CN101851612B - A kind of acid glucanase CELA and its gene and application - Google Patents

Abstract

The invention relates to the field of genetic engineering, in particular to acid glucanase CELA and a gene and application thereof. The invention provides glucanase CELA derived from Alicyclobacillus heperidumA 4(CGMCC No.3147), the amino acid sequence of which is shown in SEQ ID No.1, and the invention provides a gene CelA for coding the glucanase. The glucanase of the invention has the following properties: the optimum pH value is 3.4, and the optimum temperature is 65 ℃; better thermal stability. As a novel enzyme preparation, the enzyme preparation can be widely used in industries of animal feed, food, energy and the like.

Description

A kind of acid glucanase CELA and its gene and application

technical field

The invention relates to the field of genetic engineering, in particular, the invention relates to an acid glucanase CELA and its gene and application.

Background technique

β-glucan is widely found in the aleurone layer and endosperm cell wall of cereals (barley, oats, rye and wheat). It belongs to the structural non-starch polysaccharide in the plant cell wall. , the content and proportion of β-glucan are also different. Among them, barley and oats contain the highest proportion. Glucanase is a general term for a class of enzymes that can degrade dextran into oligosaccharides and glucose. At present, dextranase has been widely used in food, feed, beer, medicine and other fields. At present, barley is mainly used in beer brewing and feed, so the related glucanase has been most widely used in beer brewing and feed industry, especially in Europe, in the feed formula with barley and soybean meal as the main raw materials, The addition of dextranase is more extensive. During the barley germination process, the glucan in the barley cannot be completely decomposed by its own endogenous glucanase, usually only 30%-70% can be decomposed, and the residual glucan will increase the viscosity of the mash and reduce the filtration rate. Finished beer is prone to haze or early gel precipitation. In malt production, adding high-temperature-resistant β-glucanase can significantly reduce the content of β-glucan in finished malt, reduce the viscosity of wort, increase the filtration rate and yield of wort, and help improve the flavor of beer and maintain the quality of finished wine. abiotic stability. Livestock and poultry feeds based on barley, wheat, rye, and oats contain a large amount of β-glucan. Since monogastric animals do not have enzymes to digest β-glucan, they cannot hydrolyze β-glucan in the feed. Dextran. As a non-starch viscous polysaccharide, β-glucan has a high viscosity after absorbing more water in the intestine, which prevents the full contact between the intestinal digestive juice and chyme, thereby affecting the absorption of nutrients, and becomes a kind of Antinutritional Factors. The addition of β-glucanase can effectively eliminate the anti-nutritional effect of β-glucan and greatly improve the utilization efficiency of feed.

In the feed industry, the gastrointestinal tract of animals is an acidic environment (such as the gastrointestinal tract of pigs) and contains a large amount of endogenous proteases. At the same time, there is a short-term high temperature process in the process of feed processing. Therefore, it is of great significance to obtain a new type of acid glucanase with excellent temperature stability and applicability, and resistance to various proteases (especially pepsin). Cloning and isolating acid glucanase with temperature stability and protease resistance can be better applied to feed, and can reduce production costs, all of which are necessary for the application of dextranase in industrial production.

Contents of the invention

The purpose of the present invention is to provide an acid glucanase that can be used efficiently.

Another object of the present invention is to provide a gene encoding the above-mentioned acid glucanase.

Another object of the present invention is to provide a recombinant vector comprising the above gene.

Another object of the present invention is to provide recombinant strains containing the above genes.

Another object of the present invention is to provide a genetic engineering method for preparing the above acid glucanase.

Another object of the present invention is to provide the application of the above acid glucanase.

The present invention is obtained from Alicyclobacillus hesperidum A4, (preserved in the General Microbiology Center of China Microbial Strain Preservation Management Committee (Datun Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101), and its preservation number is: CGMCC No .3147, date of deposit: June 29, 2009), a new acid glucanase CELA was isolated.

The invention provides an acid glucanase CELA, the amino acid sequence of which is shown in SEQ ID NO.1.

SEQ ID NO.1:

MSPSGGVCVNRKQRTLKLGTLAATIVALSAVATPAVASADTTTAIASSTVHVTV

NAAAELGIVPNTALGVNTAVWDGHLLDAAIPSLLRGIGVTMLRYPGGSTSDE

YNWQTNTVTGGYADPNNTFDNFMGVVQKAGAQPIITVNAGTGTPSEAAAW

VQDANVTHHYGVKYWEIGNEMYGSWEAGNFANNPSGYAKEAVSFIQAMKA

VDPSIKIGVDLIAPGTGEDDWNATVLSTMHSLGVLPDFFAIVHWYAQNPGGET

DAGLLSSTNQISTMMDTLKQQLSSYGTIPVFVTETNSVSYNPGRQSTSLVNAL

FLDDDMADWLESGAQNVDWWDLHNGIVTQQAGANVDPNLYGQYNYGDY

GLLSNGSSDNGISEPAANTFPPTYYGYQMLAAVMVPGATMIGAGSNNNDLVAV

HATKLPNGAVDVMLINKDPKQAYTVDLQAEGFAAKGPAFTLFYGQGSNAVTP

GKLDNLQNVTLPPYSVTDIIIPAVPGHQPQGPQFTDKTTLSTPQVKPSANETLT

TTFTDTRGAVKDGTLDVEIYNPAGQLVGQQVQSGVTFTPGQSSQPITWNWTA

PDSPGTYTVKAFVFSQDGTSVYAADPSAATFTVTQPDPPTISATVQLSATTVK

VGTPVTITTTYTETAPTGYLNNGLLVQYAVYNNWTSSQQSNPTATLTPGQSVT

ETWTFTPEQAGTYTFPEGIFTSGWTQLQWINQNVTLTVTN

The enzyme gene encodes 715 amino acids, so the theoretical molecular weight of glucanase CELA is 75.4kDa.

The glucanase CELA of the present invention has relatively high activity in the acid range. The present invention screens out a glucanase produced by Alicyclobacillus hesperidum A4 (CGMCCNo.3147), the optimal pH value of which is 3.4 for the recombinant enzyme of Pichia pastoris, which is in the range of pH2.8-4.2 Maintain more than 80% of the enzyme activity within the range of pH 1.2 to 7.8 and keep at 37°C for 60 minutes to maintain more than 80% of the enzyme activity. The optimum temperature is 65°C, and there is more than 50% of the enzyme activity between 45°C and 75°C; when incubated at 70°C for 60 minutes, the remaining enzyme activity reaches more than 80%. Glucanases of this nature have not been reported.

The present invention provides a gene encoding the above-mentioned acid glucanase CELA. Specifically, the gene sequence of the gene is shown in SEQ ID NO.2:

SEQ ID NO.2:

ATGTCACCTTCAGGGGGAGTCTGTGTGAACCGAAAACAGCGTACGTTAAAGTT

GGGAACGCTCGCAGCAACGATTGTGGCACTCTCAGCCGTGGCCACGCCTGCGG

TCGCCAGTGCGGATACGACGACGGCCATTGCGTCATCGACAGTTCATGTCACAG

TCAATGCCGCTGCTGAACTTGGAATCGTGCCCAATACTGCACTTGGTGTGAATA

CGGCCGTCTGGGACGGGCATTTACTCGATGCAGCCATTCCATCCTCTGCTTCGTG

GCATTGGGGTAACCATGTTGCGATATCCCGGAGGATCGACTTCAGATGAGTACA

ATTGGCAAACGAATACCGTAACTGGGGGTTATGCAGATCCCAACAACACCTTTG

ACAACTTCATGGGAGTGGTCCAAAAGGCTGGTGCGCAACCCATTATTACGGTC

AACGCCGGCACGGGCACACCGAGTGAAGCTGCCGCATGGGTTCAAGATGCAA

ATGTCACGCACCACTACGGTGTCAAGTATTGGGAAATCGGAAATGAGATGTATG

GCAGCTGGGAAGCAGGGAATTTTGCAAATAACCCATCTGGTTATGCGAAAGAA

GCTGTATCCTTCATTCAGGCCATGAAAGCGGTTGATCCTTCTATTAAAATCGGCG

TGGACCTCATCGCACCTGGTACTGGAGAAGATGACTGGAATGCAACCGTGCTT

AGCACCATGCACAGCCTTGGGGTGCTTCCGGACTTCGCCATTGTGCACTGGTAT

GCGCAAAATCCGGGAGGCGAGACGGACGCTGGGCTGCTCAGTTCGACGAATC

AAATTTCGACGATGATGGATACCTTGAAGCAGCAATTGAGCTCCTATGGAACCA

TCCCGGTTTTCGTCACCGAAACGAATTCGGTTTCGTATAACCCTGGGCGCCAGA

GTACAAGTCTGGTTAATGCACTGTTCCTCGATGATGACATGGCAGACTGGCTTG

AGTCCGGAGCGCAAAACGTCGACTGGTGGGACTTGCATAACGGCATTGTTACA

CAACAGGCCGGTGCAAATGTCGATCCGAATCTGTATGGGCAGTATAACTACGGA

GACTATGGACTTTTGTCCAATGGCTCGAGTGACAATGGCATTTCAGAACCGGCT

GCCAATACCACCATTCCCAACGTATTATGGATACCAAATGCTCGCAGCCGTCATGG

TGCCAGGAGCGACGATGATCGGTGCCGGATCGAACAATGATCTGGTGGCCGTG

CATGCGACCAAGTTGCCCAATGGTGCTGTCGACGTCATGTTGATCAACAAGGAT

CCGAAACAAGCGTATACCGTCGATTTGCAAGCTGAAGGATTTGCTGCTAAGGGT

CCTGCATTCACGTTATTCTACGGGCAAGGCAGTAACGCGGTGACGCCGGGCAA

ATTGGATAATCTGCAGAATGTGACACTACCGCCCTATTCTGTGACGGACATCATC

ATACCGGCGGTGCCCGGGCATCAGCCACAAGGGCCACAGTTTACGGACAAGAC

GACGTTATCCACTCCTCAGGTAAAGCCTAGTGCCAACGAGACTTTGACCACGA

CGTTTACCGACACGCGTGGTGCGGTCAAGGATGGTACACTCGACGTGGAAATC

TACAATCCAGCAGGGCAATTGGTTGGGCAACAAGTGCAGTCTGGCGTGACGTT

TACGCCTGGGCAATCATCTCAACCGATTACCTGGAACTGGACGGCGCCCGATTC

TCCTGGGACGTATACCGTGAAGGCGTTCGTCTTCAGCCAAGACGGAACAAGCG

TGTATGCGGCAGACCCGAGTGCAGCTACGTTCACGGTCACACAGCCGGATCCG

CCCACCATTTCGGCCACCGTTCAGCTGTCCGCAACTACGGTCAAAGTGGGTAC

ACCTGTGACCATCACGACGACTTACACCGAAACCGCGCCTACGGGGTACCTGA

ACAACGGGTTGCTTGTACAGTACGCCGTGTACAATAACTGGACATCATCGCAAC

AGTCCAATCCAACTGCGACATTGACTCCTGGGCAATCGGTGACTGAGACTTGG

ACATTTACGCCAGAGCAGGCCGGAACCTACACATTCCCTGAAGGCATCTTTACC

AGTGGATGGACACAATTGCAGTGGATTAATCAGAACGTGACCTTGACTGTGAC

AAACTAA

The present invention isolates and clones the glucanase gene CELA through the PCR method, and the DNA sequence analysis result shows that the full length of the glucanase CELA structural gene CELA is 2148bp, and contains a terminator TAA. The theoretical molecular weight of the mature protein of glucanase CELA is 75.4kDa. The glucanase gene CELA sequence and the deduced amino acid sequence were compared by BLAST in GenBank. The gene has the highest amino acid sequence identity of 44% with the cellulase (CAD86595) derived from Alicyclobacillus acidocaldarius, indicating that CELA is a new glucanase.

The present invention also provides a recombinant vector comprising the above-mentioned glucanase gene, preferably pPIC9-CelA. The dextranase gene of the present invention is inserted between suitable restriction enzyme cutting sites of the expression vector, so that its nucleotide sequence is operably linked with the expression control sequence. As a most preferred embodiment of the present invention, it is preferred that the glucanase gene is inserted between SnaBI and the NotI restriction enzyme site on the plasmid pPIC9, so that the nucleotide sequence is positioned at the downstream of the AOX1 promoter and Under its regulation, the recombinant yeast expression plasmid pPIC9-CelA was obtained.

The present invention also provides a recombinant strain comprising the above glucanase gene, preferably the recombinant strain GS115/CelA.

The present invention also provides a method for preparing acid glucanase CELA, comprising the following steps:

1) Transforming host cells with the above-mentioned recombinant vectors to obtain recombinant strains;

2) cultivating the recombinant strain to induce the expression of recombinant glucanase CELA; and

3) Recover and purify the expressed glucanase CELA.

Wherein, the host cell is preferably Pichia cells, Saccharomyces cerevisiae cells or Sinusia polymorpha cells, and the recombinant yeast expression plasmid is preferably transformed into Pichia pastoris cell (Pichic pastoris) GS115 to obtain the recombinant strain GS115/CelA.

The present invention also provides the application of the above acid glucanase CELA.

The first technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a new dextranase with excellent properties and suitable for application in the food industry, especially the feed industry. The optimum pH of the glucanase of the present invention is 3.4, and all have higher enzymatic activity (more than 80%) in the scope of pH 2.8～4.2; pH stability is good under acidic conditions; Ability and resistance to various proteases (especially pepsin). Its high heat resistance can effectively reduce the loss of enzyme activity during feed processing and reduce the application cost of dextranase. The optimum pH is in the acidic range and good protease resistance (especially pepsin) can improve the conversion rate of non-starch polysaccharides in feed, reduce formulation costs, and reduce environmental pollution; it can also be used in bioenergy, such as making paper Glucan in industrial waste and agricultural waste is converted into D-glucose monomer, which is then metabolized by most microorganisms and converted into valuable fuel. Hydrolyzed products (glucose and oligoglucose) can be used in the health food industry; in the pharmaceutical industry, dextran is used in combination with other substances to delay the release of pharmaceutical ingredients.

Description of drawings

Fig. 1 SDS-PAGE analysis of recombinant glucanase expressed in Pichia pastoris, wherein, 1: low molecular weight protein Marker; 2: purified recombinant glucanase.

Figure 2 Optimum pH of recombinant dextranase.

Figure 3 pH stability of recombinant dextranase.

Figure 4 Optimum temperature of recombinant dextranase.

Figure 5 Thermostability of recombinant dextranase.

Figure 6 Various protease resistance of recombinant dextranase

Alicyclobacillus hesperidum A4 CGMCC3147, preserved in the General Microbiology Center of China Committee for Culture Collection of Microorganisms (Datun Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101), its preservation number is: CGMCCNo.3147, preserved Date: June 29, 2009.

Detailed ways

Test materials and reagents

1. Bacterial strains and carriers: Alicyclobacillus hesperidum A4 was isolated and obtained by the inventor, and was preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee (Datun Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101), Its deposit number is: CGMCCNo.3147. Pichia pastoris expression vector pPIC9 and strain GS115 were purchased from Invitrogen.

2. Enzymes and other biochemical reagents: endonucleases were purchased from TaKaRa Company, and ligases were purchased from Invitrogen Company. Barley dextran was purchased from Sigma Company, and the others were domestic reagents (all of which can be purchased from common biochemical reagent companies).

3. Medium:

(1) The culture medium of Alicyclobacillus peridum A4CGMCC3147 consists of: 0.2% peptone, 0.1% yeast extract, 0.2% glucose, pH3.0.

(2) BMGY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% glycerol (V/V).

(3) BMMY medium: replace glycerol with 0.5% methanol, and the rest of the ingredients are the same as BMGY.

Explanation: For the molecular biology experimental methods not specifically described in the following examples, all refer to the specific methods listed in the book "Molecular Cloning Experiment Guide" (Third Edition) J. Sambrook, or follow the kit and product manual.

Example 1 Isolation and purification of Alicyclobacillus peridum A4 (CGMCC No.3147) and its enzyme-producing properties

Inoculate Alicyclobacillus speridum A4 on the enzyme-producing medium plate (yeast extract 1.0g, tryptone 2.0g, oat dextran 5.0g, agar 30, Congo red 0.5g, adjust the pH to 3.0 with hydrochloric acid) at 60°C After culturing for 72 hours, it was stained with 0.5% Congo red, and it was preliminarily verified that it had glucanase activity according to the presence or absence and size of the transparent circle. Alicyclobacillus speridum A4 was cultured at 60°C for 48 hours in an enzyme-producing medium (1.0 g of yeast extract, 2.0 g of tryptone, 5.0 g of oat dextran, adjusted to pH 3.0 with sulfuric acid), and the glucose content of the supernatant was determined. Glycanase activity. Proved to have dextranase activity.

Example 2 Cloning of Alicyclobacillus peridum A4 (CGMCC No.3147) Glucanase Encoding Gene CELA

Acquisition of gene sequence

According to the conserved (YWEIGNE and AMKAVD) sequence of the 51st family glucanase gene, degenerate primers Cel51F and Cel51R were designed and synthesized as shown in Table 1

PCR amplification was performed using the total DNA of Alicyclobacillus hesperidum A4 (CGMCC No.3147) as a template. The PCR reaction parameters are: denaturation at 94°C for 5 min; the first 10 cycles, denaturation at 94°C for 30 sec, touchdown (0.5°C/cycle) at 50°C-55°C for 30 sec, extension at 72°C for 0.5 min, and the last 25 cycles at 94°C Denaturation for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 1.5 min. Finally, keep warm at 72°C for 10 minutes. A fragment of about 180bp was obtained, which was recovered and connected with the pEASY-T3 vector and sent to Sanbo Biotechnology Co., Ltd. for sequencing.

According to the nucleotide sequence obtained by sequencing, three TAIL-PCR specific nested primers were designed upstream and downstream respectively: the design direction was the direction of the unknown region to be amplified, and they were respectively named usp1, usp2, usp3 (upstream specific primers ), dsp1, dsp2, dsp3 (downstream specific primers) are shown in Table 1.

Table 1. Dextranase CelA TAIL-PCR specific primers

N stands for A, G, C or T; H stands for T, A or C; R stands for A or G; Y stands for C or T

The flanking sequence of the known gene sequence was obtained by TAIL-PCR, and the amplified product was recovered and then sequenced.

After comparing the genome sequence of glucanase, it was found that the gene was 2148bp in full length, encoded 715 amino acids and a stop codon, and the N-terminal 1-39 amino acid residues were signal peptide sequences. The amino acid sequence of the deduced gene CELA is homologously compared with the glucanase gene sequence on the GeneBank, and the highest identity is 44%, indicating that CELA is a new glucanase, which shows that it is derived from AlicyclobacillussperidumA4 (CGMCCNo.3147 The gene encoding dextranase isolated and cloned in ) is a new gene.

Alicyclobacillus peridum A4 (CGMCCNo.3147) genomic DNA was extracted:

Centrifuge the cultured bacteria for 2 days, add 1mL lysozyme, treat at 37°C for 60min, then add lysate, lyse in a water bath at 65°C for 30min, mix every 10min, and centrifuge at 10,000rpm at 4°C for 5min. The supernatant was extracted in phenol/chloroform to remove impurity proteins, and then an equal volume of isopropanol was added to the supernatant. After standing at room temperature for 5 minutes, centrifuge at 10,000 rpm for 10 minutes at 4°C. The supernatant was discarded, the precipitate was washed twice with 70% ethanol, dried in vacuum, dissolved by adding an appropriate amount of TE, and stored at -20°C for later use.

According to the sequence information of CelA, the primers CelAF and CelAR were designed and synthesized as shown in Table 1.

The total DNA of Alicyclobacillus peridum A4 (CGMCC No.3147) was used as template for PCR amplification. The PCR reaction parameters were: denaturation at 95°C for 5 min, denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 120 s, and 30 cycles. Finally, keep warm at 72°C for 10 minutes.

Example 3 Preparation of recombinant dextranase.

The expression vector pPIC9 is subjected to double digestion (SnaBI and NotI), and the gene CelA encoding glucanase is double-digested (SnaBI and NotI) at the same time, and the gene fragment encoding glucanase is cut out and connected to the expression vector pPIC9 to obtain The recombinant plasmid pPIC9-CelA containing the glucanase gene CelA was transformed into Pichia pastoris GS115 to obtain the recombinant Pichia pastoris strain GS115/CelA.

Take the GS115 strain containing the recombinant plasmid, inoculate it in 400mL BMGY culture medium, shake it at 250rpm at 30°C for 48h, and collect the bacteria by centrifugation. Then resuspend in 200mL BMMY medium, shake culture at 250rpm at 30°C. After 48 hours of induction, the supernatant was collected by centrifugation. Determination of dextranase activity. The expression level of recombinant glucanase was 20.41U/mL. SDS-PAGE results (Figure 1) showed that the recombinant glucanase was expressed in Pichia pastoris.

Activity Analysis of Embodiment 4 Recombinant Glucanase

DNS method: the specific method is as follows: at pH 3.4 (0.1M sodium dihydrogen phosphate-citric acid), at 65°C, 1 mL of reaction system includes 100 μL of appropriate diluted enzyme solution and 900 μL (1%, w/v) bottom The mixture was reacted for 10 minutes, and 1.5mL DNS was added to terminate the reaction, and boiled for 5 minutes. After cooling, the OD value was measured at 540 nm. One enzyme activity unit (U) is defined as the amount of enzyme that releases 1 μmol of reducing sugar per minute under given conditions.

The property determination of embodiment 5 recombinant glucanase CELA

1. The optimum pH and pH stability determination methods of recombinant glucanase CELA are as follows:

The recombinant glucanase purified in Example 3 was subjected to enzymatic reactions at different pHs to determine its optimum pH. The substrate dextran was buffered with different pH (0.1M glycine-hydrochloric acid pH 1.2-2.6; 0.1mol/L citric acid-disodium hydrogen phosphate pH 3.0-7.4; 0.1M Tris-HCl pH 7.8-8.6), at 65 Determination of glucanase activity was carried out at ℃. The results ( FIG. 2 ) showed that the optimum pH of CELA was 3.4, and more than 80% of the enzyme activity was maintained in the range of pH 2.8-4.2. Dextranase was treated at 37°C for 60min in the above-mentioned buffers with different pH, and then the enzyme activity was measured at 65°C in the pH3.4 buffer system to study the pH tolerance of the enzyme. The result (Fig. 3) shows that the dextranase is very stable between pH 1.2-7.8, and the remaining enzyme activity is above 80% after 60 minutes of treatment in this pH range, which shows that the enzyme has good pH stability.

2. The optimal temperature and thermostability determination method of dextranase is as follows:

Determination of optimum temperature of dextranase is carried out enzymatic reaction in citric acid-disodium hydrogen phosphate buffer (pH3.4) buffer solution system and different temperatures. The temperature resistance was measured by treating the dextranase at different temperatures for different times, and then measuring the enzyme activity at 65°C. The measurement results of the optimum temperature of the enzyme reaction (Fig. 4) showed that the optimum temperature was 65°C. The thermostability test of the enzyme showed (Fig. 5) that the recombinant enzyme had very good stability at 70°C. After incubation at 70°C for 60 minutes, the remaining enzyme activity is 84%, and the enzymes all have more than 50% enzyme activity at 45°C-75°C.

3. The influence of different metal ion chemical reagents on XYLA4 enzyme activity was determined as follows:

Add different concentrations of different metal ions and chemical reagents to the enzymatic reaction system to study their effects on enzyme activity. The final concentrations of various substances are 1 and 10mmol/L. Enzyme activity was measured at 65°C and pH 3.4. The results (Table 1) showed that, except for SDS, most ions and chemical reagents had no significant change in the activity of recombinant dextranase at a concentration of 1 mmol. Co ²⁺ , Cr ³⁺ , Fe ²⁺ , Pb ²⁺ , Cu ²⁺ , Ag ⁺ , Hg ²⁺ ' and SDS strongly inhibited its activity when the concentration was 10mmo. β-mercaptoethanol can increase the activity of recombinase to 1.16 and 1.39 times at 1mmol and 10mmol respectively.

Table 1 Effects of various chemical reagents on the activity of dextranase CELA

Example 6 Effect of Recombinant Glucanase on the Viscosity of Barley Soybean Meal Type Diet in Artificial Gastric Juice

The specific method is as follows: 1g of barley soybean meal was dissolved in 9mL of artificial gastric juice containing 1U CelA, and the mixed solution was placed on a shaker at 37°C at 180rpm for 1h. In order to determine the effect of hydrolyzate accumulation as a function of pH conditions during digestion (Haraldsson et al., 2005), CelA from barley in artificial gastric juice was tested according to the pH gradient model described by Minekus et al. (1995). The cumulative effect of hydrolysis in soybean meal-based diets was studied in experimental simulations. The entire pH gradient was carried out according to the following strategy: pH 2.0, 20min; pH 2.3, 20min; pH 2.8, 20min; pH 3.8, 10min; pH 4.6, 10min; pH 5.5, 10min. During this process the pH of the solution was adjusted by HCl or NaOH on ice. The final cumulative effect of action is expressed by measuring the viscosity. Viscosity was measured with a capillary viscometer. Specifically, pass the feed liquid through the above-mentioned treatment through Xinhua No. 1 filter paper, take 5ml of the filtrate, and measure it by a capillary viscometer.

The results showed that the viscosity of barley and soybean meal treated with enzyme solution was 36.72% lower than that of the control. It shows that CelA can be well applied in the feed industry.

sequence listing

<110> Feed Research Institute, Chinese Academy of Agricultural Sciences

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<120> A kind of acid glucanase CELA and its gene and application

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<213> Alicyclobacillus hesperidum A4

the

<400>1

MSPSGGVCVN RKQRTLKLGT LAATIVALSA VATPAVASAD TTTAIASSTV

HVTVNAAAEL 60

GIVNTALGV NTAVWDGHLL DAAIPSLLRG IGVTMLRYPG GSTSDEYNWQ

TNTVTGGYAD 120

PNNTFDNFMG VVQKAGAQPI ITVNAGTGTP SEAAAWVQDA NVTHHYGVKY

WEIGNEMYGS 180

WEAGNFANNP SGYAKEAVSF IQAMKAVDPS IKIGVDLIAP GTGEDDWNAT

VLSTMHSLGV 240

LPDFAIVHWY AQNPGGETDA GLLSSTNQIS TMMDTLKQQL SSYGTIPVFV

TETNSVSYNP 300

GRQSTSLVNA LFLDDDMADW LESGAQNVDW WDLHNGIVTQ QAGANVDPNL

YGQYNYGDYG 360

LLSNGSSNG ISEPAANTPF PTYYGYQMLA AVMVPGATMI GAGSNNDLVA

VHATKLPNGA 420

VDVMLINKDP KQAYTVDLQA EGFAAKGPAF TLFYGQGSNA VTPGKLDNLQ

NVTLPPYSVT 480

DIIIPAVPGH QPQGPQFTDK TTLSTPQVKP SANETLTTTF TDTRGAVKDG

TLDVEIYNPA 540

GQLVGQQVQS GVTFTPGQSS QPITWNWTAP DSPGTYTVKA FVFSQDGTSV

YAADPSAATF 600

TVTQPDPPTI SATVQLSATT VKVGTPVTIT TTYTETAPTG YLNNGLLVQY

AVYNNWTSSQ 660

QSNPTATLTP GQSVTETWTF TPEQAGTYTF PEGIFTSGWT QLQWINQNVT

LTVTN 715

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atgtcacctt caggggggagt ctgtgtgaac cgaaaacagc gtacgttaaa gttgggaacg 60

ctcgcagcaa cgattgtggc actctcagcc gtggccacgc ctgcggtcgc cagtgcggat 120

acgacgacgg ccattgcgtc atcgacagtt catgtcacag tcaatgccgc tgctgaactt 180

ggaatcgtgc ccaatactgc acttggtgtg aatacggccg tctgggacgg gcatttactc 240

gatgcagcca ttccatctct gcttcgtggc attggggtaa ccatgttgcg atatcccgga 300

ggatcgactt cagatgagta caattggcaa acgaataccg taactggggg ttatgcagat 360

cccaacaaca cctttgacaa cttcatggga gtggtccaaa aggctggtgc gcaacccatt 420

attacggtca acgccggcac gggcacaccg agtgaagctg ccgcatgggt tcaagatgca 480

aatgtcacgc accactacgg tgtcaagtat tgggaaatcg gaaatgagat gtatggcagc 540

tgggaagcag ggaattttgc aaataaccca tctggttatg cgaaagaagc tgtatccttc 600

attcaggcca tgaaagcggt tgatccttct attaaaatcg gcgtggacct catcgcacct 660

ggtactggag aagatgactg gaatgcaacc gtgcttagca ccatgcacag ccttggggtg 720

cttccggact tcgccattgt gcactggtat gcgcaaaatc cgggaggcga gacggacgct 780

gggctgctca gttcgacgaa tcaaatttcg acgatgatgg ataccttgaa gcagcaattg 840

agctcctatg gaaccatccc ggttttcgtc accgaaacga attcggtttc gtataaccct 900

gggcgccaga gtacaagtct ggttaatgca ctgttcctcg atgatgacat ggcagactgg 960

cttgagtccg gagcgcaaaa cgtcgactgg tgggacttgc ataacggcat tgttacacaa 1020

caggccggtg caaatgtcga tccgaatctg tatgggcagt ataactacgg agactatgga 1080

cttttgtcca atggctcgag tgacaatggc atttcagaac cggctgccaa tacaccattc 1140

ccaacgtatt atggatacca aatgctcgca gccgtcatgg tgccaggagc gacgatgatc 1200

ggtgccggat cgaacaatga tctggtggcc gtgcatgcga ccaagttgcc caatggtgct 1260

gtcgacgtca tgttgatcaa caaggatccg aaacaagcgt ataccgtcga tttgcaagct 1320

gaaggatttg ctgctaaggg tcctgcattc acgttattct acgggcaagg cagtaacgcg 1380

gtgacgccgg gcaaattgga taatctgcag aatgtgacac taccgcccta ttctgtgacg 1440

gacatcatca taccggcggt gcccgggcat cagccacaag ggccacagtt tacggacaag 1500

acgacgttat ccactcctca ggtaaagcct agtgccaacg agactttgac cacgacgttt 1560

accgacacgc gtggtgcggt caaggatggt acactcgacg tggaaatcta caatccagca 1620

gggcaattgg ttgggcaaca agtgcagtct ggcgtgacgt ttacgcctgg gcaatcatct 1680

caaccgatta cctggaactg gacggcgccc gattctcctg ggacgtatac cgtgaaggcg 1740

ttcgtcttca gccaagacgg aacaagcgtg tatgcggcag acccgagtgc agctacgttc 1800

acggtcacac agccggatcc gcccaccatt tcggccaccg ttcagctgtc cgcaactacg 1860

gtcaaagtgg gtacacctgt gaccatcacg acgacttaca ccgaaaccgc gcctacgggg 1920

tacctgaaca acgggttgct tgtacagtac gccgtgtaca ataactggac atcatcgcaa 1980

cagtccaatc caactgcgac attgactcct gggcaatcgg tgactgagac ttggacattt 2040

acgccagagc aggccggaac ctacacattc cctgaaggca tctttaccag tggatggaca 2100

caattgcagt ggattaatca gaacgtgacc ttgactgtga caaactaa 2148

Claims (9)

1. an acid glucanase CELA is characterized in that, its aminoacid sequence is shown in SEQ ID NO.1.

2. an acidic dextranase gene C elA is characterized in that, the described acid glucanase CELA of coding claim 1.

3. acidic dextranase gene C elA as claimed in claim 2 is characterized in that its base sequence is shown in SEQ ID NO.2.

4. the recombinant vectors that comprises the described acidic dextranase gene C of claim 3 elA.

5. recombinant vectors according to claim 4 is characterized in that, the described recombinant vectors that comprises acidic dextranase gene C elA is pPIC9-CelA.

6. the recombinant bacterial strain that comprises the described acidic dextranase gene C of claim 3 elA.

7. recombinant bacterial strain as claimed in claim 6 is characterized in that, described bacterial strain is a pichia spp.

8. a method for preparing acid glucanase CELA is characterized in that, may further comprise the steps:

1) with the recombinant vectors transformed host cell of claim 4, gets recombinant bacterial strain;

2) cultivate recombinant bacterial strain, induce the reorganization dextranase to express; And

3) reclaim the also expressed dextranase CELA of purifying.

9. the described acid glucanase CELA of claim 1 is in the application of fodder industry.

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