Study on the Manganese Oxidation Activity of CotA Protein of Multicopper Oxidase from Bacillus safensis

doi:10.7668/hbnxb.20195936

Abstract

Abstract:

The Bacillus safensis ST7 strain has strong manganese oxidation activity,in which the expression of the multicopper oxidase protein family gene BSL056-RS03010(cotA)is significantly upregulated under manganese stress,which may be involved in Mn(Ⅱ)oxidation and make the strain have strong manganese tolerance.In order to verify the manganese oxidation ability of the cotA in B.safensis,the cotA gene was cloned and an expression vector was constructed,and then transformed to Escherichia coli for heterologous expression to study the effect of cotA gene overexpression on the manganese oxidation activity and removal rate of E.coli,so as to validate the function of the cotA gene,and to provide a technological means for constructing highly efficient manganese oxidizing strains to treat manganese pollution.Using PCR,RT-qPCR and SDS-PAGE,the cotA gene of B.safensis ST7 was cloned,ligated into the pET28a(+)expression vector,and efficiently expressed in E.coli BL21(DE3)induced by isopropyl thiogalactoside.The growth curve,manganese tolerance concentration,manganese oxidation activity and manganese removal rate of the cotA gene transformed strain were determined.The cloning and sequence analysis showed that the coding region of the cotA gene of B.safensis ST7 was 1 533 bp,encoding 510 aa with a molecular mass of about 58.8 ku.The expression of the recombinant plasmid pET28a-cotA was significantly up-regulated in E.coli BL21(DE3)cells without inhibition of the growth of the bacterium.The cotA gene was highly efficiently expressed under 0,250,1 000 mg/L manganese stress with time and concentration dependence.The manganese oxidizing activity of the recombinant strain was 0.9-fold higher than that of the control,the manganese tolerance concentration was 1.0-fold higher.The CotA protein of B.safensis has manganese oxidizing activity,and overexpression of the cotA gene can improve the oxidizing activity,tolerance and removal efficiency of Mn(Ⅱ)in E.coli cells,which is of great significance for the construction of manganese oxidizing strains with high efficient cotA gene for manganese pollution management.

Key words: Bacillus safensis, cotA gene, Gene cloning, Manganese oxidizing, Manganese pollution remediation

CLC Number:

S939.97

SU Chengli, NIU Xi, HUANG Shihui, LI Sheng, RAN Xueqin, WANG Jiafu. Study on the Manganese Oxidation Activity of CotA Protein of Multicopper Oxidase from Bacillus safensis[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(1): 230-238. doi: 10.7668/hbnxb.20195936.

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Figures/Tables 9

Tab.1 Primer information

引物名称 Primer name	引物序列(5'— 3') Primer sequence (5'— 3')	产物长度/bp Product size
cotA-F	CGC GGATCCATGAACCTAGAAAAATTTGTTG	1 551
cotA-R	CCC AAGCTTTTACTGAATGATATCCATAGGC
cotA-qF	TCACACGTCCGCTAAAAGGG	134
cotA-qR	GGCGGCCGTATTTATCCTGT

Fig.1 Amplification and bioinformatics analysis of the cotA gene A.Amplified DNA fragment;B.DNA and amino acid sequence;C.CotA structural domain.

Fig.2 Identification of recombinant plasmid pET28a-cotA A.Colony PCR validation of recombinant plasmid;B.Plasmid PCR validation of recombinant plasmid;C.Enzymatic validation of recombinant plasmid.M1—M3.DL2000 DNA Marker;M4.1 kb DNA Ladder;1—8.cotA gene fragment;9.Expression vector empty plasmid;10.Recombinant plasmid pET28a-cotA double enzyme digestion.

Fig.3 Relative expression levels and expression-fold ratios at different times of IPTG induction A.Induction of expression at different times;B.Expression multiplicity ratio by induction of different time. Different lowercase letters indicate significant difference at 0.05 level.The same as Fig.5,6.

Fig.4 Detection of CotA protein expression by SDS-PAGE M.Protein Marker;1—2.E.coli BL21/pET28a-cotA induced supernatant and precipitation;3—4.E.coli BL21/pET28a-cotA uninduced supernatant and precipitate;5—6.E.coli BL21-pET28a uninduced holobacteria,induced holobacteria;7—9.E.coli BL21(DE3)uninduced supernatant,induced supernatant and precipitate.

Fig.5 Expression of cotA gene under different concentration manganese stresses

Fig.6 Manganese oxidizing activity test A.Liquid manganese oxidation;B.Comparison of manganese oxidizing activity between E.coli BL21(DE3)and E.coli BL21/pET28a-cotA;C,D.Solid manganese oxidation.

Fig.7 Manganese metal tolerance concentration test of different strains

Fig.8 Changes in the removal rate of Mn²⁺ by bacterial strains over cultivation time A.In medium;B.In aqueous solution;C. In manganese contaminated soil.

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