CN114107364A

CN114107364A - Construction method of laccase-producing recombinant pichia pastoris engineering strain

Info

Publication number: CN114107364A
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: 2022-03-01
Anticipated expiration: 2041-12-22
Also published as: CN114107364B

Abstract

The invention discloses a construction method of a laccase-producing recombinant pichia pastoris engineering strain, which relates to the technical field of biological engineering and comprises the following steps: s1: connecting Laccase gene to a plasmid vector to obtain a recombinant plasmid pPIC 9K-Lactase; s2: transferring the recombinant plasmid pPIC 9K-Lactase into Pichia pastoris X33 competent cells, and culturing to obtain positive transformant X33/pPIC 9K-Lactase competent cells; s3: fusing the positive transformant X33/pPIC 9K-Lacccase competent cells with a molecular chaperone Hac1, culturing, and screening the activity of the Laccase of the obtained strain to obtain the 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 (Lactase) is a copper-containing polyphenol oxidase, is ubiquitous in fungi, can catalyze the redox reaction of phenols, and plays an important role in the biodegradation of lignin. The oxidation substrates of laccases are very wide, and include phenols and derivatives thereof, arylamines and derivatives thereof, aromatic carboxylic acids and derivatives thereof, and the like.

Laccase not only has good activity, but also has great application potential, and the application of the laccase relates to a plurality of subject fields 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 self components to generate a gluing effect, and a chemical adhesive or other chemicals are replaced in the wood processing industry, namely, a seriously-polluted chemical method is replaced by an enzymological method without pollution and with low energy consumption, so that the product quality is improved, the damage to human health and the pollution to the environment can be reduced, and the product has wide market prospect nowadays increasingly paying attention to family environmental protection.

In the paper industry, the application of laccase has a great breakthrough and has gone deep into various links of the paper industry. The laccase is used for the biological bleaching of paper, can reduce the pollution of bleaching wastewater of pulping and papermaking plants, is beneficial to the final clean production of papermaking industry, is most popular and remarkably developed in the research of the laccase for the bleaching of paper pulp, and is considered as the lignin degrading enzyme with the most application prospect. The laccase is used for auxiliary pulping, has the advantages of short reaction time, mild reaction conditions and the like, is used for lignin enzyme pretreatment before chemical pulping, and can reduce the hardness of paper pulp under the condition of unchanged chemical dosage; or the consumption of chemicals and energy can be reduced under the same hardness. Laccase enzymes used in the treatment of mechanical pulp activate the lignin in the pulp, contributing to improved fiber bond strength and moderate basis weight paperboard fiber bond performance. The laccase treated mechanical pulp papermaking sheet can maintain excellent tensile strength and obtain better smoothness after supercalendering. The laccase enzyme is used for introducing a biological pre-bleaching technology into the pulping and papermaking industry to pre-bleach paper pulp, the chlorine content for subsequent chemical bleaching can be reduced by 30-40%, and the content of organic chloride and toxic substances in waste liquid is obviously reduced. And (3) squeezing the paper pulp at 180-200 ℃ after laccase treatment, so that the fibers in the paper board or the fiberboard can be bonded. The laccase has the advantages of high catalytic efficiency, mild reaction conditions, low requirements on reaction conditions and equipment and the like when used for treating the organic chloride in the papermaking waste liquid.

In the field of food processing, laccases can be used to remove turbidity caused by phenolic compounds in fruit juices, thereby improving the quality of the juice.

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

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

Laccase has wide application prospect and large market demand, but the types of strains producing laccase are limited, so that the acquisition of more strains producing laccase is a problem to be solved urgently by the technical personnel in the field.

Disclosure of Invention

In view of the above, the invention provides a construction method of a laccase-producing recombinant pichia pastoris engineering strain, and the construction method can be used for obtaining the laccase-producing engineering strain and releasing more laccase products for the market.

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

a construction method of laccase-producing recombinant Pichia pastoris engineering strains comprises the following steps:

s1: connecting Laccase gene to a plasmid vector to obtain a recombinant plasmid pPIC 9K-Lactase;

s2: transferring the recombinant plasmid pPIC 9K-Lactase into Pichia pastoris X33 competent cells, and culturing to obtain positive transformant X33/pPIC 9K-Lactase competent cells;

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

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

As a preferred technical solution of the present invention, S1 specifically is: and removing a signal peptide coding sequence of the Laccase gene, adding an EcoRI restriction endonuclease recognition site at the 5 'end, adding a NotI restriction endonuclease recognition site at the 3' end, connecting to pUC 57T to obtain a pUC57-LaccaseT cloning plasmid, and then transferring to pPIC9K to obtain a recombinant plasmid pPIC 9K-Laccasase.

As a preferred technical solution of the present invention, S2 specifically is: the recombinant plasmid pPIC 9K-Lactase is subjected to linearized enzyme digestion by SacI, 8 mu L of enzyme digestion product is taken and added into Pichia pastoris X33 competent cells, the electricity is converted, 2Kv and 4ms are quickly added into 1mLYPDS liquid culture medium, the mixture is kept still and cultured for 2 to 6 hours in a 30 ℃ incubator, then the mixture is centrifuged at 4000rpm for 5min, the supernatant is discarded, the mixture is washed by saturated normal saline, and then 500 mu L of the mixture is taken and coated on an YPD plate with the final concentration of 250 mu g/mLG418 to be cultured for 3 days, so that positive transformant X33/pPIC 9K-Lactase competent cells are obtained.

As a preferred technical solution of the present invention, S3 specifically is: the plasmid pPICZB-Hac1 is subjected to linear enzyme digestion by SfoI, fused with positive transformant X33/pPIC 9K-Lacccase competent cells obtained from S2, shocked by 2kv for 4ms, then added with precooled YPDS for static culture at 30 ℃ for 2-6h, coated on a YPD double-antibody plate with final concentration of 100 mug/mL G418 and 50 mug/mL Zeocin, placed at 30 ℃ for 3 days to obtain a strain containing transformant X33/pPIC 9K-Lacccase & Hac1, and the vitality of the obtained strain Laccase is screened to obtain the recombinant pichia pastoris engineering strain for producing Laccase.

The invention also aims to provide the laccase-producing recombinant pichia pastoris engineering strain obtained by the construction method.

Preferably, the laccase-producing recombinant Pichia pastoris engineering strains are H-1, H-5, H-13 and H-17.

According to the technical scheme, compared with the prior art, the invention discloses a construction method of laccase-producing recombinant pichia pastoris engineering strains and constructed strains thereof. 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 used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is an electrophoretogram of Lacccase gene endonuclease after digestion, provided by an embodiment of the present invention;

FIG. 2 is an electrophoretogram of pPIC9K vector endonuclease after digestion, provided by an embodiment of the present invention;

FIG. 3 is a diagram showing the complete electrophoresis of the recombinant plasmid pPIC 9K-Lactase restriction enzyme digestion verified by the embodiment 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 example of the present invention;

FIG. 5 is a SDS-PAGE result of H-1, H-5, H-13 and H-17 fermentation supernatants obtained in the example of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the 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.

Example 1

1. Linking laccase gene to cloning vector

Based on mRNA gene of laccase in Rhizopus microporus (Rigidoporus) with the accession number of AY365228.1 of the National Center for Biotechnology Information, a signal peptide coding sequence of the laccase gene is removed according to the prediction of SignalP-5.0, so that the laccase gene is heterologously expressed in Pichia pastoris, and the gene is edited, so that the corresponding amino acid sequence of the laccase gene has the mutation of 140 th G to D, the mutation of 147 th A to T, the mutation of 154 th G to D, the mutation of 231 th D to E and the mutation of 239 th A to P, and the edited amino acid sequence is shown as SEQ ID NO: 1, and the nucleotide sequence is shown as SEQ ID NO: 2, respectively. And EcoRI restriction endonuclease recognition sites are added to the 5 'end of the sequence, and NotI restriction endonuclease recognition sites are added to the 3' end of the sequence. The optimized gene sequence is then sent to Anhui general biology company for synthesis. The synthesized Lactase gene is connected to a pUC 57T cloning vector to obtain a pUC57-LaccaseT cloning vector.

2. Construction of expression vectors

Carrying out EcoRI and NotI double enzyme digestion on an artificially synthesized pUC57-LaccaseT cloning vector and a pPIC9K vector stored in a laboratory, carrying out electrophoresis separation on 1% agarose gel, cutting gel and recycling (the result of the Lactase is shown in figure 1, and the result of the pPIC9K is shown in figure 2), so as to obtain a target gene Lactase with the same cohesive end and a vector pPIC 9K; and (3) connecting and recovering the product by using T4 ligase, transforming escherichia coli DH5 alpha, sequencing the identified positive clone extraction plasmid, and comparing and analyzing the success construction of the recombinant plasmid pPIC 9K-Lacccase.

3. Construction of recombinant Pichia pastoris strain producing pPIC 9K-Lacccase

The recombinant plasmid pPIC 9K-Lacccase plasmid is subjected to linearized digestion by SacI, and a digestion product is recovered and purified. SacI linearized cleavage System (10. mu.L): recombinant plasmids pPIC 9K-Lacccase 5. mu.L, Sac10.5. mu.L, 10 XBuffer 1. mu.L and ddH₂O 3.5μL。

The enzyme digestion system reacts in a water bath kettle at 37 ℃ for 3 hours, 1% agarose gel electrophoresis is utilized to detect whether the enzyme digestion is complete, the result is shown in figure 3, and the obvious band is seen at the position of more than 10000bp and has no obvious trailing phenomenon, which indicates that the recombinant plasmid pPIC 9K-Lactase is completely digested.

Adding 8 mu L of the purified enzyme digestion product into pichia pastoris X33 competent cells, and transferring into an electric rotating cup under the electric rotating condition: 2Kv for 4ms, adding 1mLYPDS liquid culture medium rapidly, placing the transformed competent cells in an incubator at 30 ℃ for standing culture for 2-6h, centrifuging at 4000rpm for 5min, discarding the supernatant, washing with 1mL of filter-sterilized saturated physiological saline for 3 times, then spreading 500 μ L of the supernatant on YPD plates with the final concentration of 250 μ g/mLG418, and obtaining the strain containing positive transformant X33/pPIC 9K-Lactase after 3 days.

20 single colonies were picked, inoculated into 5mL YPCS liquid medium containing 100. mu.g/mLG 418, and cultured under shaking at 30 ℃ and 200r/min for 3 days while adding 1% methanol by volume at intervals of 24 hours, and the supernatant was collected after 72 hours of collection and centrifuged at 12000r/min for 5 minutes. And (3) determining the laccase activity by adopting an ABTS method, determining whether the recon is positive or not, and storing the strain with the highest enzyme activity as L-0.

Laccase enzyme activity determination method-ABTS method:

ABTS is the laccase substrate. ABTS forms ABTH free radical under the action of laccase firstly, has maximum absorption coefficient under the wavelength of 420nm, and the absorbance value is increased along with the increase of the concentration of the ABTH free radical, and the enzyme activity is calculated according to the relation that the absorbance value (OD) changes along with the time. Defined as the amount of enzyme required to oxidize 1. mu. mol of ABTS per min at 30 ℃ and pH4.0 as 1 enzyme activity unit.

4. Engineering strain for constructing fusion molecular chaperone Hac1

The pPICZB-Hac1 plasmid was linearized with SfoI restriction enzyme and fused with L-0 competent cells by electric shock at 2kv for 4 ms. Then adding pre-cooled YPDS for standing culture at 30 ℃ for 2-6h, coating the mixture on YPD double-resistant plates with final concentrations of 100 mu G/mL G418 and 50 mu G/mL Zeocin, and culturing the plates at 30 ℃ for 3 days to obtain a transformant X33/pPIC 9K-Lacccase & Hac 1.

20 groups of single colonies were picked, inoculated into 5mL of YPCS liquid medium containing 100. mu.g/mLG 418, and cultured under shaking at 30 ℃ and 200r/min for 3 days while adding 1% methanol by volume at intervals of 24 hours, followed by collection of the supernatant after 72 hours and centrifugation at 12000r/min for 5 minutes. The activity of laccase is determined by ABTS method, H-1, H-5, H-9, H-13, H-16, H-17, H-18 are obtained by screening, and the enzyme activity result is shown in figure 4.

5. The stability of laccase production 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 a 5mL test tube containing YPCS liquid culture medium of 100 mu g/mLG418, carrying out shaking culture for 3 days at the temperature of 30 ℃ and the rotating speed of 200r/min, adding methanol at intervals of 24H according to the volume ratio of 1% during the culture period for induction, collecting the strains for 72H, centrifuging at 12000r/min for 5min, collecting the supernatant, and measuring the activity of laccase by adopting an ABTS method to finally obtain the stably inherited H-1, H-5, H-13 and H-17 laccase-producing recombinant Pichia pastoris engineering strains. The enzyme activity of each group of strains was performed in triplicate, the results are shown in Table 1, and the SDS-PAGE electrophoretic analysis results of the corresponding fermentation supernatants are shown in FIG. 5.

TABLE 1

As can be seen from Table 1, the H-1, H-5, H-13 and H-17 strains all showed the characteristic of high enzyme activity in three parallel experiments, and had high stability. The recombinant strain X33/pPIC 9K-Lacccase & Hac1 constructed by the invention is proved to have high enzyme activity and high stability.

Comparative example 1

The laccase gene of NCBI accession number AY365228.1 was used to construct cloning vector and expression vector and recombinant Pichia pastoris strain X-0 without editing, the other experimental procedures were the same 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.

TABLE 2

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

Comparative example 2

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

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred 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. A construction method of laccase-producing recombinant Pichia pastoris engineering strains is characterized by comprising the following steps:

2. The construction method according to claim 1, wherein S1 specifically is: and removing a signal peptide coding sequence of the Laccase gene, adding an EcoRI restriction endonuclease recognition site at the 5 'end, adding a NotI restriction endonuclease recognition site at the 3' end, connecting to pUC 57T to obtain a pUC57-LaccaseT cloning plasmid, and then transferring to pPIC9K to obtain a recombinant plasmid pPIC 9K-Laccasase.

3. The construction method according to claim 1, wherein S2 specifically is: the recombinant plasmid pPIC 9K-Lacccase is subjected to linearized enzyme digestion by SacI, 8 mu L of enzyme digestion product is taken and added into Pichia pastoris X33 competent cells, the electricity is converted, 2Kv and 4ms are added into 1mL of YPDS liquid culture medium, the mixture is kept still and cultured for 2 to 6 hours in an incubator at the temperature of 30 ℃, then the mixture is centrifuged for 5 minutes at 4000rpm, the supernatant is discarded, the mixture is washed by saturated normal saline, and then 500 mu L of the mixture is taken and coated on an YPD plate with the final concentration of 250 mu g/mLG418 to be cultured for 3 days, so that transformant positive X33/pPIC 9K-Lacccase competent cells are obtained.

4. The construction method according to claim 1, wherein S3 specifically is: the plasmid pPICZB-Hac1 is subjected to linear enzyme digestion by SfoI, fused with positive transformant X33/pPIC 9K-Lacccase competent cells obtained from S2, shocked by 2kv for 4ms, then added with precooled YPDS for static culture at 30 ℃ for 2-6h, coated on a YPD double-antibody plate with final concentration of 100 mug/mL G418 and 50 mug/mL Zeocin, placed at 30 ℃ for 3 days to obtain a strain containing transformant X33/pPIC 9K-Lacccase & Hac1, and the vitality of the obtained strain Laccase is screened to obtain the recombinant pichia pastoris engineering strain for producing Laccase.

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