1,3/1,4-XYLANASE MLX1034, ITS GENE AND APPLICATIONS THEREOF

Abstract

MLX1034 is from Polaribacter sp. Q13, and has the amino acid sequence of the 1,3/1,4-xylanase MLX1034 is listed in SEQ ID NO.1; a nucleotide sequence of the gene is listed in SEQ ID NO.2; the 1,3/1,4-xylanase MLX1034 in the invention is capable of efficiently and specifically degrading 1,3/1,4-xylan and producing xylooligosaccharides with DP values above one; in addition, the physical and chemical properties of the 1,3/1,4-xylanase MLX1034 are stable enough to hydrolyze 1,3/1,4-xylan at room temperature; the 1,3/1,4-xylanase MLX1034 is suitable for the industrial production of red algal xylooligosaccharides at low energy costs.

Claims

1. A 1,3/1,4-xylanase MLX1034 protein having the amino acid sequence shown as SEQ ID NO.1.

2. The 1,3/1,4-xylanase MLX1034 protein according to claim 1, wherein the protein is an endolytic xylanase isolated from Polaribacter sp. Q13. Polaribacter sp. Q13 is deposited in China Center for Type Culture Collection at Jialuoshan, Bayi Road, Wuhan City, Hubei Province on Dec. 28, 2020. The deposit number is CCTCC M 2020985.

3. The 1,3/1,4-xylanase MLX1034 protein according to claim 1, wherein the protein is encoded by a gene having the nucleotide sequence shown as SEQ ID NO.2.

4. The 1,3/1,4-xylanase MLX1034 protein according to claim 3, wherein the gene is cloned from the genomic DNA of Polaribacter sp. Q13. Polaribacter sp. Q13 is deposited in China Center for Type Culture Collection at Jialuoshan, Bayi Road, Wuhan City, Hubei Province on Dec. 28, 2020. The deposit number is CCTCC M 2020985.

5. An expression vector comprising the gene of claim 3 that encodes the 1,3/1,4-xylanase MLX1034 protein.

6. The expression vector according to claim 5, wherein a recombinant cell comprising the expression vector.

7. A method for degrading 1,3/1,4-xylan and preparing ylooligosaccharides by utilizing a 1,3/1,4-xylanase MLX1034 protein having the amino acid sequence shown as SEQ ID NO.1 that is encoded by a gene having the nucleotide sequence shown as SEQ ID NO.2.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1 SDS-PAGE analysis of the recombinant 1,3/1,4-xylanase MLX1034. M, protein marker.

[0036] FIG. 2 Effect of temperature on the activity of the 1,3/1,4-xylanase MLX1034.

[0037] FIG. 3 Effect of temperature on the stability of the 1,3/1,4-xylanase MLX1034.

[0038] FIG. 4 Effect of pH on the activity of the 1,3/1,4-xylanase MLX1034.

[0039] FIG. 5 Effect of pH on the stability of the 1,3/1,4-xylanase MLX1034.

[0040] FIG. 6 High-performance liquid chromatography (HPLC) analysis of the released xylooligosaccharides from 1,3/1,4-xylan by the 1,3/1,4-xylanase MLX1034. The reaction mixture without the 1,3/1,4-xylanase MLX1034 was used as the control.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] The present invention will be further described below through drawings and embodiments. It is to be understood that the invention is not limited to the embodiments described below, and any form of modifications and/or changes made to the invention are intended to fall within the scope of the invention.

[0042] The materials used in the present invention are described below.

[0043] A bacterial strain, Polaribacter sp. Q13, has been deposited in China Center for Type Culture Collection at Jialuoshan, Bayi Road, Wuhan City, Hubei Province on Dec. 28, 2020. The deposit number is CCTCC M 2020985.

[0044] 1,3/1,4-xylan from Palmaria palmata was purchased from Elicityl (France). The proportion between ?-1,3- and ?-1,4-linkages is approximately 1:4.

[0045] 1,3-xylan was extracted from Caulerpa lentillifera.

[0046] Wheat arabinoxylan and beech wood xylan were purchased from Megazme (Ireland).

[0047] TYS broth medium contained 0.5% (w/w) tryptone and 0.1% (w/w) yeast extract in artificial seawater [3.0% (w/w) sea salts (Sigma, USA) in ddH.sub.2O]. The pH was adjusted to 7.8.

[0048] TYS agar medium contained 0.5% (w/w) tryptone, 0.1% (w/w) yeast extract and 1.5% (w/w) agar in artificial seawater. The pH was adjusted to 7.8.

[0049] Luria-Bertani (LB) medium contained 1.0% (w/w) tryptone, 0.5% (w/w) yeast extract and 1.0% (w/w) NaCl in ddH.sub.2O.

[0050] LB agar medium contained 1.0% (w/w) tryptone, 0.5% (w/w) yeast extract, 1.0% (w/w) NaCl and 1.5% (w/w) agar in ddH.sub.2O.

[0051] 1,3/1,4-xylan agar medium contained 0.2% (w/w) 1,3/1,4-xylan and 1.5% (w/w) agar in artificial seawater. The pH was adjusted to 7.8.

EXAMPLE 1 ISOLATION OF POLARIBACTER SP. Q13

[0052] Red algal samples were collected from Nelson Island, Antarctica. These algal samples were washed with sterile artificial seawater to obtain epiphytic bacteria from the algal surface. After gradient dilution (10.sup.?2-10.sup.?6), the bacteria-containing seawater was spread on TYS agar plates. Then the bacteria were incubated at 20? C. until detectable colonies formed. Morphologically different colonies were selected, purified with TYS agar, and then cryopreserved at ?80? C. in TYS broth supplemented with glycerol [TYS broth: glycerol=3:7 (v/v)]. Furthermore, the purified strains were cultivated on 1,3/1,4-xylan agar plates to screen 1,3/1,4-xylan-utilizing bacteria. Finally, one bacterial strain, designated as Q13, was found to grow on the 1,3/1,4-xylan agar plate.

[0053] After 3?4 days cultivation on the 1,3/1,4-xylan agar plate, colonies of strain Q13 were yellow, moist and circular with regular edges and smooth surfaces. Cells of strain Q13 were Gram-stain-negative. Strain Q13 was identified as a species of the genus Polaribacter (Polaribacter sp.).

[0054] Polaribacter sp. Q13 has been deposited in China Center for Type Culture Collection at Jialuoshan, Bayi Road, Wuhan City, Hubei Province on Dec. 28, 2020. The deposit number is CCTCC M 2020985.

EXAMPLE 2 CLONING OF THE GENE MLX1034 AND CONSTRUCTION OF THE RECOMBINANT PLASMID pET-22b-MLX1034

[0055] 1. Genomic DNA extraction and whole genome sequencing of Polaribacter sp. Q13. Polaribacter sp. Q13 was cultivated in TYS broth at 20? C. and 180 rpm for 24 h. Then the bacterial cells were collected and the genomic DNA was extracted using a bacterial genomic DNA isolation kit (BioTeke, China) according to the manual instruction. The whole-genome sequencing was carried out at the Beijing Genomics Institute (BGI; Beijing, China). [0056] 2. Primer design and synthesis. Based on the genomic data of Polaribacter sp. Q13, the nucleotide sequence of the gene MLX1034 and the amino acid sequence of its product (MLX1034) were obtained. Using SignalP-5.0 (http://www.cbs.dtu.dk/services/SignalP/), the signal peptide of MLX1034 was predicted as Met1-Ser27. After deleting the predicted signal peptide, a forward primer (F: 5-AAGAAGGAGATATACATATGCAAGAGGTAAAACCTCGTTTT-3) and a reverse primer (R: 5-TGGTGGTGGTGGTGCTCGAGTTTATTATTAAGGTGAATAAA-3) were designed and synthesized at BGI (Beijing, China). [0057] 3. PCR amplification and PCR product recovery. PCR amplification was performed using primers F and R with the genomic DNA of Polaribacter sp. Q13 as the template. The PCR program was determined as pre-denaturation at 95? C. for 2 min, followed by 30 cycles of PCR (denaturation at 95? C. for 30 s, annealing at 55? C. for 20 s and extension at 72? C. for 45 s), and a final extension step at 72? C. for 10 min. The 50 ?L PCR mixture contained 29 ?L sterile ddH.sub.2O, 10 ?L Trans Star Fast Pfu buffer (TransGen, China), 5 ?L dNTP, 2 ?L primer F, 2 ?L primer R, 1 ?L genomic DNA of strain Q13 and 1 ?L Trans Star Fast Pfu DNA polymerase (TransGen, China). The amplification products were purified by the 1.0% (w/w) agarose gel electrophoresis and a gel extraction kit (Omega, USA), and the MLX1034 fragment was obtained. [0058] 4. Construction of the recombinant plasmid pET-22b-MLX1034. The pET-22b vector (Novagen, USA) was digested with the restriction enzymes NdeI and XhoI and the linear product was purified by the 1.0% (w/w) agarose gel electrophoresis and a gel extraction kit (Omega, USA). Then, the obtained MLX1034 fragment was inserted into the digested pET-22b with the In-Fusion HD Cloning Kit (TaKaRa, Japan). The 2.5 ?L reaction mixture contained 1 ?L MLX1034 fragment, 1 ?L digested pET-22b and 0.5 ?L In-Fusion.

[0059] Using the heat shock method described in Molecular cloning: A laboratory manual, the assembled vector was transformed into the competent Escherichia coli DH5? cells (TransGen, China). In detail, 2.5 ?L ligation products were added into 50 ?L competent E. coli DH5? cells. After an incubation in ice for 30 min, the mixture is placed at 42? C. for 90 s and then placed back in ice for 10 min. 200 ?L LB was added and the and the transformed cells were incubated at 37? C. for 1 h. The resultant cells were spread on the LB agar plate containing 100 ?g/mL ampicillin and incubated at 37? C. overnight. Single colonies were picked and cultivated in LB containing 100 ?g/mL ampicillin at 37? C. overnight. The cells were collected and sent to BGI (Beijing, China) for plasmid extraction and gene sequencing.

[0060] The result of gene sequencing showed that MLX1034 was successfully inserted into the NdeI-XhoI restriction site of pET22b with no substitution, deletion or insertion mutations. The recombinant plasmid was named pET-22b-MLX1034.

EXAMPLE 3 EXPRESSION AND PURIFICATION OF THE 1,3/1,4-XYLANASE MLX1034

[0061] 1. Expression of the 1,3/1,4-xylanase MLX1034 in E. coli BL21(DE3). The recombinant plasmid pET-22b-MLX1034 was transformed into the competent E. coli BL21(DE3) cells (TransGen, China) with the heat shock method as mentioned above. The resultant cells were spread on the LB agar plate containing 100 ?g/mL ampicillin and cultivated at 37? C. overnight. A single colony was picked and cultivated in LB containing 100 ?g/mL ampicillin at 37? C. overnight, obtaining seed culture. The seed culture (1%, v/v) was inoculated into LB containing 100 ?g/mL ampicillin and cultivated at 180 rpm and 37? C. to an optical density (OD) at 600 nm of approximately 0.8?1.0. Then, 0.1 mM isopropyl-D-thiogalactopyranoside (IPTG) was added to the culture as an inducer, and the culture was incubated at 18? C. and 120 rpm for 16 h. The induced cells were harvested by centrifugation (7,000 rpm for 5 min at 4? C.). [0062] 2. Purification of the 1,3/1,4-xylanase MLX1034. The obtained cells were resuspended in the lysis buffer (50 mM Tris-HCl, 100 mM NaCl, pH 8.0) and lysed by a pressure crusher. After centrifugation (12,000 rpm for 60 min at 4? C.), the supernatant, namely crude enzyme solution, was collected.

[0063] The crude enzyme solution was loaded onto the nickel affinity column (GE Healthcare, USA), which was pre-equilibrated with the lysis buffer. Then, the nickel affinity column was washed with 10 column volumes of the wash buffer (50 mM Tris-HCl, 100 mM NaCl, 10 mM imidazole, pH 8.0). The target protein (MLX1034) was eluted with the elution buffer (50 mM Tris-HCl, 100 mM NaCl, 250 mM imidazole, pH 8.0).

[0064] The obtained sample was concentrated and loaded onto the gel filtration (GF) column (Superdex 200 Prep grade; GE Healthcare, USA), which was pre-equilibrated with the GF buffer (10 mM Tris-HCl, 100 mM NaCl, pH 8.0). The sample was then eluted with the GF buffer. Samples of the target protein peak were collected and stored at ?80? C. with the addition of 10% (v/v) glycerol for further use.

[0065] The purity and molecular weight of the purified MLX1034 were determined with SDS-PAGE. The result is shown in FIG. 1.

[0066] As shown in FIG. 1, the purified MLX1034 exhibited an apparent molecular weight of approximately 35.0 kDa, consistent with its theoretical molecular weight (39.1 kDa). Therefore, MLX1034 was successfully expressed in E. coli BL21 (DE3).

EXAMPLE 4 ENZYMATIC PROPERTIES OF THE 1,3/1,4-XYLANASE MLX1034

[0067] The standard reaction mixture contained 900 ?L xylan [10 mg/mL in 20 mM phosphate-buffered saline (PBS)] and 100 ?L enzyme solution of the 1,3/1,4-xylanase MLX1034.

[0068] The xylanase activity was determined by the dinitrosalicylic acid (DNS) method. The reaction mixture was incubated at 40? C. for 10 min. After incubation, the reaction was terminated by the addition of 100 ?L DNS. Then the reaction mixture was boiled at 100? C. for 5 min for coloring. After the addition of 500 ?L ddH.sub.2O, the OD.sub.550 nm was measured. The reaction mixture supplemented with the inactivated MLX1034 was used as the control. One unit of enzyme activity (1 U) is defined as the amount of enzyme required to release 1 ?mol xylose per min. A standard curve was generated with different concentrations of xylose.

[0069] Enzymatic properties of the 1,3/1,4-xylanase MLX1034 were analyzed according to the method. [0070] 1. Substrate specificity analysis. The substrate specificity of the 1,3/1,4-xylanase MLX1034 was analyzed with 1,3/1,4-xylan, 1,3-xylan and 1,4-xylans (including wheat arabinoxylan and beech wood xylan) as the substrates. The result is shown in Table 1.

[0071] As shown in Table 1, the 1,3/1,4-xylanase MLX1034 efficiently hydrolyzed 1,3/1,4-xylan (224.0 U/mg) with extremely low activity on 1,3-xylan (0.04 U/mg) and no activity on 1,4-xylans (including wheat arabinoxylan and beech wood xylan).

TABLE-US-00001 TABLE 1 The substrate specificity of the 1,3/1,4-xylanase MLX1034. Substrate Specific activity (U/mg) 1,3/1,4-xylan 224.0 ? 13.5 (100%) 1,3-xylan 0.04 ? 0.00 (0.02%) Wheat arabinoxylan Not detectable Beech wood xylan Not detectable [0072] 2. Effect of temperature on enzymatic activity and stability. To determine the effect of temperature on the activity of the 1,3/1,4-xylanase MLX1034, 1,3/1,4-xylan was used as the substrate. Reaction mixtures were separately incubated at 0? C., 10? C., 20? C., 30? C., 40? C., 50? C. and 60? C. for 10 min, and the activities of the 1,3/1,4-xylanase MLX1034 at different temperatures were determined. The maximum activity was taken as 100% to calculate the relative activities. The result is shown in FIG. 2.

[0073] To determine the effect of temperature on the stability of the 1,3/1,4-xylanase MLX1034, it was separately incubated at 40? C., 50? C. and 60? C. for 60 min. During incubation, the residual activities were determined every 10 min with 1,3/1,4-xylan as the substrate. The specific activity of the 1,3/1,4-xylanase MLX1034 without incubation (0 min) was taken as 100% to calculate the residual activities. The result is shown in FIG. 3.

[0074] As shown in FIGS. 2 and 3, the 1,3/1,4-xylanase MLX1034 exhibited more than 80% activity at 30?45? C. with the maximum activity at 40? C. It was relatively stable at 40? C., retaining more than 80% residual activities during 60 min incubation. After 60 min incubation at 50? C., the residual activity of the 1,3/1,4-xylanase MLX1034 decreased to less than 40%. It was extremely unstable at 60? C. and rapidly deactivated within 5 min. [0075] 3. Effect of pH on enzymatic activity and stability. To determine the effect of pH on the activity of the 1,3/1,4-xylanase MLX1034, reactions were carried out at 40? C. for 10 min under different pH values from 3.0 to 11.0 with 1,3/1,4-xylan as the substrate. The buffers used were 20 mM citrate buffer at pH 3.0?6.0, 20 mM PBS at pH 6.0?8.0, 20 mM Tris-HCl at pH 8.0?9.0, and 20 mM glycine-NaOH at pH 9.0?11.0. The maximum activity was taken as 100% to calculate the relative activities. The result is shown in FIG. 4.

[0076] To determine the effect of temperature on the stability of MLX1034, each of the buffers with different pH values was mixed with the 1,3/1,4-xylanase MLX1034 at a volume ratio of 49:1. The buffers used were 20 mM citrate buffer at pH 3.0?6.0, 20 mM PBS at pH 6.0?8.0, 20 mM Tris-HCl at pH 8.0?9.0, and 20 mM glycine-NaOH at pH 9.0?11.0. After incubation at 60? C. for 1 h, the residual activities of the 1,3/1,4-xylanase MLX1034 were determined with 1,3/1,4-xylan as the substrate. The maximum activity was taken as 100% to calculate the residual activities. The result is shown in FIG. 5.

[0077] As shown in FIGS. 4 and 5, the 1,3/1,4-xylanase MLX1034 exhibited the highest activity at pH 7.0. It was unstable under acidic conditions (pH 3.0?4.0) but stable at pH 5.0?11.0.

EXAMPLE 5 MODE OF ACTION ANALYSIS OF THE 1,3/1,4-XYLANASE MLX1034

[0078] A reaction mixture containing 1,3/1,4-xylan (5 mg/mL in 20 mM PBS, pH 7.0) and the 1,3/1,4-xylanase MLX1034 at a volume ratio of 99:1 was incubated at 30? C. At the reaction time of 6 min, 10 min, 1 h and 24 h, samples were taken out separately and boiled for 5 min. Then samples were filtered with a 0.22 ?M filter, obtaining the degradation products.

[0079] The degradation products were analyzed using HPLC equipped with an evaporative light scattering detector (ELSD) and a Superdex 30 Increase 10/300 GL column (GE Healthcare, USA). The mobile phase was ddH.sub.2O. A mixture of xylose and 1,4-xylooligosaccharides (1,4X2-X6) was used as the marker. The result is shown in FIG. 6.

[0080] As shown in FIG. 6, the 1,3/1,4-xylanase MLX1034 is an endolytic xylanase. The degradation products were xylooligosaccharides with DP values above one with xylohexaose as the main product. At each time point of the reaction within 24 h, only xylooligosaccharides (DP>2) were produced with no xylose.