CN111172141A

CN111172141A - A chitin deacetylase

Info

Publication number: CN111172141A
Application number: CN202010058762.2A
Authority: CN
Inventors: 王敏; 马钦元; 申雁冰; 屠琳娜; 毕心宇; 闫文芹
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2020-01-19
Filing date: 2020-01-19
Publication date: 2020-05-19

Abstract

The invention belongs to the technical field of proteases, in particular to a chitin deacetylase. The chitin deacetylase RCDA is derived from Rhodococcus equi and has the amino acid sequence shown in SEQ ID NO. The basic enzyme protein sequence similarity is low, and it has a wide substrate spectrum; it is less affected by metal ions.

Description

Chitin deacetylase

The technical field is as follows:

the invention belongs to the technical field of protease, and particularly relates to chitin deacetylase.

Background art:

chitin, also known as chitin, (1, 4) -2-acetamido-2-deoxy- β -D-glucan, is an aminopolysaccharide which is contained in natural organisms and has a content which is second to that of cellulose, and is mainly present in invertebrates such as shrimps, insects, seaweed, fungi and yeast, but chitin is insoluble in water, acid, alkali and organic solvents, so that the chitin has no great commercial value, and the product chitosan obtained after deacetylation is widely applied to industries such as medicine, food, chemical industry, cosmetics and the like.

Chitin Deacetylase (CDA) can hydrolyze and remove acetyl on Chitin to produce high-quality chitosan products with stable deacetylation degree and narrow molecular mass distribution range, and the reaction conditions are mild, so that a new way is provided for solving the problem of environmental pollution in chitosan production. Most of the CDA producing strains reported at present are fungi, and most of the CDAs derived from the fungi are glycoproteins which must be modified by glycosylation to be active; at present, the reported bacteria have less CDA, most of the CDA is chitosan oligosaccharide deacetylase, the acting substrate spectrum is narrow, the problems of low activity of produced enzyme, reduced activity of purified enzyme, unobvious improvement of enzyme activity of genetic engineering bacteria and the like exist, and the industrialization is not realized at present. Therefore, the screening of a novel CDA of excellent performance and bacterial origin is one of the important problems for industrial application of CDA, and has important practical value and academic research value.

The invention content is as follows:

aiming at the defects of the prior art, the invention aims to provide a chitin deacetylase and an application thereof, wherein the chitin deacetylase RCDA is derived from Rhodococcus equi, is a novel protein sequence reported for the first time at present, and has lower similarity with the reported chitin deacetylase protein sequence (figure 1).

The chitin deacetylase RCDA has an amino acid sequence shown in a sequence table SEQ ID NO. 1;

the invention also protects the gene of RCDA shown in SEQ ID NO. 1;

the invention also provides application of the chitin deacetylase RCDA in chitin hydrolysis.

The chitin deacetylase RCDA provided by the invention has the following enzymological properties:

(1) the optimal reaction temperature of RCDA is 45 ℃ and the tolerable temperature is less than 55 ℃;

(2) the optimal reaction pH of the RCDA is 7.0, and the pH range of the enzyme activity temperature is 5.0-9.0;

(3) when the ion concentration is 1mM or less, the metal ion has little influence on the enzyme activity, and when the ion concentration is 1mM or more, Zn²⁺、Cu²⁺、Ni²⁺Has stronger inhibition effect on the CDA activity;

(4) RCDA has a broad substrate spectrum, has deacetylation activity on low molecular weight chitooligosaccharides, colloidal chitin, ethylene glycol chitin and chitosan with a certain degree of deacetylation, and also shows deacetylation activity on various amino acids with acetyl groups.

Has the advantages that:

compared with the reported bacterial CDA, the RCDA has a wider substrate spectrum, can act on macromolecular substrates, and has the relative activity of 132.94% for deacetylation of chitosan with the deacetylation degree of 85%, 111.2% for colloidal chitin and 89.12% for ethyleneglycol chitin. In addition, RCDA showed high activity for acetyl-charged amino acid substrates, including N-acetyl-DL-methionine, N-acetyl-DL-tryptophan, N-acetyl-L-cysteine, and N-acetyl-L-leucine, which were reported for the first time.

Description of the drawings:

FIG. 1 phylogenetic analysis;

FIG. 2 enzymatic characterisation

Wherein a is an optimum temperature curve; b is the optimum pH curve; c is a temperature stability curve; d is a pH stability curve; e is metal ion tolerance.

The specific implementation mode is as follows:

in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the present invention.

The amino acid sequence of the RCDA disclosed by the invention is as follows (SEQ ID NO. 1):

MSKKIYVNFGVDVDAVAGWLGSYGGEDSPGDISRGMFAGEVGVPRLVKMFDKYDITTSWFVPGHSIETFPREIEQVVAAGHEIGIHGYSHENPIAMTRQQETDILDRSIELIESFTGSKPTGYTAPWWEFSKVTNELLIERGVKYDHSLMHNDFTPYYVRVGDSWSKIDYSQPAETWMKPLVRGQETDLVEIPANWLLDDLPPQMFIKSSPNSHGFVSPRHLEEMWRDQFDWVYREMDYAIFPVTIHPDVSGRPQSLLMLERLIEYINQHDGVEWVTFDHMVNDFKERFPRQS

example 1 substrate Spectroscopy

The RCDA substrate spectrum was studied by selecting macromolecular chitin powder, colloidal chitin, ethyleneglycol chitin, chitosan with a degree of deacetylation of 85%, small molecular weight chitooligosaccharides and several amino acids with acetyl groups as substrates, adding 5mL of RCDA crude enzyme solution to the excess substrate solution, allowing the mixture to react at 37 ℃ and pH 4.0 under stirring, and stirring for 12 hours. The sample was then boiled for 5 minutes, and the deacetylation capacity of the various substrates was characterized by the content of acetic acid formed by deacetylation by high performance liquid chromatography, with the substrate activity (157.6U/mL) of p-4-nitroacetanilide being set at 100%. The results are shown in table 1, where RCDA is active on most substrates tested, including low molecular weight chitooligosaccharides, colloidal chitin, ethyleneglycol chitin, and chitosan with a certain degree of deacetylation; in addition, RCDA exhibits deacetylation activity for a wide variety of acetyl amino acids.

Compared with other reported bacterial chitin deacetylases, RCDA shows higher activity on macromolecular substrates and also shows a broader substrate spectrum.

TABLE 1 relative enzyme Activity of RCDA on different substrates

EXAMPLE 2 enzymatic characterization

(1) In order to determine the optimal reaction temperature of the enzyme, the enzyme is reacted for 60min at different temperatures (30-60 ℃) and pH7.0, and the enzyme activity is detected;

(2) in order to research the temperature stability of the enzyme, the enzyme is preincubated for 30min at different temperatures (25-60 ℃), the pH value is 7.0, and the residual enzyme activity of the enzyme is detected after the reaction is carried out for 60min at 37 ℃;

(3) in order to determine the optimal pH value, the enzyme activity of the enzyme reacting for 60min at 37 ℃ under different pH conditions is measured;

(4) to study the pH stability of the enzyme, the enzyme was preincubated overnight in various pH2-10 buffers, and then reacted at 37 ℃ for 60 minutes at pH7.0 to detect the residual enzyme activity;

(5) simultaneously researches RCDA and different metal ions Zn²⁺、Fe²⁺、Mg²⁺、Mn²⁺、Cu²⁺、Ca²⁺、Ni²⁺And the influence of different ion concentrations (0.5, 1.0, 5.0, 10.0mM) on the enzyme activity, adding metal ions into RCDA, and reacting at 37 deg.C and pH7.0 for 60min to detect the enzyme activity;

the detection of the enzymological characteristics is carried out by taking 4-nitroacetanilide as a reaction substrate, adding 0.3mL of 200 mg/L4-nitroacetanilide aqueous solution into 0.3mL of RCDA crude enzyme solution, adding 0.9mL of buffer solution to adjust the pH, and adjusting the temperature of a water bath kettle to carry out reaction.

Relative enzyme activities of different substrates relative activity and residual activity were calculated using the enzyme activity (157.6U/mL) of 4-nitroacetanilide as a substrate as a standard (100%).

The definition of the enzyme activity unit adopted by the invention is as follows: the amount of enzyme required to produce 1. mu.g of p-nitroaniline per hour under certain reaction conditions (pH 7.0, 37 ℃ C. if not otherwise specified) is defined as one enzyme activity unit U.

The enzyme activity calculation formula is as follows:

A₄₀₀: the light absorption value of the enzymolysis liquid sample;

A₀: a blank absorbance value;

d: dilution times;

t: enzymatic reaction time, h;

k: a linear coefficient.

As a result, as shown in FIG. 2, the optimum reaction temperature and pH of RCDA were 45 deg.C (FIG. 2-a) and 7.0 (FIG. 2-b), respectively. The thermostability test showed that the enzyme activity of RCDA remained almost 100% after 30min of treatment below 40 ℃ and was completely inactivated at 55 ℃ and above (FIG. 2-c). The enzyme has stable enzyme activity between pH 5.0-9.0 (figure 2-d). When the ion concentration is 1mM or less, the metal ion has little influence on the enzyme activity, and when the ion concentration is 1mM or more, Zn²⁺、Cu²⁺、Ni²⁺Has stronger inhibitory effect on CDA activity (figure 2-e).

Example 3 application of RCDA

Pretreating chitin powder with 48% choline water solution at 90 deg.C for 12 hr, wherein the choline addition is 10mL per 5g material, filtering after pretreatment, washing the residual ionic liquid in solid phase with deionized water to pH6.8-7.0, and drying at 105 deg.C to constant weight. Weighing 1g of the dried material, adding 10mL of crude enzyme solution (the enzyme activity is 157.6U/mL) of the chitin deacetylase of the invention, reacting at 37 ℃ for 12h, and detecting by liquid chromatography that the total amount of acetic acid generated in the solution after the deacetylation reaction reaches 127mg/g of chitin powder.

In this example, choline acts to destroy the crystal structure of chitin and increase the active area of enzyme, and after pretreatment and drying to constant weight, pretreated chitin powder is added with RCDA to perform deacetylation reaction to generate acetic acid, so the yield of acetic acid is used to characterize the deacetylation capacity of RCDA on chitin powder.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not intended to limit the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Tianjin science and technology university

<120> a chitin deacetylase

<130>1

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>293

<212>PRT

<213>Rhodococcus equi

<400>1

Met Ser Lys Lys Ile Tyr Val Asn Phe Gly Val Asp Val Asp Ala Val

1 5 10 15

Ala Gly Trp Leu Gly Ser Tyr Gly Gly Glu Asp Ser Pro Gly Asp Ile

20 25 30

Ser Arg Gly Met Phe Ala Gly Glu Val Gly Val Pro Arg Leu Val Lys

35 40 45

Met Phe Asp Lys Tyr Asp Ile Thr Thr Ser Trp Phe Val Pro Gly His

50 55 60

Ser Ile Glu Thr Phe Pro Arg Glu Ile Glu Gln Val Val Ala Ala Gly

65 70 75 80

His Glu Ile Gly Ile His Gly Tyr Ser His Glu Asn Pro Ile Ala Met

85 90 95

Thr Arg Gln Gln Glu Thr Asp Ile Leu Asp Arg Ser Ile Glu Leu Ile

100 105 110

Glu Ser Phe Thr Gly Ser Lys Pro Thr Gly Tyr Thr Ala Pro Trp Trp

115 120 125

Glu Phe Ser Lys Val Thr Asn Glu Leu Leu Ile Glu Arg Gly Val Lys

130 135 140

Tyr Asp His Ser Leu Met His Asn Asp Phe Thr Pro Tyr Tyr Val Arg

145 150 155 160

Val Gly Asp Ser Trp Ser Lys Ile Asp Tyr Ser Gln Pro Ala Glu Thr

165 170 175

Trp Met Lys Pro Leu Val Arg Gly Gln Glu Thr Asp Leu Val Glu Ile

180 185 190

Pro Ala Asn Trp Leu Leu Asp Asp Leu Pro Pro Gln Met Phe Ile Lys

195 200 205

Ser Ser Pro Asn Ser His Gly Phe Val Ser Pro Arg His Leu Glu Glu

210 215 220

Met Trp Arg Asp Gln Phe Asp Trp Val Tyr Arg Glu Met Asp Tyr Ala

225 230 235 240

Ile Phe Pro Val Thr Ile His Pro Asp Val Ser Gly Arg Pro Gln Ser

245 250 255

Leu Leu Met Leu Glu Arg Leu Ile Glu Tyr Ile Asn Gln His Asp Gly

260 265 270

Val Glu Trp Val Thr Phe Asp His Met Val Asn Asp Phe Lys Glu Arg

275 280 285

Phe Pro Arg Gln Ser

290

Claims

1. A chitin deacetylase is characterized in that the amino acid sequence of the chitin deacetylase is shown in a sequence table SEQ ID NO. 1.

2. The gene encoding chitin deacetylase of claim 1.

3. Use of the chitin deacetylase of claim 1 or the gene encoding for use of claim 2 for hydrolyzing chitin.