A thermostable laccase mutant and its preparation and application

Abstract

The present invention belongs to the field of enzyme genetic engineering technology, and specifically relates to a thermostable laccase mutant and its preparation and application. bacillus amyloliquefaciens )-derived laccase gene ( lac ) were expressed in Escherichia coli BL21; the gene was PCR-PCR-enhanced lac The mutants were mutated and screened by ABTS, and the mutant T262F / T387L with improved thermal stability was selected, and expressed in Bacillus subtilis WB600, Bacillus amyloliquefaciens CGMCC No.11218, and Pichia pastoris GS115.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme genetic engineering, and specifically relates to a high temperature resistant laccase mutant and its preparation and application. Background Art

[0002] Lignocellulosic straw is a biomass resource with great potential for development and utilization. The components of straw are mainly composed of cellulose, hemicellulose and lignin. The covalent bonding of lignin with hemicellulose and cellulose hinders the effective degradation of lignin. Removing lignin is a prerequisite for the utilization of cellulose and hemicellulose. At present, the processes used in the industrial field to remove lignin are the strong alkali high-temperature pulping technology for cellulose and the strong acid high-temperature xylose (or furfural) technology for hemicellulose. These processes all have the problem of prohibition or restriction of development due to environmental pollution.

[0003] The bio-enzymatic method to replace strong acid and strong alkali processes to degrade lignin is the most effective means to solve pollution and increase benefits. However, due to the complexity of the chemical bonds between the components of lignocellulose straw, there are huge obstacles to using a single enzyme with specific properties to degrade lignin. The use of enzymes with different functions to combine into a composite enzyme in the same scenario makes it possible to degrade lignin. Lignin-degrading enzymes with specific functional properties, such as laccase, cannot function under the conditions of specific scenarios in composite enzymes (such as temperature, pH, enzyme activity, etc.) due to their natural enzymatic properties. This goal needs to be achieved through laccase mutants and their genes, engineered bacteria and preparation.

[0004] Laccase, belonging to the blue multi-copper oxidase (MCOs) family, is the largest subfamily of MCOs, widely distributed in a variety of organisms and has multiple functions. Laccase usually contains four copper atoms, one T1Cu, one T2Cu and two T3Cu. Laccase can catalyze the single-electron oxidation of phenolic compounds and their derivatives to form corresponding reactive free radicals or benzoquinone intermediates, with O2 as the final electron acceptor and H2O as the only byproduct. At the same time, the free radical intermediates spontaneously couple with each other outside the catalytic center of laccase to produce oligomers and polymers. The oxidation of laccase in lignin degradation leads to a decrease in the methyl content, an increase in conjugated carbonyl groups, and an increase in carbonyl and ester groups in the residual lignin structure, which improves the degradability and availability of lignin. This shows that laccase can be used as an environmentally friendly and widely used "green catalyst" both in vivo and in vitro. Therefore, laccase has a wide range of applications in the industrial field.

[0005] Laccase is a ubiquitous enzyme that is widely distributed in nature and is usually produced by plants, insects, fungi and bacteria. Laccase from Rhus verniciflua is the beginning of the study of laccase. Laccase can use coniferyl alcohol and other substances in plants as substrates to react and produce lignin. It can also completely degrade lignin. In addition, laccase is involved in the protection of plants and the invasion of pathogens. There are fewer studies on laccases from insects. At present, insects with laccase activity have been found to include flesh flies, tobacco hornworms, green-headed flies, mosquitoes and locusts. Laccase from fungi has become a hot topic of research because white rot fungal laccase can degrade lignin. Until now, research on laccase from fungi has not stopped, such as Lungwort , Tricholoma gigantic Laccase from bacteria. Laccase was discovered later in bacteria than in fungi. The earlier reported sources of laccase from bacteria are Spherical Bacillus , Azospirillum lipoferum and Alteromonas . Currently, the main research on bacterial laccase is on laccase from Bacillus. Most fungal laccases are unstable under high temperature and high alkali conditions, and most metal ions and inhibitors have a significant inhibitory effect on fungal laccases. Compared with fungal laccases, bacterial laccases can function at high temperatures, a wide pH range and high salt concentrations. Compared with other bacterial laccases, Bacillus laccases have better alkali resistance and thermal stability and are less affected by metal ions. In the industrial degradation process of straw, high temperature assistance is often required. Therefore, further improving the stability of wild-type Bacillus laccase is conducive to its industrial application.

[0006] Therefore, in the present invention, by Bacillus amyloliquefaciens ) laccase gene was mutated to screen out laccase mutant genes with improved thermal stability. Subsequently, the thermostable mutant laccase was expressed and prepared using Bacillus and Pichia pastoris expression systems. Summary of the invention

[0007] Based on the problem of high temperature application of laccase, in order to obtain laccase with improved thermal stability, its existing properties need to be further modified. The purpose of the present invention is to provide a high temperature resistant laccase mutant. Bacillus amyloliquefaciens )-derived laccase gene ( milk ) were expressed in Escherichia coli BL21; the gene was PCR-PCR-enhanced milk Mutations were generated and the mutants were screened using ABTS to select mutants with improved thermal stability. The mutants were expressed and prepared in Bacillus subtilis WB600, Bacillus amyloliquefaciens CGMCC No.11218, and Pichia pastoris GS115.

[0008] The technical route for achieving the purpose of the present invention is summarized as follows:

[0009] From Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) was mutated, and the mutant T262F / T387L and the encoding gene were screened using the E. coli expression system. lacm1 The half-life at 80 ℃ was increased to 172% of the wild-type laccase (LAC). The optimal mutant laccase T262F / T387L was efficiently prepared using Bacillus subtilis WB600, Bacillus amyloliquefaciens CGMCCNo.11218, and Pichia pastoris GS115.

[0010] One of the technical solutions provided by the present invention is a laccase mutant, which is obtained by mutations including T262F and T387L on the basis of the wild-type laccase shown in SEQ ID NO.1, and is named as T262F / T387L mutant;

[0011] Furthermore, the amino acid sequence of the T262F / T387L mutant is shown in SEQ ID NO.3;

[0012] Furthermore, the coding gene of the T262F / T387L mutant lacm1 , the nucleotide sequence is shown in SEQ ID NO.4.

[0013] The second technical solution provided by the present invention is a recombinant plasmid or recombinant strain containing the above mutant encoding gene;

[0014] Furthermore, the expression vector used is pET-28a(+), and the host is Escherichia coli, Bacillus subtilis, Bacillus amyloliquefaciens, or Pichia pastoris;

[0015] Furthermore, the host cell is Escherichia coli BL21;

[0016] Furthermore, the host cell is Bacillus subtilis WB600, or the host cell is Bacillus amyloliquefaciens CGMCC No.11218, or the host cell is Pichia pastoris GS115;

[0017] Preferably, the recombinant strain is obtained by connecting the mutant encoding gene to the expression vector pET-28a(+) and expressing it in the host Escherichia coli.

[0018] The third technical solution provided by the present invention is the use of the above-mentioned recombinant plasmid or recombinant strain in producing the laccase mutant described in the first technical solution.

[0019] The fourth technical solution provided by the present invention is the application of the laccase mutant described in the first technical solution, especially in the degradation of lignocellulose;

[0020] Further, it is used in degrading straw or bagasse;

[0021] Furthermore, the invention can be used in the degradation of corn straw, rice straw or wheat straw.

[0022] The following definitions are used in the present invention:

[0023] 1. Nomenclature of amino acid and DNA sequences

[0024] The generally accepted IUPAC nomenclature for amino acid residues is used, either as a single letter or three letter code. The generally accepted IUPAC nomenclature for DNA nucleic acid sequences is used.

[0025] 2. Identification of Laccase Mutants

[0026] The mutated amino acid in the LAC mutant is represented by "original amino acid + position + replaced amino acid". For example, T262F means that the amino acid at position 262 is replaced by Phe from Thr in wild-type LAC, and the position number corresponds to the amino acid sequence number of the wild-type LAC mature peptide in SEQ ID NO.1.

[0027] In the present invention, LAC represents wild-type laccase, T262F / T387L represents laccase mutants, and lowercase italics milk Indicates the gene encoding the wild-type laccase LAC, in lowercase italics lacm1 It represents the coding gene of the mutant T262F / T387L. The specific information is shown in the following table.

[0028]

[0029] Beneficial Effects

[0030] The invention utilizes error-prone PCR technology to mutate the wild-type LAC to obtain a mutant T262F / T387L having improved thermal stability at 80°C relative to the wild-type, and a half-life at 80°C increased to 172% of the wild-type laccase (LAC). BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The electrophoresis diagram of PCR amplification of wild-type laccase

[0032] Among them: M is DNA Marker, lane 1 is laccase gene milk .

[0033] Figure 2 For pET- milk Plasmid restriction enzyme cutting verification diagram

[0034] Among them: M is DNA Marker, 1 is pET- milk through No I and Xho I double enzyme digestion map.

[0035] Figure 3 The curves showing the changes in enzyme activity of wild-type and mutant T262F / T387L laccase at 80°C over time. DETAILED DESCRIPTION

[0036] The technical content of the present invention is further described below in conjunction with the embodiments, but the present invention is not limited to these embodiments, and the protection scope of the present invention cannot be limited by the following embodiments.

[0037] Some solutions and culture media used in the embodiments of the present invention are as follows:

[0038] Lysis buffer (mM): Tris 20, NaCl 500, dithiothreitol 1, imidazole 20.

[0039] Wash buffer (mM): Tris 20, NaCl 500, dithiothreitol 1, imidazole 100.

[0040] Elution Buffer (mM): Tris 20, NaCl 500, dithiothreitol 1, imidazole 500.

[0041] LB medium (g / L): 5.0% yeast extract, 10.0% tryptone, 10.0% NaCl, and the rest is water. For solid medium, add 2% agar.

[0042] BMGY medium (g / L): peptone 20.0, yeast extract 10.0, YNB 13.4, 4×10 -5 % biotin, 10% 1 mmol / L potassium phosphate buffer (pH 6.0), 0.5% glycerol.

[0043] BMMY medium (g / L): peptone 20.0, yeast extract 10.0, YNB 13.4, 4×10 -5 % biotin, 10% 1 mmol / L potassium phosphate buffer (pH 6.0), 0.5% methanol.

[0044] Fermentation medium (g / L): 64 corn flour, 40 soybean meal, add 2.7 amylase, 4 Na2HPO4, 0.3 KH2PO4, and the rest is water; keep warm at 90℃ for 30min and then sterilize at 121℃ for 20min.

[0045] In the present invention, the mature peptide sequence of the wild-type laccase LAC is shown in SEQ ID NO.1: MALEKFADELPIIETLKPQKTSNGSTYYEVTMKECFHKLHRDLPPTRLWGYNGLFPGPTIDVNQDENVYIKWMNDLPDKHFLPVDHTIHHSEGGHQEPDVKTVVHLHGGATPPDSDGYPEAWFTRDFKEKGPYFEKEVYHYPNKQRGALLWYHDHAMAITRLNVYAGLAGMYIIRERKEKQLKLPAGEYDVPLMIMDRTLNDDGSLFYPSGPDNPSETLPNPSIVPFLCGNTILVNGKAWPYMEVEPRTYRFR ILNASNTRTFSLSLNNGGRFIQIGSDGGLLPRSVKTQSISLAPAERYDVLIDFSAFDGEHIILTNGTGCGGDVNPDTDANVMQFRVTKPLKGEDTSRKPKYLSAMPDMTSKRIHNIRTLKLTNTQDKYGR PVLTLNNKRWHDPVTEAPRLGSTEIWSIINPTRGTHPIHLHLVSFQVLDRRPFDLERYNKFGDIVYTGPAVPPPPSEKGWKDTVQAHSGEVIRIAATFAPYSGRYVWHCHILEHEDYDMMRPMDVTDKQ.

[0046] In the present invention, the mature peptide sequence of the T262F / T387L mutant is shown in SEQ ID NO.3:

[0047] MALEKFADELPIIETLKPQKTSNGSTYYEVTMKECFHKLHRDLPPTRLWGYNGLFPGPTIDVNQDENVYIKWMNDLPDKHFLPVDHTIHHSEGGHQEPDVKTVVHLHGGATPPDSDGYPEAWFTRDFK EKGPYFEKEVYHYPNKQRGALLWYHDHAMAITRLNVYAGLAGMYIIRERKEKQLKLPAGEYDVPLMIMDRTLNDDGSLFYPSGPDNPSETLPNPSIVPFLCGNTILVNGKAWPYMEVEPRTYRFRILN ASNTRFFSLSLNNGGRFIQIGSDGGLLPRSVKTQSISLAPAERYDVLIDFSAFDGEHIILTNGTGCGGDVNPDTDANVMQFRVTKPLKGEDTSRKPKYLSAMPDMTSKRIHNIRTLKLTNTQDKYGRP VLLLLNNKRWHDPVTEAPRLGSTEIWSIINPTRGTHPIHLHLVSFQVLDRRPFDLERYNKFGDIVYTGPAVPPPPSEKGWKDTVQAHSGEVIRIAATFAPYSGRYVWHCHILEHEDYDMMRPMDVTDKQ.

[0048] The present invention will be further explained below through specific examples.

[0049] Example 1 Obtaining wild-type laccase gene

[0050] 1. Use the kit (OMEGA: Bacterial DNA Kit) to extract Bacillus amyloliquefaciens ( bacillus starch liquefier )TCCC 111465 genomic DNA, extraction steps are as follows:

[0051] (1) Use an inoculation loop to inoculate the strain onto a LB solid plate and culture at 37 °C overnight.

[0052] (2) Pick a single colony from the culture plate and inoculate it into liquid test tube culture medium. Incubate overnight at 37 °C and 220 rpm with shaking.

[0053] (3) Place 3 mL-5 mL of bacterial solution in a sterilized EP tube, centrifuge at 12,000 rpm for 2 min, and discard the supernatant.

[0054] (4) Add 200 μL of sterile water to the EP tube to resuspend the bacteria, then add 50 μL of lysozyme, pipette to mix, and keep at 37 °C for 20 min.

[0055] (5) Add 100 μL BTL buffer and 20 μL proteinase K to the EP tube, vortex to mix, and incubate at 55 °C for 40 min, vortexing to mix every 20 min.

[0056] (6) Add 5 μL of RNase, invert several times to mix, and leave at room temperature for 10 min.

[0057] (7) Centrifuge at 12,000 r / min for 2 min to remove the undigested portion. Transfer the supernatant to a new EP tube, add 220 μL of BDL buffer, and incubate at 65 °C in a water bath for 15 min.

[0058] (8) Add 220 μL of anhydrous ethanol and mix by pipetting.

[0059] (9) Transfer the liquid in the EP tube into the recovery column and let it stand for 1 min. Centrifuge at 12,000 r / min for 1 min. Pour the filtrate back into the recovery column. Repeat twice and discard the waste liquid.

[0060] (10) Add 500 μL HBC buffer, centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

[0061] (11) Add 700 μL DNA wash buffer, let stand for 1 min, centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

[0062] (12) Add 500 μL DNA wash buffer, let stand for 1 min, centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

[0063] (13) Incubate at 12,000 r / min for 2 min, discard the waste liquid tube, and place the recovery column in a new EP tube.

[0064] (14) Dry in a metal bath at 55 °C for 10 min.

[0065] (15) Add 50 μL of 55°C sterile water, let stand at room temperature for 5 min, centrifuge at 12,000 rpm for 2 min, discard the recovery column, and the liquid in the EP tube is the genome.

[0066] 2. Using the extracted genome of Bacillus amyloliquefaciens as a template, a pair of primers were designed upstream and downstream of the ORF frame to introduce restriction enzyme sites respectively. No I. Xho I. Laccase gene of the present invention milk The amplification primers are as follows:

[0067] Upstream primer P1:

[0068] 5'- GAAGGAGATATACCATGGGCATGGCACTGGAAAAATTTG -3'

[0069] Downstream primer P2:

[0070] 5'- TGGTGGTGGTGGTGCTCGAGCTGCTTATCCGTGACGTCC -3'

[0071] P1 and P2 were used as upstream and downstream primers, and the laccase genome of Bacillus amyloliquefaciens was used as a template for amplification.

[0072] The amplification reaction system is:

[0073]

[0074] The amplification program was as follows: 98°C pre-denaturation for 30 s; 98°C denaturation for 10 s, 54°C annealing for 20 s, 72°C extension for 8 s, 30 cycles; 72°C extension for 10 min. The PCR amplification product was subjected to 0.8% agarose gel electrophoresis, and a 1536 bp band was obtained ( Figure 1 ), using a small amount of DNA recovery kit to recover the PCR product, and obtain the wild-type laccase gene of the present invention. milk (SEQ ID NO.2), pET-28a(+) plasmid with restriction enzyme No I and Xho I was double digested and the recovered milk Homologous recombination with the vector pET vector to obtain the recombinant plasmid pET- milk , enzyme digestion verification Figure 2 As shown, it was transformed into Escherichia coli BL21 and named as the recombinant strain BL21 / pET- milk .

[0075] Example 2 LAC activity and temperature stability determination

[0076] ABTS assay method for LAC enzyme activity:

[0077] (1) Take 200 µL of pH 5.5 citric acid-sodium hydrogen phosphate buffer (50 mM, containing 5 mM Cu 2+ ) in a 96-well ELISA plate and incubate in a water bath at 80 °C for 1 min;

[0078] (2) Add 10 μL of LAC / LACM diluted to an appropriate concentration, mix well, and place in an 80 °C water bath for 1 min;

[0079] (3) Add 30 µL of ABTS (50 mM) and mix by pipetting. Incubate the mixture in a constant temperature water bath at 80 °C for 10 min. Record the initial and final OD values ​​at 420 nm.

[0080] Calculation of enzyme activity:

[0081]

[0082] Where: △OD = OD end - OD start;

[0083] V1 represents the total volume of the reaction system (µL);

[0084] △t represents the reaction time (min);

[0085] V2: volume of enzyme solution added (µL);

[0086] ε represents the molar absorption coefficient of the product at 420 nm, 36 mM -1 cm -1 ;

[0087] d represents the inner diameter / optical path thickness of the 96-well ELISA plate (cm).

[0088] Enzyme specific activity (U / mg) = enzyme activity / protein concentration.

[0089] Temperature stability of LAC was determined by placing LAC in a pH 7.0 buffer and keeping it at 80 °C for different time periods. Samples were taken at regular intervals and the residual enzyme activity was measured at pH 5.0 and 80 °C after the insulation. The enzyme activity without insulation treatment was taken as 100% to calculate the residual enzyme activity, draw the enzyme activity change curve, and further calculate the half-life.

[0090] Example 3 Construction and screening of LAC mutants

[0091] 1. Construction of mutant library

[0092] Error-prone PCR: wild-type coding gene milk Error-prone PCR was performed with 1% β-glutamic acid as the template, and the reaction system was as follows:

[0093]

[0094] Note: The above reagents are from Takara Biotech Co., Ltd.

[0095] After the system is completed, the error-prone PCR reaction is performed, and the program settings are as follows:

[0096] a. Pre-denaturation: 95 ℃ 5 min;

[0097] b. Denaturation: 95 ℃ 30 s;

[0098] c. Annealing: 56 ℃ 45 s;

[0099] d. Extension: 72 °C 50 s;

[0100] EB-D reaction for 35 cycles;

[0101] f. Extension: 72 ℃ 10 min.

[0102] After the PCR reaction was completed, the PCR amplification products were subjected to 0.8% agarose gel electrophoresis, and the PCR products were recovered using a small amount of DNA recovery kit to obtain different laccase mutant genes. tear . Recombinant plasmid pET- tear , and then the recombinant plasmid pET- tear Transformed into Escherichia coli BL21 to obtain the recombinant strain BL21 / pET- tear .

[0103] 2. Mutant Screening

[0104] (1) Take 2 mL of sterilized LB medium and add it to a 96-well plate. Add 2 µL of Kan resistance (effective concentration 50 µg / mL) and different mutant colonies. Incubate overnight in a shaker at 37 °C. The wild type is used as the control.

[0105] (2) Transfer the bacterial solution to a new 24-well plate at a 2% inoculum volume, add 1 mL of sterilized LB (containing Kan resistance) to each well, and culture at 37 °C, 600 r / min for 3 h (cultivate until OD600 is 0.6-0.8), add 0.5 mM IPTG to each well, and induce overnight culture at 16 °C in a shaker;

[0106] (3) Collect the cells using a plate centrifuge, centrifuge at 5000 r / min for 15 min, discard the supernatant, blow and mix the cells with a pH 7.0 buffer, collect the bacterial liquid into 2 mL centrifuge tubes, and perform cell disruption. After disruption, collect the supernatant by centrifugation, and divide the supernatant into two parts. One part is placed in an 80 ℃ water bath according to step 1 of Example 2 to determine the enzyme activity of different mutants; the other part is kept at 80 ℃ for 30 min, and then the thermal stability of different mutants is determined according to step 2 of Example 2;

[0107] (4) Select the mutant transformants whose supernatant catalyzed ABTS absorbance at 420 nm is higher than that of the wild type and whose absorbance at 420 nm catalyzed ABTS is still higher than that of the wild type after being incubated at 80°C for 30 min for sequencing to obtain the mutant gene lacm1 (shown in SEQ ID NO.4), and finally the mutant T262F / T387L (shown in SEQ ID NO.3) with higher activity and stability than the wild type was obtained in the preliminary screening. The transformant containing the mutant was named the recombinant strain BL21 / pET- lacm1 .

[0108] Example 4 Expression, purification and enzyme activity determination of WT and T262F / T387L

[0109] 1. Inducible expression of recombinant strains

[0110] (1) On the LB plate, pick BL21 / pET- lacm1 and BL21 / pET- milk A single colony was inoculated into a 5 mL LB tube (containing 50 µg / mL Kan) and placed in a shaker at 37 °C for 10 h.

[0111] (2) Transfer the recombinant bacterial suspension into 250 mL LB medium (final concentration of 50 µg / mL Kan) and culture in a shaker at 37 °C for 2-2.5 h.

[0112] (3) Add 125 μL of IPTG (final concentration 0.5 mmol / L) and induce the culture in a shaking incubator at 16 °C for 16-20 h;

[0113] (4) Purify the fermentation broth to prepare wild-type laccase and various laccase mutants.

[0114] 2. Ni column purification of recombinant protein

[0115] (1) Disruption of bacterial cells

[0116] The fermentation broth was collected using a centrifuge cup and centrifuged at 10,000 rpm for 15 min. The supernatant was discarded and 20 mL of Lysis buffer was added to blow the bacteria away. Ultrasonic waves were used to break the bacteria, destroy the cell wall and release the protein inside the bacteria.

[0117] After the disruption was completed, the bacterial solution was poured into a 50 mL centrifuge tube and centrifuged at 12000 rpm for 30 min at 4 °C to collect the supernatant.

[0118] (2) WT and T262F / T387L combined with nickel column

[0119] a. Before the nickel column purification process, add an appropriate amount of ddH2O to the purification column and add two column volumes of Lysis buffer to balance the resin;

[0120] b. Mix the equilibrated resin and bacterial supernatant, place in a magnetic stirrer, and stir at 80-100 r / min for 1 h, while keeping the temperature low (4 °C) throughout the process.

[0121] (3) Protein purification

[0122] a. Add the binding solution to the purification column in 2-3 times in the chromatography cabinet;

[0123] b. After the binding solution is completely filtered out, add 10 mL of Wash buffer to elute the impurities bound to the resin;

[0124] c. Finally, add 10 mL of pre-cooled Elution Buffer to the purification column to elute all the target protein bound to the resin and collect the filtrate;

[0125] d. Transfer all the eluate to an ultrafiltration centrifuge tube, centrifuge and ultrafilter until 1 mL of the solution remains, add pre-cooled 50 mM, pH 7.0 Tris-HCl buffer, and repeat the replacement twice to obtain purified WT and T262F / T387L proteins.

[0126] The specific activity was determined according to step 1 of Example 2, and the specific activity of the wild-type LAC and the mutant T262F / T387L was finally calculated as follows:

[0127]

[0128] The temperature stability was determined according to step 2 of Example 2 to obtain the thermal stability at 80°C (i.e., the enzyme activities of WT and T262F / T387L mutants after being incubated at 80°C for different times were determined according to the method of step 2 of Example 2, and the enzyme activity change curve was plotted. The enzyme activity change curve is shown in FIG. Figure 3As shown), the half-lives of wild-type LAC and mutants were finally calculated as follows:

[0129]

[0130] Example 5 Expression and preparation of laccase mutants in Bacillus subtilis

[0131] The gene encoding the laccase mutant T262F / T387L lacm1 and wild-type laccase encoding gene milk The new recombinant plasmid pLY-3- lacm1 and pLY-3- milk ;

[0132] The recombinant plasmids were transferred into Bacillus subtilis WB600, and the mutant recombinant bacteria WB600 / pLY-3- lacm1 and wild-type laccase recombinant strain WB600 / pLY-3- milk .

[0133] The recombinant strain WB600 / pLY-3- lacm1、 WB600 / pLY-3- milk The samples were inoculated into 5 mL of fermentation medium (containing kanamycin, 50 µg / mL) respectively, cultured at 37°C, 220 r / min overnight, transferred to 50 mL of fresh fermentation medium (containing kanamycin, 50 µg / mL) at a 2% inoculum amount, and cultured at 37°C, 220 r / min for 48 h (fermentation medium (g / L): corn flour 64, soybean cake powder 40, 2.7 amylase, Na2HPO44, KH2PO40.3, and the rest was water; kept at 90°C for 30 min and sterilized at 121°C for 20 min).

[0134] The ATBS method in Example 2 was used to determine the activity of the laccase obtained by fermentation of Bacillus subtilis (the fermentation broth was centrifuged and the supernatant was taken to determine the activity). In Bacillus subtilis, the activity of the wild type enzyme was 1172.3 U / mL, and the activity of the fermentation enzyme of T262F / T387L was 1452.1 U / mL.

[0135] Example 6 Expression and preparation of laccase mutants in recombinant strains of Bacillus amyloliquefaciens

[0136] The gene encoding the laccase mutant T262F / T387L lacm1 and wild-type laccase encoding gene milk The new recombinant plasmid pLY-3- lacm1 and pLY-3- milk;

[0137] The recombinant plasmids were respectively transferred into Bacillus amyloliquefaciens CGMCC No.11218, and the mutant recombinant bacteria CGMCC No.11218 / pLY-3- lacm1 and wild-type laccase recombinant strain CGMCC No.11218 / pLY-3- milk .

[0138] The recombinant strain CGMCC No.11218 / pLY-3- lacm1 、CGMCC No.11218 / pLY-3- milk The samples were inoculated into 5 mL of fermentation medium (containing kanamycin, 50 µg / mL) respectively, cultured at 37°C, 220 r / min overnight, transferred to 50 mL of fresh fermentation medium (containing kanamycin, 50 µg / mL) at a 2% inoculum amount, and cultured at 37°C, 220 r / min for 48 h (fermentation medium (g / L): corn flour 64, soybean cake powder 40, 2.7 amylase, Na2HPO44, KH2PO40.3, and the rest was water; kept at 90°C for 30 min and sterilized at 121°C for 20 min).

[0139] The ATBS method in Example 2 was used to determine the activity of the laccase obtained by fermentation of Bacillus amyloliquefaciens (the fermentation broth was centrifuged and the supernatant was taken to determine the enzyme activity). The enzyme activity of the wild type of Bacillus amyloliquefaciens was 2358.3 U / mL, and the enzyme activity of the fermentation of T262F / T387L was 2912.8 U / mL.

[0140] Example 7 Expression and preparation of laccase mutants in Pichia pastoris GS115 recombinant strain

[0141] The gene encoding the laccase mutant T262F / T387L lacm1 and wild-type laccase encoding gene milk The new recombinant plasmid pPIC9K- lacm1 and pPIC9K- milk ;;

[0142] Use restriction enzyme Salt The recombinant plasmid pPIC9K- lacm1 and pPIC9K- milk Linearization, the linearized recombinant plasmids were electroporated into Pichia pastoris GS115, and the mutant recombinant bacteria GS115 / pPIC9K- lacm1 and wild-type laccase recombinant bacteria GS115 / pPIC9K- milk .

[0143] (1) Select the recombinant expression strain GS115 / pPIC9K- lacm1 and GS115 / pPIC9K- milk A single colony was inoculated into a 5 mL YPD tube containing 50 μg / mL Kan and cultured at 30 °C, 200 rpm for 24 h;

[0144] (2) Pipette 1 mL of bacterial solution and inoculate it into BMGY enrichment medium. Cultivate at 30 °C and 220 rpm for 16-19 h.

[0145] (3) Collect the bacterial liquid in the BMGY medium in a 50-mL centrifuge tube and collect the bacteria by centrifugation; add 20 mL of BMMY medium to resuspend the bacteria and collect the bacteria by centrifugation, and repeat once; add 10 mL of BMMY medium to resuspend the bacteria and transfer the bacterial liquid to the BMMY medium with a pipette. Add methanol at a final concentration of 0.5% every 12 hours and ferment for 6 days.

[0146] The ATBS method in Example 2 was used to determine the activity of the laccase obtained from the fermentation of Pichia pastoris (the fermentation broth was centrifuged and the supernatant was taken to determine the enzyme activity). The fermentation activity of the laccase in Pichia pastoris GS115 was 1674.2 U / mL for the wild type and 2031.6 U / mL for the fermentation of T262F / T387L.

[0147] Example 8 Application of Laccase Mutants in Degrading Corn Straw and Bagasse

[0148] Preliminary treatment of corn stalks and sugarcane bagasse Corn stalks and sugarcane bagasse were sourced from local enterprises in Jiangsu, China. They were first washed with deionized water to remove surface dirt, then placed in a constant temperature drying oven at 60 °C for drying to reduce the moisture content to less than 2%. The dried raw materials were crushed with a pulverizer and filtered with a 40-mesh sieve to ensure that they became uniform powders. Finally, they were placed at room temperature for subsequent use.

[0149] 2. Laccase degradation of lignin

[0150] The treated corn stalks and sugarcane bagasse were used as samples to be degraded, and the wild-type laccase and mutant laccase T262F / T387L of the present invention were added respectively. A 20 mL system was used: 1 g of the sample to be degraded was taken in a 50 mL centrifuge tube, 50 U of WT / mutant laccase was added, and then 5 mM Cu was added to the solution at pH 5.0. 2+ The volume was filled to 20 mL with citric acid-sodium hydrogen phosphate buffer and shaken in a water bath at 50 °C and 150 r / min for 6 h.

[0151] The lignin degradation rate after treatment with wild-type laccase and mutant T262F / T387L was determined as follows:

[0152] (1) The treated corn stalks and sugarcane bagasse were extracted with benzene / alcohol for 8 h and dried to absolute dryness for later use;

[0153] (2) Weigh 0.3 g and add 3 mL of 72% sulfuric acid, stirring until the raw materials are fully mixed. After mixing, place in a 30°C water bath and keep warm for 60 min;

[0154] (3) Add 84 mL of deionized water to dilute to 4%, place in a 121 °C autoclave, take out after 45 min, use a G3 (weighed, recorded as m0) sand core funnel for vacuum filtration, take 50 mL of the filtrate to measure the absorbance of the acid hydrolyzate at 205 nm, and calculate the acid-soluble lignin content. The determination method is GB / 10337-89 Determination of acid-soluble lignin in papermaking raw materials and pulp;

[0155] Use hot deionized water to wash the filtered residue until it is neutral, dry it in an oven at 105°C, and weigh and record (m1). After drying, transfer it to a resistance furnace and burn it at 575°C for 4 hours. Take it out and cool it to room temperature, then weigh and record (m2), and calculate the content of acid-insoluble lignin. The final calculated lignin degradation rate is as follows:

[0156] 1. Degradation results of lignin in corn straw

[0157] The lignin degradation rate was 23.7% after mutant treatment and 17.2% after WT treatment.

[0158]

[0159] 2. Degradation results of lignin in bagasse

[0160] The lignin degradation rate was 22.5% after treatment with mutant T262F / T387L, and 15.3% after treatment with WT.

[0161]

[0162] The above results showed that the mutant had higher lignin degradation rates on corn straw and sugarcane bagasse than WT after laccase treatment of corn straw and sugarcane bagasse.

[0163] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A laccase mutant, characterized in that: The laccase mutant is obtained by causing T262F and T387L mutations to occur on the basis of the wild-type laccase shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

3.

2. A gene encoding the laccase mutant according to claim 1.

3. The gene encoding the laccase mutant according to claim 2, characterized in that: The nucleic acid sequence is shown in SEQ ID NO.

4.

4. A recombinant plasmid or recombinant strain comprising the mutant encoding gene according to claim 2.

5. Use of the recombinant plasmid or recombinant strain according to claim 4 in producing the laccase mutant according to claim 1, characterized in that: It is used in the degradation of lignocellulose.

6. The use according to claim 5, characterized in that The lignocellulose includes but is not limited to straw and bagasse.

7. The use according to claim 6, characterized in that The straw includes but is not limited to corn straw, rice straw and wheat straw.

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