WO2019227512A1 - 一种可高效表达角蛋白酶的重组枯草芽孢杆菌工程菌 - Google Patents

一种可高效表达角蛋白酶的重组枯草芽孢杆菌工程菌 Download PDF

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WO2019227512A1
WO2019227512A1 PCT/CN2018/089937 CN2018089937W WO2019227512A1 WO 2019227512 A1 WO2019227512 A1 WO 2019227512A1 CN 2018089937 W CN2018089937 W CN 2018089937W WO 2019227512 A1 WO2019227512 A1 WO 2019227512A1
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keratinase
bacillus subtilis
fermentation
efficiently expressing
ker
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French (fr)
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张娟
彭政
陈坚
堵国成
冒鑫哲
周恒瑞
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江南大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus

Definitions

  • the invention relates to a recombinant Bacillus subtilis engineering bacterium capable of efficiently expressing keratinase, and belongs to the technical fields of genetic engineering and fermentation engineering.
  • Keratinase is a specific protease that can degrade insoluble hard proteins--keratin substrates (such as feathers, wool, hair, dander, etc.). It is mainly composed of bacteria, fungi, actinomycetes and other microorganisms. It is obtained by keratin as a single carbon and nitrogen source.
  • keratinase As a protease with broad substrate specificity and strong hydrolytic catalysis ability, keratinase can replace traditional protease, and is widely used in feather degradation, leather textile, feed additives, organic fertilizers and detergents, etc., and has a huge market.
  • genetic engineering bacteria are often used to enhance the transcription and translation of the keratinase gene to achieve the purpose of efficient expression and active secretion of keratinase in bacteria, in order to effectively improve the performance and yield of keratinase, but at this stage it is still not Can successfully construct genetically engineered bacteria that can efficiently express keratinase.
  • keratinase there are mainly three kinds of heterologous expression hosts for keratinase, which are E. coli, Bacillus subtilis, and Pichia yeast.
  • Escherichia coli has a clear genetic background and simple expression operations, when the T7 strong promoter is used, the E. coli expression system is prone to form inclusion bodies, resulting in low keratinase production; Pichia yeasts have long fermentation cycles, and often because of codon preference and Factors such as glycosylation of enzymes have hindered the efficient production of keratinase.
  • keratinase was expressed by Bacillus subtilis, although it was successfully expressed, the production of keratinase was extremely low and its activity was almost invisible.
  • the present invention successfully constructed a keratinase gene derived from a food safety strain Bacillus licheniformis BBE11-1, pP43NMK as an expression vector, and Bacillus subtilis WB600 as an expression host, and successfully constructed A genetically engineered bacterium Bacillus subtilis WB600-pP43NMK-ker capable of efficiently expressing keratinase was developed.
  • the fermentation medium and fermentation conditions for the production of keratinase by using this engineering bacterium as a production strain were intensively studied. This is a fermentation medium that can increase the production of keratinase and the best process for fermentative production of keratinase.
  • the invention provides a recombinant Bacillus subtilis engineered bacterium capable of efficiently expressing keratinase.
  • the engineered bacterium includes a recombinant plasmid and an expression host; the recombinant plasmid includes a target gene and an expression vector; and the target gene is a keratinase gene ( ker); the expression vector is pP43NMK; the expression host is Bacillus subtilis.
  • the keratinase gene is derived from Bacillus licheniformis BBE11-1.
  • the nucleotide sequence of the keratinase gene (ker) is SEQ ID NO.1.
  • the recombinant plasmid is obtained by amplifying the keratinase gene (ker) with specific primers having nucleotide sequences of SEQ ID NO. 2 and SEQ ID ID NO. 3, to obtain the amplified keratinase gene (ker).
  • the keratinase gene (ker) was amplified with pP43NMK vector using specific primers with the nucleotide sequences of SEQ ID No. 4 and SEQ ID NO. 5 to obtain the amplified linearized pP43NMK vector.
  • the keratinase gene (ker) and the amplified linearized pP43NMK vector were obtained by homologous recombination.
  • the homologous recombination is performed by a homologous recombination kit.
  • the expression host is Bacillus subtilis WB600.
  • the invention provides a fermentation medium which can be used to produce keratinase.
  • the components of the medium include 15-25g / L of peptone, 5-15g / L of yeast powder, 15-25g / L of sucrose, and 2-4g. / L of KH 2 PO 4 , 5-7 g / L of Na 2 HPO 4 , 0.2-0.4 g / L of MgSO 4 , and the pH of the medium is 6.0-8.0.
  • the components of the culture medium include 20 g / L of peptone, 10 g / L of yeast powder, 20 g / L of sucrose, 3 g / L of KH 2 PO 4 , and 6 g / L of Na. 2 HPO 4 , 0.3 g / L of MgSO 4 , and the pH of the medium is 7.0.
  • the invention provides a method for producing keratinase.
  • the method is to use the above-mentioned recombinant Bacillus subtilis engineering bacterium which can efficiently express keratinase and the above-mentioned one which can be used for producing keratinase fermentation medium.
  • the method is to inoculate the above-mentioned recombinant Bacillus subtilis engineering bacteria capable of efficiently expressing keratinase into the above-mentioned fermentation medium that can be used to produce keratinase, and control the temperature, pH, Dissolved oxygen and fed glucose at a constant rate for fermentation.
  • the method is to inoculate the above-mentioned recombinant Bacillus subtilis engineering bacteria capable of efficiently expressing keratinase into the above-mentioned one which can be used for the production of keratinase fermentation culture.
  • the controlled temperature is 35-39 ° C
  • the pH is 6.0-8.0
  • the dissolved oxygen is 25-35%
  • the glucose is fed at a constant rate of 35-45g / L / h for fermentation, and the fermentation time is 26-30h. ;
  • the dissolved oxygen is 100% before the fermentation medium is not inoculated with the recombinant Bacillus subtilis engineering bacterial solution.
  • the method is to inoculate the above-mentioned recombinant Bacillus subtilis engineering bacteria capable of efficiently expressing keratinase into the above-mentioned one fermentation medium that can be used to produce keratinase.
  • the temperature was controlled at 37 ° C., the pH was 7.0, and the dissolved oxygen was 30%, and glucose was fed at a constant speed of 40 g / L / h for fermentation, and the fermentation time was 28 h.
  • the invention provides the above-mentioned recombinant Bacillus subtilis engineering bacteria capable of efficiently expressing keratinase, or the above-mentioned method for producing keratinase fermentation medium or the above-mentioned method for producing keratinase.
  • a keratinase gene derived from Bacillus licheniformis BBE11-1 is used as a target gene
  • pP43NMK is an expression vector
  • Bacillus subtilis WB600 is used as an expression host.
  • a highly efficient expression of keratinase was successfully constructed The genetically engineered bacterium Bacillussubtilis WB600-pP43NMK-ker, using this engineered bacterium to ferment, the enzyme activity in the fermentation supernatant can reach 3329.3U / mL;
  • the present invention makes in-depth research on the fermentation medium and fermentation conditions when using the engineered bacteria as a production strain to produce keratinase, and obtains a fermentation medium that can increase the production of keratinase and the most fermentative production of keratinase.
  • the enzyme activity in the fermentation supernatant can be as high as 31015.6 U / mL.
  • FIG. 1 SDS-PAGE analysis results of fermentation supernatants of recombinant bacteria expressing keratinase using different expression vectors
  • 1 Bacillus subtilis WB600 fermentation liquid supernatant
  • 2 Bacillus subtilis WB600-pP43NMK-ker fermentation liquid supernatant
  • Figure 2 Enzyme activity of a fermentation supernatant of a recombinant strain expressing keratinase using different expression vectors
  • 1 wild keratinase
  • 2 Bacillus subtilis WB600-pP43NMK-ker
  • 3 Bacillus subtilis WB600-pMA5-ker
  • 4 Bacillus subtilis WB600-pSTOP1622-ker
  • Figure 3 Enzyme activity of the fermentation supernatant of recombinant bacteria cultured using different carbon sources
  • Figure 4 Enzyme activity of fermentation supernatant of recombinant bacteria cultured with different concentrations of sucrose
  • Figure 5 Enzyme activity of the fermentation supernatant of recombinant bacteria cultured using different nitrogen sources
  • Figure 6 Enzyme activity of fermentation supernatant of recombinant bacteria cultured with different concentrations of peptone
  • FIG. 7 The enzyme activity trend of the fermentation supernatant of fermenting recombinant bacteria using a 3-L fermentor system.
  • Seed medium 5g / L yeast powder, 10g / L peptone, 5g / L NaCl.
  • Initial culture medium peptone 20g / L, yeast powder 10g / L, glucose 10g / L, KH 2 PO 4 3g / L, Na 2 HPO 4 6g / L, MgSO 4 0.3g / L.
  • Optimized medium peptone 20g / L, yeast powder 10g / L, sucrose 20g / L, KH 2 PO 4 3g / L, Na 2 HPO 4 6g / L, MgSO 4 0.3g / L.
  • Keratinase activity measurement Take 50 ⁇ L of a suitably diluted fermentation supernatant, add 150 ⁇ L of 50 mM Gly / NaOH solution as a buffer, and 100 ⁇ L of 2.5% water-soluble keratin (purchased from Tishila (Shanghai) Chemical Industry Development Co., Ltd. Company, product code: K0043) as substrate, after mixing, react at 40 ° C for 20min. The reaction was stopped by adding 200 ⁇ L of 4% (w / v) trichloroacetic acid (TCA), and centrifuged at 8000 r / min for 3 minutes at room temperature.
  • TCA trichloroacetic acid
  • An enzyme unit is defined as 1 ⁇ mol of tyrosine released per minute by converting keratin substrate at 40 ° C.
  • Example 1 Construction of a keratinase expression engineered strain with a strong promoter
  • PMA5 vector was used as a template to obtain linearized pMA5.
  • PCR amplification conditions were: pre-denaturation at 98 ° C for 30 min, denaturation at 98 ° C for 30 s, and annealing at 55 ° C for 10 s, 72 °C extension 7min 30s (25 cycles);
  • PCR amplification conditions are: pre-denaturation at 98 °C for 30min, denaturation at 98 °C for 30s, annealing at 55 °C for 10s, 72 °C extension for 6min 30s (25 cycles);
  • Bacillus licheniformis BBE11-1 was cultured in chicken feather medium for 48 hours, and then centrifuged to detect the keratinase activity in the supernatant of the fermentation broth.
  • Keratinase activity in the fermentation supernatant of Bacillus subtilis WB600-pP43NMK-ker was 3329.3U / mL
  • Keratinase activity in the fermentation supernatant of Bacillus subtilis WB600-pMA5-ker was 628.6.
  • the keratinase activity in the fermentation supernatant of U / mL and Bacillus subtilis WB600-pSTOP1622-ker was 574.1 U / mL, which were 27.7, 5.2, and 4.8 times the wild-type keratinase (120 U / mL), respectively.
  • Example 2 Effect of carbon source on keratinase production
  • Example 2 The engineered bacterium Bacillus subtilis WB600-pP43NMK-ker obtained in Example 1 was inoculated into the initial culture medium, cultured at 37 ° C. and 220 rpm for 28 hours, and the keratinase activity of the fermentation broth was measured (see FIG. 3 for the test results).
  • the result of enzyme activity test was: the fermentation activity of keratinase in the supernatant was 2922.4U / mL using glucose for fermentation of recombinant bacteria, and the activity of keratinase in the supernatant was 6557.5 when fermentation was performed using sucrose.
  • U / mL fermented by recombinant maltose fermentation
  • the enzyme activity of keratinase in the resulting supernatant was 3085.6 U / mL
  • fermented by soluble starch the enzyme activity of keratinase in the supernatant was 5330.2U.
  • sucrose is the optimal carbon source.
  • Example 3 Effect of carbon source concentration on keratinase production
  • Example 2 The engineered bacterium Bacillus subtilis WB600-pP43NMK-ker obtained in Example 1 was inoculated into the initial culture medium, cultured at 37 ° C. and 220 rpm for 28 hours, and the activity of the keratinase in the supernatant of the fermentation broth was measured (see FIG. 4 for the test results).
  • the enzyme activity test results were: 5g / L glucose was used for fermentation of recombinant bacteria, and the enzyme activity of keratinase in the obtained supernatant was 2687.3U / mL, and 10g / L glucose was used for fermentation of recombinant bacteria to obtain the middle angle of the supernatant.
  • the enzyme activity of the protease was 2909.1 U / mL
  • the recombinant bacteria were fermented with 15 g / L glucose
  • the enzyme activity of keratinase in the supernatant was 5212.0 U / mL
  • the recombinant bacteria were fermented with 20 g / L glucose.
  • the enzyme activity of keratinase in the supernatant is 6557.5U / mL, and the enzyme activity of keratinase in the supernatant is 2682.3U / mL using 25g / L glucose for recombinant bacteria fermentation, so the optimal concentration of sucrose is 20g / L.
  • Example 4 Effect of nitrogen source on keratinase production
  • the results of enzyme activity test were: fermentation of yeast with yeast extract, keratinase enzyme activity in the resulting supernatant was 1549.1 U / mL, and fermentation of recombinant bacteria with beef extract, keratinase enzyme activity in the supernatant obtained It was 1056.2 U / mL, and the enzyme activity of keratinase in the supernatant was 5651.9 U / mL using peptone fermentation. The enzyme activity of keratinase in the supernatant was 5651.9 U / mL.
  • Example 5 Effect of nitrogen source concentration on keratinase production
  • Example 2 The engineered bacterium Bacillus subtilis WB600-pP43NMK-ker obtained in Example 1 was inoculated into the initial culture medium, cultured at 37 ° C. and 220 rpm for 28 hours, and the keratinase activity of the fermentation broth was measured (see FIG. 6 for the test results).
  • the result of enzyme activity test was: 5 g / L peptone was used for fermentation of recombinant bacteria, and the keratinase enzyme activity in the obtained supernatant was 2360.6 U / mL; 10 g / L peptone was used for fermentation of recombinant bacteria, and the obtained supernatant was obtained.
  • the activity of keratinase was 2758.1 U / mL, and recombinant enzyme fermentation was performed using 15 g / L of peptone.
  • the resulting keratinase enzyme activity was 4307.6 U / mL, and 20 g / L of peptone was used for recombinant bacteria fermentation.
  • the enzyme activity of keratinase in the obtained supernatant was 7856.7 U / mL, and 25 g / L of peptone was used for recombinant bacteria fermentation.
  • the enzyme activity of keratinase in the supernatant was 4217.3 U / mL, so The optimal concentration is 20g / L.
  • Example 6 Batch-feed culture of a 3L fermentation tank of genetically engineered bacteria for efficient production of keratinase
  • Example 2-5 (2) Add 1.5L of the optimized medium obtained in Example 2-5 to a 3L fermentor, treat it at 121 ° C for 20 minutes, and cool it to room temperature. Adjust the initial pH to 7.0, the temperature is 37 ° C, the speed is 600rpm, and the ventilation volume is 2.0vvm. 5% of the inoculation amount is used in the secondary seed solution;
  • the initial sucrose can provide the growth requirements of the bacteria for 6h.
  • the initial sugar is depleted, glucose is fed at a constant rate of 28.8g / L ⁇ h -1 within 6h-18h, and the feeding is stopped at the 18th hour until the end of fermentation;

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Abstract

提供一种可高效表达角蛋白酶的重组枯草芽孢杆菌工程菌，其是以地衣芽孢杆菌(Bacillus licheniformis BBE11-1)来源的角蛋白酶基因为目的基因，pP43NMK为表达载体，枯草芽孢杆菌(Bacillus subtilis)WB600为表达宿主，构建得到的基因工程菌Bacillus subtilis WB600-pP43NMK-ker。还提供了一种可增加角蛋白酶产量的发酵培养基以及发酵生产角蛋白酶的最佳工艺。

Description

一种可高效表达角蛋白酶的重组枯草芽孢杆菌工程菌 技术领域
本发明涉及一种可高效表达角蛋白酶的重组枯草芽孢杆菌工程菌，属于基因工程以及发酵工程技术领域。
背景技术
角蛋白酶是一种能够降解不可溶的硬质蛋白--角蛋白类底物(例如羽毛、羊毛、头发、皮屑等)的特异性蛋白酶，其主要由细菌、真菌、放线菌等微生物在以角蛋白为单一碳氮源时生长分泌获得。
作为一种底物专一性较宽泛，水解催化能力强的蛋白酶，角蛋白酶可以替代传统蛋白酶，广泛应用于羽毛降解、皮革纺织、饲料添加剂、有机化肥和洗涤剂等领域，具有巨大的市场。
但是，野生角蛋白酶的性能拙劣、产量低下，远远不能满足市场需求，因此，它很难被真正应用于工业化生产中。
现在，常常采用基因工程菌的手段，强化角蛋白酶基因的转录和翻译，达到在菌中高效表达和活性分泌角蛋白酶的目的，以期能够有效提高角蛋白酶的性能和产量，但是，现阶段仍旧未能成功构建出可高效表达角蛋白酶的基因工程菌。
目前，角蛋白酶的异源表达宿主主要有三种菌，它们分别是大肠杆菌、枯草芽孢杆菌和毕赤酵母。大肠杆菌虽然遗传背景清晰，表达操作简单，但是采用T7强启动子时，大肠杆菌表达系统容易形成包涵体，造成角蛋白酶产量低下；毕赤酵母的发酵周期长，且往往因为密码子偏好性和酶分子糖基化等因素，阻碍了角蛋白酶的高效生产；而用枯草芽孢杆菌表达角蛋白酶时，虽然成功进行了表达，但是，角蛋白酶产量极为低下且活性几乎不可见。
因此，急需构建一种可高效表达角蛋白酶的基因工程菌以满足市场需求。
发明内容
为解决上述问题，本发明以食品安全性菌株地衣芽孢杆菌(Bacillus licheniformis BBE11-1)来源的角蛋白酶基因为目的基因，pP43NMK为表达载体，枯草芽孢杆菌(Bacillus subtilis)WB600为表达宿主，成功构建了可高效表达角蛋白酶的基因工程菌Bacillus subtilis WB600-pP43NMK-ker；同时，本发明对以该工程菌作为生产菌株生产角蛋白酶时的发酵培养基以及发酵条件进行了深入的研究，得到了一种可增加角蛋白酶产量的发酵培养基以及发酵生产角蛋白酶的最佳工艺。
本发明的技术方案如下：
本发明提供了一种可高效表达角蛋白酶的重组枯草芽孢杆菌工程菌，所述工程菌包含重组质粒和表达宿主；所述重组质粒包含目的基因以及表达载体；所述目的基因为角蛋白酶基因(ker)；所述表达载体为pP43NMK；所述表达宿主为枯草芽孢杆菌(Bacillus subtilis)。
在本发明的一种实施方式中，所述角蛋白酶基因(ker)来源于地衣芽孢杆菌(Bacillus licheniformis BBE11-1)。
在本发明的一种实施方式中，所述角蛋白酶基因(ker)的核苷酸序列为SEQ ID NO.1。
在本发明的一种实施方式中，所述重组质粒是通过以核苷酸序列为SEQ ID NO.2和SEQ ID NO.3的特异性引物扩增角蛋白酶基因(ker)，得到扩增后的角蛋白酶基因(ker)，以核苷酸序列为SEQ ID NO.4和SEQ ID NO.5的特异性引物扩增pP43NMK载体，得到扩增后的线性化pP43NMK载体，然后将扩增后的角蛋白酶基因(ker)以及扩增后的线性化pP43NMK载体进行同源重组得到的。
在本发明的一种实施方式中，所述同源重组为通过同源重组试剂盒进行同源重组。
在本发明的一种实施方式中，所述表达宿主为枯草芽孢杆菌(Bacillus subtilis)WB600。
本发明提供了一种可用于生产角蛋白酶发酵培养基，所述培养基的成分包含15-25g/L的蛋白胨、5-15g/L的酵母粉、15-25g/L的蔗糖、2-4g/L的KH 2PO 4、5-7g/L的Na 2HPO 4、0.2-0.4g/L的MgSO4，所述培养基的pH为6.0-8.0。
在本发明的一种实施方式中，所述培养基的成分包含20g/L的蛋白胨、10g/L的酵母粉、20g/L的蔗糖、3g/L的KH 2PO 4、6g/L的Na 2HPO 4、0.3g/L的MgSO4，所述培养基的pH为7.0。
本发明提供了一种生产角蛋白酶的方法，所述方法为使用上述一种可高效表达角蛋白酶的重组枯草芽孢杆菌工程菌以及上述一种可用于生产角蛋白酶发酵培养基。
在本发明的一种实施方式中，所述方法为将上述一种可高效表达角蛋白酶的重组枯草芽孢杆菌工程菌接种至上述一种可用于生产角蛋白酶发酵培养基中，控制温度、pH、溶氧，并恒速流加葡萄糖进行发酵。
在本发明的一种实施方式中，所述方法为将上述一种可高效表达角蛋白酶的重组枯草芽孢杆菌工程菌以4-6％的接种量接种至上述一种可用于生产角蛋白酶发酵培养基中，控制温度为35-39℃、pH为6.0-8.0、溶氧为25-35％，并以35-45g/L/h的速度恒速流加葡萄糖进行发酵，发酵时间为26-30h；
所述溶氧以发酵培养基未接种重组枯草芽孢杆菌工程菌菌液前的溶氧为100％。
在本发明的一种实施方式中，所述方法为将上述一种可高效表达角蛋白酶的重组枯草芽孢杆菌工程菌以5％的接种量接种至上述一种可用于生产角蛋白酶发酵培养基中，控制温度为37℃、pH为7.0、溶氧为30％，并以40g/L/h的速度恒速流加葡萄糖进行发酵，发酵时间为28h。
本发明提供了上述一种可高效表达角蛋白酶的重组枯草芽孢杆菌工程菌或上述一种可用于生产角蛋白酶发酵培养基或上述一种生产角蛋白酶的方法在制备角蛋白酶、降解角蛋白、制备饲料添加剂、制备有机化肥以及制备洗涤剂方面的应用。
有益效果：
(1)本发明以地衣芽孢杆菌(Bacillus licheniformis BBE11-1)来源的角蛋白酶基因为目的基因，pP43NMK为表达载体，枯草芽孢杆菌(Bacillus subtilis)WB600为表达宿主，成功构建了可高效表达角蛋白酶的基因工程菌Bacillussubtilis WB600-pP43NMK-ker，利用此工程菌进行发酵，可使得发酵上清液中的酶活高达3329.3U/mL；
(2)本发明对以该工程菌作为生产菌株生产角蛋白酶时的发酵培养基以及发酵条件进行了深入的研究，得到了一种可增加角蛋白酶产量的发酵培养基以及发酵生产角蛋白酶的最佳工艺条件，利用此培养基和此工艺条件，配合本发明的枯草芽孢杆菌工程菌进行发酵，可使得发酵上清液中的酶活高达31015.6U/mL。
附图说明
图1使用不同表达载体表达角蛋白酶的重组菌的发酵上清液的SDS-PAGE分析结果；
其中，1：Bacillus subtilis WB600发酵液上清、2：Bacillus subtilis WB600-pP43NMK-ker发酵液上清；
图2使用不同表达载体表达角蛋白酶的重组菌的发酵上清液的酶活；
其中，1：野生角蛋白酶、2：Bacillus subtilis WB600-pP43NMK-ker、3：Bacillus subtilis WB600-pMA5-ker、4：Bacillus subtilis WB600-pSTOP1622-ker；；
图3使用不同碳源培养的重组菌的的发酵上清液的酶活；
图4使用不同浓度蔗糖培养的重组菌的的发酵上清液的酶活；
图5使用不同氮源培养的重组菌的的发酵上清液的酶活；
图6使用不同浓度蛋白胨培养的重组菌的的发酵上清液的酶活；
图7使用3-L发酵罐体系发酵重组菌的发酵上清液的酶活趋势。
具体实施方式
以下实施例用于非限制性地解释本发明的技术方案。
下述实施例中涉及的培养基为：
种子培养基：酵母粉5g/L，蛋白胨10g/L，NaCl 5g/L。
初始培养基：蛋白胨20g/L，酵母粉10g/L，葡萄糖10g/L，KH 2PO 43g/L，Na 2HPO 46g/L，MgSO 40.3g/L。
优化培养基：蛋白胨20g/L，酵母粉10g/L，蔗糖20g/L，KH 2PO 43g/L，Na 2HPO 46g/L，MgSO 40.3g/L。
下述实施例中涉及的检测方法如下：
角蛋白酶活性测定：取50μL适当稀释的发酵上清液，加入150μL 50mM的Gly/NaOH溶液作为缓冲液和100μL浓度为2.5％的水溶性角蛋白(购自梯希爱(上海)化成工业发展有限公司，产品编码：K0043)作为底物，混匀后于40℃下反应20min。加入200μL 4％(w/v)的三氯乙酸(TCA)终止反应，室温8000r/min离心3min。取上清200μL，加入1mL 4％(w/v)的Na 2CO 3和200μL的福林酚试剂，混匀后50℃下显色10min，使用0.5cm石英比色皿于660nm下测定清液吸光值。实验组3个平行，空白对照是在加入底物之前先加入反应终止剂TCA，其余操作同上。
定义一个酶活单位为40℃下每分钟转化角蛋白底物释放1μmol酪氨酸。
实施例1：构建带有强启动子的角蛋白酶表达工程菌株
具体构建步骤如下：
(1)设计特异性引物P1、P2(见表1)从野生的地衣芽孢杆菌基因组上扩增得到角蛋白酶基因ker，PCR扩增条件为：98℃预变性30min，98℃变性30s，55℃退火10s，72℃延伸90s(34个循环)；
(2)设计特异性引物P3、P4(见表1)以pP43NMK载体为模板扩增得到线性化pP43NMK，PCR扩增条件为：98℃预变性30min，98℃变性30s，55℃退火10s，72℃延伸7min(25个循环)；
(3)设计特异性引物P5、P6(见表1)以pMA5载体为模板扩增得到线性化pMA5，PCR扩增条件为：98℃预变性30min，98℃变性30s，55℃退火10s，72℃延伸7min 30s(25个循环)；
(4)设计特异性引物P7、P8(见表1)以pSTOP1622载体为模板扩增得到线性化pSTOP1622，PCR扩增条件为：98℃预变性30min，98℃变性30s，55℃退火10s，72℃延伸6min 30s(25个循环)；
(5)使用核算定量分析仪NanoDrop 2000对纯化的基因和载体片段进行定量，并使用同 源重组试剂盒(Clonexpress II One Step Cloning Kit)得到重组质粒pP43NMK-ker、pMA5-ker、pSTOP1622-ker，将得到的重组产物转化大肠杆菌JM109，次日富集得到的阳性克隆子并提取质粒；
(6)将重组质粒pP43NMK-ker、pMA5-ker、pSTOP1622-ker转化Bacillus subtilis WB600，挑取阳性克隆子，成功构建高效表达角蛋白酶的基因工程菌株Bacillus subtilis WB600-pP43NMK-ker、Bacillus subtilis WB600-pMA5-ker、Bacillus subtilis WB600-pSTOP1622-ker。
(7)挑取阳性克隆于种子培养基中过夜培养得到种子液，以5％的接种量接种于初始培养基中，在37℃、220rpm下培养28h；
(8)于4℃、12000rpm离心发酵液得到上清，使用SDS-PAGE对角蛋白酶的表达进行检测(检测结果见图1)，同时测定发酵液上清角蛋白酶活力(检测结果见图2)；
(9)将地衣芽孢杆菌(Bacillus licheniformis BBE11-1)在鸡毛培养基中培养48h后离心，检测其发酵液上清中角蛋白酶活力。
酶活力检测结果如下：Bacillus subtilis WB600-pP43NMK-ker的发酵上清液中的角蛋白酶酶活为3329.3U/mL、Bacillus subtilis WB600-pMA5-ker的发酵上清液中的角蛋白酶酶活为628.6U/mL、Bacillus subtilis WB600-pSTOP1622-ker的发酵上清液中的角蛋白酶酶活为574.1U/mL，分别是野生型角蛋白酶(120U/mL)的27.7、5.2、4.8倍。
表1引物
Figure PCTCN2018089937-appb-000001
Figure PCTCN2018089937-appb-000002
实施例2：碳源对角蛋白酶产量的影响
(1)选取20g/L的6种碳源(葡萄糖、蔗糖、麦芽糖、可溶性淀粉、甘油和果糖)与蛋白胨20g/L，酵母粉10g/L，KH 2PO4 3g/L，Na 2HPO4 6g/L，MgSO 40.3g/L配置成初始培养基进行单因素实验；
(2)将实施例1获得的工程菌Bacillus subtilis WB600-pP43NMK-ker接种至初始培养基，在37℃、220rpm下培养28h，测定发酵液上清角蛋白酶活力(检测结果见图3)。
酶活检测结果为：使用葡萄糖进行重组菌发酵，得到的上清液中角蛋白酶的酶活为2922.4U/mL、使用蔗糖进行重组菌发酵，得到的上清液中角蛋白酶的酶活为6557.5U/mL、使用麦芽糖进行重组菌发酵，得到的上清液中角蛋白酶的酶活为3085.6U/mL、使用可溶性淀粉进行重组菌发酵，得到的上清液中角蛋白酶的酶活为5330.2U/mL、使用甘油进行重组菌发酵，得到的上清液中角蛋白酶的酶活为2662.7U/mL、使用果糖进行重组菌发酵，得到的上清液中角蛋白酶的酶活为1266.5U/mL，所以蔗糖为最优碳源。
实施例3：碳源浓度对角蛋白酶产量的影响
(1)选取5种不同浓度(5g/L、10g/L、15g/L、20g/L、25g/L)的蔗糖与蛋白胨20g/L，酵母粉10g/L，KH 2PO4 3g/L，Na 2HPO4 6g/L，MgSO 40.3g/L配置成初始培养基进行单因素实验；
(2)将实施例1获得的工程菌Bacillus subtilis WB600-pP43NMK-ker接种至初始培养基，在37℃、220rpm下培养28h，测定发酵液上清角蛋白酶活力(检测结果见图4)。
酶活检测结果为：使用5g/L葡萄糖进行重组菌发酵，得到的上清液中角蛋白酶的酶活为2687.3U/mL、使用10g/L葡萄糖进行重组菌发酵，得到的上清液中角蛋白酶的酶活为2909.1U/mL、使用15g/L葡萄糖进行重组菌发酵，得到的上清液中角蛋白酶的酶活为5212.0U/mL、使用20g/L葡萄糖进行重组菌发酵，得到的上清液中角蛋白酶的酶活为6557.5U/mL、使用25g/L葡萄糖进行重组菌发酵，得到的上清液中角蛋白酶的酶活为2682.3U/mL，所以蔗糖的最优浓度是20g/L。
实施例4：氮源对角蛋白酶产量的影响
(1)选取20g/L的6种氮源(酵母膏、牛肉膏、蛋白胨、玉米浆、尿素和硝酸铵)与蔗糖20g/L，酵母粉10g/L，KH 2PO4 3g/L，Na 2HPO4 6g/L，MgSO 40.3g/L配置成初始培养基进行单因素实验；
(2)将实施例1获得的工程菌Bacillus subtilis WB600-pP43NMK-ker接种至初始培养基，在37℃、220rpm下培养28h，测定发酵液上清角蛋白酶活力(检测结果见图5)。
酶活检测结果为：使用酵母膏进行重组菌发酵，得到的上清液中角蛋白酶的酶活为1549.1U/mL、使用牛肉膏进行重组菌发酵，得到的上清液中角蛋白酶的酶活为1056.2U/mL、使用蛋白胨进行重组菌发酵，得到的上清液中角蛋白酶的酶活为5651.9U/mL、使用玉米浆进行重组菌发酵，得到的上清液中角蛋白酶的酶活为4714.3U/mL、使用尿素进行重组菌发酵，得到的上清液中角蛋白酶的酶活为3778.3U/mL、使用硝酸铵进行重组菌发酵，得到的上清液中角蛋白酶的酶活为293.6U/mL，所以蛋白胨为最优氮源。
实施例5：氮源浓度对角蛋白酶产量的影响
(1)选取5种不同浓度(5g/L、10g/L、15g/L、20g/L、25g/L)的蛋白胨与蔗糖20g/L，酵母粉10g/L，KH 2PO4 3g/L，Na 2HPO4 6g/L，MgSO 40.3g/L配置成初始培养基进行单因素实验；
(2)将实施例1获得的工程菌Bacillus subtilis WB600-pP43NMK-ker接种至初始培养基，在37℃、220rpm下培养28h，测定发酵液上清角蛋白酶活力(检测结果见图6)。
酶活检测结果为：使用5g/L的蛋白胨进行重组菌发酵，得到的上清液中角蛋白酶的酶活为2360.6U/mL、使用10g/L的蛋白胨进行重组菌发酵，得到的上清液中角蛋白酶的酶活为2758.1U/mL、使用15g/L的蛋白胨进行重组菌发酵，得到的上清液中角蛋白酶的酶活为4307.6U/mL、使用20g/L的蛋白胨进行重组菌发酵，得到的上清液中角蛋白酶的酶活为7856.7U/mL、使用25g/L的蛋白胨进行重组菌发酵，得到的上清液中角蛋白酶的酶活为4217.3U/mL，所以蛋白胨的最优浓度是20g/L。
总结实施例2-5，即得含有20g/L蛋白胨、10g/L酵母粉、20g/L蔗糖、3g/L KH 2PO 4、6g/L Na 2HPO 4、0.3g/L MgSO4、pH为7.0的优化培养基。
利用优化培养基进行基因工程菌株Bacillus subtilis WB600-pP43NMK-ker的发酵生产角蛋白酶验证，在37℃、220rpm下培养28h后，发酵上清液中的角蛋白酶活力达到7856.7U/mL，是初始培养基的2.4倍，是野生角蛋白酶的65.5倍。
实施例6：基因工程菌3L发酵罐分批补料培养高效生产角蛋白酶
对Bacillus subtilis WB600-pP43NMK-ker在3L发酵罐上的发酵过程进行控制优化，具体发酵步骤如下：
(1)将Bacillus subtilis WB600-pP43NMK-ker的甘油菌按1/1000的比例接种到种子培养基，于37℃，220rpm培养12h得到一级种子液，再按1/100的比例转接到种子培养基中，于37℃，220rpm培养4h得到二级种子液；
(2)在3L发酵罐中加入1.5L实施例2-5得到的优化培养基，121℃处理20min后冷 却至室温，调节初始pH为7.0，温度37℃，转速600rpm，通气量2.0vvm，按照5％的接种量接入二级种子液；
(3)初始蔗糖可以提供6h的菌体生长需求，6h时初糖耗尽，在6h-18h内以28.8g/L·h -1恒速流加葡萄糖，第18h停止流加直到发酵结束；
(4)采用氨水和磷酸对发酵过程进行实时pH调节，控制pH在7.0左右，控制发酵过程温度为37℃，发酵过程通过调节搅拌转速和通气量控制体系溶氧量在30％左右；
(5)对发酵过程取样，于4℃、12000rpm离心发酵液得到上清，测定得到发酵第28h时，发酵上清液中的角蛋白酶活力最高为31015.6U/mL，是野生角蛋白酶活力(120U/mL)的258倍(检测结果如图7)。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Figure PCTCN2018089937-appb-000003
Figure PCTCN2018089937-appb-000004
Figure PCTCN2018089937-appb-000005
Figure PCTCN2018089937-appb-000006
Figure PCTCN2018089937-appb-000007

Claims (13)

  1. 一种可高效表达角蛋白酶的重组枯草芽孢杆菌工程菌，其特征在于，所述工程菌包含重组质粒和表达宿主；所述重组质粒包含目的基因以及表达载体；所述目的基因为角蛋白酶基因(ker)；所述表达载体为pP43NMK；所述表达宿主为枯草芽孢杆菌(Bacillus subtilis)。
  2. 如权利要求1所述的一种可高效表达角蛋白酶的重组枯草芽孢杆菌工程菌，其特征在于，所述角蛋白酶基因(ker)来源于地衣芽孢杆菌(Bacillus licheniformis BBE11-1)。
  3. 如权利要求1或2所述的一种可高效表达角蛋白酶的重组枯草芽孢杆菌工程菌，其特征在于，所述角蛋白酶基因(ker)的核苷酸序列为SEQ ID NO.1。
  4. 如权利要求1-3任一所述的一种可高效表达角蛋白酶的重组枯草芽孢杆菌工程菌，其特征在于，所述重组质粒是通过以核苷酸序列为SEQ ID NO.2和SEQ ID NO.3的特异性引物扩增角蛋白酶基因(ker)，得到扩增后的角蛋白酶基因(ker)，以核苷酸序列为SEQ ID NO.4和SEQ ID NO.5的特异性引物扩增pP43NMK载体，得到扩增后的线性化pP43NMK载体，然后将扩增后的角蛋白酶基因(ker)以及扩增后的线性化pP43NMK载体进行同源重组得到的。
  5. 如权利要求1-4任一所述的一种可高效表达角蛋白酶的重组枯草芽孢杆菌工程菌，其特征在于，所述同源重组为通过同源重组试剂盒进行同源重组。
  6. 如权利要求1-5任一所述的一种可高效表达角蛋白酶的重组枯草芽孢杆菌工程菌，其特征在于，所述表达宿主为枯草芽孢杆菌(Bacillus subtilis)WB600。
  7. 一种可用于生产角蛋白酶发酵培养基，其特征在于，所述培养基的成分包含15-25g/L的蛋白胨、5-15g/L的酵母粉、15-25g/L的蔗糖、2-4g/L的KH 2PO 4、5-7g/L的Na 2HPO 4、0.2-0.4g/L的MgSO4，所述培养基的pH为6.0-8.0。
  8. 如权利要求7所述的一种可用于生产角蛋白酶的发酵培养基，其特征在于，所述培养基的成分包含20g/L的蛋白胨、10g/L的酵母粉、20g/L的蔗糖、3g/L的KH 2PO 4、6g/L的Na 2HPO 4、0.3g/L的MgSO4，所述培养基的pH为7.0。
  9. 一种生产角蛋白酶的方法，其特征在于，所述方法为使用权利要求1-6任一所述的一种可高效表达角蛋白酶的重组枯草芽孢杆菌工程菌以及权利要求5或6所述的一种可用于生产角蛋白酶发酵培养基。
  10. 如权利要求9所述的一种生产角蛋白酶的方法，其特征在于，所述方法为将权利要求1-6任一所述的一种可高效表达角蛋白酶的重组枯草芽孢杆菌工程菌接种至权利要求7或8所述的一种可用于生产角蛋白酶发酵培养基中，控制温度、pH、溶氧，并恒速流加葡萄糖进行发酵。
  11. 如权利要求9或10所述的一种生产角蛋白酶的方法，其特征在于，所述方法为将权利要求1-6任一所述的一种可高效表达角蛋白酶的重组枯草芽孢杆菌工程菌以4-6％的接种量接种至权利要求7或8所述的一种可用于生产角蛋白酶发酵培养基中，控制温度为35-39℃、pH为6.0-8.0、溶氧为25-35％，并以35-45g/L/h的速度恒速流加葡萄糖进行发酵，发酵时间为26-30h。
  12. 如权利要求9-11任一所述的一种生产角蛋白酶的方法，其特征在于，所述方法为将权利要求1-6任一所述的一种可高效表达角蛋白酶的重组枯草芽孢杆菌工程菌以5％的接种量接种至权利要求7或8所述的一种可用于生产角蛋白酶发酵培养基中，控制温度为37℃、pH为7.0、溶氧为30％，并以40g/L/h的速度恒速流加葡萄糖进行发酵，发酵时间为28h。
  13. 权利要求1-6任一所述的一种可高效表达角蛋白酶的重组枯草芽孢杆菌工程菌或权利要求7或8所述的一种可用于生产角蛋白酶发酵培养基或权利要求9-11任一所述的一种生产角蛋白酶的方法在制备角蛋白酶、降解角蛋白、制备饲料添加剂、制备有机化肥以及制备洗涤剂方面的应用。
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