阅读说明：本技术 一种容受甲基化半乳糖的琼胶酶及其应用 (Agarase containing methylated galactose and application thereof ) 是由 常耀光 曹斯琦 张玉莹 薛长湖 申晶晶 王玉明 薛勇 于 2020-06-19 设计创作，主要内容包括：本发明涉及生物技术领域,尤其涉及一种容受甲基化半乳糖的琼胶酶及其应用。所述琼胶酶的氨基酸序列为SEQ ID NO.1以及经过取代、缺失或添加一个或多个氨基酸且具有相同酶活性的,由此所衍生的酶。本发明的琼胶酶能够容受甲基化半乳糖,对于甲基化的琼脂糖结构片段以及经典的琼脂糖结构片段均具有高效的催化能力；相比于普通的琼胶酶能更完全的降解琼胶,提高其生物利用率。通过控制加酶量或反应时间等条件,可快速降解琼脂糖生成分子量为6kDa-900kDa的不同分子量琼脂糖及寡糖。(The invention relates to the technical field of biology, in particular to agarase accepting methylated galactose and application thereof. The amino acid sequence of the agarase is SEQ ID NO.1 and the enzyme which is derived by substituting, deleting or adding one or more amino acids and has the same enzyme activity. The agarase disclosed by the invention can be subjected to methylated galactose, and has high-efficiency catalytic capability on methylated agarose structural fragments and classical agarose structural fragments; compared with the common agarase, the agarase can degrade the agarase more completely, and the bioavailability of the agarase is improved. By controlling the conditions of enzyme adding amount, reaction time and the like, agarose can be quickly degraded to generate agarose and oligosaccharide with different molecular weights of 6kDa to 900 kDa.)

1. An agarase accepting methylated galactose, which has an amino acid sequence of SEQ ID NO.1 and is derived from an enzyme which has one or more amino acids through substitution, deletion or addition and has the enzyme activity.

2. A gene encoding the agarase of claim 1, wherein: the nucleotide sequence is shown in SEQ ID NO.2 and can be translated into all sequences of SEQ ID NO. 1.

3. A recombinant expression vector, a transgenic cell system or a transgenic recombinant bacterium containing the nucleotide sequence shown in SEQ ID NO. 2.

4. A recombinant plasmid for expressing agarase is characterized in that: comprising the gene sequence of claim 3.

5. A recombinant strain for expressing agarase is characterized in that: the recombinant strain is introduced with the recombinant plasmid for expressing agarase in claim 4.

6. The recombinant strain of claim 5, wherein: the host bacteria of the recombinant plasmid are escherichia coli, bacillus subtilis and pichia pastoris.

7. The agarase of claim 1, wherein: the optimal reaction condition is 30 ℃ and pH 6.5.

8. The use of the agarase of claim 1 in the enzymatic digestion of agar.

Technical Field

The invention relates to the technical field of biology, in particular to agarase accepting methylated galactose and application thereof.

Background

Agar is a polysaccharide extracted from the cell wall of marine red algae, and is an important marine polysaccharide. Agarose is the main component of agar, has excellent gel property, and is widely applied to food and food related industries, cosmetics, medicines and other industries. Agarose consists of alternating (1-3) -O- β -d-galactopyranose residues (G residues) and (1-4) -O-3, 6-diether- α -l-galactopyranose residues (LA residues). Several studies have shown that the structure of agarose is highly heterogeneous, with G residues in the structure often being modified by methylation, to the extent of up to 20%.

Agarose is not easy to dissolve at room temperature and has low bioavailability. The low molecular agarose and oligosaccharide obtained by degrading agarose have the characteristics of good water solubility, low viscosity and high bioavailability, have various biological activities of resisting bacteria, moisturizing skin, whitening melanoma cells, resisting obesity, resisting diabetes and the like, are potential functional food factors, and show good application prospects in the food and medicine industries.

Glycoside hydrolases specifically cleave glycosidic bonds in the agarose backbone and do not alter substituents in their natural structure. Compared with the commonly used acidolysis, the enzyme degradation of the agarose has the characteristics of mild reaction conditions, environmental protection, low energy consumption, high efficiency of the enzyme and substrate specificity, and can effectively overcome the problems of difficult control of conditions, inhomogeneous products, difficult analysis and recovery of the products and the like in the acidolysis. Agarase is a hydrolase which can specifically degrade agarose components in agar to generate agar oligosaccharide, and according to different enzyme cutting sites, agarase can be divided into alpha-agarase and beta-agarase which respectively cut alpha-1, 3 glycosidic bonds and beta-1, 4 glycosidic bonds.

The high specificity of glycoside hydrolases and the heterogeneity of polysaccharide structures means that complete conversion of polysaccharides requires hydrolases of different specificities. The traditional agarase which is characterized at present has strong specificity, and can only degrade the classical agarase structural fragment, namely (LA-G)_nAnd not to degrade the methyl-containing polysaccharide fragment. Therefore, these agarases cannot completely degrade methyl-containing natural agarose. Finding an agarase that can tolerate methylated galactose is crucial to improving the bioavailability and conversion rate of agarose. In the current research report, only one wild-type agarase from Pseudomonas atlantic can hydrolyze methylated agarose, but the wild-type agarase can be generated under the induction of agarose, and the wild-type agarase has high preparation cost, high purification difficulty, low total enzyme yield and low activity, and is difficult to be used in industrial large-scale production application. Molecular cloning can realize high-efficiency expression and mass acquisition of enzymes according to genes, and is an ideal strategy for solving the problems. The realization of the cloning expression of the enzyme-producing sequence can provide a tool for degrading agarose by an enzyme method and provide a premise for the large-scale production and application of low-molecular-weight agarose and oligosaccharide.

Disclosure of Invention

The technical problems to be solved by the invention are that the wild agarase which is compatible with the methylated galactose has low yield, low activity and high purification difficulty, and the enzyme producing strain can produce the enzyme under the induction of an agarose substrate, so that the preparation cost of the enzyme is high, and the key tool enzyme for completely converting agarose is lacked.

To solve the above problems, the present invention is based on Wenyingzhuangiantucanavanilytica CZ1127^TA gene is obtained by discovering in a strain, the original nucleotide codes 735 amino acids, the amino acid sequence is shown in SEQ ID NO.1, and the theoretical molecular weight of the gene is predicted to be 79.73kDa by utilizing ExPASy software. According to sequence alignment, the similarity of the enzyme and the currently reported agarase of the same family is only 47 percent at most, so the enzyme is an enzyme with novel sequence. Based on the gene, the agarase gene accepting the methylated galactose and the application thereof are provided, so that the key bottlenecks of efficient acquisition and practical application of the agarase, complete conversion of agarose and large-scale preparation of low molecular weight agarose and oligosaccharide are broken through.

In order to achieve the aim, the invention provides the agarase accepting methylated galactose, the amino acid sequence of which is SEQ ID NO.1 and the enzyme which is obtained by substituting, deleting or adding one or more amino acids and has the enzyme activity of 1 and is derived from 1.

SEQ ID NO.1：

MRVKSVYKKLSVSFILVMLSASQEVNSQAKVSVNLNVKHVVGGISEFDRTKYITIHANQIENEWDGDNFTSDLRDHFLNGFDVYLGRDTGGITWNLNNMQEDASRPGFANPSNIISKGINTRNNYASKTHLHVYENRKSNHVVAAQLHPFWTGESQIATKGTGWELASPTATGEYMGRYFNEFYGGNGEPVPSWIEVINEPAYEALGGKKNFTNSLQEIADFHVEVADAIRVQNPNLKIGGYTAAFPDFETGDFQRWINRDKLFIDVAGEKMDFWSWHLYDFPVIGGKEDIRSGSNVEATFDMHDHYSMLKLGHKKPYVISEYGAQTHDFRNEGWSSYRDWLFVRAQNSLMMSFMERPEDIAMAIPFTIVKAEWGFNTDKNLPYPARLMRKANEPESYTGEWVYTDRVKFYDLWKNVKGTRIDTKSTDLDIQVDAYVDGNKGYLILNNLESEETEITLDVFEKYDSSITNILKRHLTLSSNNVVIEEETFSSSISTVQLGAGSTMILEYTFANSLTIDETSTEEKYYADSYLQPIVASQPILFAVNNVVKSATYGEAVLRLGLGRDHGKSLKPIVKVNNTEVVVPDDWRGYDQADKGRFFGTIEIPVSYDLLTTNNTVSVEFPDSSGHVSSVIMQVFNFSSDIRTLSVNDVTASDTKTLLISPNPVKDGMLNMTIPAKLKNPIASIYNVSGSLLIKQSMKHSQTSIPVNLFDKGVYLLVLQDGSKKIGESKFVIQ

The enzyme has a sequence similarity of only 47% with other known enzymes (the similarity with the Aga86C produced by Saccharophhagus degradans 2-40 is highest), and is an enzyme with novel sequence. The enzyme was compared with a sequence of the GH86 family in the CAZy database, which was characterized by the MEGA6 to construct a phylogenetic tree, and the results are shown in fig. 6: it can be seen that the enzyme is in the phylogenetic tree of GH86 family agarases. Therefore, the agarase of the present invention is a new member of GH86 family.

The nucleotide sequence of the gene for coding the agarase accepting the methylated galactose is SEQ ID NO.2 and all genes capable of being translated into SEQ ID NO. 1.

SEQ ID NO.2：

ATGAGGGTTAAATCTGTATATAAAAAACTTAGTGTGAGTTTTATTTTAGTAATGCTATCTGCTTCTCAAGAGGTAAATAGTCAAGCTAAAGTTTCTGTTAATTTAAATGTAAAACACGTTGTTGGTGGGATATCTGAATTTGATAGAACAAAGTATATCACAATTCATGCAAATCAAATTGAAAATGAGTGGGATGGTGATAATTTTACATCAGATTTAAGAGATCATTTTTTAAATGGCTTTGATGTATATTTAGGAAGAGATACAGGAGGGATTACTTGGAATTTAAATAATATGCAAGAAGATGCTTCTAGACCTGGTTTTGCAAATCCTTCTAACATAATATCAAAAGGTATAAACACTAGAAATAATTATGCTTCTAAAACGCATTTACATGTATATGAAAATAGAAAAAGCAATCATGTAGTCGCAGCACAATTACATCCGTTTTGGACAGGTGAAAGTCAAATAGCTACTAAAGGTACAGGTTGGGAATTGGCAAGTCCAACTGCAACTGGAGAATATATGGGACGTTATTTTAATGAATTTTATGGAGGTAATGGAGAGCCTGTACCTAGTTGGATAGAAGTAATTAATGAACCAGCATATGAAGCTCTTGGAGGAAAGAAAAATTTTACAAACTCACTACAAGAGATAGCAGATTTTCATGTAGAGGTAGCAGATGCTATTAGAGTACAAAATCCAAATTTAAAAATAGGAGGATACACAGCAGCATTTCCAGATTTTGAAACGGGTGATTTTCAAAGATGGATAAATAGAGATAAATTATTTATAGATGTTGCGGGTGAAAAAATGGATTTTTGGTCTTGGCATTTGTATGATTTTCCTGTAATAGGAGGAAAAGAAGATATACGATCGGGGAGTAACGTAGAGGCAACTTTTGATATGCATGATCATTATAGTATGTTAAAGTTGGGACATAAAAAACCTTATGTAATTTCAGAATATGGGGCTCAAACACACGATTTTAGAAATGAAGGTTGGTCTTCTTACAGAGATTGGTTGTTTGTAAGGGCTCAAAACTCATTAATGATGTCTTTTATGGAAAGACCAGAAGATATAGCTATGGCAATTCCATTTACAATTGTAAAAGCAGAATGGGGTTTTAATACAGATAAAAATTTACCTTATCCGGCTAGATTAATGCGTAAGGCTAATGAGCCAGAAAGTTATACAGGAGAATGGGTGTACACAGATAGAGTTAAGTTTTACGATTTATGGAAAAACGTAAAAGGAACTAGAATTGACACAAAATCTACGGATTTAGACATACAGGTAGATGCGTATGTTGATGGAAACAAAGGATATTTAATTTTAAATAATTTAGAATCTGAGGAGACTGAAATTACTTTAGATGTTTTTGAAAAATATGATAGCAGTATTACAAATATTTTAAAAAGACATTTAACACTTTCTAGTAATAACGTTGTAATAGAAGAGGAGACTTTTTCATCTTCAATTTCTACAGTCCAATTAGGAGCTGGATCTACAATGATTTTGGAGTATACCTTTGCAAATTCCCTTACCATTGATGAGACTTCTACCGAAGAAAAATATTATGCAGACAGTTATTTACAGCCTATAGTTGCTTCTCAACCTATTTTGTTTGCAGTTAATAATGTAGTTAAATCGGCTACATATGGAGAGGCTGTGTTGAGGTTAGGACTAGGTAGAGATCATGGTAAGTCTTTAAAACCAATTGTAAAAGTAAATAATACAGAAGTGGTTGTACCAGATGATTGGAGAGGTTACGATCAGGCAGATAAAGGGAGGTTTTTTGGGACTATAGAAATACCAGTCTCGTATGATTTGTTAACTACAAACAATACCGTTTCTGTTGAATTCCCAGACTCTAGCGGACATGTAAGTAGTGTAATTATGCAAGTATTTAATTTTAGTTCAGATATTAGAACATTGTCTGTGAATGATGTTACTGCATCAGATACAAAAACGCTATTGATTTCTCCAAACCCAGTAAAAGATGGAATGTTAAATATGACTATACCAGCAAAATTAAAAAATCCAATAGCTTCTATTTATAATGTTTCAGGTAGTTTGTTAATAAAACAATCAATGAAACATAGTCAAACTAGTATTCCTGTAAACTTATTTGACAAAGGAGTTTATTTATTGGTTCTACAAGATGGAAGTAAAAAAATAGGAGAATCTAAATTTGTAATACAATAA

The invention provides a preparation method of the agarase containing the methylated galactose, which is characterized in that the agarase containing the methylated galactose can be prepared in a large scale by heterogeneously expressing the agarase in systems such as escherichia coli, bacillus subtilis, pichia pastoris and the like and inducing the enzyme production. The agarase which is subjected to the methylated galactose is successfully expressed in a heterologous way, can be used for mass production and preparation of target enzymes in systems such as escherichia coli, bacillus subtilis, pichia pastoris and the like, has the highest expression activity in a pichia pastoris expression system, and can be effectively applied to the fields of chemical analysis, food industry and the like.

The invention has the beneficial effects that:

(1) the agarase gene can realize the high-efficiency preparation of agarase which is compatible with methylated galactose by a cloning expression mode;

(2) the agarase can degrade agar in an incision mode, and has high-efficiency catalytic capability on methylated agar structural fragments and classical agar structural fragments; under the condition of controlling the enzyme adding amount or reaction time, the agar can be rapidly degraded in a short time to generate the agaragar and the oligosaccharide with different molecular weights of 6kDa to 900 kDa.

Drawings

FIG. 1: the agarose enzyme target gene PCR amplified nucleic acid electrophoresis pattern;

FIG. 2: the optimal reaction condition of the agarase is shown in a schematic diagram;

FIG. 3: extracting an ion current chromatogram of a degradation final product of the agarase;

FIG. 4: the agarase of the invention generates agarose schematic diagrams with different molecular weights under the condition of controlling the enzyme adding amount;

FIG. 5: the agarase of the invention generates agarose schematics with different molecular weights under the condition of controlling the reaction time;

FIG. 6: phylogenetic analysis diagram of agarase of the invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.