KR100543470B1 - Recombinant Bacillus expressing cyclodextrinase and method for preparation thereof - Google Patents

Abstract

The present invention relates to a recombinant Bacillus strain expressing cyclodextrinase and a method for producing the same, the present invention has the effect of providing a Bacillus polyfermenticus SCD as a host strain for expression of foreign genes. In addition, the present invention has an excellent effect of providing a Bacillus polypermanticus SCD transformant expressing cyclodextrinase. Therefore, using the foreign gene expression system of the present invention, it is possible to mass-produce recombinant proteins even at low production costs, and there is an excellent effect of obtaining recombinant proteins having excellent purity and homogeneity.

Cyclodextrinase, Bacillus polyfermentus SCD, Bacillus subtilis BD 170, plasmid

Description

Recombinant Bacillus expressing cyclodextrinase and method for preparation of the recombinant bacillus strain expressing cyclodextrinase

Figure 1 shows the results of the measurement of the minimum bacterial concentration (MBC) for Bacillus polyfermentus SCD in LB agar medium containing kanamycin.

Figure 2 is a gel photograph showing the results of identifying the presence of plasmid in Bacillus polyfermentus SCD.

Figure 3 shows colonies of transformants of Bacillus polypermanticus SCD containing pRB373 and pKY1 in LB agar medium containing kanamycin.

Figure 4 is a gel photograph showing the expression pattern of pRB373 and pXyl373 in Bacillus subtilis BD 170 and Bacillus polyfermentus SCD.

5 is a gel photograph showing the expression pattern of pKY1 in Bacillus polypermanticus SCD.

Figure 6 shows the structures of the Bacillus expression plasmid and cyclodextrinase expression plasmid of the present invention.

FIG. 7 is a gel photograph showing the expression patterns of pKYCD1 and pKYCD2 in Bacillus subtilis BD 170 and Bacillus polyfermentus SCD. FIG.

8 is a transparent ring formation test results according to the I ₂ -KI staining method showing the cyclodextrinase activity of the Bacillus transformant of the present invention.

The present invention relates to a recombinant Bacillus strain expressing cyclodextrinase and a method for producing the same. More specifically, the present invention relates to a Bacillus strain transformed with a recombinant plasmid containing a cyclodextranase coding gene derived from Bifidobacterium adolescents Int 57 and a method for producing the same.

Probiotics are living microbial foods that are good for health and are the healthy human intestinal flora that is usually sold as a commodity. Generally, Bifidobacterium (Bifidobacterium), Lactobacillus genus (Lactobacillus), Enterococcus genus (Enterococcus), Clostridium subtree Com (Clostridium butricum), Bacillus subtilis (Bacillus subtilis) and Bacillus poly flops mentee Bacillus polyfermenticus and the like have been used as probiotics in humans. These probiotic bacteria can normalize intestinal flora and gastrointestinal dysfunction.

Starch, on the other hand, is one of the richest storage polysaccharides in nature. In conclusion, in many organisms starch enzymes play an important role in obtaining energy from carbon sources. In addition, starch dehydrogenase is very useful in many industrial processes, primarily the food, textile and detergent industries. Cyclomaltodextrins (CDs) belong to a cyclic, non-reducing D-glucose oligosaccharide of α-1 → 4 bonds obtained in starch by cyclomaltodextrin glucanotransferase. CDases, such as cyclodextrinases (CDases), cyclomaltodextrinases, and cyclomaltodextrin hydrolases, are enzymes that hydrolyze cyclodextrins faster than starch. CDs hydrolase is an enzyme belonging to 13 glycoside hydrolase families, and unlike general α-amylase that only hydrolyzes starch, it can hydrolyze CDs and water-soluble starch.

In addition, Bifidobacterium (Bifidobacterium) is a non-pathogenic, as well as have a beneficial effect on the host's health, Gram-positive and anaerobic bacteria and so inhabit the Minister of humans and animals, and Oregon referred to as "probiotics". Among them, species such as B. adolescentis are found in human defecation, while species such as B. thermophilum are found in organisms except animals.

Bacillus (Bacillus), it contains the major industrial species are usually used as host organism in the industry. In particular, Bacillus subtilis has been used as a useful host for protein production because of its high secretion capacity, well-known genetic systems, nonpathogenicity and extensive fermentation techniques. In other words, Bacillus subtilis is a very attractive host for foreign protein production. Although the secretion mechanism of Bacillus subtilis is not clearly known, many researchers have succeeded in using signal peptides from Bacillus species to secrete foreign proteins from protozoa. In addition, B. polyfermenticus SCD ( B. polyfermenticus SCD) has been used as a probiotic bacterium in addition to Bifidobacterium in Korea and Japan, which prevents Bacillus polypermenticus SCD from fermenting dangerous to human intestines. Not only can you do it, you can suppress it, and it creates a beneficial effect on your own health. The microorganism is commonly referred to as a "bisroot" strain, a bacterium that forms endogenous spores, such as staphylococcus aureus , Clostridium perfringens , Bacillus cereus , It produces a bacteriocin known as polyfermentincin SCD that inactivates several strains, such as Bacillus pumilis , Bacillus subtilis , and Listeria monocytogenes . Several studies have suggested that Bisruit strains are similar to Bacillus subtilis in morphological biochemical characterization. However, bisroot strains differ radically from Bacillus subtilis in that they can metabolize lactose and produce higher amounts of acetic acid and lactic acid from glucose and lactose, respectively, compared to Bacillus subtilis. Although special information has been accumulated regarding the microbial ecology, taxonomy and microbial physiology of Bacillus spp. , Relatively little has been studied about the genetic analysis of B. polyfermenticus SCD. To date, there have been no reports of cloning from Bacillus polyperientus SCD. This is because there was no proper gene delivery system.

Accordingly, an object of the present invention is to investigate the possibility of the probiotic Bacillus polyperientus SCD as a host for the expression of foreign genes.

Another object of the present invention is to introduce a plasmid vector containing a cyclodextrinase coding gene derived from Bifidobacterium adolescentis Int 57 into a Bacillus strain to prepare a transformant.

Another object of the present invention is to mass-produce cyclodextrinases from the transformants.

The object of the present invention is to identify the potential as an expression host strain of Bacillus polyfermentus SCD, and to isolate the cyclodextrinase coding gene from the chromosomal DNA of Bifidobacterium adolescentes Int 57 and then recombinant plasmid comprising the same. Was achieved by introducing the plasmid into a Bacillus strain, transforming it, and expressing cyclodextrinase therefrom.

Hereinafter, the present invention will be described in more detail.

The present invention is to identify the potential as an expression host strain of Bacillus polypermanticus SCD; Separating the cyclodextrinase coding gene from the chromosomal DNA of Bifidobacterium adolescentes Int 57; Preparing a recombinant plasmid vector comprising the gene; Preparing a recombinant Bacillus strain comprising the vector; Expressing cyclodextrinase from the recombinant strain.

Strains and plasmids used in the present invention are shown in Table 1.

Strain genotype references Bifidobacterium adolescentes Int 57 Wild type Park et al., J. Microbiol. Biotechnol. 11, 106-111 Bacillus subtilis BD 170 BGSC c1a42: trp C2, thr -5 Vyas et al. Enzyme Microbial. Technol. 16, 240-246 Bacillus polyfermentus SCD Wild type Park et al., J. Microbial. Biotechnol . 12, 657-663 Plasmid Kinds p8A1 pUC18-amyR2 (including promoter and signal sequence of B. subtilis var. natto ), Ammpr Yuuki et al., J. Biochem. 98, 1147-1156 pRB373 B. subtilis / E.coli shuttle vector, Ampr, bler, neor Lam et al., J. Biotechnol. 63, 167-177 pKY1 pRB373- (includes promoter and signal sequence from B. subtilis var. natto ), Ampr, neor pBB702K pUC19-CD1, Ampr, Lacz ' pKYCD1 pKY1-CD1, Ampr, neor, bler pKYCD2 pKY1-CD2, Ampr, neor, bler

Hereinafter, the specific configuration of the present invention will be described through Examples, but the scope of the present invention is not limited to these Examples.

EXAMPLE

Example 1 Investigation of the Possibility of Bacillus polyperientus SCD as a Foreign Gene Expression Host in an Exogenous Gene Expression System

The feasibility of B. polyfermenticus SCD as an expression host for foreign genes in foreign gene expression systems was investigated through the following procedure.

Generally, specific antibiotics are used to screen for recombinant microorganisms. Therefore, the minimum bactericidal concentration (MBC) that can completely inhibit the growth of wild-type host strain under these antibiotics was measured to determine the concentration of antibiotics required to select recombinant microorganisms. In the present invention, kanamycin was used as an antibiotic.

To determine the minimum bacterial concentration, kanamycin was prepared by serial dilution with LB medium containing agar (0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 μg / mL) and petri dishes Was dispensed on. Thereafter, the culture medium was inoculated with a culture solution of Bacillus polyfermentus SCD containing 1 × 10 ⁵ viable cell numbers. After incubation for 16 hours, MBC was measured with colonies formed and observed. The activity of kanamycin against Bacillus polyfermentus SCD was determined by its ability to inhibit microbial growth.

As shown in FIG. 1, Bacillus polypermanticus SCD showed resistance at kanamycin of less than 6.25 μg / mL (e-i), but was sensitive at higher concentrations (a-d) above. From the above results, in the present invention, 50 μg / mL of kanamycin was added to LB medium to select recombinant microorganisms.

Next, in order to check whether the Bacillus polypermanticus SCD contains its own plasmid, the Bacillus polypermanticus SCD was incubated in 5 mL of LB medium for 16 hours, followed by centrifugation, followed by miniprep method. Alkali dissolution was slightly modified to isolate the plasmid. The purified solution was electrophoresed on 8% agarose gel. At this time, Bacillus subtilis BD 170 was used as a standard strain.

As shown in FIG. 2, the plasmid was not found in Bacillus polyfermentus SCD (lane 3) as compared to Bacillus subtilis BD 170 (lane 2) as a standard strain. Thus, it was confirmed that Bacillus polyperientus SCD did not have any plasmid in the cell and confirmed the possibility of use as an expression host for foreign genes. M represents the λDNA size marker treated with Hind III, and lane 1 represents the chromosomal DNA of Bacillus polyfermentus SCD.

Next, the DNA molecule must have some features that can act as a tool for gene cloning. In other words, it must be able to replicate in the host cell to produce a large number of copies of recombinant DNA and to be able to deliver to the daughter cell. Most bacterial species, including Bacillus, can only load a limited amount of DNA under normal conditions. To transform these species effectively, Bacillus strains must be subjected to physicochemical treatment to improve DNA loading capacity. The cells treated with this treatment are called competent cells. Methods for obtaining Bacillus subtilis competent cells are already known and various modification methods have been published. We slightly modified the method of Sadiae and Kada to produce Bacillus competent cells, and to obtain a Bacillus strain (Bacillus subtilis BD 170 and Bacillus polypermanticus SCD) containing plasmid DNA (pRB373, pXy1373, pKY1). Transformation was performed.

To transform the Bacillus strain, SPMM I (0.2% ammonium sulfate, 1.4% potassium diphosphate, 0.6% potassium diphosphate, 0.1% sodium citrate, 0.02% magnesium sulfate, 0.5% glucose, 0.2% casamino acid ) And SPMM II (SPMM I with 0.5 mM CaCl ₂ and 1 mM MgCl ₂ added) were used. Receptor cells were inoculated in 5 mL of 0.5 x LB broth, then incubated for 24 hours, 2 mL of the culture was inoculated in 18 mL of SPMM I medium, and then at 37 ° C. until the culture reached the early stage of the stationary phase. Vibration was incubated for about 150 minutes at 300 rpm. Again, 20 mL of SPMM II medium, which had been pre-warmed, was added to the culture, and the culture was gently shaken at 180 rpm for 90 minutes at 37 ° C. 1 mL of cell suspension was mixed with 10 μg of plasmid DNA. The mixture was shaken at 180 rpm for 40 minutes at 37 ° C., then 2 mL of LB medium was added and shaken at 37 ° C. for 1 hour at this rate. The suspension was centrifuged at 3,000 rpm for 10 minutes, the supernatant was discarded and the pellet suspended in 100 μl of sterile water. It was then plated on an LB plate containing 50 μg / mL kanamycin and the results are shown in FIGS. 3 to 5.

As shown in FIG. 3, recombinant Bacillus polypermanticus SCD transformed with plasmids containing the kanamycin ^r gene (pRB373 (a) and pKY1 (b)) form colonies on LB agar plates containing 50 μg / mL of kanamycin. It was.

4 is a gel photograph showing the results of cleavage of Bacillus transformants containing plasmids pRB373 and pXy1373 by treatment with restriction enzyme Pst I. FIG. In FIG. 4 (a), M denotes a size marker, lane 1 includes Bacillus subtilis BD 170 including pRB373, lane 2 includes DNA of lane 1 with Pst I, and lane 3 includes pXy1373. The Bacillus subtilis BD 170 and lane 4 shows the result of cleaving the DNA of lane 3 with Pst I. In addition, in Fig. 4 (b), M is a size marker, lane 1 is a Bacillus polypermanticus SCD containing pRB373, lane 2 is a result of cutting DNA of lane 1 with Pst I, and lane 3 is pXy1373. Bacillus polypermanticus SCD containing, lane 4 shows the result of cleaving the DNA of lane 3 with Pst I.

Figure 5 shows the results of treatment of Bacillus polypermanticus SCD containing pXY1 with restriction enzymes EcoR I / Xba I (lane 1), Kpn I / Xba I (lane 2) and Bam H I / Xba I (lane 3) It is shown.

As shown in Figures 4 and 5, the vectors pRB373, pXy1373 and pKY1 were successfully transformed into Bacillus polypermanticus SCD.

From the above results, the present inventors confirmed that Bacillus polyperientus SCD can be used as a host for the expression of foreign genes.

Example 2: Preparation of plasmid vector and transformant comprising cyclodextranase coding gene

Example 1 confirmed that Bacillus polyfermentus SCD can be used as a host for expressing foreign genes. In order to prepare a recombinant Bacillus polypermanticus SCD expressing a foreign gene, a recombinant plasmid containing a foreign gene should first be prepared. In the present invention, as a foreign gene, a coding gene of cyclodextrinase derived from Bifidobacterium adolescentis Int 57 was used.

First, the phenolic extraction method was modified according to Demuyter's method to separate chromosomal DNA from Bifidobacterium adolescentes Int 57.

Second, plasmid p8A1 was used to isolate promoter and signal sequences for secretion of foreign genes in Bacillus strains. The plasmid contains an amylase promoter site (P) and a signal sequence (SS), which are known as overexpression promoters of α-amylase in B. subtilis var. Natto . In addition, Bacillus subtilis-e coli shuttle vector (pRB373) was used to prepare a Bacillus secretion plasmid. The pRB373 shuttle vector is an antibiotic resistance gene (Amp ^r , Ble ^r and Km ^r ) that can act on two replication origins, E. coli and Bacillus subtilis, T ₁ and T ₀ transcription terminators for expression of foreign genes and It contains multiple cloning sites. The two treated with restriction enzymes Xba I / BamH 1, and then the plasmid was ligated to lie to include the amylase promoter and signal sequence to produce a pKY1 Bacillus subtilis variations NATO (B. subtilis var. Natto) a plurality cloning site . From the above, the pKY1 vector obtained above was used to construct an expression plasmid system for the secretion of foreign genes in Bacillus strains.

Third, a cyclodextrinase coding gene derived from Bifidobacterium adolescents Int 57 was used as a foreign gene, and a plasmid pBB702K including the same was prepared.

Fourth, plasmids pKY1 and pBB702K were treated with EcoR I / Sac I, and then ligated to prepare pKYCD1, digested with Sma I / Eco R V restriction enzymes, and prepared pKYCD2 plasmid by self-ligation. The expression plasmid contains the kanamycin resistance gene and was used for the selection of Bacillus polyfermentus SCD.

The manufacturing process of the cyclodextrinase expression plasmids (pKYCD1 and pKYCD2) is illustrated in FIG.

The recombinant plasmid containing the cyclodextrinase coding gene prepared above was transformed by introducing into Bacillus strains according to the method of Example 1, followed by incubation for 16 hours and then 50 ㎍ / mL at 37 ° C. Transformants were selected in LB medium containing kanamycin. Plasmids pKYCD1 and pKYCD2 isolated from the transformants were digested with several restriction enzymes and analyzed by 0.8% agarose gel DNA electrophoresis.

Experimental Example 1: Investigation of transformation in the recombinant Bacillus strain of the present invention

It was examined whether the recombinant Bacillus subtilis BD 170 and Bacillus polyfermentus SCD obtained in Example 2 express cyclodextrinase.

Plasmids pKYCD1 and pKYCD2 isolated from the transformants were digested with EcoR V and BamH I and analyzed by 0.8% agarose gel DNA electrophoresis. The plasmid was isolated according to the method of Example 1 and the results are shown in a gel photograph in FIG. In FIG. 7, M denotes a size marker, lane 1 shows plasmid pKYCD1 isolated from recombinant Bacillus subtilis BD 170, lane 2 shows a plasmid of lane 1 with EcoR V, and lane 3 shows a plasmid of lane 1 As a result of cleavage with BamH I, lane 4 is the result of cleavage of plasmid pKYCD2 isolated from recombinant Bacillus subtilis BD 170, lane 5 is the plasmid of lane 4 with EcoR V, lane 6 is the plasmid of lane 4 BamH It shows the result cut by I. In addition, M 'is a size marker, lane 1' is a plasmid pKYCD1 isolated from recombinant Bacillus polypermanticus SCD, lane 2 'is a result of cutting the plasmid of lane 1' with EcoR V, lane 3 'is lane The result of cleaving the 1 'plasmid with BamH I, lane 4' is the plasmid pKYCD2 isolated from the recombinant Bacillus polyfermenticus SCD, and the lane 5 'is the result of cleaving the plasmid of the lane 4' with EcoR V. 6 'shows the result of cleavage of the plasmid of lane 4' with BamH I.

As shown in FIG. 7, the pattern of two plasmids pKYCD1 and pKYCD2 was examined to confirm that the cyclodextrinase recombinant gene was correctly constructed.

Experimental Example 2: Investigation of Enzyme Activity of Cyclodextrinase in Recombinant Bacillus Strain

The enzyme activity of cyclodextrinase expressed in the recombinant Bacillus strains obtained in Example 2 was measured.

The enzymatic activity of cyclodextrinase was determined by clear ring formation of transformants secreting cyclodextrinase according to the I ₂ -KI staining method.

In FIG. 8 a1 is negative control (Bacillus subtilis BD 170, 1% starch); a2 Bacillus subtilis BD 170 (pXyl373, 1% starch); a3, Bacillus subtilis BD 170 (pKYCD1, 1% starch); a4, Bacillus subtilis BD 170 (pKYCD2, 1% starch); b1, negative control group (Bacillus subtilis BD 170, 1% cyclodextrin); b2, Bacillus subtilis BD 170 (pXyl373, 1% cyclodextrin); b3, Bacillus subtilis BD 170 (pKYCD1, 1% cyclodextrin); b4, Bacillus subtilis BD 170 (pKYCD2, 1% cyclodextrin); c1, negative control (Bacillus polyfermentus SCD, 1% starch); c2, Bacillus polyfermentus SCD (pXyl373, 1% starch); c3, Bacillus polyfermentus SCD (pKYCD1, 1% starch); c4, Bacillus polyfermentus SCD (pKYCD2, 1% starch); d1, negative control (Bacillus polyperientus SCD, 1% cyclodextrin); d2, Bacillus polyfermentus SCD (pXyl373, 1% cyclodextrin); d3, Bacillus polyfermentus SCD (pKYCD1, 1% Cyclodextrin); d4, Bacillus polyperientus SCD (pKYCD2, 1% cyclodextrin).

As shown in FIG. 8, high cyclodextrinase activity was shown in B. subtilis BD 170 and B. polyfermenticus SCD comprising the pKYCD2 plasmid. The expression system demonstrates that it has higher enzyme activity than others.

Accordingly, the inventors have identified a recombinant strain transformed with pKYCD2 containing a cyclodextrinase coding gene derived from Bifidobacterium adolescentis Int 57, Bacillus polyfermenticus SCD pKYCD2. After that, it was deposited on April 16, 2003 with the Korean National Center for Microbiological Conservation, Accession No. KCCM-10488.

The present invention relates to a recombinant Bacillus strain expressing a cyclodextrinase and a method for producing the same, the present invention has the effect of providing a Bacillus polypermanticus SCD as an expression host strain of a foreign gene. In addition, the present invention has an excellent effect of providing a Bacillus polypermanticus SCD transformant expressing cyclodextrinase. In addition, cyclodextrinase expressed in the recombinant Bacillus strain comprising pKYCD2 of the present invention has the effect of having a high enzymatic activity. Therefore, the present invention is a very useful invention in the genetic engineering industry because it is possible to mass-produce recombinant proteins even at low production costs using the foreign gene expression system of the present invention.

Claims (3)

delete delete Recombinant Bacillus polypeptides transformed with a cyclodextrinase expression vector pKYCD2 comprising a cyclodextrinase coding gene from Bifidobacterium adolescentis Int 57 and having a cleavage map as described in FIG. Bacillus polyfermenticus SCD pKYCD2 KCCM-10488.

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