High-temperature mannase and application thereof

Abstract

The invention provides high-temperature mannase and application thereof. The amino acid sequence of the high-temperature mannase is SEQ ID NO: 1. The mannase has the strongest hydrolysis effect on konjac gum, can hydrolyze guar gum and has a wide acting temperature and pH range, the enzyme activity is kept at 95% or above within the range of 45-75 DEG C, the enzyme activity is kept at 65% or above within the range of pH 5.5-8.5, and various common metal ions can promote the enzyme activity or have little influence on the enzyme activity. The enzyme is beta-mannase with endo-activity, and has a good application prospect in the aspects of feed processing, mannan oligosaccharide preparation, biological polishing, agricultural waste utilization, plant active substance extraction and the like under medium and high temperature conditions.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a high-temperature mannanase and application thereof. BACKGROUND

[0002] Mannan is widely distributed in plant cell walls and is one of the important components of hemicellulose. The main chain of mannan is mainly composed of mannose ( / mannose and glucose) connected by a beta-1, 4-glycosidic bond, and the side chain often contains galactose, glucose or acetyl groups and other modified components. In addition, mannan is the main component of some plant tubers and seeds, such as konjac gum (glucomannan), guar gum (galactomannan) and locust bean gum (galactomannan). Beta-mannanase can specifically hydrolyze the beta-1, 4-glycosidic bond in mannan to generate functional mannose oligosaccharides, and has the advantages of green, environmental protection and high efficiency. It has great application potential in the fields of feed, textile, papermaking, food, bioenergy, pharmaceutical and health products, oil and gas exploitation (reducing the viscosity of guar gum fracturing fluid) and the like.

[0003] Mannanase is widely present in microorganisms. Due to the advantages of fast growth, simple genetic structure and easy operation, bacteria have attracted more attention. The optimal temperature of the reported mannanases is generally between 40-70℃, and most of them have a narrow temperature range, so their application scenarios are limited. Many industrial processes involve different high-temperature environments, such as feed processing, oil exploitation, food processing, etc. Mannanases with effective hydrolysis activity under wide high-temperature conditions have good application prospects. SUMMARY

[0004] The purpose of the present application is to provide a high-temperature mannanase (named Man-3) and its application, so as to make up for the shortcomings of the prior art.

[0005] The present application first provides a mannanase, which comprises an enzyme with an amino acid sequence of SEQ ID NO: 1: CKESTSPQSLDPRLEELNQAVLAGKTLLKNSAAGEGEGEFPQSAFEVCRETLNRAERFIKQAKKSTGQAKIDSVTNEVYDVLTEFEASVNSSLNELTDTRATKQTRYLYDNLKRISPQRLLFGMHEALGYGVGWSGMDTRSDVKDVSGDYPAVFSWDAYHIFNASADDLEHYTFQVKYAYQNGGVTTFCWHQYDPEKRGFYAKKVNYEVVASILPGGNYHRLYKNKLKKLARFFKRLRGEKGETIPVIFRPYHEQNGNWFWWGKGHRSEEQYIQLWRFTVHYLRDTLGVHNFIYAFSPDGNQFSNKSEYLNDYPGDDVVDILGLDFYFGRGDEEEIRRFQKRVVYAVQFAEEKNKLAALTEVGDYYGFSGDEANLKIPNWFTRCFLQPIKYHSQAKKIAYGAVWRNASKTHHFAPYPGHPSVPDFMDFYNDDFTLFLSDLPDMYRFKTPISD (SEQ ID NO: 1); A gene for encoding the above-mentioned mannanase, wherein a specific nucleotide sequence is SEQ ID NO: 2: In another aspect of the present application, a recombinant expression vector is provided, wherein a nucleic acid fragment encoding the above-mentioned mannanase is inserted into the recombinant expression vector. The recombinant expression vector is either a prokaryotic expression vector or a eukaryotic expression vector.

[0006] In another aspect of the present application, a genetically engineered strain is provided, wherein the above-mentioned recombinant expression vector is transformed into the genetically engineered strain. The genetically engineered strain can be either a eukaryotic engineered strain or a prokaryotic engineered strain. As an embodiment, the genetically engineered strain is an engineered Escherichia coli strain.

[0007] The present application also provides the use of the above-mentioned mannanase in degrading konjac gum or guar gum.

[0008] The present application also provides a method for preparing oligosaccharides, wherein the above-mentioned mannanase is used to degrade konjac gum or guar gum to prepare oligosaccharides.

[0009] The oligosaccharides are a mixture of disaccharides or oligosaccharides with more than two saccharides.

[0010] The present application also provides the use of the above-mentioned mannanase in feed processing, plant active substance extraction, and bioenergy production.

[0011] The high-temperature mannanase Man-3 of the present application has the strongest hydrolysis effect on konjac gum and can also hydrolyze guar gum. The enzyme has a wide temperature range for action, and the optimal temperature for action is 60℃. The enzyme activity is high in the range of 45~75℃, and the relative enzyme activity is maintained at more than 95%. The enzyme also has a wide pH range for action, and the optimal pH is 7.0. The enzyme activity is maintained at more than 65% in the pH range of 5.5~8.5. In addition, various common metal ions can promote the activity of the enzyme or have little effect on the activity of the enzyme. The enzyme is a β-mannanase with endo-activity, and has a good application prospect in feed processing, preparation of mannose oligosaccharides, extraction of plant active substances, and bioenergy production under medium-high temperature conditions. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 The figure is the effect of the enzyme on the hydrolysis of different substrates; Figure 2 The figure is the effect of different temperatures on the enzyme activity; Figure 3 The figure is the effect of different pHs on the enzyme activity; Figure 4 The figure is the effect of different metal ions on the enzyme activity; Figure 5 The figure is the analysis of the hydrolysis products of the enzyme. DETAILED DESCRIPTION

[0013] This invention identifies a novel mannanase, Man-3, from metagenomic sequencing data of deep-sea hydrothermal sulfide samples, comprising: 1) An enzyme with the amino acid sequence SEQ ID NO:1; 2) A gene encoding the above enzyme, the specific nucleotide sequence of which is SEQ ID NO:2, may be a nucleic acid sequence optimized based on the host bacterium.

[0014] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings.

[0015] Example 1 Preparation of mannanase Man-3 Escherichia coli ( Escherichia coli Top10 and BL21(DE3) strains (purchased from Shanghai Sangon Biotech); expression vector pET28a-SUMO (purchased from Novagen); restriction endonucleases Bam HI and Xho I (purchased from TransGen); T4 DNA ligase (purchased from Takara); LB medium (containing 10g peptone, 5g yeast extract, and 10g NaCl per liter); kanamycin (purchased from Shanghai Sangon Biotech); binding buffer (20mM phosphate, pH 7.4); wash buffer (500mM NaCl, 20mM / 50mM imidazole, 20mM phosphate, pH 7.4); elution buffer (500mM NaCl, 500mM imidazole, 20mM phosphate, pH 7.4); Protein Iso ® Ni-NTA Resin (purchased from TransGen); 30 kDa ultrafiltration tubes (purchased from Taijing Company); 3,5-dinitrosalicylic acid (DNS) (purchased from Shanghai Sangon Biotech); standards for mannose (M1), mannobiose (M2), mannotriose (M3), mannotetraose (M4) and mannopentasose (M5) (purchased from Megazyme); Konjac gum, guar gum and locust bean gum (purchased from Solarbio); sodium carboxymethyl cellulose (CMC-Na) (purchased from Megazyme); LB medium containing kanamycin used in this invention, with a kanamycin concentration of 50 μg / mL.

[0016] A mature protein sequence SEQ ID NO: 1 of a mannanase Man-3 was obtained by analyzing a deep-sea hydrothermal sulfide sample metagenome sequencing database in the northwest Indian Ocean. The gene sequence encoding the mature protein of Man-3 was optimized and synthesized in full sequence to make it suitable for expression in an Escherichia coli host, and an optimized nucleotide sequence SEQ ID NO: 2 was obtained. Analysis by NCBI online BlastP tool found that the mature protein sequence of the mannanase Man-3 has an amino acid sequence identity of 95.58% with the β-mannosidase derived from thermophilic bacteria. Calditrichota Analysis by NCBI online CD-Search tool found that Man-3 has a typical catalytic domain ManB2 of β-mannanase, and it is inferred that Man-3 may belong to a kind of endo-active β-mannanase and does not belong to the category of exo-enzyme active β-mannosidase.

[0017] The specific process of recombinantly expressing the enzyme is described below.

[0018] (1) Construction of recombinant plasmid The 5' and 3' ends of the artificially synthesized mannanase Man-3 gene sequence were introduced with Bam HI and Xho I two enzyme cutting sites, respectively. After the artificially synthesized DNA fragment of the mannanase Bam gene with the enzyme cutting sites of Xho HI and Man-3 I was cut by restriction enzymes Bam HI and Xho I, it was connected to a pET28a-SUMO vector cut by the same restriction enzymes using T4 DNA ligase. The ligation product was mixed with Escherichia coli E. coli Top10 competent cells, and placed on ice for 30 min. After heat shock at 42°C for 90 s, 150 μL of LB liquid medium was added, and the cells were recovered at 37°C and 100 rpm for 1 h. After centrifugation, the cells were spread on LB solid medium containing kanamycin and cultured overnight at 37°C. The recombinant plasmid was screened, and the recombinant plasmid was verified by double enzyme digestion and sequencing to obtain a recombinant plasmid containing the mannanase gene.

[0019] (2) Induced expression of the gene and preparation of crude enzyme solution The recombinant plasmid containing the mannanase Man-3 gene was transformed into E. coliThe recombinant strain containing the mannanase gene was obtained in BL21 (DE3). The recombinant strain was inoculated in 5 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured overnight, inoculated in 500 mL of LB liquid medium containing kanamycin at an inoculation amount of 1%, and cultured at 37°C, 180 rpm until the OD 600 was about 0.6, and IPTG was added to a final concentration of 0.5 mmol / L for induction, and the expression was induced overnight at 18°C, 180 rpm. The bacterial cells were collected by centrifugation at 8000 rpm for 10 min. Then the bacterial cells were resuspended in 25 mL of binding buffer, and an appropriate amount of protease inhibitor was added. The ultrasonic crushing was performed at a power of 300 W, and the working / gap time was 3 s / 5 s for 30 min. The supernatant was collected by centrifugation at 4°C, 5000 rpm for 10 min, and the crude enzyme solution was obtained.

[0020] (3) Purification of the enzyme The prepared crude enzyme solution was added to the pre-equilibrated His purification column Ni-NTA Resin, mixed at 4°C for 3 h, and then the waste liquid was discarded. Then, 10 mL of rinse buffer containing 20 mM imidazole was used to rinse twice, and 10 mL of rinse buffer containing 50 mM imidazole was used to rinse twice. After the rinsing was completed, 500 mM imidazole was used to elute 5 times (elution volumes were 3 mL, 4 mL, 5 mL, 5 mL and 3 mL, respectively), and the eluate was collected in batches. The 30 kDa ultrafiltration tube was used for ultrafiltration to remove imidazole and high-concentration NaCl, and the purified enzyme solution was obtained.

[0021] Example 2: Detection of the enzymatic properties of mannanase (1) Method for determining the activity of mannanase The DNS method was used to determine the content of reducing sugar. 20 μL of diluted enzyme solution and 80 μL of 0.25% konjac gum solution were mixed and reacted at 60°C for 10 min. After the reaction was completed, 200 μL of DNS reagent was added, boiled for 5 min, immediately placed in ice water for cooling, and after a short centrifugation, the supernatant was taken for determination of OD 540 value (using inactivated enzyme solution as a control), and the amount of reducing sugar and enzyme activity were calculated according to the standard curve. The definition of enzyme activity unit: the amount of enzyme required to hydrolyze 1 μmol of konjac gum per minute under the above determination conditions is defined as one enzyme activity unit (U).

[0022] (2) Effect of the enzyme on the hydrolysis of different substrates Enzyme activity was determined at 60℃ and pH 7.0 using 0.25% konjac gum, guar gum, locust bean gum, and sodium carboxymethyl cellulose as substrates. The relative hydrolytic activity of mannanase on different substrates was calculated with maximum enzyme activity defined as 100%. The results showed that the enzyme exhibited the strongest hydrolytic activity against konjac gum, and could also hydrolyze guar gum, but could not hydrolyze locust bean gum or sodium carboxymethyl cellulose, indicating that this enzyme is a mannanase. (See attached results.) Figure 1 .

[0023] (3) Effect of different temperatures on enzyme activity Enzyme activity was measured using 0.25% konjac gum prepared with 50 mM Na-phosphate buffer (pH 7.0) as a substrate within the enzyme solution range of 30–100 °C. The maximum enzyme activity was taken as 100%, and the relative enzyme activity at different temperatures was calculated. The results showed that this enzyme has a wide operating temperature range, with an optimal reaction temperature of 60 °C, making it a thermophilic mannanase. Its enzyme activity remained high within the range of 45–75 °C, maintaining a relative enzyme activity above 95%. Within the ranges of 30–40 °C and 80–90 °C, its relative enzyme activity still remained above 58%, but the enzyme activity decreased rapidly above 90 °C. (See attached results). Figure 2 .

[0024] (4) Effect of different pH values ​​on enzyme activity Enzyme activity was measured at 60°C using 0.25% konjac gum prepared with different buffers (50mM Na-acetate buffer, pH 4.5~6.0; 50mM Na-phosphate buffer, pH 6.0~8.5; 50mM Glycine-NaOH buffer, pH 8.5~9.5) as substrates. The relative activity of the enzyme at different pH values ​​was calculated with the maximum enzyme activity as 100%.

[0025] The results showed that the enzyme has a relatively wide pH range, with an optimal pH of 7.0. Within the pH range of 5.5–8.5, the enzyme activity is maintained at over 65%. The enzyme activity is relatively high in 50 mM Na-acetate buffer at pH 5.5–6.0, maintaining over 95% activity. In 50 mM Na-phosphate buffer at pH 6.5–7.5, the enzyme activity is also maintained at over 88%. The enzyme protein is essentially inactivated below pH 5.0 and above pH 9.0. (See attached results). Figure 3 .

[0026] (5) Effects of different metal ions on enzyme activity Under conditions of 60℃ and pH 7.0, different metal ions with final concentrations of 1 mM and 5 mM were added to the reaction system, and then enzyme activity was measured. The relative enzyme activity under the influence of different metal ions was calculated, with the relative enzyme activity under the condition of no added metal ions as 100%. The results showed that Mn 2+ and Ba 2+ It has a significant promoting effect on the activity of this enzyme, and low concentrations of Mg 2+ It also promotes enzyme activity; Na + K + Zn 2+ Fe 2+ and Ca 2+ It has little effect on enzyme activity; Cu 2+ High concentrations of Cu significantly inhibited the activity of this enzyme. 2+ The enzyme activity was almost completely inhibited. See the results below. Figure 4 .

[0027] (6) Analysis of enzyme hydrolysis products Thin-layer chromatography (TLC) was used to analyze mannanase products. 200 μL of purified enzyme solution was added to 1.8 mL of 10 mM PBS buffer (pH 7.0) containing 1% konjac gum, mixed well, and reacted overnight at 45℃, 60℃, and 75℃, respectively. The reaction mixture was ultrafiltered using a 30 kDa ultrafiltration tube, centrifuged at 5000 rpm for 10 min at 4℃, and the filtrate was subjected to TLC analysis with a loading volume of 2 μL. The same concentration of konjac gum solution was treated with inactivated enzyme solution to obtain a control sample. The plates were developed twice in the same direction using n-butanol:acetic acid:water (2:1:1, v / v / v). After the silica gel plates dried, they were stained with 10% sulfuric acid ethanol and heated at 110℃ for 15 min to develop the sugars.

[0028] The results showed that the enzyme could effectively hydrolyze konjac gum to produce oligosaccharides at 45℃, 60℃, and 75℃. The amount of oligosaccharides produced in the hydrolysis products was significantly increased compared with the control group, further indicating that the enzyme has a wide operating temperature range and can effectively hydrolyze mannan under medium and high temperature conditions. This suggests promising applications in feed processing, mannan oligosaccharide preparation, extraction of plant bioactive substances, and bioenergy production. Furthermore, the products of this enzyme's hydrolysis of konjac gum were mainly a mixture of disaccharides and higher oligosaccharides, with almost no obvious monosaccharide components detected, indicating that this enzyme is an endoglucanase with endoglucanase activity. (See attached results.) Figure 5 .

Claims

1. A mannanase, characterized in that, The mannanase comprises an enzyme with an amino acid sequence of SEQ ID NO:

1.

2. A gene, characterized in that, The gene encodes the mannanase of claim 1, wherein a nucleotide sequence is SEQ ID NO:

2.

3. A recombinant expression vector, characterized in that, The recombinant expression vector has inserted therein a nucleic acid fragment for encoding the mannanase of claim 1.

4. The recombinant expression vector of claim 3, wherein, The recombinant expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

5. A genetically engineered bacterial strain, characterized in that, The genetically engineered bacterial strain has transformed therein the recombinant expression vector of claim 3.

6. Use of the mannanase of claim 1 in degrading konjac gum or guar gum.

7. A method of preparing oligosaccharides, characterized by, The method is for preparing oligosaccharides by using the mannanase of claim 1 to degrade konjac gum or guar gum.

8. The method of claim 7, wherein, The oligosaccharides are disaccharides or a mixture of oligosaccharides with more than two disaccharides.

9. Use of the mannanase of claim 1 in feed processing, plant active substance extraction, and bioenergy production.

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