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An endo-type alginate lyase, its coding gene and application

A technology of alginate lyase and coding gene, which is applied in the field of endo-type alginate lyase and its coding gene and application, can solve the problems of low exonuclease activity, etc., and achieve high degradation rate, high specific activity, and broad bottom The effect of biodegradability

Active Publication Date: 2022-05-20
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the present study, most exo-type alginate lyases can produce unsaturated monosaccharide products, but the low exonuclease activity becomes the rate-limiting step

Method used

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  • An endo-type alginate lyase, its coding gene and application
  • An endo-type alginate lyase, its coding gene and application
  • An endo-type alginate lyase, its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Preparation of endo-type alginate lyase TsAly7C

[0044] 1. Cloning and acquisition of endo-type alginate lyase TsAly7C coding gene

[0045]The full-length amino acid sequence of the endo-type alginate lyase TsAly7C provided by the present invention is shown in SEQ ID No.7. The full-length sequence of endo-type alginate lyase TsAly7C coding gene (SEQ ID No.1) was used as the template of PCR reaction, and the following primers containing pET-24a homology arms were used for PCR amplification:

[0046] F: 5'-TAAGAAGGAGATATACATATGGGCTCAACAGCACCAAATAACG-3' (SEQ ID No.2);

[0047] R: 5'-GTGGTGGTGGTGGTGCTCGAGTTCTGGTTTAGTTGCGTCACTTAATA-3' (SEQ ID No. 3).

[0048] The PCR reaction was carried out according to the following conditions: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s; annealing at 58°C for 15 s; extension at 72°C for 1 min, for a total of 28 cycles, followed by extension at 72°C for 5 min. After the obtained PCR product is sequenced...

Embodiment 2

[0059] Example 2: Enzymatic properties of endo-type alginate lyase TsAly7C

[0060] 1. Effect of pH on endo-alginate lyase TsAly7C

[0061] (1) Add 100 μL of an appropriate concentration of alginate lyase TsAly7C to 900 μL of 0.3% alginate substrate (20mM PB buffer, pH=8.0), 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 50 mM Na 2 HPO 4 -NaH 2 PO 4 (pH6.0~8.0), glycine-NaOH (pH8.6~10.6), Na 2 HPO 4 - Citric acid (pH3.0~8.0), Tris-HCl (pH7.05~8.95), calculate the relative enzyme activity of endo-type alginate lyase TsAly7C under different pH conditions. The result is as figure 2 As shown in A, the endo-alginate lyase TsAly7C is suitable for the reaction pH of 7.7-8.9, and has the highest enzyme activity at pH 8.0.

[0062] (2) Store the pure enzymes at appropriate concentrations in the above-mentioned different pH buffers at 4 °C for 12 h, mix 100 μL wit...

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Abstract

The invention discloses an endo-type alginate lyase, its coding gene and application. The amino acid sequence of the endo-type alginate lyase TsAly7C is shown in SEQ ID No.7, and its nucleotide sequence is shown in SEQ ID No.1. The degradation mode of the endo-type alginate lyase TsAly7C is endo-type, which can significantly degrade alginate, polyM and polyG, and then obtain saturated alginate monosaccharide, unsaturated alginate monosaccharide and unsaturated alginate disaccharide, In addition, the enzyme belongs to a cold-adapted alginate lyase, suitable for a reaction temperature of 20-30°C, and is NaCl-dependent. The present invention also constructs a recombinant vector and a recombinant strain comprising the gene encoding the endo-type alginate lyase TsAly7C, which can provide a good tool for industrial application and production of bioethanol.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to an endo-type alginate lyase, its coding gene and application. Background technique [0002] Bioethanol has attracted attention as an alternative to petroleum-derived fuels. The presence of lignin in lignocellulosic biomass complicates the extraction of carbohydrates during bioconversion, leading to inefficient bioethanol production. Therefore, using raw materials with low lignin content is an effective way to produce bioethanol at low cost. Brown algae are good sources for bioethanol production due to their low lignin content. Moreover, the algae are rich in variety, fast in growth rate, and do not require arable land, which also significantly reduces the production cost of bioethanol. [0003] Algin is the main polysaccharide in the cell wall of brown algae, accounting for 40% of the dry weight of brown algae. Alginate is a kind of β-D-mannuronic acid (β-D...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12P19/02C12P19/12C12P19/14
CPCC12N9/88C12Y402/02003C12Y402/02011C12P19/12C12P19/02C12P19/14Y02W10/10
Inventor 于文功韩峰孙家霞王海楠傅政
Owner OCEAN UNIV OF CHINA
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