一株烟用产纤维素酶菌株的筛选、鉴定及酶学特性研究

吴旭东; 黄仕新; 吴鹏; 徐长安; 龙腾; 唐旭

doi:10.19303/j.issn.1008-0384.2022.003.017

一株烟用产纤维素酶菌株的筛选、鉴定及酶学特性研究

doi: 10.19303/j.issn.1008-0384.2022.003.017

吴旭东^{1, 2,},
黄仕新²,
吴鹏²,
徐长安²,
龙腾^1, ,,
唐旭^2, ,

1.
福建中烟工业有限责任公司技术中心，福建厦门，361021
2.
自然资源部第三海洋研究所，福建厦门，361005

基金项目: 福建中烟工业有限责任公司技术合作项目（FJZYHZJH2020003）

详细信息

作者简介:
吴旭东（1998−），女，硕士研究生，主要从事海洋微生物研究（E-mail：wuxudong@tio.org.cn）

通讯作者:
龙腾（1983−），男，硕士，工程师，主要从事烟草原料研究（E-mail：lt10625@fjtic.cn）

唐旭（1974−），男，博士，正高级工程师，主要从事海洋微生物技术研究（E-mail：tangxu@tio.org.cn）

中图分类号: Q 93
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- 被引次数: 0
出版历程
- 收稿日期: 2021-09-10
- 修回日期: 2022-02-15
- 网络出版日期: 2022-03-21
- 刊出日期: 2022-03-28

A High Cellulase-producing Bacterium from Tobacco Lamina

WU Xudong^{1, 2
,},
HUANG Shixin²,
WU Peng²,
XU Changan²,
LONG Teng^{1
, ,},
TANG Xu^{2
, ,}

1.
Technology Center, Fujian China Tobacco Industry Co., Ltd., Xiamen, Fujian 361021, China
2.
Third Institute of Oceanography, Ministry of Natural Resources, Xiamen, Fujian 361005, China

摘要

摘要: 目的从不同品种烟叶原料中分离筛选高产纤维素酶细菌菌株，为提高烟叶原料使用价值提供优秀菌种资源。方法采用稀释涂布法从37份片烟样品中分离纯化菌株，利用刚果红染色法结合摇瓶复筛进行筛选，测定菌株纤维素酶活性，通过形态学观察与16S rRNA序列鉴定功能菌的系统发育学地位，并对功能菌所产纤维素酶进行酶学性质研究。结果筛选到1株烟用纤维素酶活性高的菌株FX-1，其在37 ℃发酵48 h纤维素酶酶活力最高，达56.261 U·mL⁻¹。FX-1菌体呈杆状，可产芽孢，革兰氏阳性，其16S rRNA序列与Bacillus subtilis相似度最高（100%），系统发育树中与枯草芽孢杆菌位于同一分支中，初步鉴定为枯草芽孢杆菌（Bacillus subtilis）。通过酶学性质分析，酶反应的最适温度为60 ℃，最适pH为5.0，在温度30～55 ℃或pH值3.0～7.0保温30 min后仍有较高活力，相对酶活有90%以上，具有良好的热稳定性和耐酸能力。结论从37份片烟样品中筛选获得1株高产纤维素酶菌株FX-1，初步鉴定为枯草芽孢杆菌。FX-1所产纤维素酶的反应条件较宽，酶学性质稳定，在烟草行业中具有良好的产业化开发利用前景。
- 烟用 /
- 纤维素酶 /
- 筛选 /
- 鉴定 /
- 酶学特性
Abstract: Objective Bacteria with a high cellulase-producing capacity were isolated from different varieties of tobacco for the potential of upgrading the economic value of tobacco raw materials. Methods Candidate bacterial strains were isolated and purified from 37 flue-cured tobacco strip samples using dilution coating method. They were screened by the Congo red staining combined with shaking flask culture and cellulase activity determination. Phylogenetic property of a strain identified by morphological observation was confirmed with 16S rRNA sequencing. The cellulase activities were investigated. Results FX-1 was preliminarily identified as a strain that produced high cellulase activity of 56.261 U·mL⁻¹ after 48h fermentation at 37 ℃. It was a gram-positive, rod-shape, spore-producing bacterium that showed a perfectly matching 16S rRNA sequence in a branch of Bacillus subtilis phylogenetic tree. The cellulase from FX-1 displayed a peak activity at 60 ℃ and pH 5.0. Greater than 90% of its relative activity was retained after being held at 30-55 ℃ or pH 3.0-7.0 for 30 min. Conclusion A high cellulase-producing bacterium screened from 37 flue-cured tobacco strip samples was preliminarily identified as a strain of B. subtilis. The cellulase produced by the strain FX-1 exhibited a wide-range temperature and pH stability, and thus, had a considerable potential to be developed for new commercial use in tobacco manufact.
- tobacco /
- cellulase /
- screening /
- identification /
- enzymatic property

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图 1 菌株FX-1纤维素酶水解圈

Figure 1. Cellulase hydrolysis circle of FX-1

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图 2 菌株FX-1 形态特征

注：A：菌落形态；B：透射电镜图（20 000×）；C：革兰氏染色（1 000×）；D：芽孢染色（1 000×）。

Figure 2. Morphological characteristics of FX-1

Note: A: colony morphology of FX-1; B: TEM image of FX-1 (20 000×); C: gram stained FX-1 (1 000×); D: bacteriophage staining of FX-1 (1 000×).

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图 3 FX-1菌株的系统发育树

Figure 3. Phylogenetic tree of FX-1

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图 4 葡萄糖标准曲线

Figure 4. Standard curve of glucose

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图 5 培养时间对菌株生长和产酶的影响

Figure 5. Effect of incubation time on growth and enzyme production of FX-1

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图 6 FX-1纤维素酶不同反应温度条件下酶活变化情况

Figure 6. FX-1 cellulase activity at different reaction temperatures

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图 7 FX-1纤维素酶不同pH条件下酶活变化情况

Figure 7. FX-1 cellulase activity under different pH conditions

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图 8 FX-1纤维素酶在不同温度环境下的耐受情况

Figure 8. Tolerance of FX-1 cellulase to elevated temperatures

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图 9 FX-1纤维素酶在不同酸碱环境下的耐受情况

Figure 9. Tolerance of FX-1 cellulase to acidic and alkaline conditions

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表 1 样品片烟来源

Table 1. Source of flue-cured tobacco strip samples

编号 Number	等级 Grade	编号 Number	等级 Grade
1	云南马沾YLC1Y-2019片烟 Yunnan Mazhan YLC1Y-2019 flue-cured tobacco strips	20	湖南永州宁远C3F-2019片烟 Hunan Yongzhou Ningyuan C3F-2019 flue-cured tobacco strips
2	云南马沾C4FY-2018片烟 Yunnan Mazhan C4FY-2018 flue-cured tobacco strips	21	湖南永州宁远B2F-2019片烟 Hunan Yongzhou Ningyuan B2F-2019 flue-cured tobacco strips
3	云南保山AB-2019香料烟 Yunnan Baoshan AB-2019 spice tobacco	22	辽宁YELC3F-2019片烟 Liaoning YELC3F-2019 flue-cured tobacco strips
4	云南楚雄YLC1-2019片烟 Yunnan Chuxiong YLC1-2019 flue-cured tobacco strips	23	辽宁B23F-2019片烟 Liaoning B23F-2019 flue-cured tobacco strips
5	云南大理祥云YLC1Y-2019片烟 Yunnan Dali Xiangyun YLC1Y-2019 flue-cured tobacco strips	24	贵州遵义铜仁YLC2Y-2019片烟 Guizhou Zunyi Tongren YLC2Y-2019 flue-cured tobacco strips
6	云南马沾ELC1Y-2019片烟 Yunnan Mazhan ELC1Y-2019 flue-cured tobacco strips	25	贵州遵义铜仁YLC1Y-2019片烟 Guizhou Zunyi Tongren YLC1Y-2019 flue-cured tobacco strips
7	云南楚雄ELC1-2019片烟 Yunnan Chuxiong ELC1-2019 flue-cured tobacco strips	26	山东潍坊诸城B2F-2019片烟 Shandong Weifang Zhucheng B2F-2019 flue-cured tobacco strips
8	云南大理祥云ELC1Y-2019片烟 Yunnan Dali Xiangyun ELC1Y-2019 flue-cured tobacco strips	27	山东潍坊诸城C3F-2019片烟 Shandong Weifang Zhucheng C3F-2019 flue-cured tobacco strips
9	云南曲楚大F03-2019片烟 Yunnan Quchu Da F03-2019 flue-cured tobacco strips	28	重庆彭水B2F-2019片烟 Chongqing Pengshui B2F-2019 flue-cured tobacco strips
10	云南楚雄F03-2019片烟 Yunnan Chuxiong F03-2019 flue-cured tobacco strips	29	黑龙江哈尔滨C3L-2019片烟 Heilongjiang Harbin C3L-2019 flue-cured tobacco strips
11	福建龙岩三明YLC2YCB-1-2018片烟 Fujian Longyan Sanming YLC2YCB-1-2018 flue-cured tobacco strips	30	巴西TMB1O-FJ-2019片烟 Brazil TMBIO-FJ-2019 flue-cured tobacco strips
12	福建三明尤溪YLC1CB-2019片烟 Fujian Sanming Youxi YLC1CB-2019 flue-cured tobacco strips	31	巴西B1O/C-2019片烟 Brazil B1O/C-2019 flue-cured tobacco strips
13	福建龙岩永定YLC1Y-2019片烟 Fujian Longyan Yongding YLC1Y-2019 flue-cured tobacco strips	32	巴西L1O-2019片烟 Brazil L1O-2019 flue-cured tobacco strips
14	福建龙岩三明YLC2YCB-1-2019片烟 Fujian Longyan Sanming YLC2YCB-1-2019 flue-cured tobacco strips	33	津巴布韦L1O/C(天泽)-2019片烟 Zimbabwe L1O/C(Tianze)-2019 flue-cured tobacco strips
15	福建三明尤溪ELC1CB-2019片烟 Fujian Sanming Youxi ELC1CB-2019 flue-cured tobacco strips	34	津巴布韦L1O/C（英美）-2019片烟 Zimbabwe L1O/C (Anglo-American)-2019 flue-cured tobacco strips
16	福建龙岩永定ELC1Y-2019片烟 Fujian Longyan Yongding ELC1Y-2019 flue-cured tobacco strips	35	津巴布韦L1OA（环球）-2019片烟 Zimbabwe L1OA (Global)-2019 flue-cured tobacco strips
17	福建龙岩南平SLC4-2019片烟 Fujian Longyan Nanping SLC4-2019 flue-cured tobacco strips	36	赞比亚L1OL-3（CNT）-2019片烟 Zambia L1OL-3 (CNT)-2019 flue-cured tobacco strips
18	福建龙岩三明CF03-2019片烟 Fujian Longyan Sanming CF03-2019 flue-cured tobacco strips	37	阿根廷ASBOCF-1-2019片烟 Argentina ASBOCF-1-2019 flue-cured tobacco strips
19	福建龙三南X2F03-2019片烟 Fujian Long Sanan X2F03-2019 flue-cured tobacco strips

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表 2 葡萄糖标准曲线制作参数

Table 2. Parameter of glucose standard curve

管号 Tube number	葡萄糖溶液 Glucose solution/mg·mL⁻¹	蒸馏水 Distilled water/mL	DNS/mL
0	0.0	2.0	1.5
1	0.2	1.8	1.5
2	0.4	1.6	1.5
3	0.6	1.4	1.5
4	0.8	1.2	1.5
5	1.0	1.0	1.5
注：葡萄糖溶液为1.5 mg·mL⁻¹。 Note: glucose solution was 1.5 mg·mL⁻¹.

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表 3 FX-1菌株刚果红染色结果

Table 3. Congo red staining of FX-1

菌株 Strain	菌落直径 Colony diameter/mm	水解圈直径 Hydrolysis circle diameter/mm	H/C
FX-1	5.89±0.41 （SD=0.3141）	23.58±0.38 （SD=0.3138）	4.01±0.23 （SD=0.2529）

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表 4 菌株FX-1的生理生化特征

Table 4. Physiological and biochemical characteristics of FX-1

测定指标 Measurement item	结果 Result	测定指标 Measurement item	结果 Result
革兰氏染色 Gram stain	+	硝酸盐还原试验 Nitrate reduction test	+
接触酶试验 Exposure to enzyme test	+	V-P试验 V-P test	+
柠檬酸盐试验 Citrate test	+	吲哚试验 Indole test	−
注：+：阳性；−：阴性。 Note: +: Positive; −: Negative.

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