CN100453646C - A gene sequence of β-mannanase and preparation of its encoded recombinase - Google Patents

Abstract

The invention discloses an alkaline β-mannanase gene (β-mannanase gene) obtained from total DNA of alkaliphilic Bacillus N16-5 and a preparation method thereof, constructs a prokaryotic expression plasmid, and transforms Escherichia coli to express β-mannanase. According to the comparison of nucleotide sequence and amino acid sequence, the enzyme is a new type of β-mannanase. A series of oligosaccharides are produced by hydrolyzing konjac powder with this enzyme, and the total conversion rate of oligosaccharides is over 80%. The manno-oligosaccharides produced thus have important application value in functional foods.

Description

A gene sequence of β-mannanase and preparation of its encoded recombinase

The invention relates to a gene sequence of β-mannanase, the preparation of the recombinant enzyme coded by the gene and the product containing the enzyme. Specifically, the present invention relates to a β-mannanase DNA molecule encoding alkaliphilic Bacillus (alkaliphilic Bacillus) N16-5, a recombinant plasmid containing the enzyme gene and a recombinant bacterial strain expressing the corresponding enzyme.

β-mannanase (β-mannanase, EC 3.2.1.78) is a hemicellulase that can hydrolyze plant polysaccharides such as mannan, glucomannan, galactomannan and galactoglucomannan (See Tipdon, R.S. et al.: Advances in Carbohydrate Chemistry and Biochemistry, 32:299-316, Academic Press, New York, 1976.). Mannan is a relatively abundant hemicellulose resource in nature, second only to cellulose in quantity. Leguminous plant seeds, conifer wood, green coffee beans, etc. all contain a large amount of mannan, and some plant gums, such as tianqing gum, carob gum, konjac powder, etc., are almost entirely composed of galactose or glucose and mannose mannan. The use of β-mannanase to further process and comprehensively utilize the above-mentioned plant materials has great application potential, especially in food and medicine, because β-mannanase can degrade plant mannan Mannan oligosaccharides can promote the growth and development of human intestinal normal flora (see Akino, T. et al.: Agric. Biol. Chem., 52: 773-779, 1988), which can improve human body Fitness level matters.

β-mannanase has been isolated from organisms of different origins, such as Bacillus, Aeromonas, Xanthomonas, Clostridium, Penicillium, Trichoderma, and Streptomyces, etc. ⁽⁷⁾ . The starting point of relevant patents and documents is mainly to deal with hemicellulose in the paper industry (seeing Buchert et al.: USP5,661,021 Aug.26,1997; Ratto, M.et al.: Biotechnol.Letters, 10:661-664 , 1988; and Christgau et al.USP5,795,764 Aug.18, 1998), there is not yet a kind of β-mannanase product suitable for the production of mannan oligosaccharides, and its main problem is that the enzyme activity is complex and the substrate level is low, Directly affect the yield and yield of mannan oligosaccharides. The hydrolysis reaction of plant mannan by β-mannanase is carried out under alkaline conditions, which can increase the swelling property of hemicellulose in water, thereby facilitating the action of the enzyme. The optimum pH of the currently known microbial β-mannanase is generally in the acidic or neutral range. The facultative alkalophilic Bacillus AM001 studied in Japan has an optimum pH of 8.5-9.0, and attempts to enzymatically process mannan oligosaccharides Production, the conversion rate of mannan oligosaccharides is 29%. In addition, there are many patents and literature reports on mannanase genes in recent years (see USP5,661,021 and USP5,795,764), but there are few reports on alkaliphilic alkaline β-mannanase genes.

An object of the present invention is to provide a β-mannanase gene.

Another object of the present invention is to provide a method for preparing β-mannanase.

Another object of the present invention is to provide recombinant expression plasmids and recombinant strains containing the β-mannanase gene, and use the gene expression products to efficiently produce mannan oligosaccharides.

The present invention provides a DNA molecule encoding the β-mannanase of alkalophilic bacillus N16-5, the amino acid sequence of the β-mannanase is SEQ ID NO: 2.

According to the DNA molecule of the present invention, wherein, said DNA molecule comprises SEQ NO: 1 DNA sequence.

The present invention also provides recombinant expression plasmids containing the above-mentioned DNA sequences.

According to the recombinant expression plasmid of the present invention, said recombinant expression plasmid is a recombinant expression plasmid pMAN1 and a recombinant expression plasmid pMAN2 containing the above-mentioned DNA sequence.

The present invention also relates to recombinant bacterial strains containing the above-mentioned recombinant expression plasmids, including Bacillus alkalophilus N16-5 strain, Escherichia coli JM109 strain JM109MAN1 and Escherichia coli JM109 strain JM109MAN2.

According to another aspect of the present invention, the present invention provides a method for preparing a β-mannanase with enzymatic activity, comprising the steps of:

(1) Total DNA was extracted from the Bacillus alkalophilus N16-5 strain, partially hydrolyzed by restriction enzymes to obtain a DNA fragment, which was connected to the pUC19 vector and transformed into Escherichia coli JM109 to obtain a gene containing β-mannanase Recombinant expression plasmid pMAN1 and recombinant Escherichia coli strain JM109MAN1;

(2) Isolate the DNA fragment containing the β-mannanase gene from the recombinant expression plasmid pMAN1, and then partially hydrolyze it with restriction enzymes to obtain a small fragment DNA, which is connected to the pUC19 vector and transformed into Escherichia coli JM109 to obtain the β-mannanase-containing gene - Recombinant expression plasmid pMAN2 of mannanase gene and recombinant Escherichia coli strain JM109MAN2.

Brief description of the drawings

Fig. 1 is a construction pattern diagram of the alkaline β-mannanase gene recombinant plasmid pMAN1 of the present invention.

Fig. 2 is a construction pattern diagram of the alkaline β-mannanase gene recombinant plasmid pMAN2 of the present invention.

Fig. 3 is the expression of the alkaline β-mannanase gene of the present invention in Escherichia coli.

Fig. 4 is the DNA sequence of β-mannanase from Bacillus alkalophilus N16-5 of the present invention.

Fig. 5 is the amino acid sequence of the β-mannanase of the present invention.

The present invention will be described in detail below in conjunction with the accompanying drawings.

The present invention was accomplished based on the following discovery by the inventors of the present invention: Bacillus alkalophilus N16-5 produces a large amount of alkaline β-mannanase under alkaline conditions, which is particularly suitable for hydrolyzing plant polysaccharides to form manna Oligosaccharides, such as enzymatic konjac flour, etc., the conversion rate of oligosaccharides is over 80%.

The present invention obtains the gene of beta-mannanase from the isolation of Bacillus alkalophilus N16-5, and it is the DNA of 1479bp, and its DNA sequence diagram is as shown in Figure 4 (SEQ ID NO: 1) coding one is made up of 493 amino acids The protein, its amino acid sequence diagram is shown in Figure 5 (SEQ ID NO: 2). A recombinant expression plasmid containing the gene was obtained by a conventional method, and Escherichia coli was transformed to make the recombinant strain express β-mannanase. Therefore the present invention provides a kind of possibility simultaneously, promptly by genetic engineering or molecular biology means, the enzyme gene that the present invention relates to is cloned into Bacillus alkalophilus or other recipient bacterium, by Bacillus alkalophilus N16-5 bacterial strain or Other strains or other culture conditions produce the β-mannanases involved in the present invention. The β-mannanase involved in the invention is particularly suitable for the enzymatic hydrolysis production of mannan oligosaccharides, and also provides a possibility for mass production of mannan oligosaccharides using konjac polysaccharides and the like as raw materials.

The β-mannanase involved in the invention is particularly suitable for the enzymatic hydrolysis production of mannan oligosaccharides, and provides a possibility for establishing a process for mass production of mannan oligosaccharides using konjac polysaccharides and the like as raw materials.

In order to achieve the purpose of the present invention, the method for preparing β-mannanase of the present invention and its recombinant expression plasmid and recombinant bacterial strain comprises the steps:

(1) Total DNA was extracted from Bacillus alkalophilus N16-5, partially hydrolyzed by restriction enzymes to obtain DNA fragments, connected to pUC19 vector and transformed into Escherichia coli JM109 to obtain recombinant plasmid pMAN1 containing β-mannanase gene and recombinant Escherichia coli strain JM109MAN1;

(2) Isolate the DNA fragment containing the β-mannanase gene from the recombinant plasmid pMAN1, and then hydrolyze it with restriction enzymes to obtain a small fragment of DNA, which is connected to the pUC19 vector and transformed into Escherichia coli JM109 to obtain the DNA fragment containing β-mannan The recombinant plasmid pMAN2 of the enzyme gene and the recombinant Escherichia coli JM109MAN2.

Cultivate the recombinant bacterium under the condition that the above-mentioned β-mannanase gene can be expressed. On the medium containing β-mannan, the colony forms a transparent circle, which proves that the recombinant bacterium expresses β-mannanase. active. The protein has mannanase activity, pI is 4.3, optimum pH is 9.5, optimum temperature is 70°C, and can efficiently hydrolyze plant mannan to produce oligosaccharides. Sequencing showed that the structural gene of the enzyme was a 1479bp DNA encoding a protein consisting of 493 amino acids.

The expression product of the above-mentioned gene is hydrolyzed into konjac flour and converted into oligosaccharides. The substrate concentration is 5-15%, the reaction temperature is 40-60°C, the reaction pH is 9-10, and the time is 8-24 hours. After the reaction, adjust the pH to 5-6 with acetic acid, citric acid or hydrochloric acid, and add 0.5-2% powder Or granular activated carbon for decolorization. Mannan oligosaccharide syrup can be obtained by filtering. The conversion rate of oligosaccharides is more than 80%, and the total yield is more than 70%, in which 2-8 sugars account for more than 80% of the total sugars. The oligosaccharides play an important role in promoting the growth of bifidobacteria in the body and improving human health.

The β-mannanase involved in the present invention is a novel β-mannanase. According to the amino acid sequence similarity of enzymes, the currently known β-mannanases belong to the 5th and 26th families of glycoside hydrolases (see Ethier, N.et al.: Appl.Environ.Microbiol.64:4428- 4432, 1998). Through the comparative analysis of the deduced amino acid sequence of the β-mannanase gene involved in the present invention, the β-mannanase involved in the present invention belongs to a member of the 5th family of glycoside hydrolase, and is different from other reported β-mannanases. Compared with the amino acid sequences of carbohydrases, the similarity is less than 60%.

The performance of the β-mannanase involved in the present invention is different from the known β-mannanase, and its optimum pH and temperature are the highest among the β-mannanases found so far. The invention provides the structural gene, recombinant expression plasmid and recombinant cell model of the novel β-mannanase obtained by conventional methods, so that the β-mannanase of the invention can be produced by other bacterial strains or under other culture conditions. At the same time, the invention provides an effective method for producing mannan oligosaccharides by enzymolysis using konjac powder as raw material, and the conversion rate of mannan oligosaccharides can reach 80%.

The β-mannanase of the present invention has the following characteristics:

(1) Produced by alkalophilic Bacillus N16-5 or its derivatives, the derivatives refer to recombinant strains transformed with the DNA fragments involved in the present invention;

(2) have the DNA sequence code shown in Figure 4;

(3) have the amino acid sequence shown in Figure 5;

(4) It has at least one of the following characteristics: 1) has mannanase activity, pI is 4.3, optimum pH 9.0, optimum temperature 70°C, molecular weight 51000 Daltons; 2) has mannan Enzyme activity, pI is 2.5, optimum pH 10.0, optimum temperature 70°C, molecular weight 38000 Daltons; 3) has mannanase activity, pI is 2.5, optimum pH 9.0, optimum temperature 70°C, Molecular weight 34700 Daltons;

(5) It can efficiently hydrolyze plant polysaccharides to produce oligosaccharides.

Further, the present invention relates to a DNA molecule encoding the β-mannanase involved in the present invention, the DNA nucleotide sequence:

(1) consists of the encoding part of the DNA sequence shown in Figure 4;

(2) Encode a protein whose amino acid sequence is as shown in the figure.

The present invention is described in further detail below by way of examples. It should be understood that the described examples are only for illustration and not limitation of the invention.

The extraction of embodiment 1 alkalophilic bacillus N16-5 total DNA

Using 20 grams of fresh wet bacteria of the alkaliphilic Bacillus N16-5 isolated from Udu Naojian Lake in Inner Mongolia, China, suspended in 10 ml of 50 mM Tris buffer (pH 8.0), adding a small amount of lysozyme and 8 ml of 0.25 mMEDTA (pH8.0), mix well and place it at 37°C for 20min, then add 2 ml of 10% SDS, place it at 55°C for 5min, extract once with equal volumes of phenol and chloroform respectively, take the last supernatant solution, add 2 times the volume of ethanol, recover the DNA, wash with 70% and absolute ethanol respectively, dissolve the precipitate in 0.5 ml TE buffer (pH8.0, 10mM Tris, 1mM EDTA), add 10mg/ml RNase 3μl, and incubate at 37°C for 1 hour, Extract once with equal volumes of phenol and chloroform respectively, add 2 times the volume of ethanol to the supernatant, recover DNA, wash with 70% and absolute ethanol respectively, dry in vacuum, and dissolve with deionized water. The UV spectrophotometer measurement results of the DNA solution were A260/A280=1.98, A260/A230=2.18.

Cloning of embodiment 2β-mannanase gene

Take 10 μl (about 50 μg DNA) of the total DNA solution described above, partially hydrolyze it with the restriction enzyme EcoRI, and perform agarose gel electrophoresis to obtain a 2-10 kb DNA fragment. Take 2 μl (5 μg) of EcoRI-digested DNA fragments and 1 μl (1 μg) of EcoRI-digested plasmid pUC19DNA for ligation in a 20 μl ligation system, which contains 2 μl (10X ligation buffer), 1 μl T4 DNA ligase, and 14 μl of water. The ligation system was reacted at 16°C for 16 hours, transformed into competent Escherichia coli JM109, and spread on LB solid medium containing Amp (ampicillin), IPTG and X-gal. Cultivate at 37°C for 16-18 hours, pick white spot in liquid medium containing Amp and mannan or solid medium containing Amp and mannan, culture at 37°C for 18-20 hours, it can liquefy mannan or contain Colonies with transparent circles formed on the solid medium of β-mannan were determined as positive clones (see Figure 3). Extract recombinant plasmids from positive colonies with alkaline method, hydrolyze recombinant plasmids with various restriction enzymes, and confirm that a DNA fragment is inserted into the plasmid according to electrophoresis results, and its size is about 8.0kb. The recombinant plasmid containing this DNA fragment is called pMAN1 (see Figure 1), and the recombinant Escherichia coli containing this recombinant plasmid pMAN1 is called Escherichia coli JM109MAN1.

The recombinant plasmid pMAN1 can transform Escherichia coli with high frequency to express β-mannanase activity and ampicillin resistance. The DNA insert fragment in the recombinant plasmid was labeled with Digoxigenin DNA Labeling Detection Kit, and Southern blot DNA hybridization experiment was carried out with the chromosomal DNA of Bacillus alkalophilus N16-5, and it was confirmed that the DNA fragment inserted in the recombinant plasmid pMAN1 was from Bacillus alkalophilus Chromosomal DNA from N16-15.

Example 3 Subcloning and sequence of the β-mannanase gene

Take 10 μl of the inserted DNA fragment in the plasmid pMAN1 in a 50 μl system, and perform various restriction enzyme digestion reactions, such as single or double digestion reactions of AccI, HindIII, PstI, EcoRI and XbalI, etc., and incubate at 37°C for 1.5 hours. The recovered DNA fragments were purified by agarose gel electrophoresis. As mentioned above, connect the single or double digested DNA fragments to the plasmid pUC19DNA or pGEM-4Z to form a series of subcloning plasmids, transform the competent Escherichia coli JM109, and obtain a series of recombinant Escherichia coli accordingly. The enzyme activity was measured, and the result showed that the recombinant Escherichia coli containing the HincII enzyme-cut DNA fragment had mannanase activity, and the HincII enzyme-cut DNA fragment was about 3.5 kb. The recombinant plasmid containing the DNA fragment is called pMAN2 (see Figure 2), and the recombinant Escherichia coli containing the recombinant plasmid pMAN2 is called Escherichia coli JM109MAN2. The DNA fragment was sequenced by Sanger dideoxy method. The sequencing results are shown in Figure 4. The HincII digested DNA fragment has a full length of 3419bp and contains a 1479bp open reading frame (ORF), starting from the ATG start codon and ending with the ATT stop codon. The complete ORF encodes a protein consisting of 492 amino acids.

Example 4. Purification and Characterization of Recombinant β-Mannanase

The cells of the recombinant bacterium E.coli JM109MAN2 were suspended in 10mM glycine buffer (pH9.6), the cells were disrupted by ultrasonic waves, and the centrifuged supernatant was the crude enzyme solution of the recombinant β-mannanase. The supernatant enzyme solution was purified by DEAE-Sephadex A-25 ion exchange column chromatography, hydroxyapatite adsorption column chromatography and PAGE preparative electrophoresis, and the obtained enzyme preparation showed a band on SDS-PAGE. The basic properties of this recombinant β-mannanase were determined using standard methods known in protein chemistry. The molecular weight of the recombinase measured by SDS-PAGE is 51000 Daltons, which is close to the theoretically calculated molecular weight (54000 Daltons); the isoelectric point pI of the recombinase measured by PAGEIEF is 4..3; The optimum pH for the enzyme reaction is 9..5, and the optimum temperature is 70°C. A series of oligosaccharides such as 2-8 sugars were produced by the hydrolysis of konjac flour by recombinant β-mannanase by high pressure liquid chromatography.

Example 5 Recombinant β-mannanase hydrolyzes konjac powder for oligosaccharide conversion

Put 1.8L of water in a 2.5L reactor, raise the temperature to 55°C, add 4.8 grams of Na ₂ CO ₃ , 3 grams of NaHCO ₃ , 1.8×10 ⁴ units of β-mannanase, and 180 grams of konjac flour. Incubate at 55°C for 16 hours. Use HCl to adjust the pH to 5.5-6.0, add 10 grams of granular activated carbon, raise the temperature to 100°C and keep it for 5 minutes, cool to 70°C and filter (common industrial filter cloth). The 2-8 sugars in the obtained product account for 80% of the total sugars. % or more, the oligosaccharide conversion rate is more than 80%, and the yield is more than 70% (Table 1).

Table 1, Mannan oligosaccharide composition changes (g/100ml)

Claims (10)

1. The coding 'beta '-mannase dna molecular, the aminoacid sequence of this 'beta '-mannase is SEQ ID NO:2.
2. 2. dna molecular as claimed in claim 1, wherein, the nucleotides sequence of this dna molecular is classified SEQ ID NO:1 as.
3. 3. the recombinant expression plasmid that contains the described dna molecular of claim 1.
4. 4. recombinant expression plasmid as claimed in claim 3, wherein, the sequence of described dna molecular is the sequence shown in the SEQ ID NO:1.
5. 5, recombinant expression plasmid as claimed in claim 4, wherein, the carrier that forms this recombinant expression plasmid is pUC19 or pGME-4Z.
6. 6. the recombinant bacterial strain that contains claim 3,4 or 5 described recombinant expression plasmids.
7. 7. recombinant bacterial strain as claimed in claim 6, wherein, this recombinant bacterial strain is the recombinant bacterial strain of e. coli jm109 bacterial strain.
8. 8. a reorganization 'beta '-mannase is characterized in that the aminoacid sequence of this reorganization 'beta '-mannase is SEQ ID NO:2.
9. 9. reorganization 'beta '-mannase as claimed in claim 8, wherein, this reorganization 'beta '-mannase is expressed by the described recombinant bacterial strain of claim 6.
10. 10. a method for preparing 'beta '-mannase is characterized in that, this method is included in and cultivates the described recombinant bacterial strain of claim 6 under the condition that is fit to the beta-mannase gene expression.

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