WO2004113538A1

WO2004113538A1 - Gene coding mannanase and recombinant mannanase expressed from transformant thereof

Info

Publication number: WO2004113538A1
Application number: PCT/KR2004/001520
Authority: WO
Inventors: Ki-Hong Yoon; Ki-Haeng Cho; Joon-Ho Choi; Mi-Sung Lee; Kyung-Sook Ryu; Min-A Kweun; Hyun-Suk Kim
Original assignee: Ctc Bio, Inc.
Priority date: 2003-06-26
Filing date: 2004-06-24
Publication date: 2004-12-29
Also published as: KR100560375B1; KR20050001413A

Abstract

Disclosed is a novel gene coding for mannanase, derived from Bacillus licheniformis WL-12, a recombinant plasmid carrying the gene, and an E. coli transformant harboring the recombinant plasmid. With ability to degrade galactomannan into mannose, mannobiose and mannotriose, the mannanase is highly activity at neutral pH values and moderate temperatures, similar to the physiological conditions in animals.

Description

DESCRIPTION

GENE CODING MANNANASE AND RECOMBINANT MANNANASE EXPRESSED PRO! TRANSFORMANT THEREOF

Technical Field

The present invention relates to gene coding for mannanase and the production of the enzyme. More particularly, the present invention relates to a gene sequence encoding the mannanase derived from Bacillus licheniformis WL-12 which is isolated as a strain having high activity of hydrolyzing galactomannan from bacteria cultured in soybean paste, and an amino acid sequence corresponding to the gene sequence.

Background Art

Mannanase, which enzymatically hydrolyzes mannan, has found increasing variety of applications. Mannan is a major component of polysaccharides typically referred to as hemicelluloses. Second in abundance to cellulose on earth, hemicelluloses are plant cell wall polysaccharides which exist in association with cellulose and lignin. Other hemicellulosic polysaccharides include galactomannan, arabinogalactan, glucomannan, and galactoglucomannan. Galactomannan is mostly found in seeds of leguminous plants and glucomannan in acicular trees.

Hemicellulases, including xylanase, glucanase and mannanase, are being extensively studied for a possible ability to convert hemicelluloses, which are among the most abundant renewable plant resources on earth, into a carbon source available to humans, but the conversion has not yet been realized.

Now, the fields in which hemicellulases find applications include food and pulp industries, such as coffee, chocolate, cocoa, tea, cereal, etc.

For example, the treatment of hemicellulose-containing cereals with hemicellulases affords easily digestible feedstuff or foods. The enzymatic treatment increases energy efficiency and decreases the mucilage content of the cereals. Increasing the available energy content of feedstuffs decreases the production cost of livestock.

Soybean proteins are useful as feedstuffs for domestic animals including dogs, cats, pigs, fishes, chickens, etc. Soybean oil cakes, though not a main energy source, can be important protein sources having a high content of necessary amino acids. Amounting to about 10 % of the carbohydrate content of soybean oil cakes, galactan and pentosan are excreted undigested by monogastric animals. Mannanase degrades galactan into oligo- or mono-saccharides which can be easily metabolized by monogastric animals.

Mannanase is produced from a group of bacteria including Bacillus spp., Aeromonas spp., Enterococcus spp., Pseudomonas spp. and Streptomyces spp., and from other microorganisms such as fungi and yeasts . Some higher plants and animals are also found to produce the enzyme.

Now, microorganisms utilized in producing mannanases in practice are fungi belonging to the families Trichoderma and Aspergillus. Fungus-derived mannanases have maximal activities in acidic conditions. The organs of pigs or chickens where mannanases are required to perform their activity are the small intestine and subsequent digestive tracts. These digestive organs are maintained at pH 6.5. Thus, mannanases which are active at neutral pH conditions, unlike those showing high enzymatic activity at pH 3.6-5.5 which are derived from fungus, are required as feedstuff additions . Three enzymes endo-β-mannanase, exo-β-mannanase and β- mannosidase must work in cooperation in order to degrade mannan to complete saccharification. The endomannanase catalyze the random hydrolysis of the β-D-1, 4-mannopyranosyl linkages within the main chain of mannans and various polysaccharides consisting mainly of at least four mannose units. In practice, the biodegradation of mannan, a major polysaccharide component of the ubiquitous hemicellulose, is achieved in nature by the catalytic action of the endo- mannanases . The present invention chiefly pays attention to endo- β-mannanase which will be referred to just as "mannanase" below, for convenience. Leading to the present invention, the intensive and thorough research on mannanase, conducted by the present inventors, resulted in the finding that Baccillus licheniformis WL-12 produces mannanase with high activity in neutral pH conditions and dwells in the Korean traditional food soybean paste. Accordingly, the bacteria isolated and identified from soybean paste was provided for cloning a gene coding for mannanase, followed by DNA sequencing. A transformant with a recombinant plasmid harboring the gene was cultured to produce the mannanase.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide a nucleotide sequence coding for a polypeptide having high mannanase activity in neutral pH conditions, derived from Bacillus licheniformis WL-12.

It is another object of the present invention to provide a polypeptide having mannanase activity, produced from E. coli having a recombinant plasmid carrying the mannanase gene.

It is a further object of the present invention to provide feedstuff comprising the polypeptide having mannanase activity.

In the present invention, chromosomal DNA extracted from Bacillus licheniformis WL-12, which is isolated as a strain highly active in degrading galactomannan from soybean paste, is used to construct a DNA library. Next, this is introduced into E. coli . Colonies of the transformant grown on agar plates are cultured under an overlay medium containing galactomannan from seeds of Acacia leucophloea to observe the development of degradation circles. Purification of the recombinant plasmid from the transformant is followed by determining the base sequence of the gene coding for the mannanase. With the restriction enzyme map constructed for the gene of interest, the mannanase produced by the transformed E. coli is partially purified. Effects of temperature and pH on the activity of the enzyme are analyzed. To determine the availability of the mannanase as a foodstuff addition, hydrolysates resulting from the catalytic hydrolysis of various substrates including galactomannan, mannotriose, mannotetraose, mannopentose, and mannohexose with the enzyme are also analyzed.

Accordingly, the present invention is described with the following steps.

First, Bacillus licheniformis WL-12 which is highly active in degrading galactomannan is isolated and identified.

Second, a DNA library of Bacillus lichniformis WL-12 is constructed. In this regard, chromosomal DNA is extracted from the isolated Bacillus licheniformis WL-12, treated with restriction enzymes, and inserted into plasmids which are then introduced to E. coli .

Third, bacteria having a mannanase gene is selected. In this step, the transformed E. coli is spread on agar plates and the colonies are cultured under a coverlay medium containing galactomannan from the seeds of Acacia leucophloea . The colonies at galactomannan degradation circles are selected. Fourth, DNA sequencing of a mannanase gene is conducted with the recombinant plasmid which is extracted from the E. coli transformant containing the gene.

Fifth, after the expression of the mannanase of interest, it is purified and analyzed for activity according to temperature, pH conditions and substrates.

Sixth, through thin layer chromatography, lysates from the digestion of galactomannan, mannobiose, mannotriose, mannotetraose, mannopentose and mannohexose with the enzyme are analyzed. As a coverlay medium for selecting a strain capable of degrading galactomannan from the transformed E. coli, an LB- galactomannan agar medium containing galactomannan from the seeds of Acacia leucophloea 2 g/L, yeast extract 5 g/L, tryptone 10 g/L, NaCl 5 g/L, ampicillin 50 mg/L, agar 8 g/L, adjusted to pH 7.0, is used.

As a culturing medium for the E. coli transformant, an LB complex liquid medium containing yeast extract 5 g/L, tryptone 10 g/L, NaCl 5 g/L, added with ampicillin 50 mg/L, is used. In order to isolate microorganisms producing mannanases, a suspension of soybean paste is spread over a nutrient agar plate. Selection is executed for the colonies which are surrounded by galactomannan degradation circles.

Identification of the selected strains is achieved through morphological and biochemical properties and by determining the base sequence of a gene coding for 16S rRNA. With the properties of being aerobic and Gram positive, producing spores and having catalase activity, the selected strain is believed to belong to Bacillus.

Analysis of the Bacillus bacteria for biochemical properties is conducted with the aid of a VITEK reader and BAC card, showing that the bacteria of interest has 97% similarity to Bacillus licheniformis. DNA sequencing data reveals that the 16S rRNA gene from the selected bacteria shares 99% homology with that of Bacillus licheniformis. Accordingly, the selected strain is identified as a Bacillus licheniformis mutant and named Bacillus licheniformis WL-12.

For gene cloning of mannanase, a DNA library of the

Bacillus licheniformis WL-12 is created, followed by the selection of an E. coli transformant with the mannanase gene.

After being cultured to the exponential phase, Bacillus licheniformis WL-12 is harvested by centrifugation. Chromosomal DNA is extracted from the biomass, digested with Sau3AI, and electrophoresed to obtain DNA fragments with a size of 1-10 kb. These DNA fragments are inserted into pUC19 to create a gene library. The recombinant plasmids thus obtained are introduced into E. coli which is spread over LB agar plates to obtain transformants having a mannanase gene. As for the host bacteria, any may be used if it allows the introduction, gene expression and maintenance of the recombinant plasmids. For example, XL-1, C600, Y1088, Y1090, NM522, K802 and JM105 may be used. Colonies grown on the LB agar plates are developed under an LB-galactomannan coverlay medium. After culturing for five hours, the transformants surrounded by galactomannan degradation circles are selected.

For the DNA sequencing of a gene coding for mannanase, the E. coli transformant is cultured in an LB liquid medium with ampicillin added. The biomass obtained from the medium by centrifugation is lysed with alkali to prepare the recombinant plasmid.

Analysis with various restriction enzymes shows that the recombinant plasmid carries a foreign 1.3 kb DNA fragment. It is named pMWLl3. To determine the base sequence of the cloned 1.3 kb DNA fragment, it is digested with various restriction enzyme and the digests of various sizes are inserted into pUC19 which is then provided for dideoxynucleotide sequencing of the 1,300 bp DNA fragment of interest.

In this DNA sequence, there is an open reading frame with a size of 1,083 bp corresponding to the structural gene of mannanase (Sequence No. 1) . Inferred from the base sequence of the structural gene, the amino acid sequence of mannanase consists of 361 amino acid residues as given in Sequence No. 2.

Comparing the amino acid sequence inferred from the DNA sequence of Bacillus licheniformis WL-12 to the amino acid sequences of mannanases of known Bacillus spp., the highest homology shared therebetween is found to be 80 % . Therefore, the annanase-encoding gene of Bacillus licheniformis WL12 can be determined to be novel.

Gene expression in the E. coli transformant carrying pMWLl3 is followed by examining the recombinant mannanase for activity according to temperature and pH conditions . For gene expression, E. coli transformed with pMWL13 is cultured in an LB liquid medium added with ampicillin. Through centrifugation, lysis and concentration, the enzyme is partially isolated from the biomass. The enzyme is measured for mannanase activity in various temperature and pH conditions . Data, as shown in FIG. 3, exhibits that the enzyme has maximal activity at 65°C and pH 6.5, with 90% of the maximal activity at pH 5.5-6.5. Particularly, at pH 6.5, which is a physiological condition in the small intestine of animals, the activity of the enzyme is as high as 93 % of the maximum. With regard to galactomannan from seeds of Acacia leucophloea, guar gum glucomannan and konjac, the enzyme is examined for relative activity. The highest activity is found for the galactomannan from seeds of Acacia leucophloea.

The products resulting from the catalytic hydrolysis of the mannanase of the present invention are also analyzed. In this regard, hydrolysates obtained by incubating the mannanase with galactomannan from seeds of Acacia leucophloea under optimal conditions are subjected to TLC analysis. FIG. 4 shows that mannose, mannobiose and mannotriose are detected as the final products as measured by TLC. With mannobiose, mannotriose, mannotetraose, mannopentose and mannohexose serving as substrates, the enzyme is incubated. The TLC analysis of the resulting hydrolysates shows that mannotriose, mannotetraose, mannopentose and mannohexose are degraded into mannose, mannobiose and mannotriose without further degradation of mannobiose, as seen in FIG. 5. Consequently, it is determined that the mannanase encoded by the mannanase gene of Bacillus lichenformis WL-12 according to the present invention can degrade mannan materials to smaller oligosaccharides .

Brief Description of Drawings

FIG. 1 is a restriction enzyme map illustrating a recombinant plasmid pMWL13 harboring a gene encoding the mannanase of Bacillus licheniformis WL-12.

FIG. 2 is a cleavage map illustrating a recombinant plasmid pMWL13 harboring a gene encoding the mannanase of Bacillus licheniformis WL-12.

FIG. 3 is a graph in which the activity of the mannanase produced by an E. coli transformant carrying the recombinant plasmid pMWL13 of the present invention is plotted versus pH and temperature.

FIG. 4 is a TLC photograph showing hydrolysates resulting from the catalytic hydrolysis of galactomannan from seeds of Acacia leucophloea with mannanase produced by the E. coli transformant carrying pMWLl3.

FIG. 5 is a TLC photograph showing hydrolysates resulting from the catalytic hydrolysis of mannobiose, manotriose, mannotetraose, mannopentose and mannohexose with the mannanase produced by the E. coli transformant carrying pMWL13.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.

EXAMPLE 1: Isolation and Identification of Bacillus sp. WL-12

1 g of soybean paste was suspended in 10 mL of 0.85% NaCl solution and an appropriate amount of the suspension was spread over a nutrient plate (beet extract 3g; bacto-pentone 5g; water 1 liter) supplemented with 0.5 % locust bean gum. After incubation at 37°C, colonies formed with galactomannan degradation circles were selected as mannanase-producing strains. From them, one strain able to grow at 60°C was finally selected and named WL-12. The strain was also examined for determining whether WL-12 can degrade polymeric materials other than galactomannan. Using agar plates supplemented with skim milk 1%, starch 0.2%, tributylin 1% or carboxymethyl cellulose 0.5%, the strain was found to hydrolyze these polymeric materials as determined by the formation of degradation circles around the colonies. Identification of the isolated strain was achieved by examining morphological and biochemical properties and a DNA sequence of 16S rRNA. Gram and spore staining was carried out for the determination of morphological properties and VITEK (bioMerieux Inc. France) was used for the determination of biochemical properties. Found to be aerobic and Gram positive, form spores and have catalase activity, the stain WL-12 was believed to belong to Bacillus.

Using a BAG Card, the strain WL-12 was examined for carbohydrate availability. The strain WL-12 was determined to utilize sucrose, glucose, inositol, arabinose, mannose, salicin, amygdalin, maltose, trehalose and palatinose, but not other carbohydrates.

From a morphological and biochemical property point of view, the strain WL-12 was the most similar to Bacillus licheniformis among Bacillus spp., with 97 % similarity therebetween.

In addition, the DNA sequence of the 16S rRNA-encoding gene of the strain WL-12 was compared with those of other strains. In this regard, PCR was conducted using conservative sequences as primers, with the chromosomal DNA from the strain WL-12 serving as a template. The base sequence of the amplified DNA fragment was compared with those of 16S rRNA from other strains. As a result, the strain WL-12 was found to share the highest homology with Bacillus licheniformis. In consequence, the strain WL-12 belongs to Bacillus licheniformis .

EXAMPLE 2: Cloning of Mannanase Gene of Bacillus licheniformis WL-12

Stage 1: Preparation of DNA library of Bacillus licheniformis WL-12

Bacillus licheniformis WL-12, isolated and identified in Example 1, was inoculated in 200 mL of an LB liquid medium and cultured to the exponential phase. After being harvested by centrifugation from the culture, the biomass was washed with TEN buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 10 mM NaCl) , suspended in 20 ml of an SET buffer (20% sucrose, 50 mM Tris-HCl, pH 7.6, 50 mM EDTA) supplemented with lysozyme, and incubated at 37 °C for 30 ins. To the suspension was added 2 ml of 10% sodium dodecylsulfate (SDS) so as to dissolve the biomass, followed by treatment with an appropriate amount of protease K. The suspension was allowed to stand for 1 hour at 37°C.

After phenol addition and centrifugation, the supernatant thus obtained was combined with two volumes of cold ethanol to precipitate DNA which was then wound around a glass rod. The DNA was washed with 70%, 80% and 90% ethanol in that order and dissolved in an appropriate volume of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

50 μg of the separated chromosomal DNA was partially digested with Sau3AI and electrophoresed on agar gel to obtain a pool of DNA fragments with a size of 1-10 kb. The plasmid pUC19 which was completely cut with BamHI was mixed with the DNA fragment pool in the same amount. Ligation at 14°C for 15 hours in the presence of T4 DNA ligase produced a DNA library of Bacillus WL-12.

Stage 2: Selection of E. coli transformant carrying mannanase gene

E. coli XL-1 was transformed with the DNA library of Bacillus licheniformis WL-12 and spread over an LB agar plate to give about 10,000 colonies. Alternatively, instead of E. coli XL-1, any one selected from among C600, Y1088, Y1090, NM522, K802 and JM105 could be used. They were further cultured under a coverlay medium to observe the formation of galactomannan degradation circles around the colonies. Only one colony was found to be surrounded by such a circle.

EXAMPLE 3: DNA Sequencing of Gene Coding for Mannanase

After the transformant obtained in Example 3 was cultured in an LB liquid medium with ampicillin added (50 mg/L) , the biomass obtained from the medium by centrifugation f was lysed by alkali to separate a plasmid (Birnboim, H. C. and J. Doly, Nucleic Acid Res. 7, 1513-1523 (1979)). Following treatment with various restriction enzymes, the plasmid was analyzed and found to carry a foreign 1.3 kb DNA fragment. It was named pMWLl3. A restriction enzyme map and a cleavage map for pMWL13 are given in FIGS. 1 and 2, respectively.

In FIGS. 1 and 2, the symbol "Man" represents a DNA fragment harboring the mannanase gene of the present invention.

To determine the base sequence of the cloned 1.3 kb DNA fragment, it was digested with various restriction enzymes and the digests of various sizes were inserted into pUCl9 which was then provided for dideoxynucleotide sequencing of the 1,300 bp DNA fragment of interest. The DNA sequence was found to contain an open reading frame with a size of 1,083 bp corresponding to the structural gene of mannanase (Sequence No. 1) . Inferred from the base sequence of the structural gene, the amino acid sequence of the mannanase consists of 361 amino acid residues as given in Sequence No. 2. Comparing of the amino acid sequence inferred from the

DNA sequence of Bacillus licheniformis WL-12 with the amino acid sequences of mannanases of known Bacillus spp., the highest homology shared therebetween was found to be 80 %. Therefore, the mannanase-encoding gene of Bacillus licheniformis WL12 was determined to be novel.

EXAMPLE 4: Activity of Mannanase Expressed in the Transformed E. coli.

The recombinant mannanase expressed in the E. coli transformant carrying pMWLl3 was examined for activity according to temperature and pH conditions. For the gene expression, E. coli transformed with pMWL13 was inoculated in an LB liquid medium with ampicillin added, and incubated at 37°C for 16 hours with agitation. The biomass obtained from the culture was lysed by sonication and the cell lysate was subjected to ammonium sulfate fraction ion chromatography to partially purify the enzyme of interest. The enzyme was measured for mannanase activity in various temperature and pH conditions, with galactomannan from seeds of Acacia leucophloea serving as a substrate. Data shown in FIG. 3 exhibits that the enzyme has maximal activity at 65°C and pH 6.5, with 90% of maximal activity at pH 5.5-6.5. Particularly, at pH 6.5 which is the physiological condition in the small intestine of animals, the activity of the enzyme was measured to amount to as much as 93 % of the maximum. With regard to galactomannan from' seeds of Acacia leucophloea, guar gum glucomannan and konjac, the enzyme was examined for relative activity. The highest activity was found for the galactomannan from seeds of Acacia leucophloea, the next for konjac, and the lowest for guar resin glucomannan, as shown in Table 1 below.

When these results were taken into account, the mannanase produced by the E. coli transformant harboring the mannanase gene derived from Bacillus licheniformis WL-12 was determined to be suitable for use in foodstuff addition. TABLE 1 : Relative Activity of Mannanase for Substrates

EXAMPLE 5: Mannan Hydrolysate of Mannanase Produced by Transformed E. coli

Products of the catalytic hydrolysis of the mannanase obtained by partial purification in Example 4 were analyzed. In this regard, hydrolysates obtained by incubating the mannanase with galactomannan from seeds of Acacia leucophloea under optimal conditions were subjected to TLC analysis. The analysis results are given in FIG. 4. As shown in FIG. 4, mannose, mannobiose and mannotriose were detected as the final products.

With mannobiose, mannotriose, mannotetraose, mannopentose and mannohexose serving as substrates, the enzyme was incubated, followed by the TLC analysis of the hydrolysates thus obtained. The TLC results are given in FIG. 5. As shown, the enzyme of the present invention degraded mannotriose, mannotetraose, mannopentose and mannohexose into mannose, mannobiose and mannotriose without further degradation of mannobiose.

Consequently, it was determined that the mannanase encoded by the mannanase gene of Bacillus lichenformis WL-12 according to the present invention can degrade mannan materials to smaller oligosaccharides.

Advantageous Ef ects

The mannanase expressed in the E. coli transformed with the Bacillus licheniformis WL-12-derived mannanase gene has high activity of hydrolyzing mannans from seeds of Acacia leucophloea, guar gum and konjac. In addition, the mannanase of the present invention is very useful in the food industry because the hydrolysates of the enzyme are oligosaccharides including mannose, mannobiose and mannotriose, which act as growth factors for useful intestinal bacteria.

Claims

1. A nucleotide sequence encoding a polypeptide having mannanase activity, given in SEQ ID NO:l.

2. A recombinant vector harboring the nucleotide sequence of claim 1, given in the cleavage map of FIG. 2.

3. fin I coli strain carrying the recombinant vector of claim 2.

. A polypeptide having mannanase activity, wherein the amino acid sequence of the polypeptide comprises SEQ ID NO: 2.

5. A method for producing mannanase, comprising culturing the E. coli strain of claim 3.

6. Feedstuff for livestock, comprising the polypeptide of claim 4.