR 12/2 have the same characteristics: at 65 ° C and in the presence of calcium ions, 50% of the enzyme activity is reached 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, microorganism strains are particularly useful for economical industrial production or those themselves related to Bacillus subtilis or Bacillus otnyloliquefaciens, since they secrete the beta-glucanase into the culture medium and eliminate the difficulties which inevitably arise 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; it was successfully used for the production of Bacillus amyloliquefaciens bata-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 in the viscosity of the brewing is sufficient addition of inventively prepared beta-glucanase of less than 50% of the activity of mesophilic beta-glucanases.

embodiments

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, Sill for SaulllA, Pl for PstI, El for EcoRI and BII for BglII) and Fig. 3: the construction of a recombinant plasmid pBMG 1 from the vector plasmid pDB 101 and the plasmid pUC 19/1 (with the abbreviations HpII for Hpall, HpI for Hpal, Hill for HindIII and IR for, inverted repetitive sequence ').

Bet game 1

Cloning of the gene for thermostable beta-glucanase Chromosomal DNA from Bacillus macerans F. 138 is partially digested with a Sau3A restriction enzyme 20

of this DNA are ligated with 5 micrograms of the vector plasmid pBR 322 (linearized with BamH 1) and ligated into donkey ierichia coli DH5-

Cells transformed. There are about 20,000 ampicillin-resistant transformants; Of these, about 20% are seisitive Tetracycline due to the inactivation of the resistance gene by inserted foreign DNA. The presence of gins for the Glucanase is tested by plating the tetracycline-sensitive transformants on lichenin agar. Nai h shifts

with 0.1% solution of Congo red, colonies are found which have a hydrolysis site. Part of the, leta-glucanase-positive clones are isolated and the plasmid DNA is prepared. The DNA of the vector plasmids pBR 11/4 ur.'o pBR IJ 72 is digested with various restriction enzymes, and the resulting DNA fragments are analyzed by agarose-Qol electrophoresis.

Both vector plasmids have a homologous region with the restriction sites Pst 1, EcoR 5 and Hpa 1. First, a subcloning of the EcoR 1 -Bgl 2 fragment of pBR 12/2 is performed in a vector plasmid ρ DC19 / 10 / du

a resulting clone pUC 19/1 is obtained, which has a union-in-hydrolyzing activity, ti'i by a? 600-

Base pair DNA fragment is encoded. Furthermore, if various members of this DNC fragment are further subcloned, then

In conclusion , the beta-qiukanase gene is located on a 1,800 base pair Pst 1 -Bg112 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 in 1 minute

Intervals, 2 microliter aliquots are removed from the batch. Each sample is mixed at 70 ^° 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 15 microliters of S1 buffer and incubated for 10 minutes at room temperature. Subsequently, the

Reaction stopped by addition of 5 microliter O, 8M Tris / HCl buffer pH 8. The deleted vector plasmids become

recircularized and transformed into Escherichia coli. Apart from beta-glucanase negative clones, positive results are also found with a DNA insert of only 850 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 sites Pvuli (Bp.34), Hinfl (Bp.92 and 146), Hpall (3p.130), Acci

(Bp 213 and 420), Eco RV (bp 376), Mbol (bp 505), Haell (bp 701) and Dral (bp 789). Subcloning with different

Restriction fragments show that the genetic information for beta-glucanase is between base pairs 34 and 789

That is, 755 nucleotides are sufficient for the expression of Bacillus macerans beta-glucanase {Fig. 1).

After SDS polyacrylamide gel electrophoresis of the purified beta-glucanase, the molecular weight thereof increases

about 23kD. Antibodies produced by purified Bacillus amyloliquefaciens beta-glucanase show little or no cross-reaction with the Bacillus macerans enzyme as well as beta-glucanase antibodies

Bacillus macerans are only very weakly reactive with the Bacillus amyloliquefaciens enzyme. The temperature optimum of the beta-glucanase of Bacillus macerans is at 60 to 65 ⁰ C, about 10grd above that

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 gene for the beta-glucanase from Bacillus macerans may be used (FIG. 2). 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 ultrasound treatment becomes in the usual way with Lichonin as substrate certainly.

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 Bac. macerans E138 0.49 Bac. amylotiquefa- - 19.3 ciens BE 20/78 Escher, coli DH5 pBR322 0 Escher, coli DH5 pBR11 / 4 28.7 Escher, coli DH5 PBR12 / 2 20.0 Escher, coli DH5 pUC19 / 1 11.3

For comparison, the activity is indicated in the culture fluid of the donor strain E138 of Bacillus macerans and of an industrially used strain BE 20/78 of Bacillus amyloliquefaciens / 2 / which is obtained under the same conditions. It can be seen that strains with recombinant vector plasmids 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

As a vector plasmid for the expressions in Bacillus the streptococcal plasmid pDB 101 is used iFig.3). 0.5 micrograms of plasmid DNA are cut with the restriction enzymes EcoRI and Hpall. A DNA fragment with the Bacillus macorans 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 ügationsgemisch - the recombinant plasmid pBMG 1 - transformed into competent Bacillus subtilis cells, which are prepared after SPIZIZEN / 11 /. The host strain used is a glucanase-deficient strain BQ314 / 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 werrian used for the preparation of 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 Nänr agar so that up to 200 colonies grow on a Petri dish. Then it is over-stamped 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 are inoculated in 5CmI glucose broth and shaken at 37 ⁰ C for 24 h. It is found that the activity for beta-glucanase in the culture medium is about 3.0 U / ml h, whether or not erythromycin is added thereto. However, the activity is about 6 times higher than in the donor strain E318 from Bacillus macerans.

literature

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

2 DD-PS 226012.

3 Borriss, R., i-.Zemek, J., Zbl.Bakt.Il Abt., 136 (1981), pp. 63-69.

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

5 Sergey's Miinual 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), p.431-442.

-90 -80 -70 -60 -50 -40 -30 -20 -IO

til ItI lit

AGC TCG GAT ATA CTA TAA TTA CCC AGG TAA AAT ATT CCA ACA CCG TGG CTC CAT AAC TTC GTT CAT ATT TAA AAT CAT TIT GGA OVER GCA I IO 20 30 40 50 60 70 80

I I I ill I i

TTA TGC ATT ATG AAA AAG AAG TCC TGT TTT ACA CTG CTR ACC ACfi TTT GCG TTT TCT TTG ATI TTT TCT GTA AGC GCT Ui »GCG 6BG AGT Het Lys Lys Lys Ser Cys fhe Thr Leu VaI Ibt Your Phe Ala Phe Ser Leu Ue Phe Ser VaI Ser Ala Leu Ala GIy Ser

90 100 HO 120 I30 HO ISO 160 170

6TG TTC TGG 6AA CCA TTA AGT ACT TTT AAT CCG AGT AC6 GAA AAG 6CA 6TG 6TA TCC AAT 666 GGG TGT TCA ATf GCA CAT GCT GCC AAC

VaI Phe Trp GIu Pro Leu Ser Tyr Phe Asn Pro Ser Thr GIu Lys Ala VaI VaI Ser Asn G! Y Gly Cys Ser ile Ala His Ala Ala Aso

180 190 200 210 220 230 240 250 260

I lit lit Ii

AAT GTT AAT TTT ACG AAT GAT GfA AAG CTC AAG CTG GGC TTA ACG ABT TCT GCG TAC AAC AAA TTT FaC TGC GCG GAG TAC CGA TCA ACG Asn VaI Asn Phe Thr Asn Asp GIy 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

I III ill II

AAC ATT TAC GGA TAC 6GC CTG TAC GAG GTC AGT ATG AA6 CCA GCC AftA AAT ACA GGA ATT oTC TCA TCC TTT TTC ACG ΪΑΤ ACA GGA CCT Asr. He Tyr GIy Tyr GIy Leu Tyr Giu VaI Ser Hat Lys Pro Ala lys Asn Thr GIy II «VaI Ser Ser Phe Phe Thr Ty Thr Gl / Pro

360 370 380 390 400 410 420 430 440

• I I I I I I I

GCT CAT GGC ACA CAA T66 GAT 6AA ATA MT ATC GAA TTT CTA GGA AAA 6AC AHb ACA AAA GTC CAG TTT AAC ACT TAT ACC AAT GGG GfT Ala His GIy Thr Gin Trp (Up Gulu Asp He Giu Phe Leu Giy Lys Asp Her Thr Lys Vai Gin Phe Asn Tyr Tyr Thr Asn Giy Vai

450 460 470 480 'WO 500 510 520 530

I III III Il

G6C G6T CAT GAA AAG GTT ATC TCT CTT GGC TTT GAT GCA TCA AAP GSC TTC CAT ACC TAT 6CT TTC CAT TGG CAB CCA GGG ATT AAA GIy GIy His GIu Lys VaI He Ser Leu GIy Phe Asp Ala Ser i. ^ GIy Phe His Thr Tyr Ala Phe Asp Tip Gin Pro GIy Tyr! Le Lys

540 550 560 570 580 590 600 610 620

Ilit III Il

T66 TAT 6TA 6AC 66T GTT TTG AAA CAT ACC GCC ACf. riCG mAT ATT CC6 AGT ACG CCA GGC ΑΛΑ ATT ATG ATG AAT CTA TGB NC GGA ACC Trp Tyr VaI Asp GIy VaI Leu Lys Hio Thr Ala τ ;, _Γ AIa Asn He Pro Ser Thr Pro GIy Lys He ilet HeI Asn Lau Γ; ρ Asn GIy Thr

630 640 · 650 660 670 680 690 700 710

I II til Il

GGA GTG GAT GAC T6G TTA 66! TCT TAT AAT GGA GC6 AAT CCG TTG TAC GCT GAA TAT GAC TGG GTA AAA IAI ACG AGC AAT TAA TAT GAT GIy VaI Asp Asp Trp L ° u GIy Ser Tyr Asn GIy Ala Asn Pro Leu Tyr Ala GIu Tyr Asp Tip VaI Lys Tyr Thr Ser Asn

720 7JO 740 750

'til

TGC AGC TGG GCA TGA GCi TTT TAG TCC ACT CCA GGC ATG C

Claims (6)

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 brought to expression using a microorganism strain which is close to or belongs to the species Bacillus subtilis. 2. The method according to claim 1, characterized in that the gene for the beta-glucanase is obtained from a Donar strain E138 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 an Escherichia cou plasmid pBR322 is used as the vector. 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.