Sodium alginate catenase gene algl and its preparation method and application

Abstract

An algin lyase gene algl able to code marine pseudomonads QDA is prepared from the QDA genome DNA library by cloning, and cna be used to prepare the recombinant algin lyase. It can degradate the homopolymannuronic acid to obtain algin oligose. Said recombinant algin lyase features high stability of temp and pH value, and activity, high expression level in colibacillus 24%, and low cost.

Description

technical field

[0001] The invention relates to alginate lyase gene algL of marine pseudomonas (Pseudomonas sp.) QDA. The invention also discloses the production method and application of the vector containing the gene, the Escherichia coli recombinant strain and the expression product thereof. Background technique

[0002] Alginate is a linear polysaccharide molecule composed of two sugar units, α-L-guluronic acid and β-D-mannuronic acid, connected continuously or alternately. In recent years, with the rapid development of glycobiology and glycochemistry research, it has been found that mixtures of alginate oligosaccharides with different degrees of polymerization have important medicinal value, such as: lowering blood fat, anti-tumor, anti-virus, etc., and are expected to be developed into A new generation of marine medicines. There are many methods for the preparation of algin oligosaccharides, such as acid degradation, oxidative degradation, sonication and enzymatic de...

Examples

Embodiment 1

[0016] Example 1 Cloning of alginate lyase gene algL

[0017] Use degenerate polymerase chain reaction (PCR) primers to screen the genome library of Pseudomonas sp.) QDA, and clone the alginate lyase gene algL. The specific operation is: in Luria-Bertani ( Pseudomonassp.QDA was cultured in LB) medium to the end logarithmic phase, and genomic DNA was extracted. The genomic DNA was digested with Sau3AI, electrophoresed on agarose gel, and a 5-8 kb fragment was recovered, which was ligated with the pBluescript II KS (+) plasmid fragment that was completely digested with BamHI and dephosphorylated. Then, the ligation product was transferred into Escherichia coli DH5α competent cells according to the standard calcium chloride method, and cultured on LB solid medium (containing ampicillin, X-gal, IPTG). Pick the white spot recombinant colony, culture it in LB liquid medium (containing ampicillin), and use the degenerate primer pair UpperA (5'AACCACACCGGCAARTCCAT3') and LowerA (5'GC...

Embodiment 2

[0018] Example 2 Construction of Escherichia coli expression vector pLyase-A

[0019] Design upstream primers (5'CCGAAT TCA TCC CCC ACA GG GTT ACT AC G A3') and downstream primers (5'CCC AAGCTT GCT TTT TTG CTC TCT GCG TTC G3') according to the obtained complete sequence of the alginate lyase gene algL; The complete sequence of the alginate lyase gene was amplified by PCR. The PCR conditions were as follows: pre-denaturation at 94°C for 3 minutes, followed by 30 cycles of 94°C for 30s, 60°C for 30s, and 72°C for 60s, and finally extension at 72°C for 10 minutes. Agarose gel electrophoresis showed a specific band at 1.0 kb, which was excised from the agarose gel, digested with Hind III and EcoR I, and a 1.0 kb DNA fragment was recovered after agarose gel electrophoresis.

[0020] The Escherichia coli expression vector pBAD gIII / A was also digested with Hind III and EcoR I, separated by agarose electrophoresis, and a 4.1kb DNA fragment was recovered, which was connected to the a...

Embodiment 3

[0021] Example 3 Construction of Escherichia coli engineering strain pLyase-A / Top10F' capable of highly expressing alginate lyase

[0022] The expression vector pLyase-A was transformed into Escherichia coli Top10F' according to the standard calcium chloride method, and transformants with ampicillin resistance were screened. The plasmid was extracted by a standard alkaline lysis method, and two fragments of 1.0 kb and 4.1 kb were obtained by Hind III and EcoR I digestion, which were respectively combined with the full-length fragment of the alginate lyase gene algL and the expression vector pBAD gIII / A, proving that the energy Escherichia coli recombinant strain pLyase-A / Top10F' highly expressing alginate lyase.

technical field

[0001] The invention relates to alginate lyase gene algL of marine pseudomonas (Pseudomonas sp.) QDA. The invention also discloses the production method and application of the vector containing the gene, the Escherichia coli recombinant strain and the expression product thereof. Background technique

[0002] Alginate is a linear polysaccharide molecule composed of two sugar units, α-L-guluronic acid and β-D-mannuronic acid, connected continuously or alternately. In recent years, with the rapid development of glycobiology and glycochemistry research, it has been found that mixtures of alginate oligosaccharides with different degrees of polymerization have important medicinal value, such as: lowering blood fat, anti-tumor, anti-virus, etc., and are expected to be developed into A new generation of marine medicines. There are many methods for the preparation of algin oligosaccharides, such as acid degradation, oxidative degradation, sonication and enzymatic de...

Examples

Embodiment 1

[0016] Example 1 Cloning of alginate lyase gene algL

[0017] Use degenerate polymerase chain reaction (PCR) primers to screen the genome library of Pseudomonas sp.) QDA, and clone the alginate lyase gene algL. The specific operation is: in Luria-Bertani ( Pseudomonassp.QDA was cultured in LB) medium to the end logarithmic phase, and genomic DNA was extracted. The genomic DNA was digested with Sau3AI, electrophoresed on agarose gel, and a 5-8 kb fragment was recovered, which was ligated with the pBluescript II KS (+) plasmid fragment that was completely digested with BamHI and dephosphorylated. Then, the ligation product was transferred into Escherichia coli DH5α competent cells according to the standard calcium chloride method, and cultured on LB solid medium (containing ampicillin, X-gal, IPTG). Pick the white spot recombinant colony, culture it in LB liquid medium (containing ampicillin), and use the degenerate primer pair UpperA (5'AACCACACCGGCAARTCCAT3') and LowerA (5'GC...

Embodiment 2

[0018] Example 2 Construction of Escherichia coli expression vector pLyase-A

[0019] Design upstream primers (5'CCGAAT TCA TCC CCC ACA GG GTT ACT AC G A3') and downstream primers (5'CCC AAGCTT GCT TTT TTG CTC TCT GCG TTC G3') according to the obtained complete sequence of the alginate lyase gene algL; The complete sequence of the alginate lyase gene was amplified by PCR. The PCR conditions were as follows: pre-denaturation at 94°C for 3 minutes, followed by 30 cycles of 94°C for 30s, 60°C for 30s, and 72°C for 60s, and finally extension at 72°C for 10 minutes. Agarose gel electrophoresis showed a specific band at 1.0 kb, which was excised from the agarose gel, digested with Hind III and EcoR I, and a 1.0 kb DNA fragment was recovered after agarose gel electrophoresis.

[0020] The Escherichia coli expression vector pBAD gIII / A was also digested with Hind III and EcoR I, separated by agarose electrophoresis, and a 4.1kb DNA fragment was recovered, which was connected to the a...

Embodiment 3

[0021] Example 3 Construction of Escherichia coli engineering strain pLyase-A / Top10F' capable of highly expressing alginate lyase

[0022] The expression vector pLyase-A was transformed into Escherichia coli Top10F' according to the standard calcium chloride method, and transformants with ampicillin resistance were screened. The plasmid was extracted by a standard alkaline lysis method, and two fragments of 1.0 kb and 4.1 kb were obtained by Hind III and EcoR I digestion, which were respectively combined with the full-length fragment of the alginate lyase gene algL and the expression vector pBAD gIII / A, proving that the energy Escherichia coli recombinant strain pLyase-A / Top10F' highly expressing alginate lyase.

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