CN111718946A

CN111718946A - Codon-optimized lipase gene, engineering bacterium and textile application thereof

Info

Publication number: CN111718946A
Application number: CN201910161637.1A
Authority: CN
Inventors: 郑宏臣; 宋诙; 谭明; 马红丽; 付晓平; 徐健勇
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS
Priority date: 2019-03-04
Filing date: 2019-03-04
Publication date: 2020-09-29
Anticipated expiration: 2039-03-04
Also published as: CN111718946B

Abstract

The invention discloses a codon-optimized lipase gene, engineering bacteria and textile application thereof. The lipase gene (NCBI access No. XM _ 001261900.1) derived from Aspergillus fischeri NRRL 181 is optimized aiming at the codon of a Pichia pastoris host to obtain a gene sequence shown as SEQ ID NO.1, and the lipase enzyme activity produced by the Pichia pastoris engineering bacteria constructed by applying the sequence can reach 524.3U/ml which is 5.2 times of the original gene. And the lipase is used as a new component of the desizing and refining compound enzyme preparation, is applied to the grey cloth dyeing pretreatment, can obviously improve the refining effect, and respectively improves the capillary effect and the desizing rate of the treated cloth by 50 percent and 5 percent. Therefore, the lipase has good industrial production and textile application potential.

Description

Codon-optimized lipase gene, engineering bacterium and textile application thereof

Technical Field

The invention belongs to the fields of biotechnology and textile application, and relates to a codon-optimized lipase gene, engineering bacteria and textile application thereof.

Background

Lipases, also known as triphenolyl glycerol hydrolases (EC 3.1.1.3), catalyze the breakdown of triglycerides into diglycerides, monoglycerides, glycerol and fatty acids, and are a special class of ester bond hydrolases. The lipase has a plurality of sources, wherein the lipase from microorganisms can catalyze hydrolysis, alcoholysis, coolization, cool exchange, synthesis and the like of ester compounds, and has the advantages of no need of coenzyme for reaction, mild conditions, low energy consumption, low requirement on substrate raw material quality and few byproducts. The lipase of the microorganisms is various, about 65 genera of microorganisms produce lipase, wherein 28 genera are derived from bacteria, 4 genera are derived from actinomycetes, 10 genera are derived from yeasts, and other fungi are 23 genera. In fact, lipase-producing microorganisms in nature are far from these and will be discovered continuously. With the continuous expansion of the application range of lipase in recent years, new requirements on the characteristics of high temperature resistance, acid resistance, alkali resistance and the like of lipase are provided, and the lipase is an important direction for the research of lipase at home and abroad. However, the conditions for culturing the lipase-producing bacteria which are resistant to high temperature, acid and alkali are harsh, and the screening of the strains is difficult. With the rapid development and expansion of biotechnology and biological information, researchers can search for proper enzyme coding genes in the existing gene database, so that a high-efficiency expression system is constructed, enzyme resources with excellent performance are rapidly developed, large-scale production and application are promoted as soon as possible, and the development cycle of an enzyme preparation can be greatly shortened. Therefore, efficient protein expression technology becomes a key technical problem in the development of enzyme preparations. In recent years, the expression system aiming at the foreign protein has been well developed, and more enzyme preparations realize large-scale production through foreign expression and are widely applied to industry.

Lipase is widely applied to the industrial fields of food flavor improvement, grease hydrolysis and modification, leather degreasing, drug modification, biodiesel, medicine and the like at present, for example, in the medical industry, acid lipase can be used for preparing lipase medicines which are mainly used for treating diseases such as gastrointestinal disorder, dyspepsia and the like; in the food industry, the acid lipase can be used for improving the flavor of food, generating flavor components such as lower fatty acid and the like under proper conditions, and enhancing the flavor of the food; the enzyme has good application prospect in preparing biodiesel, is suitable for preparing biodiesel from free fatty acid and oil with high water content, and has high yield; in the feed industry, the addition of the acid lipase can enable animals to better digest and utilize fatty substances in the feed. However, in the textile industry, the application of lipase is only rarely reported, the natural waxiness and the colloid on the surface of the cotton fabric fiber can seriously affect the hydrophilicity of the fabric, so that the hair effect is low, the contact and the treatment of the enzyme are not facilitated, and the pretreatment effect is affected. At present, the types of lipases are various, and the specificity of catalytic substrates is various, so that the screening and the development of the lipase suitable for textile pretreatment are key to solve the problems.

Disclosure of Invention

The invention aims to provide a codon-optimized lipase gene, engineering bacteria and textile application thereof.

Aiming at the enzymatic textile dyeing pretreatment process in the earlier stage, in the process of developing a desizing and refining complex enzyme preparation, the accessibility of cotton fibers can be further improved on the basis of removing the colloid on the fiber surface by the existing pectinase, the contact efficiency of the enzyme preparation and a substrate is improved, the desizing and refining effects of the complex enzyme preparation are further enhanced, and the desizing rate and the capillary effect of fabrics are improved. Therefore, the method screens the lipase which possibly has the degradation effect on the wax on the surface of the cotton fabric, firstly screens various commercially available lipase preparations, screens 1-2 lipase preparations with certain refining effect according to the result, and then searches the included lipid in the NCBI database according to the enzymological property of the lipaseThe lipase coding gene is finally discovered to be derived from the expression and application screening of more than 200 target genesAspergillus fischeriThe heat-resistant acid lipase coded by the lipase gene (NCBI access number XM _ 001261900.1) of NRRL 181 has the best refining effect, and the sequence obtained by performing codon optimization on the sequence coding the mature peptide of the lipase according to the codon preference of pichia pastoris is shown as SEQ ID No. 1.

The gene has a full length of 1632bp, encodes 543 amino acids, and the amino acid sequence is shown in SEQ ID NO. 2.

The invention inserts the encoding gene of the heat-resistant acidic lipase shown in SEQ ID NO.1 into the EcoRI and NotI of a Pichia pastoris expression vector pPIC9k, and successfully integrates the encoding gene into the corresponding position of a Pichia pastoris GS115 genome to obtain a recombinant strain P. pastoris GS115(AOX-lip605) with single copy, and the recombinant strain P. pastoris GS115(AOX-lip605) is identified as a methanol utilization rapid type.

The invention further optimizes the fermentation enzyme production conditions of the engineering bacteria P. pastoris GS115(AOX-lip605), and the optimal enzyme production conditions are 2.18 percent of methanol addition, 128.34 hours of culture time and 4 mg/L of biotin. On the basis of a shake flask, an amplified fermentation enzyme production experiment of a 10L fermentation tank is carried out, when recombinant Pichia pastoris P.pastoris GS115(AOX-lip605) is fermented for 172 hours, the growth of thalli tends to be stable, the wet weight of cells reaches 395 g/L, and the enzyme production amount reaches 524.3.3U/ml. And the recombinant enzyme protein accounts for more than 90 percent of the pichia pastoris expression extracellular protein, has little foreign protein and has good industrial production potential.

In addition, the invention also carries out enzymology property determination on the purified thermostable acid lipase Lip605, the optimum action temperature is 60 ℃ and the optimum action pH is 5.0, the enzyme activity can be kept above 95% after 1h of heat preservation under the conditions of pH3.0-7.0 and temperature 50 ℃, and the method is suitable for the application conditions of enzyme method spinning.

The lipase is added into a desizing refining complex enzyme preparation prepared in the early stage according to a certain proportion, and the treatment is carried out according to the enzymatic pre-dyeing treatment process disclosed in the early stage, so that the final effect is improved by 50% and 5% respectively compared with a control group without the lipase. Therefore, the present invention provides a method for improving the effect of the textile enzymatic pretreatment before dyeing by using lipase Lip605 as an effective scouring enzyme.

Drawings

FIG. 1 is the SDS gel electrophoresis of recombinant Pichia pastoris extracellular lipase.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 codon optimization of thermostable acid lipase and expression vector construction

According to the codon preference of pichia pastoris, under the premise of not changing an amino acid sequence, codon optimization software is used for carrying out codon optimization on a mature peptide coding sequence of the gene by referring to an Aspergillus fischeri NRRL 181-derived lipase gene sequence (NCBIACCESS number XM-001261900.1) in GenBank, an EcoRI enzyme cutting site is designed at the 5 'end of the sequence, and a NotI enzyme cutting site is designed at the 3' end of the sequence. And (3) sending the optimized and constructed gene sequence to Suzhou Hongsn biotechnology limited company for synthesis, directly connecting the synthesized gene between corresponding enzyme cutting sites of a pPIC9k vector, electrically converting the gene into pichia pastoris GS115 competent cells, selecting positive cloning, and carrying out sequencing verification to obtain the engineering bacterium pichia pastoris GS115(AOX-lip 605). Meanwhile, synthesizing an original gene fragment which is not subjected to codon optimization to obtain a control engineering bacterium Pichia pastoris GS115(AOX-lip 24).

Example 2 expression and fermentation of thermostable acid lipase Lip605 in Pichia pastoris

Respectively inoculating the recombinant pichia pastoris to YPD solid culture media by a plate marking method, culturing at 30 ℃ until monoclonals appear, picking the monoclonals to BMGY culture media, and culturing at 30 ℃ at 250 r/min to a certain concentration. The supernatant was discarded by centrifugation, and the cells were transferred to an equal volume of BMMY medium and cultured at 28 ℃ at 250 r/min. Sampling 1 mL every 24 hours, adding 2.18% methanol, measuring the biotin content at 4 mg/L, centrifuging the sample, diluting the supernatant by a proper multiple to measure the enzyme activity, wherein when the culture time is 120 hours, the highest enzyme activity of lipase Lip24 coded by an original sequence is 51.2U/mL, the highest enzyme activity of Lip605 optimized by a codon can reach 207.3U/mL, and the enzyme activity is improved by 4 times. The extracellular protein electrophoresis of the two bacteria is shown in figure 1, and the expression level of Lip605 protein is obviously higher than that of Lip 24.

Fermenting in a 10L fermentation tank: the method comprises the steps of respectively picking and culturing the monoclonal colonies of the recombinant pichia pastoris in 100 ml of YPD liquid culture medium at 30 oC 250 r/min until the OD600nm =10 is about, adding seed liquid into a 10L fermentation tank containing 3L of initial culture medium, and performing fermentation in three different stages, namely glycerol single-batch culture, glycerol fed-batch culture and methanol fed-batch culture. Sampling and measuring OD600nm, cell wet weight and enzyme activity at intervals, wherein the thallus growth tends to be stable when fermenting for 172 h, the cell wet weight reaches 395 g/L, the enzyme activity of the recombinant lipase Lip605 reaches 524.3U/mL, and the enzyme activity of the lipase Lip24 without codon optimization is only 101.2U/mL, so that the expression level of the lipase coding gene in pichia pastoris can be improved by 5.2 times through the codon optimization.

Example 3 application of recombinant thermostable acid lipase Lip605 in textile enzyme pretreatment process

In this embodiment, the enzymatic pretreatment of textile by the following method specifically includes the following steps:

(1) padding biological compound enzyme liquid: padding all-cotton grey cloth (specification 20 multiplied by 16) in biological compound enzyme liquid at 50 ℃, and carrying out two-time padding and two-time padding, wherein the liquid carrying rate is 100%; the biological compound enzyme liquid comprises a desizing and refining compound enzyme preparation (the addition amount is 10ml/L) and heat-resistant acid lipase Lip605 (the addition amount is 100U/L), and the pH value is 6.0-7.0;

(2) steaming at 55 deg.C for 1 h;

(3) washing 2 times with hot water at the temperature of 90 ℃ and 1 time with cold water, and then carrying out oxygen bleaching by using oxygen bleaching liquid, wherein the oxygen bleaching liquid comprises the following components: 15g/L of oxygen bleaching auxiliary agent, 20g/L of hydrogen peroxide, 5g/L of chelating agent and 8g/L of penetrating agent, the liquid carrying rate is 100%, steaming for 30 min under saturated steam at 100 ℃, cleaning for 2 times by hot water at 70 ℃, and drying.

Comparative example 1

The difference from example 3 is that in this comparative example, a commercially available acid lipase having the same enzyme activity was used in place of the lipase Lip605, and the contents of the remaining components and the operation procedure were the same as in example 3.

Comparative example 2

The difference from example 3 is that lipase Lip605 was not used in this comparative example, and the contents of the remaining components and the operation procedure were the same as in example 3.

The desizing rate, capillary effect and whiteness test results of the treated cotton cloth are shown in table 1.

TABLE 1

As can be seen from Table 1, when the heat-resistant acid lipase Lip605 is applied to the enzymatic pre-dyeing treatment process of the pure cotton cloth, compared with comparative examples 1 and 2, the capillary effect and the desizing rate of the treated cotton cloth are obviously improved, and it can be seen that the capillary effect and the desizing rate of the treated cotton cloth can be respectively improved by 50% and 5% by adding 100U/L lipase Lip605 (the improvement effect on polyester cotton treatment is similar to that of the pure cotton), and in addition, compared with commercial lipase screened in earlier stage, the heat-resistant acid lipase Lip605 also shows obvious advantages, which indicates that the heat-resistant acid lipase Lip605 is very suitable for the application of enzymatic spinning, can be developed into a novel spinning refined enzyme preparation, and has good market application prospect.

The foregoing description is intended to be illustrative rather than limiting, and it will be apparent to those of ordinary skill in the art that numerous modifications, variations or equivalents may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

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aacctgggtt tgttggacca aagattggcc ttgcagtggg ttgccgataa cattgcagct 600

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Ala Pro Ala Lys Pro Ala Ala Pro Thr Val Thr Ile Ala Ser Pro Ala

1 5 10 15

Ala Thr Ile Val Gly Ser Ser Gly Lys Val Glu Lys Phe Asn Ala Ile

20 25 30

Pro Phe Ala Gln Pro Pro Thr Gly Pro Leu Arg Leu Lys Pro Pro Gln

35 40 45

Pro Ile Gln Lys Ser Leu Gly Thr Ile Asp Gly Thr Gly Ser Ala Lys

50 55 60

Ser Cys Pro Gln Phe Phe Phe Ser Thr Asp Asn Ser Glu Phe Pro Gly

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

85 90 95

Asn Ala Gly Glu Asp Cys Leu Thr Leu Asn Val Ala Arg Pro Ser Gly

100 105 110

Thr Thr Pro Gly Ser Lys Leu Pro Val Leu Val Trp Ile Tyr Gly Gly

115 120 125

Gly Phe Glu Leu Gly Ser Thr Ala Met Tyr Asp Ala Thr Ser Leu Val

130 135 140

Ala Ser Ser Ile Asp Leu Gly Met Pro Ile Val Phe Val Ala Met Asn

145 150 155 160

Tyr Arg Thr Gly Gly Phe Gly Phe Met Pro Gly Lys Glu Ile Leu Ala

165 170 175

Asp Gly Ala Ala Asn Leu Gly Leu Leu Asp Gln Arg Leu Ala Leu Gln

180 185 190

Trp Val Ala Asp Asn Ile Ala Ala Phe Gly Gly Asp Pro Asp Lys Val

195 200 205

Thr Ile Trp Gly Glu Ser Ala Gly Ser Ile Ser Val Phe Asp His Met

210 215 220

Ile Leu Tyr AspGly Asp Asn Thr Tyr Lys Glu Lys Pro Leu Phe Arg

225 230 235 240

Gly Gly Ile Met Asn Ser Gly Ser Val Ile Pro Ala Asp Pro Val Asp

245 250 255

Gly Val Lys Gly Gln Gln Val Tyr Asp Ala Val Val Asp Tyr Ala Gly

260 265 270

Cys Ser Ser Ala Ala Asp Thr Leu Glu Cys Leu Arg Arg Leu Asp Tyr

275 280 285

Thr Asp Phe Leu Asn Ala Ala Asn Ala Val Pro Gly Ile Leu Ser Tyr

290 295 300

His Ser Val Ala Leu Ser Tyr Leu Pro Arg Pro Asp Gly Lys Ala Ile

305 310 315 320

Thr Ala Ser Pro Asp Ile Leu Val Lys Thr Gly Lys Tyr Ala Ala Val

325 330 335

Pro Ile Ile Ile Gly Asp Gln Glu Asp Glu Gly Thr Leu Phe Ala Leu

340 345 350

Phe Gln Ser Asn Ile Thr Thr Thr Lys Gln Val Val Asp Tyr Leu Ala

355 360 365

Lys Tyr Tyr Phe Phe Gly Ala Thr Arg Asp Gln Leu Glu Glu Leu Val

370 375 380

Ala Thr Tyr Pro Asp ValThr Thr Asp Gly Ser Pro Phe Arg Thr Gly

385 390 395 400

Ile Phe Asn Asn Trp Tyr Pro Gln Phe Lys Arg Leu Ala Ala Leu Leu

405 410 415

Gly Asp Leu Thr Phe Thr Leu Thr Arg Arg Ala Tyr Leu Lys Tyr Val

420 425 430

Thr Glu Leu Lys Pro Asp Leu Pro Cys Trp Ser Tyr Leu Ser Ser Tyr

435 440 445

Asp Tyr Gly Thr Pro Ile Met Gly Thr Phe His Gly Ser Asp Ile Leu

450 455 460

Gln Val Phe Tyr Gly Ile Leu Pro Asn Tyr Ala Ser Arg Ala Phe His

465 470 475 480

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

485 490 495

Gly Ser Leu Met Glu Trp Pro Arg Trp Asn Glu Asp Gln Gln Leu Met

500 505 510

Gln Phe Phe Asn Asn Arg Gly Ala Leu Leu Ala Asp Asp Phe Arg Asn

515 520 525

Asp Thr Tyr Asn Phe Ile Leu Glu Asn Val Asp Ser Phe His Ile

530 535 540

Claims

1. A lipase gene is characterized in that the gene sequence is shown as SEQ ID NO. 1.

2. An engineered bacterium obtained by introducing the lipase gene according to claim 1 into a starting strain.

3. The engineered bacterium of claim 2, wherein the producer strain is pichia pastoris GS 115.

4. The lipase gene as set forth in claim 1, wherein the encoded amino acid sequence is shown in SEQ ID NO.2, and the enzyme with the amino acid sequence is applied to the process of enzyme method dyeing pretreatment.

5. Use of the engineered bacteria or enzymes of any of claims 2, 3 or 4 in a pretreatment process with enzymatic staining.