Protein, coding gene of the protein, and use of the protein in petroleum hydrocarbon degradation

A technology for encoding genes and proteins, which is applied in the application field of proteins and their encoding genes and the degradation of petroleum hydrocarbons, can solve the problems of limiting the effective utilization of high-efficiency biological resources, and achieve the effect of improving the degradation ability

Active Publication Date: 2014-09-17
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the molecular level research on the alkane degradation mechanism and related functions of Dietzia, which limits the effective utilization as a high-efficiency biological resource.

Method used

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  • Protein, coding gene of the protein, and use of the protein in petroleum hydrocarbon degradation
  • Protein, coding gene of the protein, and use of the protein in petroleum hydrocarbon degradation
  • Protein, coding gene of the protein, and use of the protein in petroleum hydrocarbon degradation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1, the cloning of gene YMF4383

[0030] A new gene was discovered from a strain of Dietzia, and sequence 1 was artificially synthesized as the nucleotide sequence of the gene.

[0031] Using the artificially synthesized sequence 1 as a template, using the alkBr upstream primer and alkBr downstream primer as primers,

[0032] alkBr upstream primer 5'-GGAATTC Catatgt ccagcaccgaatacat-3' (sequence 3), containing Nde I restriction site;

[0033] alkBr downstream primer 5'-CCC AAGCTT gctacttcacgggcaggaag-3' (SEQ ID NO: 4), containing a HindIII restriction site;

[0034] Using TOYOBO's KOD-plus-high-fidelity enzyme, perform PCR to obtain PCR products.

[0035] The reaction system is as follows:

[0036] 10×PCR buffer 5μl

[0037] Deionized water 37μl

[0038] dNTP mix 5μl

[0039] KOD-plus- 0.5μl

[0040] DNA template 1μl

[0041] Primer 1 (alkBr upstream primer) 1 μl

[0042] Primer 2 (alkBr downstream primer) 1 μl

[0043] Total 50μl

[0044] The P...

Embodiment 2

[0055] Example 2, Functional Research of YMF4383

[0056] 1. Construction of expression vector

[0057] Select pCom8 shuttle vector (Smits and T, 2001, New Alkane-Responsive Expression Vectors for Escherichia coli and Pseudomonas, Plasmid 46 (1): 16-24, the public can obtain from Peking University.) as expression plasmid, containing gentamicin resistance. The expression host is Pseudomonas fluorescens KOB2Δ1 (Smits, T.H.M., S.B.Balada, et al., 2002, Functional Analysis of Alkane Hydroxylases from Gram-Negative and Gram-Positive Bacteria, Journal of Bacteriology 184(6) : 1733-1742., publicly available from Peking University.).

[0058] The PCR product obtained in Example 1 and the pCom8 plasmid were subjected to double digestion with Nde I and Hind III respectively, and the digested products were purified with the kit. The system is as follows:

[0059] PCR recovery product pCom8

[0060] 10×M buffer 5μl 5μl

[0061]Deionized water 33μl 41μl

[0062] NdeI...

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Abstract

The invention discloses a protein, a coding gene of the protein, and a use of the protein in petroleum hydrocarbon degradation. The protein is a protein 1 which is composed of an amino acid sequence 2 shown in the sequence table, or a protein 2 which is derived from the protein 1 through replacement and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence 2 and is related to alkane degradation. Experiments of the invention prove that the invention provides an alkane degradation-associated protein of Dietzia and a coding gene YMF4383 thereof; after being introduced into a microbial host, the coding gene YMF4383 can be stably expressed under induction when an alkane exists; a recombined bacterial strain has an alkane degradation capability and especially, greatly improves a capability of degrading a long-chain alkane having carbon chain length more than 16; and a bacterial strain expressing the protein can be used in a petroleum-polluted environment restoration and microbial enhanced oil recovery technology and has an important application prospect.

Description

technical field [0001] The invention relates to the field of microbial biotechnology, in particular to a protein and its coding gene and the application of degrading petroleum hydrocarbons. Background technique [0002] The microbial degradation of alkanes is a key mechanism for microbial oil recovery technology and microbial remediation of petroleum-contaminated environments. The biodegradation of long-chain alkanes is one of the bottlenecks that limit the application of these technologies, and is an important scientific issue in the study of petroleum microorganisms. The biodegradation of long-chain alkanes can reduce the viscosity, wax and asphaltene content of oil, increase fluidity, and greatly improve the efficiency of microbial oil recovery and microbial remediation of oil-polluted environments. However, my country's crude oil has the characteristics of high viscosity and high waxiness. The application prospect is very huge. [0003] Dietzia is an actinomycete discove...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/36C12N15/31C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12N15/11A62D3/02A62D101/20
Inventor 聂勇吴晓磊汤岳琴谭燕
Owner PEKING UNIV
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