阅读说明：本技术 一种利用扩张蛋白预处理黄原胶降低其黏度的应用 (Application of xanthan gum pretreated by expansin to reduce viscosity of xanthan gum ) 是由 李蓉 李宪臻 刘珊君 于 2021-07-28 设计创作，主要内容包括：本发明公开了一种利用扩张蛋白预处理黄原胶降低其黏度的应用方法,属于降低黄原胶黏度的预处理技术领域。本发明通过使用扩张蛋白预处理黄原胶解聚,促使黄原胶黏度下降。采用本发明的方法,克服了传统化学法处理黄原胶时使用大量的强碱溶液,对设备要求高、产品质量差等现有技术中的不足。节约了成本,不造成环境污染,符合环境友好型。同时,这种方法简单易行,利于工业推广应用。(The invention discloses an application method for reducing viscosity of xanthan gum by utilizing expansin to pretreat the xanthan gum, and belongs to the technical field of pretreatment for reducing viscosity of the xanthan gum. The invention promotes the viscosity reduction of the xanthan gum by pretreating the xanthan gum for depolymerization by using the expansin. The method overcomes the defects of the prior art that the traditional chemical method uses a large amount of strong alkali solution when processing the xanthan gum, has high requirements on equipment, poor product quality and the like. Saves cost, does not cause environmental pollution and is environment-friendly. Meanwhile, the method is simple and easy to implement and is beneficial to industrial popularization and application.)

1. The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

2. The expansin encoded by the gene of claim 1 and having an amino acid sequence shown in SEQ ID No. 2.

3. A recombinant expression strain comprising the expansin according to claim 2.

4. Use of the expansin of claim 2 to pre-treat xanthan gum to reduce its viscosity, wherein the viscosity of xanthan gum is reduced by pre-treating xanthan gum with the expansin of claim 2.

5. The use according to claim 4, wherein the optimum temperature for pre-treating xanthan gum is 50 ℃ and the reaction time is 72 h.

Technical Field

The invention belongs to the technical field of pretreatment for reducing the viscosity of xanthan gum, and particularly relates to an application method for reducing the viscosity of xanthan gum by utilizing expansin to pretreat the xanthan gum.

Background

Xanthan gum is a microbial polysaccharide, which is a polysaccharide substance with a main chain of beta- (1,4) glucan and a side chain as a basic structural unit, wherein the side chain is formed by beta-D-mannose, beta-D-glucuronic acid and alpha-D-mannose on one glucose group at intervals. The xanthan gum which is not subjected to any treatment has abundant hydrogen bonds among molecules to form a complex secondary structure, so that the aqueous solution of the xanthan gum has very high viscosity. The viscosity reduction of xanthan gum can only be promoted by pretreating the xanthan gum with strong alkali, ionic liquid or high temperature, and then the xanthan gum is degraded by hydrolytic enzyme to generate xanthan gum oligosaccharide. The product xanthan gum oligosaccharide has the general properties of bioactive oligosaccharides such as chitosan oligosaccharide, seaweed oligosaccharide and the like, and has the potential of clearing free radicals in vitro and inhibiting the growth of pathogenic bacteria. Has greater application value in the fields of medicine, food health products, planting and the like. This makes the lowering of viscosity of xanthan gum an important key step to increase its hydrolysis efficiency.

At present, the main method for reducing the viscosity of xanthan gum is to dissolve the xanthan gum by using a strong alkali solvent at a high temperature to reduce the viscosity of the xanthan gum, and then the pH of a xanthan gum solution needs to be adjusted to be neutral for enzymolysis to generate xanthan gum oligosaccharide. The method needs high temperature in the production process, uses strong base solvent in the production, has high requirements on production equipment and is easy to cause environmental pollution. Therefore, it is increasingly important to find a method for pretreating xanthan gum to reduce its viscosity in a green and efficient manner.

Expansins are derived from bacteria and plants, are non-hydrolytic accessory proteins, have a destructive effect on substrates with polysaccharide networks, and break hydrogen bonds between polysaccharides without hydrolyzing them, opening the dense crystal structure of the polysaccharide substrate. Thereby allowing water molecules or hydrolytic enzymes to enter the polysaccharide substrate, causing it to form a water-soluble form.

Disclosure of Invention

The invention aims to provide an application method for depolymerizing xanthan gum by utilizing expansin to pretreat the xanthan gum; aims to overcome the defects of environmental pollution caused by chemical reagents, poor product quality and the like in the prior art when xanthan gum is hydrolyzed by a traditional chemical method, and provides an application of the economical, efficient and environment-friendly enzyme method for pretreating the xanthan gum.

The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

The amino acid sequence of the expansin coded by the gene is shown in SEQ ID NO. 2.

A recombinant expression strain of the expansin.

An application of xanthan gum pretreated by expansin for reducing its viscosity features that the expansin is added to xanthan gum for pretreatment.

Furthermore, the optimum temperature for pretreating xanthan gum is 50 ℃, and the reaction time is 72 h.

Compared with the reported method for reducing the viscosity of the xanthan gum, the application method for reducing the viscosity of the xanthan gum by pretreating the xanthan gum through the expansin adopts a biological method for efficiently hydrolyzing hydrogen bonds in the xanthan gum and destroying a dense grid structure of the xanthan gum to pretreat the xanthan gum for reducing the viscosity of the xanthan gum. The method overcomes the defects of the prior art that the traditional chemical method uses a large amount of strong alkali solution when processing the xanthan gum, has high requirements on equipment, poor product quality and the like. Saves cost, does not cause environmental pollution and is environment-friendly. Meanwhile, the method is simple and easy to implement and is beneficial to industrial popularization and application.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of purified expansin according to the present invention; in the figure, the first lane: a protein Marker; second lane: empty bacterium pET-28a/BL21 full thallus; a third lane: empty bacterium pET-28a/BL21 expression supernatant; a fourth lane: the recombinant bacterium pET-28a-HcEX/BL21 expresses the supernatant.

FIG. 2 is the optimum temperature for depolymerization of expansin pretreated xanthan gum of the present invention.

FIG. 3 is the optimum reaction time for depolymerization of expansin pretreated xanthan gum of the present invention.

Fig. 4 is a viscosity comparison of depolymerized untreated xanthan gum and the expansin-pretreated xanthan gum of the present invention.

Detailed Description

The process of the present invention is further illustrated by the following specific examples.

The present invention is further illustrated by the following examples, which are intended to be illustrative only and not to be limiting in scope, and the experimental procedures in the examples, unless otherwise specified, are conventional. The reagents used in the present invention are commercially available unless otherwise specified.

Example 1 expansin recombinant bacterium construction

According to the nucleotide sequence shown in SEQ ID NO.1, a third party company is entrusted to synthesize the expansin, namely, the expansin gene sequence is shown in SEQ ID NO.1, and the coding region is 660bp in length. The amino acid sequence coded by the expansin gene is shown in SEQ ID NO. 2. The gene sequence of SEQ ID NO.1 is synthesized and connected with plasmid pET-28a to obtain recombinant pET-28a-HcEX plasmid (synthetic gene of Jilin province, U.S. Biotech, Inc.). Escherichia coli BL21 competence is prepared, recombinant pET-28a-HcEX plasmid is electrically transferred into BL21, the plasmid is coated on an LB flat plate containing 30 mu g/ml kanamycin for screening, a plurality of single colonies are selected to be cultured in an LB liquid culture medium containing 30 mu g/ml kanamycin at 30 ℃ and 200rpm for 12 hours, colony PCR verification is carried out by using a T7 universal primer, an amplification product with correct size is obtained, a correctly constructed recombinant expression bacterium is proved, and the recombinant bacterium is named as pET-28a-HcEX/BL 21.

SEQ ID NO.1

GAAAACCGTGTTTCTGCGACTCACACCTCCGCCGCGCTGCATGAGGGTGAAGGTACTTACTACTTCTACAACGGCGGCGGCCATTGCAGCGTTCCGGTGCCGGCGATGTTCACTGCAGCGATGAACCAGACCGACTATAACGGTTCCCAGGCTTGTGGCGGTTGCGTTAAGGTGACCAACCGCAACAACGGCAAGTCCGTGGTGGCGCGCGTTGACGACTCTTGTCCGGGCTGCAACCCGGGTGACGTGGATCTGACCGACGCCGCCTTCGCGCAGATTTCTCCACTGGAGGCGGGTCGCATTCCGATCAGCTGGGATTATGTTCCGTGCGATTATCCGTCTGTGCTGCTGTACTTCATGGAAGGTTCTAGCCAGTGGTGGACCGCCGTGCAAGTACGCGAACAGCGTTATCCGGTAAGCTCTCTGGCGTACCGTGAATCTGGTTCTACCGGCTCCTATCAGGAGATCGCCCGTGAAGACTACAACTACTTTGTTGAACGTTCCGGCATGGGTACCGGCCCGTTTGATTTTCGCATCACCGACATCTATGGTCATGTGCTGGAAGCAGGTAACATCACCCTGCAGTCTGGCGTTCCGATCAACACCCAGCAACAGTTTCCGTCTATGGGTACCTCCGGCGTTATTAACCAGGCAGACAAA

SEQ ID NO.2

Example 2 expansin expression in recombinant bacteria

The recombinant strain identified correctly in example 1 was streaked on pET-28a-HcEX/BL21 on LB plates with 30. mu.g/ml kanamycin; selecting single colony in 5mL LB liquid of 30 mug/mL kanamycin, culturing for 12h, and using the single colony as seed liquid; the cells were inoculated into LB liquid medium containing 30. mu.g/ml kanamycin at a ratio of 1:50 (by volume) until OD600 became 1, IPTG was added to a final concentration of 0.6mM, and the cells were cultured at 16 ℃ and 200rpm for 20 hours. The expression of the expansin was detected by polyacrylamide gel electrophoresis, and the results are shown in FIG. 1, where the expansin was clearly expressed under IPTG induction.

Example 3 investigation of expansin pretreatment Xanthan Gum depolymerization conditions

The expansin pET-28a-HcEX/BL21 obtained in example 1 was subjected to measurement of the depolymerization conditions of the pretreated xanthan gum using xanthan gum as a substrate, including the optimum temperature and reaction time

(1) Determination of optimum temperature

In the experimental group, 0.5 wt% xanthan gum was used as a substrate, expansins were reacted at different temperatures (40 ℃, 50 ℃, 60 ℃) at 200rpm for 72 hours, the viscosity of the xanthan gum was measured by an NDJ-79A rotational viscometer (Shanghai Changji geological instruments Co., Ltd.), 0.5 wt% xanthan gum was used as a control group, and as a result, as shown in FIG. 2, the viscosity of the xanthan gum was decreased from 214mPa.s to 150mPa.s at a reaction temperature of 50 ℃, the decrease in viscosity was most significant, and therefore the optimum temperature of the expansins was 50 ℃.

(2) Determination of optimum reaction time

The same system was reacted at different times (12 hours, 24 hours, 36 hours, 48 hours, 72 hours) under the above optimum temperature conditions, and the viscosity of xanthan gum was measured by NDJ-79A rotational viscosity. As shown in FIG. 3, the viscosity of xanthan gum was unchanged at 84h, and the optimum reaction time of the extended protein xanthan gum was 72h in consideration of the stability of the protein.

Example 4 expansin pretreatment of xanthan gum to reduce viscosity

15mg of the expansin pET-28a-HcEX/BL21 obtained in example 1 was reacted with 20ml of 0.5 wt% xanthan gum at 50 ℃ and 160rpm for 72 hours, and the viscosity change of the xanthan gum was measured by NDJ-79A rotational viscosity, as a control, 20ml of 0.5 wt% xanthan gum was reacted at 50 ℃ and 200rpm for 72 hours, and the viscosity change of the xanthan gum was measured by NDJ-79A rotational viscosity. The relative viscosity ratio was obtained by comparing the viscosity of the reacted xanthan gum with the viscosity of the xanthan gum before the reaction, as shown in fig. 4, the relative viscosity ratio of the control xanthan gum was unchanged, while the viscosity of the xanthan gum after the expansin pretreatment was reduced by 30%.

SEQUENCE LISTING

<110> university of Dalian Industrial university

<120> application of xanthan gum pretreated by expansin to viscosity reduction

<130> 2021

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 660

<212> DNA

<213> Artificial Sequence (Artifical Sequence)

<400> 1

gaaaaccgtg tttctgcgac tcacacctcc gccgcgctgc atgagggtga aggtacttac 60

tacttctaca acggcggcgg ccattgcagc gttccggtgc cggcgatgtt cactgcagcg 120

atgaaccaga ccgactataa cggttcccag gcttgtggcg gttgcgttaa ggtgaccaac 180

cgcaacaacg gcaagtccgt ggtggcgcgc gttgacgact cttgtccggg ctgcaacccg 240

ggtgacgtgg atctgaccga cgccgccttc gcgcagattt ctccactgga ggcgggtcgc 300

attccgatca gctgggatta tgttccgtgc gattatccgt ctgtgctgct gtacttcatg 360

gaaggttcta gccagtggtg gaccgccgtg caagtacgcg aacagcgtta tccggtaagc 420

tctctggcgt accgtgaatc tggttctacc ggctcctatc aggagatcgc ccgtgaagac 480

tacaactact ttgttgaacg ttccggcatg ggtaccggcc cgtttgattt tcgcatcacc 540

gacatctatg gtcatgtgct ggaagcaggt aacatcaccc tgcagtctgg cgttccgatc 600

aacacccagc aacagtttcc gtctatgggt acctccggcg ttattaacca ggcagacaaa 660

<210> 2

<211> 220

<212> PRT

<213> Artificial Sequence (Artifical Sequence)

<400> 2

Glu Asn Arg Val Ser Ala Thr His Thr Ser Ala Ala Leu His Glu Gly

1 5 10 15

Glu Gly Thr Tyr Tyr Phe Tyr Asn Gly Gly Gly His Cys Ser Val Pro

20 25 30

Val Pro Ala Met Phe Thr Ala Ala Met Asn Gln Thr Asp Tyr Asn Gly

35 40 45

Ser Gln Ala Cys Gly Gly Cys Val Lys Val Thr Asn Arg Asn Asn Gly

50 55 60

Lys Ser Val Val Ala Arg Val Asp Asp Ser Cys Pro Gly Cys Asn Pro

65 70 75 80

Gly Asp Val Asp Leu Thr Asp Ala Ala Phe Ala Gln Ile Ser Pro Leu

85 90 95

Glu Ala Gly Arg Ile Pro Ile Ser Trp Asp Tyr Val Pro Cys Asp Tyr

100 105 110

Pro Ser Val Leu Leu Tyr Phe Met Glu Gly Ser Ser Gln Trp Trp Thr

115 120 125

Ala Val Gln Val Arg Glu Gln Arg Tyr Pro Val Ser Ser Leu Ala Tyr

130 135 140

Arg Glu Ser Gly Ser Thr Gly Ser Tyr Gln Glu Ile Ala Arg Glu Asp

145 150 155 160

Tyr Asn Tyr Phe Val Glu Arg Ser Gly Met Gly Thr Gly Pro Phe Asp

165 170 175

Phe Arg Ile Thr Asp Ile Tyr Gly His Val Leu Glu Ala Gly Asn Ile

180 185 190

Thr Leu Gln Ser Gly Val Pro Ile Asn Thr Gln Gln Gln Phe Pro Ser

195 200 205

Met Gly Thr Ser Gly Val Ile Asn Gln Ala Asp Lys

210 215 220