CN114107364B

CN114107364B - Construction method of laccase-producing recombinant pichia pastoris engineering strain

Info

Publication number: CN114107364B
Application number: CN202111579365.0A
Authority: CN
Inventors: 高庆华; 罗同阳; 王玥; 董聪; 王庆庆; 王云鹏; 马清河; 马金国
Original assignee: Hebei Institute Of Microbiology Co ltd
Current assignee: Hebei Institute Of Microbiology Co ltd
Priority date: 2021-12-22
Filing date: 2021-12-22
Publication date: 2023-07-28
Anticipated expiration: 2041-12-22
Also published as: CN114107364A

Abstract

The invention discloses a construction method of a recombinant pichia pastoris engineering strain for producing laccase, which relates to the technical field of bioengineering and comprises the following steps: s1: connecting Laccase genes to a plasmid vector to obtain a recombinant plasmid pPIC9K-Laccase; s2: transferring the recombinant plasmid pPIC9K-Laccase into pichia pastoris X33 competent cells, and culturing to obtain positive transformant X33/pPIC9K-Laccase competent cells; s3: fusing positive transformant X33/pPIC 9K-lacase competent cells with molecular chaperone Hac1, culturing, and screening the activity of Laccase of the obtained strain to obtain Laccase-producing recombinant pichia pastoris engineering strain. The engineering strain laccase constructed by the method has high activity and good stability.

Description

Construction method of laccase-producing recombinant pichia pastoris engineering strain

Technical Field

The invention relates to the technical field of bioengineering, in particular to a construction method of a laccase-producing recombinant pichia pastoris engineering strain.

Background

Laccase (lacase) is a copper-containing polyphenol oxidase that is ubiquitous in fungi and catalyzes the redox reaction of phenols and plays an important role in the biodegradation of lignin. The oxidation substrates of laccase are very wide-ranging and include phenols and derivatives thereof, aromatic amines and derivatives thereof, aromatic carboxylic acids and derivatives thereof, and the like.

Laccase has good activity and huge application potential, and the application of laccase relates to a plurality of disciplines such as biology, chemistry, physics, medicine, environment and the like.

In the field of wood processing, laccase is adopted to treat wood, so that the surface of the wood is activated under the action of enzyme through the components of the laccase to generate a gluing effect, thereby replacing chemical adhesive or other chemicals in the wood processing industry, namely replacing a chemical method with serious pollution by using a pollution-free low-energy-consumption enzymology method, improving the quality of products, reducing the damage to human health and the pollution to the environment, and the products have wide market prospect in the day of increasingly paying attention to family environmental protection.

In the paper industry, laccase has been used with great breakthroughs and has been advanced into various links of the paper industry. The lignin degrading enzyme is used for biological bleaching of paper, can reduce pollution of bleaching wastewater of pulp and paper mill, is favorable for the paper industry to finally realize clean production, and is considered as lignin degrading enzyme with the most application prospect, and the laccase is the hottest in research on pulp bleaching at present. The laccase has the advantages of short reaction time, mild reaction conditions and the like, is used for assisting pulping, and can reduce the hardness of paper pulp under the condition that the dosage of chemicals is unchanged by carrying out lignin enzyme pretreatment before chemical pulping; or the consumption of chemicals and energy is reduced under the same hardness. The laccase is used for treating mechanical pulp laccase, can activate lignin in paper pulp, and is helpful for improving the bonding strength of fibers and the bonding performance of medium-quantitative paperboard fibers. The mechanical pulp paper sheet processed by laccase can keep excellent tensile strength after super-calendering, and better smoothness is obtained. The laccase can reduce the chlorine consumption for subsequent chemical bleaching by 30% -40% and the content of organic chloride and toxic substances in waste liquid is obviously reduced. The paper pulp is squeezed at 180-200 ℃ after laccase treatment, so that the fibers in the paper board or the fiber board can be bonded. The laccase treatment of organic chloride in papermaking waste liquid has the advantages of high catalytic efficiency, mild reaction condition, no harsh requirements on reaction condition and equipment, and the like.

In the field of food processing, laccase enzymes can be used to remove haze caused by phenolic compounds in juice, thereby improving the quality of the juice.

In the textile industry, laccase can be used for decolorization treatment during indigo dyeing of denim, for indigo bleaching, and has unique treatment effect (different from chlorine bleaching effect). Can also be used for removing lignin on the cloth blank.

In the fields of environmental protection and bioremediation, chlorophenols organic compounds are important industrial raw materials, and are used for producing chemical products such as dyes, preservatives, herbicides, pesticides and the like, and the environment can be polluted after the chlorophenols organic compounds are used; many industrial wastewaters contain chlorinated aromatic compounds, which have a certain toxicity, whereas laccase can oxidize chlorophenols and their derivatives, reducing their toxicity and reducing environmental pollution.

The laccase has wide application prospect and large market demand, however, the variety of laccase-producing strains is limited, so that obtaining more laccase-producing strains is a problem to be solved by those skilled in the art.

Disclosure of Invention

In view of the above, the invention provides a construction method of recombinant pichia pastoris engineering strain for producing laccase, and by using the construction method, the engineering strain for producing laccase can be obtained, and more laccase products can be released for the market.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

a construction method of laccase-producing recombinant pichia pastoris engineering strain comprises the following steps:

s1: connecting Laccase genes to a plasmid vector to obtain a recombinant plasmid pPIC9K-Laccase;

s2: transferring the recombinant plasmid pPIC9K-Laccase into pichia pastoris X33 competent cells, and culturing to obtain positive transformant X33/pPIC9K-Laccase competent cells;

s3: fusing positive transformant X33/pPIC 9K-lacase competent cells with molecular chaperone Hac1, culturing, and screening the activity of Laccase of the obtained strain to obtain Laccase-producing recombinant pichia pastoris engineering strain;

wherein S1 edits the laccase gene to mutate the 140 th G of the corresponding amino acid sequence into D, 147 th A into T, 154 th G into D, 231 th D into E and 239 th A into P.

As a preferable technical scheme of the invention, S1 specifically comprises the following steps: removing the signal peptide coding sequence of the Laccase gene, adding EcoRI restriction endonuclease recognition site at the 5 'end, adding NotI restriction endonuclease recognition site at the 3' end, connecting to pUC 57T to obtain pUC57-laccaseT cloning plasmid, and transferring to pPIC9K to obtain recombinant plasmid pPIC9K-Laccase.

As a preferable technical scheme of the invention, S2 specifically comprises the following steps: the recombinant plasmid pPIC9K-Laccase is subjected to linearization enzyme digestion by SacI, 8 mu L of enzyme digestion product is added into pichia pastoris X33 competent cells, electric conversion is carried out, 2Kv and 4ms are carried out, 1mLYPDS liquid culture medium is rapidly added, the culture is carried out for 2 to 6 hours in a 30 ℃ incubator for static culture, centrifugation is carried out at 4000rpm for 5 minutes, supernatant is discarded, saturated physiological saline is used for washing, 500 mu L of the supernatant is coated on YPD plates with the final concentration of 250 mu g/mLG418, and positive transformant X33/pPIC9K-Laccase competent cells are obtained after 3 days of culture.

As a preferable technical scheme of the invention, the S3 specifically comprises the following steps: carrying out linearization enzyme digestion on the pPICZB-Hac1 plasmid by using SfoI, fusing the plasmid with positive transformant X33/pPIC 9K-Lacccase competent cells obtained in S2, electric shocking for 2kv and 4ms, adding precooled YPDS, standing and culturing for 2-6h at 30 ℃, coating the mixture on YPD double-resistant plates with final concentration of 100 mug/mL G418 and 50 mug/mL Zeocin, culturing for 3 days at 30 ℃, obtaining strain containing transformant X33/pPIC 9K-Lacccase & Hac1, screening the activity of Laccase of the obtained strain, and obtaining the Laccase-producing recombinant pichia pastoris engineering strain.

The invention also provides a laccase-producing recombinant pichia pastoris engineering strain obtained by the construction method.

Preferably, the laccase-producing recombinant Pichia pastoris engineering strain is H-1, H-5, H-13 or H-17.

Compared with the prior art, the invention discloses a construction method of a recombinant pichia pastoris engineering strain for producing laccase and the constructed strain. The engineering strain laccase constructed by the method has high activity and good stability.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.

FIG. 1 is a diagram showing electrophoresis after enzyme digestion of the enzyme of the lacase gene provided by the embodiment of the invention;

FIG. 2 is a diagram showing electrophoresis after incision of the pPIC9K vector endonuclease provided by the embodiment of the invention;

FIG. 3 is a diagram showing complete electrophoresis of the plasmid pPIC9K-Laccase of the present invention;

FIG. 4 is a graph showing laccase activity results of H-1, H-5, H-9, H-13, H-16, H-17 and H-18 strains obtained in the examples of the invention;

FIG. 5 is a diagram showing SDS-PAGE results of H-1, H-5, H-13, H-17 fermentation supernatants obtained in examples of the present invention.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

1. Ligation of laccase Gene onto cloning vector

Based on the mRNA gene of laccase of small Kong Yingkong bacteria (Rigidoporus microporus) with AY365228.1 in American national center for biotechnology information (National Center for Biotechnology Information, NCBI), the signal peptide coding sequence of laccase gene is predicted to be removed according to SignalP-5.0, so that the laccase gene is heterologously expressed in pichia pastoris, and the gene is edited to ensure that the 140 th G mutation, the 147 th A mutation, the 154 th G mutation, the 231 th D mutation and the 239 th A mutation of the corresponding amino acid sequence are D, T, D, E and P, and the edited amino acid sequence is shown in SEQ ID NO:1, the nucleotide sequence is shown as SEQ ID NO: 2. Then EcoRI restriction enzyme recognition site is added at the 5 'end of the sequence, and NotI restriction enzyme recognition site is added at the 3' end. And then sending the optimized gene sequence to Anhui general biological company for synthesis. The synthesized lacase gene is connected to pUC 57T cloning vector to obtain pUC 57-lacaseT cloning vector.

2. Construction of expression vectors

Respectively carrying out EcoRI and NotI double digestion on an artificially synthesized pUC 57-LacbaseT cloning vector and a pPIC9K vector stored in a laboratory, separating by 1% agarose gel electrophoresis, and recovering gel by gel digestion (the Lacbase result is shown in figure 1, the pPIC9K result is shown in figure 2), thereby obtaining a target gene Lacbase with the same sticky end and the vector pPIC9K vector; and (3) connecting and recovering the product by using T4 ligase, transforming escherichia coli DH5 alpha, sequencing the identified positive clone extraction plasmid, and successfully constructing the comparative analysis recombinant plasmid pPIC 9K-lacase.

3. Construction of recombinant Pichia pastoris Strain producing pPIC9K-Laccase

The recombinant plasmid pPIC9K-Laccase plasmid is subjected to linearization digestion with SacI, and the digested product is recovered and purified. SacI linearization cleavage System (10)μL): recombinant plasmid pPIC 9K-lacase 5. Mu.L, sac10.5. Mu.L, 10 XBuffer 1. Mu.L and ddH ₂ O 3.5μL。

The digestion system reacts for 3 hours in a water bath kettle at 37 ℃, and whether the digestion is complete is detected by using 1% agarose gel electrophoresis, and the result is shown in a figure 3, wherein obvious strips are arranged at a position of more than 10000bp, and no obvious tailing phenomenon exists on the strips, so that the recombinant plasmid pPIC9K-Laccase is completely digested.

Taking 8 mu L of the purified enzyme digestion product, adding the purified enzyme digestion product into pichia pastoris X33 competent cells, transferring the cells into an electrorotating cup, and carrying out electrorotating under the condition: 2Kv,4ms, rapidly adding 1mLYPDS liquid culture medium, standing and culturing the electrotransferred competent cells in a 30 ℃ incubator for 2-6h, centrifuging at 4000rpm for 5min, discarding the supernatant, washing 3 times with 1mL of saturated physiological saline for filtration and sterilization, and then coating 500 mu L of the supernatant on a YPD plate with a final concentration of 250 mu g/mLG418, wherein the strain containing positive transformant X33/pPIC9K-Laccase is obtained after 3 days.

20 single colonies were picked up, inoculated into 5mL tubes containing YPCS liquid medium of 100. Mu.g/mLG 418, shake-cultured at 30℃and a rotational speed of 200r/min for 3 days, induced by adding methanol at 1% by volume ratio every 24 hours during the culture, harvested for 72 hours, and the supernatant was collected by centrifugation at 12000r/min for 5 minutes. The activity of laccase is determined by ABTS method, and whether recombinants are positive is determined by the method, and the strain with the highest enzyme activity is stored and marked as L-0.

Laccase enzyme activity determination method-ABTS method:

ABTS is the laccase substrate. ABTS forms ABTH free radical under laccase first, has maximum absorbance coefficient under 420nm wavelength, and with increasing concentration of ABTH free radical, absorbance value increases, and enzyme activity is calculated according to time-varying relationship of absorbance value (OD). Defined as the amount of enzyme required to oxidize 1. Mu. Mol of ABTS per minute at a temperature of 30℃and a pH of 4.0, is 1 enzyme activity unit.

4. Construction of engineering strains fused with molecular chaperones Hac1

The pPICZB-Hac1 plasmid was linearized with SfoI restriction enzyme and fused with L-0 competent cells by electric shock with a shock program of 2kv,4ms. Then adding precooled YPDS, standing and culturing for 2-6h at 30 ℃, coating on YPD double-antibody plates with final concentration of 100 mug/mL G418 and 50 mug/mL Zeocin, and culturing at 30 ℃ for 3 days to obtain transformant X33/pPIC 9K-lacase & Hac1.

The single colony 20 groups are selected, inoculated into 5mL of YPCS liquid culture medium containing 100 mug/mLG 418 respectively, cultured for 3 days under shaking at 30 ℃ and a rotating speed of 200r/min, methanol is added for induction at intervals of 24h according to a volume ratio of 1%, bacteria are collected for 72h, and supernatant is collected after centrifugation for 5min at 12000 r/min. The activity of laccase is determined by ABTS method, and H-1, H-5, H-9, H-13, H-16, H-17 and H-18 are obtained by screening, and the enzyme activity results are shown in figure 4.

5. The stability of the laccase produced by H-1, H-5, H-9, H-13, H-16, H-17 and H-18 is verified

Respectively inoculating H-1, H-5, H-9, H-13, H-16, H-17 and H-18 strains into 5mL test tubes containing YPCS liquid culture medium of 100 mug/mLG 418, carrying out shake culture for 3 days at the temperature of 30 ℃ and the rotating speed of 200r/min, adding methanol according to the volume ratio of 1% every 24H for induction during the culture period, collecting bacteria after 72H, centrifuging for 5min at 12000r/min, collecting supernatant, measuring the activity of laccase by adopting an ABTS method, and finally obtaining the recombinant Pichia pastoris engineering strain for producing laccase by four strains of stable inheritance, namely H-1, H-5, H-13 and H-17. The results of three parallel experiments on the enzyme activities of each group of strains are shown in Table 1, and the results of SDS-PAGE electrophoresis analysis of the corresponding fermentation supernatants are shown in FIG. 5.

TABLE 1

As shown in Table 1, the H-1, H-5, H-13 and H-17 strains all show high enzyme activity in three parallel experiments, and have high stability. Proved by the invention, the recombinant strain X33/pPIC9K-Laccase & Hac1 constructed by the invention has high enzyme activity and high stability.

Comparative example 1

The laccase gene of NCBI accession number AY365228.1 was subjected to the same experimental procedures as in example 1, and the enzyme activities of X-0 and L-0 obtained in example 1 were compared, and the results are shown in Table 2, without editing, to construct a cloning vector and an expression vector and to construct a recombinant Pichia pastoris strain X-0.

TABLE 2

	X-0	L-0	Rate of increase in enzyme activity
				Enzyme activity 1 (U/mL)	0.35	0.95	1.71 times
Enzyme activity 2 (U/mL)	0.45	0.96	1.13 times
				Enzyme activity 3 (U/mL)	0.49	0.97	0.98 times

Comparative example 2

The constructed recombinant Pichia pastoris strain L-0 and molecular chaperone PDI are fused, the influence of different molecular chaperones on laccase activity expression of positive strains is examined, other test operations are the same as in example 1, and the results are shown in Table 3.

TABLE 3 Table 3

According to the embodiment, the laccase activity of the positive strain constructed by the construction method of the laccase-producing recombinant pichia pastoris engineering strain is high, the stability is good, and a large amount of laccase-producing engineering strains can be provided for the market.

In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

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Claims

1. The construction method of the laccase-producing recombinant pichia pastoris engineering strain is characterized by comprising the following steps:

s1 edits the laccase gene to enable the 140 th G mutation to D, 147 th A mutation to T, 154 th G mutation to D, 231 th D mutation to E and 239 th A mutation to P of the corresponding amino acid sequence; the amino acid sequence of the laccase after mutation is shown as SEQ ID NO: 1.

2. The construction method according to claim 1, wherein S1 is specifically: removing the signal peptide coding sequence of the Laccase gene, adding EcoRI restriction endonuclease recognition site at the 5 'end, adding NotI restriction endonuclease recognition site at the 3' end, connecting to pUC 57T to obtain pUC57-laccaseT cloning plasmid, and transferring to pPIC9K to obtain recombinant plasmid pPIC9K-Laccase.

3. The construction method according to claim 1, wherein S2 is specifically: the recombinant plasmid pPIC9K-Laccase is subjected to linearization enzyme digestion by SacI, 8 mu L of enzyme digestion product is added into pichia pastoris X33 competent cells, electrotransformation is carried out, 2Kv and 4ms are carried out, 1mL of YPDS liquid culture medium is added, 2-6h is subjected to stationary culture in a 30 ℃ incubator, centrifugation is carried out at 4000rpm for 5min, supernatant is discarded, washing is carried out by saturated normal saline, 500 mu L of the supernatant is coated on YPD plates with the final concentration of 250 mu g/mLG418, and positive transformant X33/pPIC9K-Laccase competent cells are obtained after 3 days of culture.

4. The construction method according to claim 1, wherein S3 is specifically: carrying out linearization enzyme digestion on the pPICZB-Hac1 plasmid by using SfoI, fusing the plasmid with positive transformant X33/pPIC 9K-Lacccase competent cells obtained in S2, electric shocking for 2kv and 4ms, adding precooled YPDS, carrying out stationary culture for 2-6h at 30 ℃, coating the mixture on YPD double-resistant plates with final concentration of 100 mug/mL G418 and 50 mug/mL Zeocin, culturing at 30 ℃ for 3 days to obtain strain containing transformant X33/pPIC 9K-Lacccase & Hac1, and screening the activity of Laccase of the obtained strain to obtain the Laccase recombinant pichia pastoris engineering strain.

5. The recombinant Pichia pastoris engineering strain producing laccase obtained by the construction method according to any one of claims 1 to 4.