美洲大蠊肠道产7-木糖紫杉烷糖基水解酶菌株的筛选及鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2021-0620

摘要/Abstract

摘要：

旨在筛选美洲大蠊肠道具有产7-木糖紫杉烷糖基水解酶性能的内生菌,并对其进行生物学鉴定。以实验室保存的178株美洲大蠊肠道内生菌为研究对象,采用刚果红透明圈法和荧光显色法筛选产木聚糖酶菌株,进一步利用薄层色谱法筛选产7-木糖紫杉烷糖基水解酶菌株。在此基础上,选取一株高产7-木糖紫杉烷糖基水解酶菌株进行形态学、生理生化和分子生物学鉴定,并采用高效液相色谱法测定其对7-木糖-10-去乙酰基紫杉醇的转化率。结果显示,178株美洲大蠊肠道内生菌中有55株菌产木聚糖酶,其中8株链霉菌和2株贪铜菌可产生7-木糖紫杉烷糖基水解酶。高产菌株WA11-2-9经形态学特征、生理生化试验和16S rDNA序列分析鉴定为Streptomyces enissocaesilis。WA11-2-9可将7-木糖-10-去乙酰基紫杉醇转化为10-去乙酰巴卡亭Ⅲ和10-去乙酰紫杉醇,主要产物10-去乙酰紫杉醇的转化率为21.52%。美洲大蠊肠道内生菌WA 11-2-9（GenBank Accession：MZ411692）具有分泌7-木糖紫杉烷糖基水解酶的能力,它为后续生物转化制备紫杉醇奠定了坚实基础。

关键词: 美洲大蠊肠道, 7-木糖紫杉烷糖基水解酶, 7-木糖-10-去乙酰基紫杉醇, 10-去乙酰紫杉醇

Abstract:

The objective of this work is to screen endophytes that can produce 7-β-xylosyltaxanes glycoside hydrolases and to identify its biological characteristics. Taking total 178 strains of intestinal endophytes from Periplaneta americana preserved in the laboratory as the research object,the Congo red transparent circle method and fluorescent color method were used to screen the xylanase-producing strains,and the thin-layer chromatography was further used to screen strains producing the 7-β-xylosyltaxanes glycoside hydrolases. On the basis of this,a strain producing high 7-β-xylosyltaxanes glycoside hydrolases was selected for morphological,physiological,biochemical and molecular biological identification,and the conversion rate of 7-β-xylosyl-10-deacetylpaclitaxel by the strain was measured by high performance liquid chromatography. Among the 178 strains of intestinal endophytes in P. americana,total 55 endophytes produced xylanase,of which 8 strains of Streptomyces and 2 strains of Cupriavidus produced 7-β-xylosyltaxanes glycoside hydrolases. The high-yield strain WA11-2-9 was identified as Streptomyces enissocaesilis by bacterial morphology,physiological and biochemical experiments,combined with 16S rDNA sequence analysis. WA11-2-9 converted 7-β-xylosyl-10-deacetylpaclitaxel to 10-deacetylbaccatin III and 10-deacetylpaclitaxel. The conversion rate of the main product 10-deacetylpaclitaxel was 21.52%. In conclusion,the intestinal endophytes WA11-2-9（GenBank accession number：MZ411692）from P. americana secreted 7-β-xylosyltaxanes glycoside hydrolases,which lays a solid foundation for the subsequent biotransformation of paclitaxel.

Key words: Periplaneta americana gut, 7-β-xylosyltaxanes glycoside hydrolases, 7-β-xylosyl-10-deacetylpac-litaxel, 10-deacetylpaclitaxel

唐彬, 刘文彬, 李小波, 王宁, 金小宝. 美洲大蠊肠道产7-木糖紫杉烷糖基水解酶菌株的筛选及鉴定[J]. 生物技术通报, 2022, 38(3): 139-148.

TANG Bin, LIU Wen-bin, LI Xiao-bo, WANG Ning, JIN Xiao-bao. Screening and Identification of Strains Producing 7-β-xylosyltaxanes Glycoside Hydrolases from the Periplaneta americana Gut[J]. Biotechnology Bulletin, 2022, 38(3): 139-148.

图/表 12

图1 产木聚糖酶菌株种属分布 A：产木聚糖酶放线菌种属分布;B：产木聚糖酶非放线菌种属分布

Fig. 1 Distribution of xylanase-producing strains A：Distribution of xylanase-producing actinomycetes. B：Distribution of xylanase-producing endophytes

图2 产木聚糖酶菌株的刚果红水解圈

Fig.2 Congo red transparent circles of xylanase-producing strains

图3 产木聚糖酶菌株的荧光显色图 A-F：WA1-37,WA2-17,WA3-2-36,WA4-65,WA11-2-9和WA14-2-7。左边为荧光显色,右边为自身对照

Fig. 3 Fluorescence color image of xylanase-producing strain A to F：WA1-37,WA2-17,WA3-2-36,WA4-65,WA11-2-9 and WA14-2-7. The left is fluorescent color,and the right is self-control

表1 产木聚糖酶菌株的菌落直径、刚果红透明圈直径及比值

Table 1 Colony diameters,Congo red transparent circles and their ratios of xylanase-producing strains（n=3）

菌株编号 Strain code	菌落直径 Diameter of colony（d/cm）	透明圈直径 Diameter of transparent circle of clony（D/cm）		比值 Ratio（D/d）
菌株编号 Strain code	菌落直径 Diameter of colony（d/cm）	内圈Inner circle	外圈Outer circle	内圈Inner circle	外圈Outer circle
1-37	0.75±0.02	1.67±0.12	3.68±0.18	2.21±0.11	4.88±0.12
2-17	0.83±0.05	1.54±0.05	2.66±0.04	1.87±0.16	3.23±0.17
3-2-36	0.64±0.06	1.33±0.07	2.89±0.02	2.13±0.06	4.63±0.17
4-65	0.68±0.08	1.72±0.12	3.50±0.17	2.56±0.16	5.03±0.13
11-2-9	0.62±0.04	1.33±0.05	3.72±0.04	2.07±0.12	5.55±0.17
14-2-7	0.62±0.04	1.50±0.03	2.92±0.04	2.44±0.10	4.80±0.15

图4 菌株WA11-2-9转化产物TLC结果 1-4：WA 10-1-2、WA 11-2-9、WA 11-1-3和WA 23-1-5的转化产物;5-7：7-XDT、10-DAT和10-DAB标准品

Fig. 4 TLC result of transformed product of strain WA11-2-9 1st to 4th are：WA 10-1-2,WA 11-2-9,WA 11-1-3 and WA 23-1-5 transformation products,the points from 5th to 7th are：7-XDT,10-DAT and 10-DAB standards

图5 菌株WA11-2-9的形态学特征 A：菌株WA11-2-9菌落形态;B：菌株WA11-2-9的革兰染色图（100×）

Fig. 5 Morphological characteristics of strain WA11-2-9 A：The colony morphology of strain WA11-2-9. B：The gram staining diagram of strain WA11-2-9（100×）

表2 菌株WA11-2-9的生理生化特征

Table 2 Physiological and biochemical characteristics of strain WA11-2-9

检测项目Test item	结果Result	检测项目Test item	结果Result	检测项目Test item	结果Result
淀粉水解	+	H₂S产生	+	葡萄糖	+
明胶液化	+	肌醇	+	阿拉伯糖	+
硝酸盐还原	+	果糖	+	生长温度范围	20-40℃
石蕊牛奶	+	甘露醇	+	生长pH范围	4-9
尿素酶	+	木糖	+	生长耐受NaCl范围	2%-8%

图6 菌株WA11-2-9 分子生物学鉴定 A：菌株WA11-2-9 16S rDNA扩增产物的电泳图;B：菌株WA11-2-9 16S rDNA基因序列系统发育树

Fig. 6 Molecular biology identification of strain WA11-2-9 A：The 16S rDNA amplified products of strain WA11-2-9. B：The phylogenetic tree of strain WA11-2-9 based on 16S rDNA gene sequence

图7 10-DAT的标准曲线

Fig.7 10-DAT standard curve

图8 菌株WA11-2-9转化产物的HPLC分析图谱 A：7-XDT标准品的HPLC图谱;B：10-DAT标准品的HPLC图谱;C：10-DAB标准品的HPLC图谱;D：菌株WA11-2-9的HPLC图谱;E：菌株WA11-2-9对照组（未加7-XDT）的HPLC图谱

Fig. 8 HPLC analyzing spectrum of transformed product of strain WA11-2-9 A：The HPLC spectrogram of standard 7-XDT. B：The HPLC spectrogram of standard 10-DAT. C：The HPLC spectrogram of standard 10-DAB. D：The HPLC spectrogram of strain WA11-2-9. E：The HPLC spectrogram of WA11-2-9 contrast group（no 7-XDT added）

图9 7-β-xyl的最适反应温度和pH A：最适反应温度;B：最适反应pH

Fig. 9 Optimal reaction temperature and pH of 7-β-xyl A：Optimal reaction temperature. B：Optimal reaction pH

图10 7-β-xyl的温度稳定性、酸碱稳定性和金属离子稳定性 A：温度稳定性;B：酸碱稳定性;C：金属离子稳定性

Fig.10 Thermal stability,pH stability and metal ion stab-ility of 7-β-xyl A：Thermal stability. B：pH stability. C：Metal ion stability

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