CN115838705B - A lipase mutant - Google Patents

Abstract

The invention relates to the technical fields of genetic engineering and protein engineering, in particular to a novel lipase mutant and application thereof. The amino acid sequence of the mutant is SEQ ID NO:4, comprising three mutation sites of T63A, N103,103, 103R, A176,176S. Compared with wild lipase, the mutant has the advantages that the expression enzyme activity of the mutant in Trichoderma reesei is improved by 40%, the shake flask fermentation enzyme activity is 683U/ml, unexpected technical effects are achieved, the production cost of the enzyme is reduced, and the mutant is widely applied to the industrial fields of foods, feeds, medicines and the like.

Description

Lipase mutant

Technical Field

The invention relates to the technical fields of genetic engineering and protein engineering, in particular to a lipase mutant and application thereof.

Background

Lipase (Lipase, EC 3.1.1.3) is a common biological macromolecular substance, widely existing in animal and plant kingdoms, and in fungi and bacteria, the Lipase has the function of hydrolyzing triglyceride to generate free fatty acid, diglyceride, monoglyceride and glycerol, and is a biological catalyst with high catalytic activity and very wide application in the field of biotechnology. Lipases are naturally water-soluble biological macromolecules, but their catalytic substrates are poorly water-soluble compounds, and it is reported that lipases are activated at the oil-water interface, thus hydrolyzing the ester bonds of water-insoluble glycerides, which can be attributed to the unique structural features of such enzymes.

Lipases are one of the most important industrial enzyme preparations for their numerous applications in ester hydrolysis, transesterification, alcoholysis, esterification and ammonolysis, etc., due to their various properties, they are commonly used in various fields such as detergents, foods, bioenergy, perfumes, pharmaceuticals, as well as fine chemicals and agrochemicals. Although not as widely used as amylase and protease, lipases have immeasurable development potential, and it is apparent from the current demands of various industries on various enzymes that lipases have become an integral part of modern industrial production. Lipase is widely used in selective biocatalysis due to its good site specificity and stereoselectivity.

Lipases are widely found in the animal and plant kingdoms, as well as in fungi and bacteria, and have a broad biological origin. It is known from examination of the literature that animal lipases which have been found so far are mainly produced in the pancreatic tissues of animals, and the activities of pancreatic lipase and gastric lipase have been found and reported successively in 1834 and 1854, respectively. After ten years of fumbling research by researchers, researchers have found and reported lipase activity in plant seeds in 1871, the seeds of oil crops are the main sites of lipase accumulation in plants, and the lipase content of oil crops has obvious advantages compared with other organs and tissues, such as rapeseeds. Compared with the research of animal and plant lipases, the activity of the microbial lipase is found relatively later, in 1967, researchers of the institute of microbiology of China academy of sciences screen a candida lipolytica strain with higher lipase yield through a large number of screening works, and the lipase produced by the candida lipolytica strain is made into a commercial enzyme preparation to be put in the market after two years of research works, so that the application of the lipase is more and more wide after years of research and development.

Lipases are known as natural biocatalysts, have a highly efficient catalytic action, and most lipases which are used industrially today are produced by microbial fermentation. The literature reports that about 2% (65 genera) of microorganisms in nature produce lipases, mainly bacteria, fungi and actinomycetes, with the most fungi (33 genera), the less bacteria (28 genera) and the least actinomycetes (4 genera). Numerous researchers have made extensive research efforts in the production and use of lipases from different sources, and have also made extensive research in terms of physicochemical properties and catalytic properties.

The lipase produced by microbial fermentation is a main way for obtaining lipase at present, and has the characteristics of rich sources, high enzyme activity yield, multiple catalytic reaction types, simple operation, short production period and the like, so that the lipase is wider in application range than the lipase produced by plants and animals. In addition, the production of the microbial lipase is not limited by season fluctuation, and the microbial lipase can regularly grow on a cheap culture medium, so that the production process is more stable and the production cost is lower compared with the production process of the animal and plant lipase. However, the naturally-produced lipase has low expression level, which is unfavorable for modern industrial production, and the biotechnology means can effectively improve the yield of lipase and the enzymatic properties, which is the focus of research in the field.

Disclosure of Invention

The invention aims to provide a novel lipase mutant. Compared with the wild type, the enzyme activity level of the mutant is obviously improved, and the production cost of lipase is reduced.

One aspect of the invention relates to a lipase having the amino acid sequence of SEQ ID NO:1.

One aspect of the invention relates to a lipase mutant, which has an amino acid sequence of SEQ ID NO:1 from Thr to Ala, from Asn to Arg, and from Ala to Ser, from amino acid 176.

The amino acid sequence of the lipase mutant is SEQ ID NO:4.

The invention also relates to a gene for encoding the lipase mutant.

The nucleotide sequence of the gene is SEQ ID NO:3.

The invention also relates to a recombinant plasmid carrying the lipase mutant coding gene.

The invention also relates to a host cell carrying the recombinant plasmid.

The host cell is Trichoderma reesei (Trichoderma reesei).

The lipase mutant provided by the invention comprises three mutation sites of T63A, N103R, A S, and the expressed enzyme activity of the lipase mutant in Trichoderma reesei is obviously improved. The recombinant expression of the lipase mutant Trichoderma reesei engineering bacteria constructed by the invention has shake flask fermentation enzyme activity reaching 683U/ml, and improves 40% of the recombinant expression of the wild lipase engineering bacteria, thus achieving unexpected technical effects, being beneficial to reducing the production cost of the enzyme and promoting the wide application of the enzyme in the industrial fields of food, feed, medicine and the like.

Detailed Description

The present invention uses conventional techniques and methods used in the fields of genetic engineering and molecular biology, such as those described in MOLECULAR CLONING: A LABORATORY MANUAL, 3nd Ed (Sambrook, 2001) and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003). These general references provide definitions and methods known to those skilled in the art. However, those skilled in the art may adopt other methods, experimental schemes and reagents which are conventional in the art on the basis of the technical scheme described in the present invention, and are not limited to the specific embodiments of the present invention.

Enzyme and kit: the DNA polymerase is purchased from NEB company, T4 ligase and restriction endonuclease are purchased from Fermentas company, the plasmid extraction kit and the gel purification recovery kit are purchased from Omega company, and the GeneMorph II random mutagenesis kit is purchased from Beijing Bomeis Biotechnology Co.

The method for detecting lipase enzyme activity in the embodiment of the invention comprises the following steps:

1. Definition of enzyme Activity

The amount of enzyme required to produce 1. Mu. Mol of titratable fatty acid per minute at pH 7.5 and 40℃is 1 enzyme activity unit (IU).

The method mainly uses olive oil emulsion as substrate solution and polyvinyl alcohol as emulsifier, and the measurement principle is that lipase catalyzes olive oil to generate fatty acid, sodium hydroxide titration is used for making phenolphthalein turn red, and the fatty acid content is reacted according to the consumption of sodium hydroxide.

2. Substrate configuration:

(1) 40g (accurate to 0.1 g) of polyvinyl alcohol (PVA) is weighed, 800ml of water is added, the mixture is heated in a boiling water bath and stirred until the mixture is completely dissolved, the volume is fixed to 1000ml after cooling, and the mixture is filtered by clean double-layer gauze, and filtrate is taken for standby.

(2) 150Ml of the filtrate is measured, 50ml of olive oil is added, the mixture is treated by a high-speed refiner for 6min (two treatments are carried out for 5min at intervals and 3min for each treatment), and the milky PVA emulsion is obtained, and the solution is prepared immediately.

3. The specific measurement method comprises the following steps:

Taking 2 conical flasks of 100mL, namely a blank control (A) and a sample (B) to be tested, respectively adding 4 mL substrate solution and 5 mL NaHPO4-KH ₂ PO4 buffer (pH=7.5) into the two conical flasks, adding a small amount of high-volume fraction ethanol into the control flask, preheating 5 min under the water bath condition of 40 ℃, adding 1 mL enzyme solution to be tested into the two conical flasks, immediately mixing, timing, immediately adding equal amount of ethanol into the sample flask after the reaction is 15 min to terminate the reaction, shaking, and taking out. A small amount of phenolphthalein solution was added dropwise to each of the 2 conical flasks, and the mixture was titrated with 0.05 mol/L NaOH standard solution, and the volume of NaOH standard solution consumed was recorded as the end point of titration after reddening was observed and 30 s was kept intact.

X =[(V1 - V2) × c × 50×n]/( 0.05×15)。

X-is the enzyme activity of the sample, IU;

v1- -is the consumption of the NaOH standard solution for titrating the sample, and mL;

v2- -is the consumption of the NaOH standard solution in titration blank, and mL;

c- -is the concentration of NaOH standard solution and mol/L;

1ml of 50-0.05 mol/L sodium hydroxide solution corresponds to 50 mu mol of fatty acid;

n-is the dilution of the sample;

0.05- -sodium hydroxide standard solution conversion coefficient;

15 is the reaction time 15 min, the time conversion coefficient.

The invention is further described in conjunction with the following detailed description.

EXAMPLE 1 Gene cloning

The applicant named ZFCB wild-type lipase gene from Leuconostoc mesenteroides (Moesziomyces rugulosus) and the amino acid sequence thereof was SEQ ID NO. 1. According to the codon preference of Trichoderma reesei, the Trichoderma reesei is subjected to codon optimization, and the Hua-delivery megagene is subjected to gene synthesis. The nucleotide sequence of the wild type lipase ZFCB gene is SEQ ID NO. 2.

Designing upstream and downstream primers and reaction conditions, and cloning a lipase ZFCB gene fragment by using a PCR technology. The primer sequences and reaction conditions are as follows:

Upstream primer 1 (F): GTACGGTACCACGCCCCTCGTCAAGCGCCTGCC (underlined is KpnI cleavage site);

downstream primer 1 (R): CTGATCTAGATTAGGGCGTGACGATGCCGCTGCACG (underlined is XbaI cleavage site).

The PCR conditions were: denaturation at 98℃for 1min; denaturation at 98℃for 10s, renaturation at 56℃for 15s, extension at 72℃for 1min,30 cycles, and incubation at 72℃for 5min. The full length of the wild lipase ZFCB gene is 972bp.

EXAMPLE 2 screening of Lipase mutant and construction of Trichoderma reesei engineering bacteria

The applicant has carried out a large number of mutated screens of the gene of the wild-type lipase ZFCB by directed evolution techniques in order to further increase the expressed enzyme activity of the enzyme.

Using the optimized lipase gene as a template, and using an upstream primer 1 (F) and a downstream primer 1 (R) of the primers to carry out PCR amplification by using a GeneMorph II random mutation PCR kit (Stratagene); and (5) recovering PCR products by using a gel. The PCR product is the lipase mutant gene fragment obtained by random mutation.

2.1 Construction of expression plasmids

The wild-type lipase ZFCB gene fragment and the PCR product obtained above were digested simultaneously with restriction enzymes Kpn I and XbaI, and the Trichoderma expression vector pKL was digested simultaneously with restriction enzymes Kpn I and XbaI. The double digested product, i.e.the cloned gene, was then ligated with the expression vector using T4 ligase overnight at 22 ℃. Finally, the ligation product was introduced into E.coli DH5a, spread on LB+Amp plates, and cultured upside down at 37 ℃. After the transformants appeared, a number of 96-well plates were picked one by one with toothpicks, and the plasmids were then extracted for Trichoderma reesei transformation.

2.2 Preparation of Trichoderma reesei protoplast

Inoculating Trichoderma reesei hyphae to a PDA plate for 7 days; colonies of about 3cm diameter were excised and placed in about 60ml of YEG (0.5% yeast powder, 1% glucose) liquid medium, and shake-cultured at 30℃overnight at 200 rpm; filtering with multi-layer gauze to collect mycelium; placing mycelium in 20 ml lyase (0.2 g/10ml, 0.7M NaCl solution, sigma L1412) for enzymolysis for 2h; taking out the enzymolysis liquid, slightly shaking, pouring the enzymolysis liquid into three layers of sterilizing mirror wiping paper for filtering, collecting filtrate, 3000 rpm, and centrifuging for 10 min; the supernatant was discarded, 5ml solution 2 was added to suspend, then 3000 rpm, and centrifuged 10 min; adding a proper amount of solution 2, suspending and split charging (200 mul/tube, 10 ⁸/ml).

2.3 Conversion

Adding 10 ul of each plasmid DNA into 200 mu l of protoplast, then adding 50 mu l of 25% PEG solution, gently mixing, and carrying out ice bath for 20min; then adding 2 ml of 25% PEG, gently mixing, standing at room temperature for 5min, adding protoplast into upper semi-solid culture medium (0.1% MgSO ₄,1%KH₂PO4,0.6%(NH₄)₂SO₄, 1% glucose, 18.3% sorbitol, 0.35% agarose) of which the temperature is 45-55 ℃ after melting about 50 ml, pouring into a lower basal culture medium plate (2% glucose ,0.5%(NH4)₂SO₄,1.5%KH₂PO₄,0.06%MgSO₄,0.06%CaCl₂,1.5% agar) containing 100 mug/ml hygromycin after gently mixing, and culturing in the dark at 30 ℃ for several days until transformants grow out.

The obtained positive transformant is the Trichoderma reesei engineering strain for recombinant expression of the wild lipase ZFCB and the mutant thereof.

2.4 Shake flask fermentation verification

The positive transformants obtained by the above construction were inoculated into MM fermentation medium (1.5% glucose, 1.7% lactose, 2.5% corn steep liquor, 0.44% (NH 4) 2SO4,0.09% MgSO4,2% KH2PO4,0.04% CaCl2,0.018% Tween-80,0.018% trace elements, 0.018% polypropylene glycol-2000), respectively, cultured at 30℃for 48 hours, and then induced with lactose at 25℃for 72 hours. And centrifuging to obtain fermentation supernatant, and performing lipase enzyme activity assay.

The results show that compared with engineering bacteria for recombinant expression of wild lipase ZFCB, the engineering bacteria for recombinant expression of lipase mutants constructed by the invention have no obvious change in fermentation enzyme activity of most strains, have obviously reduced fermentation enzyme activity of 8 strains, and only have obviously improved enzyme activity of 2 strains. The applicant named Trichoderma reesei ZFCB-768 (Trichoderma reesei ZFCB-768) the engineering bacterium with the highest enzyme activity. The lipase activity in the shake flask fermentation supernatant of the strain is as high as 683U/ml, and the lipase activity is improved by 40% with the engineering bacteria for recombinant expression of the wild lipase ZFCB, so that unexpected technical effects are achieved.

EXAMPLE 4 determination of Lipase mutant sequences

4.1 Extraction of Total DNA

Culturing Trichoderma reesei ZFCB-768 with the highest enzyme activity level overnight, placing a proper amount of thalli into a centrifuge tube, centrifuging 13000-rpm for 5-min, and discarding the supernatant; 400 μl extraction buffer (100 mM Tris-HCl,100 mM EDTA,250 mM NaCl,1%SDS) was added; then adding 100mg of quartz sand or glass beads, and shaking vigorously in a bead beating instrument for about 2 min; after 20min of water bath at 65 ℃, 200 μl of 10M NH4AC is added, and the ice bath is carried out for 10min; centrifuging at 13000rpm for 10min, and collecting supernatant; adding 2 times volume of absolute ethyl alcohol, and standing at-20deg.C for 30min;13000 Centrifuging at rpm for 10min, and discarding supernatant; washing with 70% ethanol for 2 times; air drying, dissolving in water, and storing at-20deg.C.

4.2 Sequencing analysis

PCR was performed using the upstream and downstream primers described in example 1 to amplify the gene of interest. The PCR amplification conditions were: 94 ℃ for 3min;94 ℃ for 30S;56 ℃ 30S,72 ℃ 60S 30 cycles; and at 72℃for 5min. And (5) recovering PCR amplification products by using a gel recovery kit. And (3) performing TA cloning on the PCR recovered product, and selecting positive transformants to send to Shanghai biological engineering Limited company for sequencing analysis.

Sequencing results show that the nucleotide sequence of the lipase mutant gene amplified from Trichoderma reesei ZFCB-768 is SEQ ID NO:3, the encoded amino acid sequence is SEQ ID NO:4.

The amino acid sequences of the wild type lipase ZFCB and the lipase mutants are compared, and the lipase mutants comprise four mutation sites which are respectively T63A, N103R, A176S.

The lipase mutant provided by the invention can obviously improve the expression level of the lipase mutant in Trichoderma reesei, is beneficial to reducing the production cost of the lipase and promotes the wide application of the lipase mutant in the industrial fields of foods, feeds, medicines and the like.

Claims (5)

1. A lipase mutant, characterized in that the mutant has the amino acid sequence of SEQ ID NO:1 from Thr to Ala, from Asn to Arg, and from Ala to Ser, from amino acid 176.

2. A gene encoding the lipase mutant of claim 1.

3. The gene of claim 2, wherein the nucleotide sequence of the gene is SEQ ID NO:3.

4. A recombinant plasmid is characterized in that, the recombinant plasmid carries the lipase mutant gene of claim 3.

5. A host cell carrying the recombinant plasmid of claim 4, wherein the host cell is trichoderma reesei (Trichoderma reesei).