Isolation and Identification of a Cellulose-degrading Strain of Olivibacter jilunii and Analysis of Its Degradability

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

Abstract

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

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.

Figures/Tables 9

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

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

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

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

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

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）

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

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

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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