一株Olivibacter jilunii 纤维素降解菌株的分离鉴定与降解能力分析

doi:10.13560/j.cnki.biotech.bull.1985

摘要/Abstract

摘要：

为了获得常温新型纤维素降解细菌，在园林堆肥中分离纯化得到一株具有纤维素降解能力的细菌菌株18B。通过刚果红染色实验和滤纸降解实验验证其纤维素降解能力。通过16S rRNA序列比对和全基因组比对确定其属于Olivibacter属，且与Olivibacter jilunii 14-2A^T最为接近，进一步生理生化特征比较分析发现，其与O. jilunii 14-2A^T在生长温度、氧化酶和糖酵解等方面存在差异。菌株18B在12-48℃范围内能生长，最适生长温度在30-37℃。O. jilunii 14-2A^T的生长温度在4-42℃；菌株18B的氧化酶检测为阳性，糖酵解为阴性，O. jilunii 14-2A^T则不具有氧化酶活性同时糖酵解为阳性。结合基因组共线性比对结果可知，菌株18B与O. jilunii 14-2A^T存在一定的进化关系。纤维素降解能力检测发现，在37℃，200 r/min培养至第5天时，菌株18B的纤维素酶活最高可达82.14^+0.99_-9.90 U/L，同时在以羧甲基纤维素钠为唯一碳源，仅添加无机氮源的培养条件下可维持酶活不退化。O. jilunii 14-2A^T则不具有纤维素降解能力，也无法在羧甲基纤维素钠为唯一有机碳源的培养环境下存活。筛选全基因组测序结果可知菌株18B基因组上存在纤维素酶系基因。在大肠杆菌中异源表达筛选所得纤维素酶系基因，并检验表达产物的降解能力，结果表明筛选所得纤维素酶系基因确有酶活。综合以上结果，最终确定菌株18B属于具有纤维素酶活的O. jilunii新生理株。

关键词: 纤维素降解, Olivibacter jilunii, 生理株, 分离鉴定, 16S rRNA比对, 纤维素酶活, 全基因组序列分析

Abstract:

In order to obtain the new cellulose-degrading bacteria at normal temperature, a bacterial strain named as 18B with cellulose-degrading ability was isolated and purified from garden compost in this study. Congo red staining experiment and filter paper degradation experiment were conducted to verify its cellulose-degradation ability. 16S rRNA sequence alignment and whole genome alignment confirmed that it belonged to the genus Olivibacter, and was the closest to Olivibacter jilunii 14-2A^T. Further comparative physiological and biochemical characterization revealed differences with O. jilunii 14-2A^T in terms of growth temperature, oxidase and glycolysis. The strain 18B grew in the range of 12-48℃, and the optimal growth temperature was 30-37℃. O. jilunii 14-2A^T grew at 4-42℃. Strain 18B was positive for oxidase and negative for glycolysis, while O. jilunii 14-2A^T had no oxidase activity and was positive for glycolysis. Combined with the results of the genome-wide covariance alignment, it was known that strain 18B had a certain evolutionary relationship with O. jilunii 14-2A^T. The cellulose-degradation ability was found that the cellulase activity of 18B was up to 82.14^+0.99_-9.90 U/L at 37℃, 200 r/min until the 5th d. Meanwhile, the enzyme activity could be maintained without degradation under the incubation conditions with sodium carboxymethyl cellulose as the only carbon source and only inorganic nitrogen source added. O. jilunii 14-2A^T had no cellulose degrading ability and could not survive in a culture environment where sodium carboxymethylcellulose was the only organic carbon source. The results of whole genome sequencing showed that there were cellulase genes in the genome of strain 18B. The cellulase genes were heterologous expressed in Escherichia coli, and the degradation ability of the expressed products was tested. The results showed that the cellulase genes were active. The above results finally identified strain 18B as a new physiological strain of O. jilunii with cellulase activity.

Key words: cellulose degrading, Olivibacter jilunii, physiological strain, isolation and identification, 16S rRNA alignment, cellulase activity, genome-wide sequence analysis

饶紫环, 谢志雄. 一株Olivibacter jilunii 纤维素降解菌株的分离鉴定与降解能力分析[J]. 生物技术通报, 2023, 39(8): 283-290.

RAO Zi-huan, XIE Zhi-xiong. Isolation and Identification of a Cellulose-degrading Strain of Olivibacter jilunii and Analysis of Its Degradability[J]. Biotechnology Bulletin, 2023, 39(8): 283-290.

图/表 9

图1 菌株18B纤维素降解能力检测 A：滤纸降解；B：刚果红褪色圈检测

Fig. 1 Detection of cellulose degradation ability of strain 18B A: Degradation of filter paper. B: Congo red fading circle

图2 基于16S rRNA序列构建的非加权组平均法进化树（以Albibacterium bauzanense BZ42为外群）

Fig. 2 UPGMA-tree based on 16S rRNA (Albibacterium bauzanense BZ42 used as the outgroup)

图3 基于16S rRNA序列构建最大简约树（以Albibacte-rium bauzanense BZ42为外群）

Fig. 3 MP-tree based on 16S rRNA (Albibacterium bauzan-ense BZ42 used as the outgroup)

图4 菌株18B和O. jilunii 14-2AT的全基因组共线性比对图中上方为菌株18B，下方为O. jilunii 14-2AT

Fig. 4 Genome-wide covariance alignment of strain 18B and O. jilunii 14-2AT It in the upper part of the figure it is strain 18B and in the lower part is O. jilunii 14-2AT

图5 Olivibacter属内基因组比对从NCBI上下载目前Olivibacter属内相关菌株全基因组序列信息，与菌株18B一同进行比对。图中sitiens指菌株O. sitiensis AW-6T，domesticus指菌株O. domesticus DC186T，LS_1指菌株O. sp. LS-1，jilunii指菌株O. jilunii 14-2AT

Fig. 5 Genome alignment within the genus Olivibacter Complete genome sequence information of related strains in Olivibacter genus was downloaded from NCBI and compared with strain 18B. In the figure, sitiens indicates the strain O. sitiensis AW-6T, domesticus indicates O. domesticus DC186T, and LS_1 indicates O. sp.LS-1. jilunii refers to strain O. jilunii 14-2AT

表1 18B和O. jilunii 14-2AT常见生理生化特征比对

Table 1 Comparison of common physiological and bioche-mical characteristics between 18B and O. jilunii 14-2AT

项目 Item	18B	O. jilunii 14-2A^T
生长温度/℃	12-48	4-42
生长pH	6.0-8.0	6.0-9.0
NaCl耐受/%	0-6	0-5
氧化酶	+	-
硝酸盐还原	+	+
糖酵解	-	+
VP	+	+
β-半乳糖苷酶	+	+
革兰氏染色	-	-
MacConkey agar	+（Red）	+（Red）

图6 18B纤维素酶活随培养时间变化灰线为CMC-Na培养环境中转接后的菌株纤维素酶活，红线为从CMC-Na培养环境转接至LB中培养一代后的菌株纤维素酶活；O. jilunii 14-2AT的酶活未测得

Fig. 6 Variation of 18B cellulase activity with incubation time The gray line is the cellulase activity of the strain after transfer from CMC-Na culture environment, and the red line is the cellulase activity of the strain after transfer from CMC-Na culture environment to LB culture for one generation. The enzyme activity of O. jilunii 14-2AT was not detected

图7 诱导表达后基因检测 Marker为Trans2k plusII；D为内切葡聚糖酶D前体基因，长度为1 812 bp；M为纤维素酶M基因，长度为1 119 bp；Z为内切葡聚糖酶Z前体基因，长度为1 542 bp；P为纯内切葡聚糖酶基因，长度为1 395 bp

Fig. 7 Gene amplification after induction of expression Marker is Trans2k plusII. D is the precursor gene of endoglucanase D, the length is 1 812 bp. M is cellulase M gene with length of 1 119 bp. Z is the precursor gene of endoglucanase Z, the length of which is 1 542 bp. P is a pure endoglucanase gene with a length of 1 395 bp

图8 纤维素酶基因诱导表达蛋白检测 0：Maker；1：D蛋白表达后破细胞全液；2：D蛋白表达后破细胞上清；3：D蛋白表达后破细胞沉淀；4：Z蛋白表达后破细胞全液；5：Z蛋白表达后破细胞上清；6：Z蛋白表达后破细胞沉淀；7：空载诱导表达后破细胞全液；8：空载诱导表达后破细胞上清；9：空载诱导表达后破细胞沉淀；10：marker；11：M蛋白表达后破细胞全液；12：M蛋白表达后破细胞上清；13：M蛋白表达后破细胞沉淀；14：P蛋白表达后破细胞全液；15：P蛋白表达后破细胞上清；16：P蛋白表达后破细胞沉淀；17：空载诱导表达后破细胞全液；18：空载诱导表达后破细胞上清；19：空载诱导表达后破细胞沉淀。红框圈出的为目的蛋白区段

Fig. 8 Protein assay of cellulase gene induced expression 0: Maker. 1: The whole solution broken after D protein expression; 2: the supernatant broken after D protein expression; 3: the precipitation broken after D protein expression; 4: the whole fluid broken after Z protein expression; 5: the supernatant broken after Z protein expression; 6: the precipitation broken after Z protein expression; 7: the whole solution of cell breaking after no-load induction expression; 8: the supernatant of cell breaking after no-load induction expression; 9: the precipitation of cell breaking after no-load induction expression; 10: marker; 11: the whole solution broken after M protein expression; 12: the supernatant broken after M protein expression; 13: the precipitation broken after M protein expression; 14: the whole fluid broken after P protein expression; 15: the supernatant broken after P protein expression; 16: the precipitation broken after P protein expression; 17: the whole solution of cell breaking after no-load induction expression; 18: the supernatant of cell breaking after no-load induction expression; 19: the precipitation of cell breaking after no-load induction expression. The red box marked the target protein region

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