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Externally tangent type algin lyase VsAly7D and recombinant strain and application thereof

A technology of alginate lyase and recombinant strain, which is applied in exo-type alginate lyase VsAly7D and its recombinant strain and application field, can solve the problem of low activity and achieve high degradation rate, high specific activity, and wide substrate degradability Effect

Active Publication Date: 2020-04-03
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the activity of exo-algin lyase is low at present, and the activity is even lower at low temperature, which has become a limiting condition for bioethanol production.

Method used

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  • Externally tangent type algin lyase VsAly7D and recombinant strain and application thereof
  • Externally tangent type algin lyase VsAly7D and recombinant strain and application thereof
  • Externally tangent type algin lyase VsAly7D and recombinant strain and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1: Preparation of exo-type alginate lyase VsAly7D

[0040] 1. Cloning of gene encoding exo-alginate lyase VsAly7D

[0041] Codon optimization is performed on the alginate lyase gene whose nucleotide sequence is shown in SEQ ID No.1 to obtain the alginate lyase gene whose nucleotide sequence is shown in SEQ ID No.2.

[0042] The alginate lyase gene whose nucleotide sequence is shown in SEQ ID No.2 was subjected to a PCR amplification reaction experiment, and the amplification primers used were as follows:

[0043] F: 5'-GGAGATATACATATGAGCGATCTGAATAATGGC-3' (SEQ ID No.3);

[0044] R: 5'-GTGGTGGTGCTCGAGTTTGCCATTCAGCTTCAG-3' (SEQ ID No. 4).

[0045]The PCR reaction was carried out according to the following conditions: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 50°C for 20 s; extension at 72°C for 60 s, for a total of 30 cycles, followed by extension at 72°C for 10 min. After the obtained PCR product is sequenced, the partial ...

Embodiment 2

[0055] Example 2: Enzymatic properties of exo-type alginate lyase VsAly7D

[0056] 1. Effect of pH on exo-alginate lyase VsAly7D

[0057] (1) Add 100 μl of alginate lyase VsAly7D of appropriate concentration to 900 μl of 0.3% alginate substrate (20 mMPB buffer, pH=8.6), react in buffers with different pH for 10 min, and measure with a spectrophotometer A 235 Values, taking the highest enzyme activity as 100%; the buffer used was 20 mM Na 2 HPO 4 -NaH 2 PO 4 (pH 6.0~8.0), Glycine-NaOH (pH 8.6~10.6), Na 2 HPO 4 - Citric acid (pH 3.0~8.0), Tris-HCl (pH 7.05~8.95), calculate the relative enzyme activity of exo-alginate lyase VsAly7D under different pH conditions. The result is as Figure 4 As shown in A, the exo-type alginate lyase VsAly7D has the highest enzyme activity at pH 7.6, and its suitable reaction pH is 7.4-8.2.

[0058] (2) Store the pure enzymes at appropriate concentrations in the above-mentioned different pH buffers at 4°C for 24 hours, mix 100 μl with 900 μ...

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Abstract

The invention discloses externally tangent type algin lyase VsAly7D and a recombinant strain and application thereof. The amino acid sequence of the externally tangent type algin lyase disclosed by the invention is as shown in SEQ ID No.6, and the nucleotide sequence of the coding amino acid sequence is as shown in SEQ ID No.5. The suitable reaction temperature of the externally tangent type alginlyase is 20-35 DEG C, and the externally tangent type algin lyase has high stability at 0-30 DEG C, so that the enzyme can effectively degrade substrate at low temperature and belongs to cold-adaptedalgin lyase; the externally tangent type algin lyase has degradation capacity on algin, polyG and polyM, a degradation manner is an externally tangent type, and a final degraded product is unsaturated monose and has NaCl dependence. The invention further constructs a recombinant carrier and recombinant strain containing externally tangent type algin lyase VsAly7D coded genes, and a favorable basecan be provided for industrialized application and production of biologic ethanol.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to an exo-type alginate lyase VsAly7D and its recombinant strain and application. Background technique [0002] With the ever-increasing demand for energy and the depletion of fossil fuel resources, bioethanol as a renewable and clean energy source has received increasing attention. Brown algae have become an important source of bioethanol production due to their excellent properties, including wide distribution, high productivity, no need for arable land, fresh water, and fertilizers, and no lignin. [0003] Alginate is the main polysaccharide in the cell wall of brown algae, which can reach 40% of the dry weight of brown algae. Alginate is a linear polysaccharide linked by β-D-mannuronic acid (M) and its C5 epimer α-L-guluronic acid (G), and arranged into poly β-D- Mannucuronic acid (polyM), poly alpha-L-guluronic acid (polyG) and heteropolymer (polyMG). Algi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P19/02C12R1/19
CPCC12N9/88C12N15/70C12P19/02C12Y402/02011
Inventor 于文功韩峰傅政张凤超张哲伦唐璐瑶
Owner OCEAN UNIV OF CHINA
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