高知芽孢杆菌<i>Cytobacillus kochii</i> H产蛋白酶条件优化及抑菌效应

王学文; 于存; 潘洪祥

doi:10.19303/j.issn.1008-0384.2023.01.007

高知芽孢杆菌Cytobacillus kochii H产蛋白酶条件优化及抑菌效应

doi: 10.19303/j.issn.1008-0384.2023.01.007

贵州大学林学院，贵州　贵阳　550025

基金项目: 国家自然科学基金项目( 32160375)；黔科合平台人才项目（［2018］5261）

详细信息

作者简介:
王学文（1998−），男，硕士研究生，研究方向：马尾松立枯病防治（E-mail：2252346912@qq.com）

通讯作者:
于存（1988−），男，博士，副教授，研究方向：植物病理（E-mail：chifengyucun@163.com）

中图分类号: Q 939
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- 被引次数: 0
出版历程
- 收稿日期: 2022-08-29
- 修回日期: 2022-11-21
- 网络出版日期: 2023-02-08
- 刊出日期: 2023-01-28

Process Optimization and Antimicrobial Effect of Cytobacillus kochii H Protease

Forestry College, Guizhou University, Guiyang, Guizhou　550025, China

摘要

摘要: 目的对筛选到具有抑菌效果且产蛋白酶的菌株H进行分类地位的确定，同时对其产蛋白酶的条件进行优化，以期为其在植物病害防治上的应用奠定基础。方法通过生理生化、电子显微镜扫描及16sRNA测序相结合的方法明确菌株H的分类地位；通过单因素和正交试验对菌株H产蛋白酶条件进行优化，并检测其优化前后对病原真菌的抑菌效果。结果菌株H鉴定为高知芽孢杆菌（Cytobacillus kochii）；菌株H产蛋白酶的优化条件为：pH8.0、葡萄糖20.0 g·L⁻¹、蛋白胨8.0 g·L⁻¹、MgSO₄ 1.0 g·L⁻¹、CaSO₄·2H₂O 0.1 g·L⁻¹，在装有50 mL发酵培养基的250 mL三角瓶中接种2.5 mL10⁸ CFU·mL⁻¹的种子液，培养24 h后蛋白酶活力达到402.2 U·mL⁻¹，较初始培养条件下的蛋白酶活力提高13.92倍；优化后菌株H对尖孢镰刀菌（Fusarium oxysporum）和辣椒疫霉病菌（Phytophthora capsici）的抑菌率分别为67.32%和44.87%，较优化前抑菌率提升值分别为9.15%和12.82%。结论明确菌株H为高知芽孢杆菌（C. kochii），优化了高知芽孢杆菌产蛋白酶条件及抑菌效果。
- 高知芽孢杆菌 /
- 蛋白酶 /
- 产酶条件优化 /
- 病原菌抑菌率
Abstract: Objective Taxonomy of antimicrobial protease-producing Cytobacillus kochii H was studied for the development of a natural disease control agent on plants. Method Taxonomy of C. kochii H was determined by physiological, biochemical, electron microscopy scanning, and 16sRNA sequencing methods. A process of producing the antimicrobial protease secreted by the bacterium was optimized in single-factor and orthogonal experiments. The efficacy of the culture broth on 4 selected pathogenic fungi was applied in an in vitro test as the evaluation criterium. Result The optimized C. kochii H protease-producing process was conducted in a 250 mL triangular flask using 50 mL of pH 8.0 medium, which contained 20.0 g·L⁻¹ of glucose, 8.0 g·L⁻¹ of peptone, 1.0 g·L⁻¹ of MgSO₄, and 0.1g·L⁻¹ of CaSO₄-2H₂O, inoculated with 2.5 mL of 10⁸ CFU·mL⁻¹ C. kochii H fermentation liquid. After 24 h of incubation, the protease activity reached 402.2 U·mL⁻¹, which was 13.92 times greater than it was prior to the optimization. The antimicrobial rates of the broth against Fusarium oxysporum and Phytophthora capsici were 67.32% and 44.87%, respectively, i.e., 9.15% and 12.82%, respectively, increases over those before the optimization. Conclusion The antimicrobial protease-producing C. kochii H was identified taxonomically. The culture process of the enzyme production was optimized to deliver significant in vitro inhibition rates on F. oxysporum and P. capsici.
- Cytobacillus kochii /
- protease /
- optimized fermentation /
- antimicrobial rate on pathogens

HTML全文

图 1 菌株H形态学特征

A：革兰氏染色；B：菌落形态；C：菌株形态。

Figure 1. Morphological characteristics of C. kochii H

A：Gram's stain, B：Colony morphology, C：Strain morphology.

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图 2 H菌株与其他相近菌株基于16S rRNA基因序列的系统发育树

Figure 2. Phylogenetic trees of C. kochii H and other similar strains based on 16S rRNA gene sequence

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图 3 高知芽孢杆菌(C. kochii) H产蛋白酶活性检测

A：对照，B：箭头所示为产蛋白酶透明圈。

Figure 3. Determination of C. kochii H protease activity

A: control; arrow in B: transparent circle produced by protease.

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图 4 培养时间对高知芽孢杆菌(C. kochii) H产蛋白酶能力的影响

Figure 4. Effect of C. kochii H culture time on protease production

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图 5 单因素和培养基成分优化对高知芽孢杆菌(C. kochii) H产蛋白酶活力的影响

不同小写字母表示差异显著(P<0.05)。下同。

Figure 5. Effects of single factor and medium optimization on C. kochii H protease activity

Different lowercase letters indicate significant difference (P<0.05).Same for below.

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图 6 高知芽孢杆菌(C. kochii) H产蛋白酶正交试验验证

Figure 6. Verification of orthogonal test results on C. kochii H protease production

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图 7 高知芽孢杆菌(C. kochii) H产蛋白酶优化前后对4种病原菌的抑菌效果

A：尖孢镰刀菌（F. oxysporum）；B：脐橙青霉病菌（P. italicu）； C：辣椒疫霉病菌（P. capsici）； D：辣椒炭疽病菌（C. capsici）； A～D为对照； E～H为优化前； J～M为优化后。

Figure 7. Bacteriostatic effects of C. kochii H culture broth on 4 pathogens before and after process optimization

A: F. oxysporum; B: P. italic; C: P. capsica; D: C. capsica; Lines A-D: control; Lines E-H: before optimization; Lines J-M: after optimization.

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图 8 高知芽孢杆菌(C. kochii) H优化前后对4种植物病原真菌的抑制效果

差异性分析对象为单个病原菌的基础抑菌率和优化抑菌率。

Figure 8. Inhibition effects of C. kochii H culture broth on 4 pathogens before and after process optimization

The differential analysis targets are the basic and optimized antibacterial rates of a single pathogen.

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表 1 单因素优化设计

Table 1. Single factor optimization experimental design

接种量 Inocuiation amount/mL	装液量 Refilled amount/mL	pH
2.5	20	5.0
5.0	30	6.0
7.5	50	7.0
10	70	8.0
12.5	90	9.0
15	110	10.0
17.5	－	－
20	－	－

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表 2 正交试验优化组合试验因子及水平

Table 2. Factors and levels of optimization experiment

水平 Level	A:葡萄糖 Glucose/ （g·L⁻¹）	B:蛋白胨 Peptone/ （ g·L⁻¹）	C:Mg²⁺/ （ g·L⁻¹）	D:Ca²⁺/ （ g·L⁻¹）
1	10	8	0.1	0.1
2	15	10	0.5	0.5
3	20	12	1.0	1.0

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表 3 菌株H生理生化试验

Table 3. Physiological and biochemical test results on C. kochii H

试验名称　　　　　 Test names	试验现象　　　　　 Experimental phenomenon	试验结果 Results
甲基红试验 Methylred test	反应液呈黄色 The reaction solution is yellow	−
革兰氏染色 Gram staining test	菌体呈紫色 The cell is purple	+
V-P试验 V-P test	培养液呈非红色 The culture medium is not red	−
靛基质试验 Imdole test	反应液呈红色 The reaction solution is red	+
触酶试验 Contact enzyme test	有气泡 Bubble	+
柠檬酸盐利用试验 Citrate test	培养基呈深蓝色 The culture medium is navy blue	+
+：阳性；−：阴性。 +: positive; −: negative.

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表 4 高知芽孢杆菌(C. kochii) H产蛋白酶优化组合正交试验结果

Table 4. Orthogonal optimization of C. kochii H protease-producing process

试验编号 Test No	因子 Factor				酶活力 Protease activity/（U·mL⁻¹）
试验编号 Test No	葡萄糖 Glucose	蛋白胨 Peptone	Mg²⁺	Ca²⁺	酶活力 Protease activity/（U·mL⁻¹）
1	1	1	1	1	215.370
2	1	2	2	2	51.584
3	1	3	3	3	122.418
4	2	1	2	3	15.460
5	2	2	3	1	176.814
6	2	3	1	2	100.002
7	3	1	3	2	360.028
8	3	2	1	3	107.773
9	3	3	2	1	221.348
K1	129.791	196.953	141.048	204.511	－
K2	97.426	112.057	96.130	170.538	－
K3	229.716	147.923	219.753	81.884	－
R	132.291	84.896	123.623	122.627	－
优化组合 Optimization grouping	3	1	3	1	－

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表 5 蛋白酶抑菌验证

Table 5. Validation on bacteriostasis of C. kochii H protease

病原菌 Pathogen	粗酶液抑菌率 Antibacterial rate of crude enzyme solution/%	粗酶液灭活后抑菌率 Antibacterial rate of crude enzyme solution after inactivation/%
尖孢镰刀菌 Fusarium oxysporum	21.89±1.40 a	6.26±1.23 b
辣椒疫霉病菌 Phytophthora capsici	36.67±3.10 a	29.63±3.40 a
脐橙青霉病菌 Penicillium italicu	16.90±2.70 a	1.98±1.31 b
辣椒炭疽病菌 Colletotrichum capsici	17.57±9.69 a	0.40±0.00 b

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