解淀粉芽孢杆菌高产纤维素酶菌株的筛选与鉴定

何深宏; 程方俊; 罗干; 张耕; 杜亚楠; 康霞梅; 王晓涵; 赵自亮; 任绍科; 郭建华

doi:10.19303/j.issn.1008-0384.2020.07.012

解淀粉芽孢杆菌高产纤维素酶菌株的筛选与鉴定

doi: 10.19303/j.issn.1008-0384.2020.07.012

西南大学动物科学学院，重庆 402460

基金项目: 重庆市社会民生科技创新专项（cstc2016shmszx80068）

详细信息

作者简介:
何深宏（1996−），男，硕士研究生，主要从事兽医微生物研究（E-mail：2577437282@qq.com）

通讯作者:
程方俊（1962−），男，博士，副教授，主要从事预防兽医学研究（E-mail：cfjxndx@163.com）

郭建华（1977−），男，博士，副教授，主要从事微生物研究（E-mail：guo0619@163.com）

中图分类号: Q 93
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出版历程
- 收稿日期: 2020-02-04
- 修回日期: 2020-04-14
- 刊出日期: 2020-07-31

Screening and Identifying Cellulase-producing Bacillus amyloliquefaciens

College of Animal Science, Southwest University, Chongqing 402460, China

摘要

摘要: 目的筛选高产纤维素酶菌株，为牛粪快速无害化降解提供菌剂。方法采用平板稀释法从重庆三峡库区3个牛粪堆肥厂样品分离纯化菌株，利用水解圈法和羧甲基纤维素（Carboxymethyl cellulose, CMC）酶糖化力法筛选、测定菌株纤维素酶活性，通过菌株形态特征观察、生理生化及分子生物学方法鉴定菌株，采用分子生物学软件MEGA6.0对菌株16S rRNA基因进行遗传系统发育树构建。结果从牛粪堆肥样品中分离得到的57株菌株中筛选出10株有较高纤维素酶活性，其中菌株X10纤维素酶活最高，为31.9 U·mL⁻¹。菌株X10鉴定为解淀粉芽孢杆菌（Bacillus amyloliquefaciens），其在16S rRNA基因遗传系统发育树中与解淀粉芽孢杆菌位于同一簇。结论筛选的菌株X10为高产纤维素酶解淀粉芽孢杆菌，可用于牛粪无害化降解。
- 牛粪发酵 /
- 纤维素酶 /
- 解淀粉芽孢杆菌
Abstract: Objective To screen and isolate a cellulase-producing microbe for efficient, rapid and natural composting cattle waste. Method At 3 cow manure composting plants in the Three Gorges Reservoir area in Chongqing, samples for microbial analysis were collected. The agar dilution method was applied to isolate bacillus strains from the compost specimens. Cellulase activity of the strains was determined for the screening by the hydrolytic circle method and the CMC enzyme saccharification method. Selected strains were morphologically examined and further identified by physiological, biochemical, and molecular biological methods. Based on 16S rRNA sequence, phylogenetic tree of the final selection was constructed using the molecular biological software MEGA6.0 for confirmation. Result Out of 57 isolates from the compost specimens, 10 strains showed varying degrees of cellulase activity. Among them, X10 had the highest activity at 31.9 U·mL⁻¹ and was identified on the 16S rRNA phylogenetic tree to be Bacillus amyloliquefaciens. Conclusion X10 was a species of B. amyloliquefaciens that was highly productive in secreting cellulase to enhance the degradation of cow manure in compost.
- Cow manure compost /
- cellulase /
- Bacillus amyloliquefaciens

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图 1 基于16S rRNA基因序列构建的系统发育树

Figure 1. Phylogenetic tree based on 16S rRNA gene sequence

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图 2 菌株X10普通营养琼脂培养基上的生长特性

Figure 2. Growth of strain X10 on general nutrient agar medium

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图 3 菌株X10革兰氏染色（10×100倍）

Figure 3. Gram stained strain X10（10×100 times）

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图 4 菌株X10纤维素酶水解圈

Figure 4. Cellulase hydrolysis circle by strain X10

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图 5 高产纤维素酶菌株X10多重PCR扩增结果

注：M：2 000 DNA Marker；1：菌株X10。

Figure 5. Multiplex PCR amplifications of cellulase-producing strain X10

Note: M: 2 000 DNA Marker; 1: strain X10.

下载: 全尺寸图片幻灯片

表 1 种特异性引物

Table 1. Species specific primers

细菌名称 Bacteria name	引物名称 Primer	引物序列（5'−3'）Primer sequences（5'−3'）	PCR产物 PCR product/bp	基因 Gene
枯草芽孢杆菌 Bacillus subtilis	BSL72	CGTAGAGCCACTTGAGCG	256	rpoA
枯草芽孢杆菌 Bacillus subtilis	BSR328	CTGCCGTTACAGTTCCTT	256	rpoA
解淀粉芽孢杆菌 Bacillus amyloliquefaciens	L100	AAATCTGCCCGTATCGTCG	736	gyrA
解淀粉芽孢杆菌 Bacillus amyloliquefaciens	R836	GCGTCACGGCGR（AG）ATCTCAA	736	gyrA
地衣芽孢杆菌 Bacillus licheniformi	L168	TGGGATGACAAGTGATAAGC	346	gyrA
地衣芽孢杆菌 Bacillus licheniformi	R514	CTCCGTTGACAAGCAAGTTCG	346	gyrA
短小芽孢杆菌 Bacillus pumilus	L354	AGGGAAGAACAAGTGC（GA）AGAG	321	16S rRNA
短小芽孢杆菌 Bacillus pumilus	R674	GCTCCTCAGCGTCAGTTACA	321	16S rRNA

下载: 导出CSV

表 2 水解圈直径与菌落直径

Table 2. Hydrolysis circle and colony diameter tests

菌株 Strain	水解圈直径与菌落直径比值 Ratio of hydrolysis circle diameter to colony diameter	菌株 Strain	水解圈直径与菌落直径比值 Ratio of hydrolysis circle diameter to colony diameter
S1	3.21±0.26	FJ7	3.44±0.42
S6	3.09±0.41	LP5	3.70±0.30
S14	3.57±0.09	LP6	3.22±0.24
X10	4.24±0.72	LP10	3.11±0.36
X14	3.32±0.18	LP15	3.30±0.20

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表 3 CMC酶活测定结果

Table 3. CMC enzyme activity assay results

菌株 Strain	酶活力 Enzyme activity/（U·mL⁻¹）	菌株 Strain	酶活力 Enzyme activity/（U·mL⁻¹）
S1	4.48±0.12	FJ7	1.57±0.12
S6	1.23±0.31	LP5	3.57±0.24
S14	7.90±0.00	LP6	3.48±0.24
X10	31.90±0.20	LP10	6.82±0.24
X14	6.98±0.12	LP15	3.48±0.12

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表 4 高产纤维素酶菌株X10生化鉴定结果

Table 4. Biochemical identification of cellulase-producing strain X10

项目 Item	结果 Result	项目 Item	结果 Result	项目 Item	结果 Result
MR试验 MR test	−	水杨酸 Salicylic acid	+	蔗糖 sucrose	+
VP试验 VP test	+	甘露醇 Mannitol	−	乳糖 lactose	−
靛基质 Indigo matrix	−	麦芽糖 maltose	−	七叶苷 Aescin	+
枸橼酸盐 Citrate	−	葡萄糖 glucose	+	硫化氢试验 Hydrogen sulfide test	−
注：“+”表示阳性；“−”表示阴性。 Note:“+”means positive; “−”means negative.

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