l-arabinose isomerase, mutants and applications thereof

An arabinose and mutant technology, applied in the field of L-arabinose isomerase and its mutants, can solve the problems of restricting the production level of D-tagatose, unfavorable industrial production, low substrate concentration, etc., and achieve ultra-high substrate Conversion rate, improvement of industrialization level, effect of ultra-high enzyme activity

Active Publication Date: 2021-10-15
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the substrate concentration of D-tagatose prepared from D-galactose by biological method is low, all below 100g / L, which is not conducive to the application in industrial production
In addition, there are few enzyme sources that can be developed, which restricts the production level of D-tagatose

Method used

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  • l-arabinose isomerase, mutants and applications thereof
  • l-arabinose isomerase, mutants and applications thereof
  • l-arabinose isomerase, mutants and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1: the screening of novel enzyme

[0022] 1. Source of enzyme and construction of recombinant bacteria

[0023] The key catalytic regions of isomerases were analyzed, and three unstudied sugar isomerases were obtained from the NCBI database, which were derived from Pocillopora damicornis (GenBank No. XP_027045535.1), Vignaunguiculata (GenBank No. Candidatus Paracaedibacteracanthamoebae (GenBank accession number WP_075261590.1), and named EPoda, EViun and ECapa. According to the amino acid sequence, the codon was optimized according to the codon preference of Escherichia coli, and three selected nucleotide sequences were synthesized by the method of total synthesis through the conventional operation of genetic engineering, such as SEQ ID NO.2, SEQ ID NO.4 and shown in SEQ ID NO.6; the amino acid sequences encoding the enzyme are shown in SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5 respectively. Add 6×His-tag tags at the end of the nucleic acid sequence, add res...

Embodiment 2

[0037] Example 2: Construction and screening of EPoda single point mutants

[0038] 1. Mutant construction

[0039] According to the parental sequence of EPoda (the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2), the mutation primers for site-directed mutation were designed, and the recombinant vector pET28b / EPoda was used as a template by using rapid PCR technology. To introduce a single mutation at position 107, the primers are:

[0040] Forward primer 107I: AACTGGATGTGGCG NNN GATGGCGCGGATGATGTG (the underline is the mutated base)

[0041] Reverse primer 107I: CACATCATCCGCGCCATC NNN CGCCACATCCAGTT (the underline is the mutated base)

[0042] PCR reaction system: 2×Phanta Max Buffer (containing Mg 2+ ) 25 μL, dNTPs 10 mM, forward primer 107I 2 μL (5 pmol / μL, the same below), reverse primer 107I 2 μL (5 pmol / μL, the same below), template DNA 1 μL (20ng / μL, the same below), Phanta Max Super- Fidelity DNA Polymerase 50U ...

Embodiment 3

[0054] Example 3: Construction and screening of EPoda double-site mutants

[0055] According to the single mutant EPoda-1 sequence constructed in Example 2, the mutation primers for site-directed mutation were designed, and the rapid PCR technology was used to use the recombinant vector pET28b / EPoda-1 as a template to introduce a single mutation at position 199. The primers were:

[0056] Forward primer 199D: GGCCCGGTGGTGACC NNN AACAGCAACTTTATTA (the underline is the mutated base)

[0057] Reverse primer 199D: TAATAAAGTTGCTGTT NNN GGTCACCACCGGGCC (the underline is the mutated base)

[0058] PCR reaction system: 2×Phanta Max Buffer (containing Mg 2+ ) 25μL, dNTPs 10mM, forward primer 199D 2μL, reverse primer 199D 2μL, template DNA 1μL, Phanta Max Super-Fidelity DNA Polymerase 50U, add ddH 2 0 to 50 μL.

[0059] The PCR amplification conditions were 95°C for 3min; (95°C for 15s, 63°C for 15s, 72°C for 6.5min) for 30 cycles; 72°C for 5min.

[0060] The PCR product was tra...

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Abstract

The invention relates to a novel L-arabinose isomerase (LAI) and its mutant, and its application in continuously catalyzing D-galactose isomerization to prepare D-tagatose. The isomerase mutant is obtained by site-directed mutation of the amino acid shown in SEQ ID NO.1, and the point mutation site is one or more of the following: (1) 107th position, (2) 199th, (3) 59th. The beneficial effects of the present invention are mainly reflected in: the present invention provides a brand-new LAI and its mutants, and the product yield is as high as 89.2% under the reaction system of 85° C. and 700 g / L D-galactose. The recombinant enzyme was immobilized and continuously catalyzed for 200 batches, and the conversion rate was still greater than 89%. The invention solves the problem of low efficiency of the existing enzyme-catalyzed preparation of D-tagatose, and obtains a continuous conversion effect better than that reported in the literature, which is of great significance for improving the industrialization level of D-tagatose.

Description

(1) Technical field [0001] The invention relates to an L-arabinose isomerase and its mutant, and its application in preparing D-tagatose by catalyzing the isomerization of D-galactose by microorganisms. (2) Background technology [0002] D-tagatose is a rare ketohexose found in nature and is an isomer of D-galactose. It was first found in the gum of a tropical evergreen plant, and later found in yogurt and cheese. D-tagatose has the properties and effects of high sweetness, low calorie, low absorption rate, effectively lowering blood sugar, anti-caries, and improving intestinal flora. The US Food and Drug Administration has passed the safety certification of D-tagatose, allowing it to be used in food. The production methods of D-tagatose mainly include chemical method and biological method. The chemical method has problems such as high cost, high acid and alkali consumption, serious pollution, complex product components, and difficult separation and purification. In cont...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/90C12P19/24C12P19/02
CPCC12N9/90C12P19/02C12P19/24C12Y503/01004
Inventor 柳志强贾东旭郑裕国孙晨奕金利群彭晨陈德水廖承军程新平李勉毛宝兴
Owner ZHEJIANG UNIV OF TECH
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