信号肽筛选优化提高耐热α-环糊精酶在枯草芽胞杆菌中的表达

陈龙军; 林陈强; 贾宪波; 方宇; 张慧; 陈济琛

doi:10.19303/j.issn.1008-0384.2022.003.018

信号肽筛选优化提高耐热α-环糊精酶在枯草芽胞杆菌中的表达

doi: 10.19303/j.issn.1008-0384.2022.003.018

福建省农业科学院土壤肥料研究所，福建　福州　350003

基金项目: 福建省农业科学院对外合作项目（DWHZ2021-17）；福建省科技计划公益类专项（2022R1022-5、2018R1022-7）；福建省农业科学院探索创新项目（AA2018-13）；

详细信息

作者简介:
陈龙军（1983−），男，硕士，助理研究员，主要从事生物化工、酶工程及发酵工程等方面的研究（E-mail：monkeyirene@163.com）

通讯作者:
陈济琛（1964−），男，研究员，主要从事微生物菌剂研究（E-mail：chenjichen2001@163.com）

中图分类号: Q 786
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出版历程
- 收稿日期: 2021-08-26
- 修回日期: 2021-10-27
- 网络出版日期: 2022-03-21
- 刊出日期: 2022-03-28

Enhanced Thermophilic α-Cyclodextrin Glycosyltransferase Expression by Optimizing Target Signal Peptide in Bacillus subtilis

Soil and Fertilizer Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350003, China

摘要

摘要: 目的探索嗜热α-环糊精葡萄糖基转移酶（α-CGTase）在枯草芽胞杆菌（Bacillus subtilis RIK1285）中的高效胞外表达条件。方法以来源于枯草芽胞杆菌的173种信号肽为基础构建信号肽文库，筛选获得9条表达效率更高的信号肽，其中citH信号肽引导分泌效率最高。在此基础上，为进一步优化α-CGTase的分泌表达，对信号肽citH的Gly2、Asn3及Thr4进行饱和突变，并比较不同突变体的引导分泌效率。结果突变体G2R-N3K-T4L-CGT的引导分泌效率最高，重组枯草芽胞杆菌的胞外α-CGTase活力高达（14.2±0.11 ）U·mL⁻¹，相比未突变信号肽[（9.6±0.29 ）U·mL⁻¹]，分泌效率提高了47.9%；是野生菌株嗜热地芽胞杆菌Geobacillus caldoxylosilyticus.CHB1（0.66 U·mL⁻¹）的21.5倍。重组α-CGTase的最适反应pH值为6.0，最适反应温度为60 ℃，在50 ℃以内稳定；Mg²⁺、Ca²⁺对α-环糊精酶活性具有一定的激活作用。结论信号肽对环糊精酶在芽胞杆菌中的高效表达具有重要影响，为外源蛋白在芽胞杆菌中的表达提供一定借鉴。
- 信号肽 /
- α-环糊精葡萄糖基转移酶 /
- 饱和突变 /
- 枯草芽胞杆菌
Abstract: Objective Conditions to achieve high-efficiency secretory expression of the thermophilic α-cyclodextrin glucosyltransferase (α-CGTase) in Bacillus subtilis RIK1285 were explored to understand the role played by signal peptide. Method A library based on 173 signal peptides from B. subtilis was constructed to identify the highest secretion efficiency candidate. Using the saturation mutagenesis of segments in the selected signal peptide, the secretion expression of α-CGTase was enhanced. Result There were 9 signal peptides with high expression efficiency identified from the library. Among them, the signal peptide citH showed the highest secretion efficiency. Subsequently, the saturation mutagenesis of Gly2, Asn3, and Thr4 of citH produced a mutant, G2R-N3K-T4L-CGT, with an extracellular activity of α-CGTase as high as 14.2 U·mL⁻¹ and a secretion efficiency increased by 47.9% over the non-mutated signal peptide (9.6 U·mL⁻¹), which was 21.5 times higher than that of the wild-type Geobacillus caldoxylosilyticus CHB1 (0.66 U·mL⁻¹). The purified α-CGTase had a maximal activity at pH 6.0 and 60 ℃, was thermally stable within 50 ℃, and could be activated by Mg²⁺ and Ca²⁺. Conclusion The important effect signal peptide had on the high-efficiency expression of cyclodextrinase in B. subtilis was verified. The result provided a new and valuable reference for studies on the expression of extraneous proteins in the bacteria.
- Signal peptide /
- α-cyclodextrin glycosyltransferase /
- saturation mutagenesis /
- Bacillus subtilis

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图 1 不同信号肽对目的蛋白的影响

注：图中小写字母表示不同处理在P＜0.05水平下差异显著。下图同。

Figure 1. Effects of signal peptides on target protein production

Note: Data with different letters indicate significant differences among treatments at P＜0.05. Same for following figures.

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图 2 不同信号肽SDS-PAGE 电泳结果

注：M：protein marker；1：对照；2：aprE；3：citH；4：ybdG；5：amyE； 6：nprE； 7：bpr； 8：bglS； 9：bglC； 10：sacB； 11：yweA。

Figure 2. SDS-PAGE analysis on signal peptides

Note: M: protein marker; 1: control; 2: aprE; 3: citH; 4: ybdG；5: amyE; 6: nprE； 7: bpr; 8: bglS； 9：bglC； 10：sacB； 11：yweA.

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图 3 citH信号肽Gly2饱和突变对α-环糊精酶分泌表达的影响

Figure 3. Effect of Gly2 saturation mutation of citH on α-CGTase activity

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图 4 citH信号肽Asn3饱和突变对α-环糊精酶分泌表达的影响

Figure 4. Effect of Asn3 saturation mutation of citH on α-CGTase activity

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图 5 citH信号肽Thr4饱和突变对α-环糊精酶分泌表达的影响

Figure 5. Effect of Thr4 saturation mutation of citH on α-CGTase activity

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图 6 citH信号肽多重突变对α-环糊精酶分泌表达的影响

Figure 6. Effect of multiple mutation of citH on α-CGTase activity

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图 7 不同信号肽的SDS-PAGE 电泳图分析

注：M：Protein Marker；1：对照；2：citH；3：G2R；4：N3K；5：T4L；6：G2R/N3K；7:G2R/T4L；8:N3K/ T4L；9：G2R/N3K/T4L。

Figure 7. SDS-PAGE analysis on signal peptide mutant

Note: M: Protein Marker; 1: control; 2: citH; 3: G2R; 4: N3K；5:T4L; 6: G2R/N3K； 7:G2R/T4L; 8:N3K/ T4L；9：G2R/N3K/T4L.

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图 8 pH对α-环糊精酶活力的影响

Figure 8. pH dependence of α-CGTase activity

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图 9 温度对α-环糊精酶活力的影响

Figure 9. Temperature dependence of α-CGTase activity

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图 10 不同温度下α-环糊精酶的热稳定性

Figure 10. Thermal stability of α-CGTase activity

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图 11 不同金属离子及抑制剂对α-环糊精酶活力的影响

Figure 11. Effect of metal ions and inhibitors on α-CGTase activity

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表 1 citH信号肽第二位Gly饱和突变引物

Table 1. Primers for saturated mutation of G2

突变体 Muntants	引物名称 Primer name	引物序列 Primer sequence(5′–3′)
G2F	G2F-F G2F-R	AGAGGGACGCGTATGTTCAATACTCGTAAAAAAG CTTTTTTACGAGTATTGAACATACGCGTCCCTCT
G2M	G2M-F G2M-R	AGAGGGACGCGTATGATGAATACTCGTAAAAAAG CTTTTTTACGAGTATTCATCATACGCGTCCCTCT
G2P	G2P-F G2P-R	AGAGGGACGCGTATGCCGAATACTCGTAAAAAAG CTTTTTTACGAGTATTCGGCATACGCGTCCCTCT
G2A	G2A-F G2A-R	AGAGGGACGCGTATGGCAAATACTCGTAAAAAAG CTTTTTTACGAGTATTTGCCATACGCGTCCCTCT
G2Y	G2Y-F G2Y-R	AGAGGGACGCGTATGTATAATACTCGTAAAAAAG CTTTTTTACGAGTATTATACATACGCGTCCCTCT
G2H	G2H-F G2H-R	AGAGGGACGCGTATGCATAATACTCGTAAAAAAG CTTTTTTACGAGTATTATGCATACGCGTCCCTCT
G2K	G2K-F G2K-R	AGAGGGACGCGTATGAAGAATACTCGTAAAAAAG CTTTTTTACGAGTATTCTTCATACGCGTCCCTCT
G2D	G2D-F G2D-R	AGAGGGACGCGTATGGATAATACTCGTAAAAAAG CTTTTTTACGAGTATTATCCATACGCGTCCCTCT
G2C	G2C-F G2C-R	AGAGGGACGCGTATGTGCAATACTCGTAAAAAAG CTTTTTTACGAGTATTGCACATACGCGTCCCTCT
G2Q	G2Q-F G2Q-R	AGAGGGACGCGTATGCAGAATACTCGTAAAAAAG CTTTTTTACGAGTATTCTGCATACGCGTCCCTCT
G2L	G2L-F G2L-R	AGAGGGACGCGTATGCTAAATACTCGTAAAAAAG CTTTTTTACGAGTATTTAGCATACGCGTCCCTCT
G2I	G2I-F G2I-R	AGAGGGACGCGTATGATTAATACTCGTAAAAAAG CTTTTTTACGAGTATTAATCATACGCGTCCCTCT
G2V	G2V-F G2V-R	AGAGGGACGCGTATGGTGAATACTCGTAAAAAAG CTTTTTTACGAGTATTCACCATACGCGTCCCTCT
G2S	G2S-F G2S-R	AGAGGGACGCGTATGTCTAATACTCGTAAAAAAG CTTTTTTACGAGTATTAGACATACGCGTCCCTCT
G2T	G2T-F G2T-R	AGAGGGACGCGTATGACCAATACTCGTAAAAAAG CTTTTTTACGAGTATTGGTCATACGCGTCCCTCT
G2N	G2N-F G2N-R	AGAGGGACGCGTATGAATAATACTCGTAAAAAAG CTTTTTTACGAGTATTATTCATACGCGTCCCTCT
G2E	G2E-F G2E-R	AGAGGGACGCGTATGGAAAATACTCGTAAAAAAG CTTTTTTACGAGTATTTTCCATACGCGTCCCTCT
G2R	G2R-F G2R-R	AGAGGGACGCGTATGCGGAATACTCGTAAAAAAG CTTTTTTACGAGTATTCCGCATACGCGTCCCTCT
G2W	G2R-F G2R-R	AGAGGGACGCGTATGTGGAATACTCGTAAAAAAG CTTTTTTACGAGTATTCCACATACGCGTCCCTCT
注：带下划线的碱基序列为突变氨基酸的碱基密码子。 Note: the underlined base is the base codon of the mutant amino acid.

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表 2 信号肽氨基酸序列表

Table 2. Amino acid sequence of signal peptide

信号肽名称 Signal peptide	信号肽氨基酸序列 Signal peptide amino acid sequence
pBE-aprE-CGT	MRSKKLWISLLFALTLIFTMAFSNMSVQA
pBE-citH-CGT	MGNTRKKVSVIGAGFTGATTAFLIAQKELADV
pBE-ybdG-CGT	MKTLWKVLKIVFVSLAALVLLVSVS
pBE-amyE-CGT	MFAKRFKTSLLPLFAGFLLLFHLVLAGPAAASA
pBE-nprE-CGT	MGLGKKLSVAVAASFMSLSISLPGVQA
pBE-bpr-CGT	MRKKTKNRLISSVLSTVVISSLLFPGAAGA
pBE-bglS-CGT	MPYLKRVLLLLVTGLFMSLFAVTATASA
pBE-bglC-CGT	MKRSISIFITCLLITLLTMGGMIASPASA
pBE-sacB-CGT	MNIKKFAKQATVLTFTTALLAGGATQAFA
pBE-yweA-CGT	MLKRTSFVSSLFISSAVLLSILLPSGQAHA

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