High temperature mannanase and use thereof

Abstract

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

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a high-temperature mannanase and its applications. Background Technology

[0002] Mannans are widely distributed in plant cell walls and are an important component of hemicellulose. Their main chain is composed primarily of mannose (or mannose and glucose) linked by β-1,4-glycosidic bonds, while the side chains often contain modifying components such as galactose, glucose, or acetyl groups. Furthermore, mannans are a major component of some plant tubers and seeds, such as konjac gum (glucomannan), guar gum (galactomannan), and locust bean gum (galactomannan). β-Mannanase can specifically hydrolyze the β-1,4-glycosidic bonds in mannans to generate functional mannooligosaccharides, which have advantages such as being green, environmentally friendly, and highly efficient. They have great application potential in fields such as feed, textiles, papermaking, food, bioenergy, pharmaceuticals, and oil and gas extraction (reducing the viscosity of guar gum fracturing fluids).

[0003] Mannanases are widely found in microorganisms and have attracted increasing attention due to their advantages such as rapid bacterial growth, simple genetic structure, and ease of operation. The optimal temperatures for most reported mannanases are generally between 40 and 70°C, with a narrow operating temperature range limiting their applications. Many industrial processes involve varying high-temperature environments, such as feed processing, oil extraction, and food processing. Mannanases exhibiting effective hydrolytic activity under broadly defined high-temperature conditions show promising application prospects. Summary of the Invention

[0004] The purpose of this invention is to provide a high-temperature mannanase (named Man-3) and its applications, thereby overcoming the shortcomings of the prior art.

[0005] This invention first provides a mannanase, wherein the mannanase comprises an enzyme with the amino acid sequence SEQ ID NO:1:

[0006] CKESTSPQSLDPRLEELNQAVLAGKTLLKNSAAGEGEGEFPQSAFEVCRETLNRAERFIKQAKKSTGQAKIDSVTNEVYDVLTEFEASVNSSLNELTDTRATKQTRYLYDNLKR ISPQRLLFGMHEALGYGVGWSGMDTRSDVKDVSGDYPAVFSWDAYHIFNASADDLEHYTFQVKYAYQNGGVTTFCWHQYDPEKRGFYAKKVNYEVVASILPGGNYHRLYKNKLK KLARFFKRLRGEKGETIPVIFRPYHEQNGNWFWWGKGHRSEEQYIQLWRFTVHYLRDTLGVHNFIYAFSPDGNQFSNKSEYLNDYPGDDVVDILGLDFYFGRGDEEEIRRFQKR VVYAVQFAEEKNKLAALTEVGDYYGFSGDEANLKIPNWFTRCFLQPIKYHSQAKKIAYGAVWRNASKTHHFAPYPGHPSVPDFMDFYNDDFTLFLSDLPDMYRFKTPISD (SEQ ID NO:1);

[0007] A gene encoding the aforementioned mannanase, wherein a specific nucleotide sequence is SEQ ID NO:2:

[0008]

[0009] In another aspect, the present invention provides a recombinant expression vector in which a nucleic acid fragment encoding the above-mentioned mannanase is inserted;

[0010] The recombinant expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

[0011] Another aspect of the present invention provides a genetically engineered strain, wherein the genetically engineered strain is transformed with the above-mentioned recombinant expression vector;

[0012] The genetically engineered strain can be a eukaryotic engineered strain or a prokaryotic engineered strain; as a specific example, it is an Escherichia coli engineered strain.

[0013] The present invention also provides the application of the mannanase in the degradation of konjac gum or guar gum.

[0014] The present invention also provides a method for preparing oligosaccharides, wherein the method comprises using the above-mentioned mannanase to degrade konjac gum or guar gum to prepare oligosaccharides.

[0015] The oligosaccharide is a mixture of disaccharides or more.

[0016] The present invention also provides the application of the mannanase in feed processing, extraction of plant active substances, and bioenergy production.

[0017] The high-temperature mannanase Man-3 of this invention exhibits the strongest hydrolytic activity against konjac gum and can also hydrolyze guar gum. It has a wide operating temperature range, with an optimal temperature of 60°C. Enzyme activity remains high within the range of 45–75°C, maintaining a relative activity above 95%. This enzyme also has a relatively wide pH range, with an optimal pH of 7.0. Within the pH range of 5.5–8.5, enzyme activity remains above 65%. Furthermore, many common metal ions can promote enzyme activity or have little effect on it. This enzyme is an endokinase with β-mannanase activity, showing promising applications in feed processing, mannan oligosaccharide preparation, extraction of plant bioactive substances, and bioenergy production under medium- and high-temperature conditions. Attached Figure Description

[0018] Figure 1 This is a diagram showing the effect of enzymes on the hydrolysis of different substrates;

[0019] Figure 2 Figure showing the effect of different temperatures on enzyme activity;

[0020] Figure 3 The graph shows the effect of different pH values ​​on enzyme activity.

[0021] Figure 4The graph shows the effect of different metal ions on enzyme activity.

[0022] Figure 5 This is a chromatogram showing the analysis of enzyme hydrolysis products. Detailed Implementation

[0023] This invention identifies a novel mannanase, Man-3, from metagenomic sequencing data of deep-sea hydrothermal sulfide samples, comprising:

[0024] 1) An enzyme with the amino acid sequence SEQ ID NO:1;

[0025] 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.

[0026] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings.

[0027] Example 1 Preparation of mannanase Man-3

[0028] 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.

[0029] By analyzing metagenomic sequencing databases of sulfide samples from deep-sea hydrothermal vent areas in the Northwest Indian Ocean, the mature protein sequence of a mannanase, Man-3, was obtained (SEQ ID NO:1). The gene sequence encoding the mature Man-3 protein was optimized and the full sequence synthesized to suit expression in *E. coli*, yielding the optimized nucleotide sequence (SEQ ID NO:2). Analysis using NCBI's online BlastP tool revealed that the mature protein sequence of mannanase Man-3 is similar to that derived from thermophilic bacteria. Calditrichota The amino acid sequence identity of bacterial β-mannosidase was 95.58%. Analysis using NCBI's online CD-Search tool revealed that Man-3 possesses a typical catalytic domain, ManB2, characteristic of β-mannanases. This suggests that Man-3 may belong to an endonuclease-like β-mannanase, rather than an exonuclease-like β-mannosidase.

[0030] The specific process of recombinant expression of this enzyme is described below.

[0031] (1) Construction of recombinant plasmids

[0032] Artificial synthesis of mannanase Man-3 Introduced at the 5' and 3' ends of the gene sequence Bam HI and Xho I. Two restriction enzyme sites will be used to cleave the artificially synthesized [product / product]. Bam HI and Xho Mannanase at cleavage site I Man-3 DNA fragments of the gene are processed by restriction endonucleases Bam HI and Xho After digestion with restriction enzyme I, the DNA was ligated into the pET28a-SUMO vector, which had been digested with the same restriction endonuclease, using T4 DNA ligase. The ligation product was then used with *E. coli*. E. coli Top 10 competent cells were mixed, placed on ice for 30 min, heat-shocked at 42℃ for 90 s, and then 150 µL of LB liquid medium was added. The cells were then recovered at 37℃ and 100 rpm for 1 h. After centrifugation, the cells were spread on LB solid medium containing kanamycin and cultured overnight at 37℃. Recombinant plasmids were screened and verified by double enzyme digestion and sequencing to obtain recombinant plasmids containing the mannanase gene.

[0033] (2) Induction of gene expression and preparation of crude enzyme solution

[0034] Contains mannanase Man-3 Recombinant plasmid of gene transformed into E. coliA recombinant strain containing the mannanase gene was obtained from BL21(DE3). The recombinant strain was inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured overnight. Then, it was inoculated at a 1% inoculation rate into 500 mL of LB liquid medium containing kanamycin and cultured at 37°C and 180 rpm until OD (dose retardation). 600 The concentration was approximately 0.6. IPTG was added to achieve a final concentration of 0.5 mmol / L for induction. Expression was induced overnight at 18°C ​​and 180 rpm. The cells were collected by centrifugation at 8000 rpm for 10 min. The cells were then resuspended in 25 mL of binding buffer, and an appropriate amount of protease inhibitor was added. The cells were sonicated at 300 W with a work / interval time of 3 s / 5 s for 30 min. The cells were then centrifuged at 4°C and 5000 rpm for 10 min, and the supernatant was collected to obtain the crude enzyme solution.

[0035] (3) Enzyme purification

[0036] The prepared crude enzyme solution was added to a pre-equilibrated His purification column (Ni-NTA Resin) and mixed at 4°C for 3 hours, then the waste liquid was discarded. The solution was then washed twice with 10 ml of rinsing buffer containing 20 mM imidazole, followed by two washes with 10 ml of rinsing buffer containing 50 mM imidazole. After rinsing, the solution was eluted five times with elution buffer containing 500 mM imidazole (elution volumes of 3 mL, 4 mL, 5 mL, 5 mL, and 3 mL, respectively). The eluent was collected in batches and ultrafiltered using a 30 kDa ultrafiltration tube to remove imidazole and high concentrations of NaCl, yielding the purified enzyme solution.

[0037] Example 2: Detection of the enzymatic properties of mannanase

[0038] (1) Method for determining mannanase activity

[0039] The reducing sugar content was determined using the DNS method. 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 complete, 200 μL of DNS reagent was added, and the mixture was boiled for 5 min. It was immediately cooled in ice water, briefly centrifuged, and the supernatant was collected for OD measurement. 540 The reducing sugar content and enzyme activity were calculated based on the standard curve (using inactivated enzyme solution as a control). Enzyme activity unit definition: Under the above test conditions, the amount of enzyme required to hydrolyze konjac gum to produce 1 µmol of reducing sugar per minute is defined as one enzyme activity unit (U).

[0040] (2) Effects of enzymes on the hydrolysis of different substrates

[0041] 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 .

[0042] (3) Effect of different temperatures on enzyme activity

[0043] 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 .

[0044] (4) Effect of different pH values ​​on enzyme activity

[0045] 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%.

[0046] 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 .

[0047] (5) Effects of different metal ions on enzyme activity

[0048] 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 .

[0049] (6) Analysis of enzyme hydrolysis products

[0050] 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.

[0051] 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 amino acid sequence of the mannanase is that of SEQ ID NO:

1.

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

2.

3. A recombinant expression vector, characterized in that, The recombinant expression vector contains an inserted nucleic acid fragment encoding the mannanase of claim 1.

4. The recombinant expression vector as described in claim 3, characterized in that, The recombinant expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

5. A genetically engineered strain, characterized in that, The genetically engineered strain is transformed with the recombinant expression vector of claim 3.

6. The use of the mannanase according to claim 1 in the degradation of konjac gum or guar gum.

7. A method for preparing oligosaccharides, characterized in that, The method described in claim 1 involves using the mannanase described in claim 1 to degrade konjac gum or guar gum to prepare oligosaccharides.

8. The method as described in claim 7, characterized in that, The oligosaccharide is a mixture of disaccharides or more.

9. The application of the mannanase described in claim 1 in feed processing, extraction of plant active substances, and bioenergy production.

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