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Thermophilic esterase derived from aquifex aeolicus strain and functional verification of thermophilic esterase

A thermophilic ester and esterase technology, applied in the field of bioengineering, can solve problems such as difficult acquisition and slow growth of thermophilic bacteria

Active Publication Date: 2017-05-10
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most thermophilic bacteria grow slowly and the culture conditions are strict, so it is extremely difficult to obtain a large amount of thermophilic enzymes by cultivating wild bacteria

Method used

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  • Thermophilic esterase derived from aquifex aeolicus strain and functional verification of thermophilic esterase
  • Thermophilic esterase derived from aquifex aeolicus strain and functional verification of thermophilic esterase
  • Thermophilic esterase derived from aquifex aeolicus strain and functional verification of thermophilic esterase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Obtaining and Gene Sequence Analysis of A.aeolicus Recombinant Thermophilic Esterase Gene

[0060] According to the characteristics of the conserved sequence "Gly-Xaa-Ser-Xaa-Gly" in the primary structure of esterase and the key enzyme active site "Ser, His, Asp", two hypothetical protein sequences were selected from the genome of the A.aeolicus strain, Blast analysis was performed on it in the SWISS-PROT database, and the Aaeo1 sequence was found to have 27% similarity with a methylester esterase (Reference sequence: Q82SL8.2) derived from Nitrosomonas europaea. The sequence of Aaeo2 has 25% similarity with a mycophenolic acid acyl-glucuronide esterase (Reference sequence: Q5E9H9.1) derived from Bos taurus. Therefore, it can be preliminarily guessed that these two sequences may encode esterase proteins.

[0061] Five thermophilic esterases / lipases that have been studied were selected and compared with two thermophilic esterases of A. aeolicus for sequence c...

Embodiment 2

[0065] Example 2 Obtaining the three-dimensional predicted structures of two thermophilic esterases from A. aeolicus by using the "homology modeling" method

[0066] A thermophilic esterase gene Aaeo1 derived from A. aeolicus is 621bp in length, encodes 207 amino acids, and has a molecular weight of 23.4kDa. The nucleic acid sequence and protein sequence of the protein are shown in SEQ ID NO:3 and SEQ ID NO:1. Another thermophilic esterase gene Aaeo2 is 678bp in length, encodes 226 amino acids, and has a molecular weight of 26.8kDa. The nucleic acid sequence and protein sequence of the protein are shown in SEQ ID: 4 and SEQ ID NO: 2.

[0067] According to the SignalP prediction, the N-terminus of the hypothetical protein Aaeo2 has no possibility of signal peptide (see figure 2 ).

[0068] Submit the amino acid sequences of the two thermophilic esterases of A.aeolicus to the I-TASSER protein online modeling server (http: / / zhanglab.ccmb.med.umich.edu / I-TASSER / ) for homology mo...

Embodiment 3

[0069] Example 3 Construction of eukaryotic expression vectors for two kinds of aeolicus thermophilic esterases, recombinant expression and protein expression thereof

[0070] 1. Construction of eukaryotic expression vector

[0071] According to the codon preference of Pichia pastoris, the two thermophilic esterase genes of A. aeolicus introduced 6xHis tag at the C-terminus of the DNA coding frame, and introduced AvrII and NotI restriction enzyme sites on the 5' and 3' sides, respectively , the optimized sequences, namely SEQ ID NO: 5 (Aaeo1) and SEQ ID NO: 6 (Aaeo2), were synthesized by the biological company, and connected to the expression vector pPIC9K to obtain the recombinant expression vectors pPIC9K-Aaeo1 and pPIC9K-Aaeo2.

[0072] The recombinant expression vector plasmid pPIC9K-Aaeo2 was digested with AvrII and NotI, and digested at 37°C for 30min. Double enzyme digestion for simple identification, 1% agarose nucleic acid gel such as Figure 4 As shown, the positive ...

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Abstract

The invention discloses thermophilic esterase derived from an aquifex aeolicus strain and functional verification of the thermophilic esterase, and belongs to the field of bioengineering technology. According to the invention, a novel thermophilic esterase gene is discovered, three expression systems are constructed, and efficient expression and research of enzymatic properties are achieved. Construction of an eukaryotic expression system: preferably, a vector pPIC9K is adopted for expression vector construction, and a pichia pastoris host, preferably GS115, is transformed, so that efficient expression is achieved; construction of a prokaryotic escherichia coli expression system: preferably, a vector MBP3 is adopted for expression vector construction, and escherichia coli hosts, preferably BL21 and Origami2, are transformed, so that efficient expression is achieved; and construction of a prokaryotic bacillus megaterium expression system: preferably, a vector pHIS1525 is adopted for expression vector construction, and a bacillus megaterium host, preferably YYBm1, is transformed, so that efficient expression is achieved. The recombinant enzyme (the thermophilic esterase) has the advantages of esterase activity, ,thermophilic characteristic, thermal stability and the like; and the recombinant enzyme has a great potential in industrial application under a high-temperature condition.

Description

technical field [0001] The invention relates to a thermophilic esterase derived from Aquifex aeolicus strain and its function verification, belonging to the technical field of bioengineering. Background technique [0002] Extremophiles are the general term for microorganisms suitable for living in extreme environments, including thermophilic, psychrophilic, acidophilic, alkaliphilic, barophilic, halophilic, metal-loving, radiation-resistant, desiccation-resistant and extreme anaerobic and other types . Thermophilic bacteria (high-temperature bacteria, also known as thermophilic microorganisms) are a class of microorganisms that live in high-temperature environments above 50°C. Common sources of thermophilic microorganisms include hot springs, craters, marine sediments, waste water, geothermal soils, etc. In the past 30 years, after the discovery of bacteria that can live at and above the boiling point of water, the research on thermophilic microorganisms has been promoted....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N1/19C12N9/18C12N15/55C12N15/70C12N15/75C12N15/81C12R1/84C12R1/19C12R1/11
Inventor 喻晓蔚徐岩
Owner JIANGNAN UNIV
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