CN108753756B

CN108753756B - Hyperthermophilic lipase LipD and related biological material and application thereof

Info

Publication number: CN108753756B
Application number: CN201810596461.8A
Authority: CN
Inventors: 顾金刚; 王宇洲; 马锐; 李世贵; 龚明波; 向杰; 陈敬师
Original assignee: Institute of Agricultural Resources and Regional Planning of CAAS
Current assignee: Institute of Agricultural Resources and Regional Planning of CAAS
Priority date: 2018-06-11
Filing date: 2018-06-11
Publication date: 2021-09-14
Anticipated expiration: 2038-06-11
Also published as: CN108753756A

Abstract

The invention discloses a hyperthermophilic lipase LipD and related biological materials and application thereof. The hyperthermophilic lipase LipD is any protein in A1) -A3): A1) a protein having the amino acid sequence of SEQ ID No. 2; A2) a protein having an amino acid sequence of positions 53-598 of SEQ ID No. 2; A3) the amino acid sequence is the protein at positions 54-598 of SEQ ID No. 2. The optimum temperature of the enzyme reaction of the hyperthermophilic lipase is 80 ℃, the optimum pH of the enzyme reaction is 9.5, the lipase activity under the conditions of 37 ℃ and pH value of 9-10 is more than 94% of the lipase activity under the conditions of 37 ℃ and pH value of 9.5, and the lipase activity under the conditions of 80-90 ℃ and pH value of 9.5 is more than 87% of the lipase activity under the conditions of 80 ℃ and pH value of 9.5.

Description

Hyperthermophilic lipase LipD and related biological material and application thereof

Technical Field

The invention relates to a hyperthermophilic lipase LipD and related biomaterials and application thereof in the field of biotechnology.

Background

Lipases (EC 3.1.1.3) are considered to be one of the most important commercial enzymes, and have attracted considerable attention in the rapidly growing field of biotechnology. Lipases can catalyze the hydrolysis of triacylglycerols at the interface of oil and water, releasing diacylglycerides, long chain fatty acids (>10 carbons) and glycerol. During hydrolysis, the lipase binds to the substrate acyl group to form a lipase-acyl complex, which then transfers the acyl group to the hydroxyl group of a water molecule to effect hydrolysis. Under water-insoluble conditions, lipases can transfer the acyl group of a carboxylic acid to a nucleophile.

Lipases of microbial origin are widely used in various fields including industrial, food, feed and medical fields. Compared with the traditional chemical catalyst, the lipase has the advantages of wider application field, reusability, environmental friendliness and the like, but most of the lipases are sensitive to extreme temperature and harsh reaction pH, and the lipase with specific enzymology property must be selected according to specific industrial environment. Lipases can be classified into low-temperature, medium-temperature, thermophilic and hyperthermophilic ones according to the optimum reaction temperature, and most of industrial production is currently carried out under medium-high temperature alkaline conditions (above 45 ℃ and above pH 7). The chemical catalyst usually used in practical application has no biological activity, which is a great advantage of enzyme preparation in industrial reaction, and lipase which stably exists under severe industrial conditions and continuously exerts catalytic activity appears to have phoenix feather bone, especially hyperthermophilic lipase with optimum temperature of more than 80 ℃ is difficult to find. Many industrial catalytic reaction processes involving lipases require high reaction temperatures and good thermal stability, such as paper-frame degreasing and deinking, fur degreasing, and feed production, and therefore hyperthermophilic lipases are of great importance in the industrial field.

Disclosure of Invention

The invention aims to solve the technical problem of providing the hyperthermophilic lipase.

The hyperthermophilic lipase provided by the invention is any protein in A1) -A5):

A1) a protein having the amino acid sequence of SEQ ID No. 2;

A2) a protein having an amino acid sequence of positions 53-598 of SEQ ID No. 2;

A3) a protein having an amino acid sequence of positions 54-598 of SEQ ID No. 2;

A4) fusion protein obtained by carboxyl-terminal or/and amino-terminal fusion protein labels of the protein shown in A2) or A3);

A5) the protein with the activity of the hyperthermophilic lipase is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in A2) or A3).

In the above hyperthermophilic lipase, the protein name of A1) was RTLipD-his, the protein name of A2) was RTLipD, and the protein name of A3) was TLipD. TlipD is a mature protein derived from Trichoderma lentiform ACCC 30425. RTLipD is a recombinant protein obtained by adding a methionine residue to the amino terminus of TlipD while keeping the other amino acid residues of TlipD unchanged. RTLipD-his is a recombinant protein obtained by adding amino acid residues 1 to 53 of SEQ ID No.2 to the amino terminus of TlipD, and adding amino acid residues 599-606 of SEQ ID No.2 to the carboxy terminus of TlipD, while keeping the other amino acid residues of TlipD unchanged.

In the above hyperthermophilic lipase, SEQ ID No.2 consists of 606 amino acid residues.

In the hyperthermophilic lipase, the protein tag is a polypeptide or protein which is fused and expressed with a target protein by using a DNA in vitro recombination technology so as to be convenient for expression, detection, tracing, purification and the like of the target protein. Experiments prove that the protein has lipase activity. The protein has the highest enzyme activity of lipase at 80 ℃, namely the optimum temperature of the lipase reaction of the protein is 80 ℃. The protein has the highest enzyme activity of lipase at a pH value of 9.5, namely the optimum pH value of the lipase reaction of the protein is 9.5. The lipase activity of RTLipD-his at 37 ℃ and pH value of 9-10 is more than 94% of that of lipase at 37 ℃ and pH value of 9.5. The lipase activity of RTLipD-his at 80-90 ℃ and pH value of 9.5 is more than 87% of that of lipase at 80 ℃ and pH value of 9.5.

The biological material related to the hyperthermophilic lipase also belongs to the protection scope of the invention.

The biological material related to the hyperthermophilic lipase can be at least one of the following B1) -B7):

B1) a nucleic acid molecule encoding said hyperthermophilic lipase;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic plant cell line containing B1) the nucleic acid molecule, or a transgenic plant cell line containing B2) the expression cassette, or a transgenic plant cell line containing B3) the recombinant vector;

B6) a transgenic plant tissue containing B1) the nucleic acid molecule, or a transgenic plant tissue containing B2) the expression cassette, or a transgenic plant tissue containing B3) the recombinant vector;

B7) a transgenic plant organ containing B1) the nucleic acid molecule, or a transgenic plant organ containing B2) the expression cassette, or a transgenic plant organ containing B3) the recombinant vector.

In the above biological material, the nucleic acid molecule of B1) may be a gene represented by any one of the following B11) -B13):

B11) the coding sequence is a DNA molecule of SEQ ID No. 1;

B12) the DNA molecule with the nucleotide sequence of 157 th and 1794 th sites of SEQ ID No. 1;

B13) the nucleotide sequence is the DNA molecule at position 160-1794 of SEQ ID No. 1.

In the above biomaterial, B11) is an optimized gene of RTLipD-his, which is named as RTLipD-his-Y gene; B12) is an optimized gene of RTLipD, and is named as RTLipD-Y gene; B13) is an optimized gene of TLipD, and is named as TLipD-Y gene. Wherein, the sequence 1(SEQ ID No.1) in the sequence table consists of 1821 nucleotides.

In the above-mentioned biological materials, the recombinant vector described in B3) may be specifically pET30a (+) -RTLipD-his-Y, pET30a (+) -RTLipD-his-Y is a recombinant expression vector obtained by replacing a fragment (small fragment including an EcoRI recognition site and an XhoI recognition site) between EcoRI and XhoI recognition sites of pET30a (+) with a DNA molecule having the nucleotide sequence of the 151 th-1800 th site of SEQ ID No.1, and leaving the other sequences of pET30a (+) unchanged.

In the above biological material, the recombinant microorganism of B4) may be a recombinant microorganism in which a gene encoding the protein is introduced into a recipient microorganism, and the recipient microorganism may be any one of C1) to C4):

C1) a prokaryotic microorganism;

C2) bacteria of the enterobacteriaceae family;

C3) an Escherichia bacterium;

C4) coli, e.g. e.coli BL21(DE 3).

Among the above-mentioned biomaterials, the recombinant microorganism of B4) may be specifically a recombinant escherichia coli expressing a recombinant protein (named RTLipD-his) having an amino acid sequence of SEQ ID No.2, which is obtained by introducing the pET30a (+) -RTLipD-his into escherichia coli e.coli BL21(DE 3).

Of the above-mentioned biological materials, B5) to B7) may or may not include propagation material.

The application of the protein as lipase (such as hyperthermophilic lipase) and the application of the biological material in the preparation of lipase (such as hyperthermophilic lipase) also belong to the protection scope of the invention.

The invention also provides a method for preparing the lipase.

The method for preparing the lipase provided by the invention comprises the following steps: expressing the coding gene of the protein in an organism to obtain lipase; the organism is a microorganism, a plant or a non-human animal.

In the above method, the microorganism may be any one of C1) -C4):

C1) a prokaryotic microorganism;

C2) bacteria of the enterobacteriaceae family;

C3) an Escherichia bacterium;

C4) coli, e.g. e.coli BL21(DE 3).

In the above method, the expression of the gene encoding the protein in the organism comprises introducing the gene encoding the protein into a recipient microorganism to obtain a recombinant microorganism expressing the protein, and culturing the recombinant microorganism to express the protein.

In the above method, the recipient microorganism may be any one of the above C1) -C4).

In the above method, the recombinant microorganism may be specifically a recombinant escherichia coli expressing a recombinant protein (named RTLipD-his) having an amino acid sequence of SEQ ID No.2, which is obtained by introducing the pET30a (+) -RTLipD-his into escherichia coli e.coli BL21(DE 3).

In the above, all applications may be directed towards non-disease treatment purposes, non-disease prognosis purposes and/or non-disease diagnosis purposes.

Herein, the hyperthermophilic lipase refers to a lipase having an optimum temperature for enzymatic reaction of 80 ℃ or higher, which is an optimum temperature for enzymatic reaction time of 15 minutes.

Experiments prove that the optimum temperature of the lipase reaction of the hyperthermophilic lipase is 80 ℃, and the optimum pH of the lipase reaction is 9.5. The lipase activity of RTLipD-his at 37 ℃ and pH value of 9-10 is more than 94% of that of lipase at 37 ℃ and pH value of 9.5. The lipase activity of RTLipD-his at 80-90 ℃ and pH value of 9.5 is more than 87% of that of lipase at 80 ℃ and pH value of 9.5. The hyperthermophilic lipase of the present invention is an alkaline hyperthermophilic lipase. Compared with the gene RTLipD-his-W before codon optimization, the gene RTLipD-his-Y after codon optimization improves the yield of lipase RTLipD-his by 4 times; compared with the gene RTLipD-W before codon optimization, the gene RTLipD-Y after codon optimization improves the yield of lipase RTLipD-his by 4 times; compared with the gene TlipD-W before codon optimization, the gene TlipD-Y after codon optimization improves the yield of lipase RTLipD-his by 4 times. The invention can be used for paper frame degreasing and deinking, fur degreasing and feed production.

Drawings

FIG. 1 is a graph of the effect of pH on RTLipD-his activity.

FIG. 2 is a graph of the effect of temperature on RTLipD-his activity.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

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

Trichoderma (Trichoderma lentiform) ACCC 30425 in the following examples was collected at 2008. 6/8 in the agricultural microorganism center of China Committee for culture Collection of microorganisms (also known as the China agricultural culture Collection, ACCC for short, address: southern Avenue 12 of Guancun in the Haishen district, Beijing, institute of agricultural resources and agriculture, postal code 100081), and was publicly available from the agricultural microorganism center of China Committee for culture Collection of microorganisms since the date of collection.

In the following examples, the lipase expression vector pET30a (+) and the competent cell e.coli BL21(DE3) are products of the TransGen Biotech Co.

Example 1 preparation of Lipase

1 preparation of recombinant bacterium

1.1 preparation of recombinant bacterium E.coli BL21(DE3)/pET30a (+) -RTLipD-his-Y containing codon-optimized Gene

The obtained lipase gene cDNA was annotated according to Trichoderma lentiform ACCC 30425 Whole genome sequencing technology, and the signal peptide was manually deleted to obtain TlipD gene, which was named TlipD-W gene. The TlipD-W gene is a DNA molecule obtained by deleting nucleotides 1 to 159 of SEQ ID No.3 and deleting nucleotides 1795-1818 of SEQ ID No.3 and keeping other nucleotides of SEQ ID No.3 unchanged. The TlipD-W gene is the original sequence of Trichoderma (Trichoderma lentiform) ACCC 30425, and was not codon optimized. The TlipD-W gene encodes a protein (named TLipD) whose amino acid sequence is positions 54-598 of SEQ ID No. 2. TlipD is a wild-type protein.

Selecting proper restriction enzyme cutting sites (EcoRI and XhoI), deleting the original stop codon of the TlipD-W gene, sending the DNA sequence information of the mature peptide chain to be synthesized to Shanghai Czeri biological company, and synthesizing a nucleotide sequence (synthetic product) which is optimized by the codon and is suitable for prokaryotic expression. Carrying out enzyme digestion on the synthetic product by using EcoRI and XhoI, recovering a target fragment, carrying out enzyme digestion on pET30a (+) by using EcoRI and XhoI, and recovering a large carrier fragment; and connecting the recovered target fragment with the recovered large fragment of the vector to obtain a connecting product, and transforming the connecting product into a competent cell E.coli BL21(DE3) to obtain the recombinant escherichia coli. The recombinant plasmid in the recombinant E.coli was isolated and sequenced. The sequencing result showed that the fragment between the EcoRI and XhoI recognition sites of pET30a (+) (small fragment including the EcoRI recognition site and the XhoI recognition site) was replaced with a DNA molecule having the nucleotide sequence of position 151-1800 of SEQ ID No.1, and the other sequences of pET30a (+) were kept unchanged, and the resulting recombinant expression vector was named pET30a (+) -RTLipD-his-Y. The recombinant E.coli containing pET30a (+) -RTLipD-his-Y was named E.coli BL21(DE3)/pET30a (+) -RTLipD-his-Y.

pET30a (+) -RTLipD-his-Y contains RTLipD-his gene, the RTLipD-his gene is named as RTLipD-his-Y gene, the nucleotide sequence of the RTLipD-his-Y gene is SEQ ID No.1, which is a codon optimized gene, and the RTLipD-his-Y gene encodes a protein (the name of which is RTLipD-his) whose amino acid sequence is SEQ ID No. 2; pET30a (+) -RTLipD-his-Y contains an RTLipD gene, the RTLipD gene is named as RTLipD-Y gene, the nucleotide sequence of the RTLipD-Y gene is 157 th and 1794 th positions of SEQ ID No.1 and is a codon optimization gene, and the RTLipD-Y gene encodes a protein (the name of which is RTLipD) the amino acid sequence of which is 53 th to 598 th positions of SEQ ID No. 2; pET30a (+) -RTLipD-his-Y contains a TLipD gene, which is named as TlipD-Y gene, the nucleotide sequence of the TlipD-Y gene is the 160 nd 1794 th position of SEQ ID No.1, and is a codon optimized gene, and the TlipD-Y gene encodes a protein (named as TLipD) with the amino acid sequence of 54 th to 598 th positions of SEQ ID No. 2.

pET30a (+) -RTLipD-his-Y expresses a protein (named RTLipD-his) whose amino acid sequence is SEQ ID No.2 in E.coli BL21(DE 3). BL21(DE3)/pET30a (+) -RTLipD-his-Y can produce lipase RTLipD-his.

1.2 preparation of recombinant bacterium E.coli BL21(DE3)/pET30a (+) -RTLipD-his-W containing non-codon-optimized Gene

Recombinant E.coli containing pET30a (+) -RTLipD-his-W was constructed according to the method of step 1.1 and named E.coli BL21(DE3)/pET30a (+) -RTLipD-his-W.

pET30a (+) -RTLipD-his-W is a recombinant expression vector named pET30a (+) -RTLipD-his-W obtained by replacing the fragment between the EcoRI and XhoI recognition sites of pET30a (+) (a small fragment including the EcoRI recognition site and the XhoI recognition site) with a DNA molecule having the nucleotide sequence of position 151-1800 of SEQ ID No.3, keeping the other sequences of pET30a (+) unchanged.

pET30a (+) -RTLipD-his-W contains an RTLipD-his gene, which is named as RTLipD-his-W gene, the nucleotide sequence of the RTLipD-his-W gene is SEQ ID No.3, which is a gene not subjected to codon optimization, and the RTLipD-his-W gene encodes a protein (named as RTLipD-his) whose amino acid sequence is SEQ ID No. 2; pET30a (+) -RTLipD-his-W contains an RTLipD gene, which is named as RTLipD-W gene, the nucleotide sequence of which is 157 th and 1794 th positions of SEQ ID No.3 and is a gene which is not subjected to codon optimization, and the RTLipD-W gene encodes a protein (named as RTLipD) whose amino acid sequence is 53 th to 598 th positions of SEQ ID No. 2; pET30a (+) -RTLipD-his-W contains the TLipD gene, which is named as TlipD-W gene, the nucleotide sequence of the TlipD-W gene is the 160 nd 1794 th position of SEQ ID No.3, and is a codon optimized gene, and the TlipD-W gene encodes a protein (named as TLipD) whose amino acid sequence is the 54 th to 598 th positions of SEQ ID No. 2.

pET30a (+) -RTLipD-his-W expresses a protein (named RTLipD-his) whose amino acid sequence is SEQ ID No.2 in E.coli BL21(DE 3). BL21(DE3)/pET30a (+) -RTLipD-his-W can produce lipase RTLipD-his.

1.3 preparation of recombinant bacterium containing empty vector E.coli BL21(DE3)/pET30a (+)

pET30a (+) was transformed into competent cell E.coli BL21(DE3) to obtain recombinant E.coli. The recombinant plasmid in the recombinant E.coli was isolated and sequenced. The recombinant E.coli containing pET30a (+) was named E.coli BL21(DE3)/pET30a (+) as an empty vector control recombinant strain.

2 expression of alkaline hyperthermophilic Lipase

Three strains, E.coli BL21(DE3)/pET30a (+) -RTLipD-his-Y, E.coli BL21(DE3)/pET30a (+) -RTLipD-his-W and E.coli BL21(DE3)/pET30a (+), were inoculated separately in an inoculum size of 0.5% to 30mL of LB vial liquid medium (containing 50. mu.g/mL kanamycin sulfate), and were cultured and activated for 12 to 16 hours in a shaking table at 37 ℃ and 220 rpm. Then, an appropriate amount of activated bacterial liquid is taken according to the inoculation amount of 1 percent and inoculated into a 300mL large bottle LB culture solution (containing 50 mu g/mL kanamycin sulfate), in a shaking table at 37 ℃ and 220rpm, the continuous culture is carried out for 2.5 to 3 hours (the OD600 value of the bacterial liquid is determined to be 0.8 by an ultraviolet spectrophotometer, an LB liquid culture medium containing 50 mu g/mL kanamycin sulfate is used as a blank control), IPTG (filtration sterilization through a 0.22 mu m filter membrane) is added until the content of the IPTG is 0.6mM, and the induction culture is carried out for 6 hours in the shaking table at 30 ℃ and 220 rpm. Transferring the induced culture solution to a centrifugal cup, centrifuging at the rotating speed of 4000rpm for 10 minutes, removing supernatant, re-suspending the thalli by using 5mL of buffer solution, recovering the thalli, and collecting the re-suspended thalli into a 10mL centrifugal tube. And (3) carrying out ultrasonic cell disruption on the heavy suspension by using an ultrasonic cell disruptor under the ice-water bath condition. The power of the crushing instrument is set to be 200W, the working time of ultrasonic waves is 4 seconds, the interval time is 3 seconds, and the crushing time is 30 minutes. And after the crushing is finished, immediately centrifuging the bacterial liquid at the rotating speed of 12,000rpm and the temperature of 4 ℃ for 10 minutes, retaining the supernatant, and removing impurities such as cell fragments, wherein the supernatant is the crude enzyme liquid obtained by induction expression.

The crude enzyme solution is dissolved in 20mM Tris-HCl buffer solution with the pH value of 8.0 to obtain the enzyme solution to be detected.

The lipase activity of the enzyme solution to be tested was measured by the p-nitrophenol (p-NP) method. The specific method comprises the following steps: preparing a p-NP standard solution (the concentration of the p-NP is 8mM, and the p-NP is prepared by 20mM Tris-HCl buffer solution with the pH value of 8.0); the p-NP standard solution was diluted to an appropriate gradient with 20mM Tris-HCl buffer solution having a pH of 8.0, and the absorbance was measured and plotted as an absorbance-concentration relationship. Taking 2.4mL of substrate solution (weighing a proper amount of p-nitrophenol palmitate p-NPP, dissolving the p-nitrophenol palmitate p-NPP in 20mM Tris-HCl buffer solution with the pH value of 8.0, wherein the concentration of the p-NPP is 0.8mM), preheating at 37 ℃ for 5min, adding 0.1mL of enzyme solution to be tested, reacting at 37 ℃ for 15min, immediately adding 2.5mL of absolute ethyl alcohol, uniformly mixing, standing for 5min, and stopping the reaction; 0.1mL of the enzyme solution to be assayed was replaced with 0.1mL of 20mM Tris-HCl buffer solution having a pH of 8.0, and the other conditions were not changedObtaining a blank; absorbance was measured at 410 nm. And (5) calculating the concentration of the generated p-nitrophenol by contrasting with a standard curve, and further calculating the enzyme activity. Enzyme activity definition and calculation formula: the lipase activity unit is defined as: the amount of enzyme that released 1. mu. mol of p-nitrophenol per minute at 37 ℃ and pH8.0 was defined as 1 lipase activity unit (U). Calculating the formula: the lipase yield of E.coli BL21(DE3)/pET30a (+) -RTLipD-his-Y, E.coli BL21(DE3)/pET30a (+) -RTLipD-his-W and E.coli BL21(DE3)/pET30a (+) is calculated according to the number of bacteria from which the enzyme solution to be tested comes, and the result shows that the lipase RTLipD-his yield produced by BL21(DE3)/pET30a (+) -RTLipD-his-Y is 0.10U/10U/mL⁸The yield of lipase RTLipD-his produced by cfu BL21(DE3)/pET30a (+) -RTLipD-his-Y, E.coli BL21(DE3)/pET30a (+) -RTLipD-his-W is 0.02U/10⁸cfu E. coli BL21(DE3)/pET30a (+) -RTLipD-his-W, E.coli BL21(DE3)/pET30a (+) did not produce lipase. The lipase RTLipD-his yield of BL21(DE3)/pET30a (+) -RTLipD-his-Y is 5 times that of E.coli BL21(DE3)/pET30a (+) -RTLipD-his-W.

The results show that compared with the gene RTLipD-his-W before codon optimization, the gene RTLipD-his-Y after codon optimization improves the yield of lipase RTLipD-his by 4 times; compared with the gene RTLipD-W before codon optimization, the gene RTLipD-Y after codon optimization improves the yield of lipase RTLipD-his by 4 times; compared with the gene TlipD-W before codon optimization, the gene TlipD-Y after codon optimization improves the yield of lipase RTLipD-his by 4 times.

Example 2 RTLipD-his is alkaline hyperthermophilic Lipase

E.coli BL21(DE3)/pET30a (+) -RTLipD-his-Y was inoculated at an inoculum size of 0.5% into 30mL LB vial liquid medium (containing 50. mu.g/mL kanamycin sulfate), and cultured and activated at 37 ℃ for 12 to 16 hours on a shaking table at 220 rpm. Then, an appropriate amount of activated bacterial liquid is taken according to the inoculation amount of 1 percent and inoculated into a 300mL large bottle LB culture solution (containing 50 mu g/mL kanamycin sulfate), in a shaking table at 37 ℃ and 220rpm, the continuous culture is carried out for 2.5 to 3 hours (the OD600 value of the bacterial liquid is determined to be 0.8 by an ultraviolet spectrophotometer, an LB liquid culture medium containing 50 mu g/mL kanamycin sulfate is used as a blank control), IPTG (filtration sterilization through a 0.22 mu m filter membrane) is added until the content of the IPTG is 0.6mM, and the induction culture is carried out for 6 hours in the shaking table at 30 ℃ and 220 rpm. Transferring the induced culture solution to a centrifugal cup, centrifuging at the rotating speed of 4000rpm for 10 minutes, removing supernatant, re-suspending the thalli by using 5mL of buffer solution, recovering the thalli, and collecting the re-suspended thalli into a 10mL centrifugal tube. And (3) carrying out ultrasonic cell disruption on the heavy suspension by using an ultrasonic cell disruptor under the ice-water bath condition. The power of the crushing instrument is set to be 200W, the working time of ultrasonic waves is 4 seconds, the interval time is 3 seconds, and the crushing time is 30 minutes. And (3) immediately centrifuging the bacterial liquid at the rotating speed of 12,000rpm and the temperature of 4 ℃ for 10 minutes after the crushing is finished, reserving a supernatant, and removing impurities such as cell fragments, wherein the supernatant is crude enzyme liquid containing the lipase RTLipD-his obtained by induction expression.

1. Effect of pH on Lipase RTLipD-his Activity

And dissolving the crude enzyme solution containing lipase RTLipD-his in 20mM Tris-HCl buffer solutions with the pH values of 7,8,8.5,9,9.5 and 10 respectively to obtain the enzyme solution to be detected with the corresponding pH value.

The optimum pH for the lipase RTLipD-his activity was determined at 37 ℃ according to the p-nitrophenol method of example 1 in the following buffer: 20mM Tris-HCl at pH7, 8,8.5,9,9.5 or 10. The substrate solution and the p-NP standard solution were prepared using the buffer solution as a solvent. Wherein the substrate is p-nitrophenol palmitate (p-NPP). The experiment was repeated three times. The lipase activity unit is defined as: the amount of enzyme that released 1. mu. mol of p-nitrophenol per minute at 37 ℃ and pH9.5 was defined as 1 lipase activity unit (U). The other operations were the same as in the p-nitrophenol process of example 1.

The results show 10⁸The lipase RTLipD-his produced by cfu BL21(DE3)/pET30a (+) -RTLipD-his-Y has lipase activity of 0.90 + -0.01U at 37 ℃ and pH 9.5. Lipase activity of lipase RTLipD-his at 37 ℃ and pH value of 9.5Defined as 100%, to compare the effect of pH on enzyme activity. The lipase RTLipD-his was in a poor state under neutral and acidic conditions, with a steady increase starting from pH7 until the pH reached an optimum pH of 9.5. The relative enzyme activity of the lipase RTLipD-his at 37 ℃ and pH9 was 97.96%, and the relative enzyme activity at 37 ℃ and pH 10 was 94.78% (FIG. 1). The lipase RTLipD-his has the enzyme activity of over 94 percent at the temperature of 37 ℃ and the pH value of 9-10 and is alkaline lipase.

2. Effect of temperature on Lipase RTLipD-his Activity

The crude enzyme solution containing lipase RTLipD-his was dissolved in 20mM Tris-HCl buffer solution at pH9.5 (pH9.5) to obtain an enzyme solution to be assayed.

And (3) determining the lipase activity of the enzyme solution to be detected by adopting a p-nitrophenol method under the condition that the pH value is 9.5.

The substrate solution and the p-NP standard solution were prepared using 20mM Tris-HCl buffer solution, pH9.5, as a solvent. Wherein the substrate is p-nitrophenol palmitate (p-NPP).

Taking 2.4mL of substrate solution, preheating for 5min at the temperature to be measured (40 ℃,50 ℃,60 ℃,65 ℃,70 ℃,80 ℃ and 90 ℃ respectively), adding 0.1mL of enzyme solution to be measured, reacting for 15min at the corresponding temperature to be measured, immediately adding 2.5mL of absolute ethyl alcohol, uniformly mixing, and standing for 5min to terminate the reaction; replacing 0.1mL of enzyme solution to be detected with 0.1mL of 20mM Tris-HCl buffer solution with pH9.5, and obtaining blank when other conditions are unchanged; absorbance was measured at 410 nm. And (5) calculating the concentration of the generated p-nitrophenol by contrasting with a standard curve, and further calculating the enzyme activity. The experiment was repeated three times. The lipase activity unit is defined as: the amount of enzyme that released 1. mu. mol of p-nitrophenol per minute at 80 ℃ and pH9.5 was defined as 1 lipase activity unit (U).

The results show 10⁸The lipase RTLipD-his produced by cfu BL21(DE3)/pET30a (+) -RTLipD-his-Y has lipase activity of 1.09 + -0.03U at 80 ℃ and pH 9.5. The effect of temperature on the enzyme activity was compared by defining the lipase activity of the lipase RTLipD-his at 80 ℃ and pH9.5 as 100%. The optimum temperature of the lipase RTLipD-his is 80 ℃, the lipase activity of the lipase is in a poor state under the condition of low temperature, the temperature is increased from 40 ℃,the enzyme activity is gradually increased along with the temperature increase, when the temperature reaches 80 ℃, the enzyme activity reaches the highest activity, and the enzyme activity is kept stable in an ultrahigh temperature region with the optimal temperature, but due to super-heat inactivation, the enzyme activity begins to be reduced after the temperature exceeds 80 ℃, and the relative enzyme activity of the lipase RTLipD-his is 87.46 percent at 90 ℃ and the pH value of 9.5 (figure 2). The lipase RTLipD-his has the enzyme activity of more than 87 percent at the temperature of 80-90 ℃ and the pH value of 9.5 and is alkaline hyperthermophilic lipase.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

<110> institute of agricultural resources and agricultural regionalism of Chinese academy of agricultural sciences

<120> hyperthermophilic lipase LipD and related biological material and application thereof

<130> GNCFH181282

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 1821

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgcaccatc atcatcatca ttcttctggt ctggtgccac gcggttctgg tatgaaagaa 60

accgctgctg ctaaattcga acgccagcac atggacagcc cagatctggg taccgacgac 120

gacgacaagg ccatggctga tatcggatcc gaattcatga gcccgaccgt tcataccatt 180

aatggtacct atagcggcct gaatattccg agctttaaac aggaagcctt ttatggcatt 240

ccgtatgcac aggctcctgt gggcgatctg cgcttacgca aaagtctgcc gtataatcag 300

tcttggagtg gcgtgcgcaa tgccaccgtt cgtagcgata gttgtccggg ctataatggt 360

tttccgctga ccttagtggg cggctcactg attgatggcc tgaccttagg cgaagattgt 420

ctgaccattg atattgtgcg cccggccaat gttaatccgc tggataaact gccggtgttt 480

gtgtggtttt atggtggcgg ctttattgca ggtggtagtg cagatccgaa atataatatg 540

agctatattg ttcagaactc agttgaaatg aataaaccga ttatgggtgc cattattaat 600

tatcgcacca tgtgttttgg ctttctggca agtaaagaag tgctggatgc caatgtgggc 660

aatattggtc tgtttgatca gcgtcgcggt ctgaaatgga ttcaggaaaa tattaaaggt 720

tttggtggcg atccggaaaa agttaccatt gcaggtgaat cagcaggcgg ttctagtacc 780

ggctatcatc tgattggctt taaaggtaat aatgatggtc tgtttcgcgg cgccattatg 840

gaaagcgcct cactgttagg cgccccgatt aataccccgg aacagttaca gcgtagctat 900

cagggcatgt atgataatat taccgaaacc gtgggttgta gtacctctaa tgattctcta 960

gcttgtctgc gtagcgtgcc gtatgatacc ctgtataatg cctgtattgg ctttcgccag 1020

accccgatta tggatggcga atttatttca cagctgccgt cacagagcat tcagaaaggt 1080

gaaattgcag atgtgagcat tattatgggt accaataccg atgaaggcac cgcaattttt 1140

ctgggtccgc gcgccaatcc gttaaatacc gatgaagatg tgtttaaata tgttcaggca 1200

ctgggtagcg gcttagataa taaaaccgtt gaaaccgtga tgaaactgta tccggatgat 1260

ccgacctggg gttgtccgtt tggtaccggc ccggaacgct ttgcagatca gggttttcag 1320

tataaacgcg gtgcagcaat tgcaggcgat tattttatgc acgcaggtcg tcgcttttat 1380

gccaattctc atagcacccg ctctcataaa ccgatttata cctatcgttt tgatcaggcc 1440

ccgtgggata tgcgtgaacc gtcaattatg attgtgccgc cggtgtatgt gacccatttt 1500

agtgaaattg tgtatgtgtt tgataatccg aataataatt caaattttat tggtccgtat 1560

ccgagctatg cacgcttaca gtcatttatg tcacgttctt gggcatcttt tgttcatgat 1620

ctgaatccga ataatcatgg cttacaggac ccgaatctgc cgaaatggcc ggaatatgat 1680

ccgaaacagc cgcagaatat tgtgtttcgc gaaggcggct cttttctgga aaatgatgat 1740

tatcgcaaac cgcagttagc cttttggggc accatttggc cggaattgca aaccctcgag 1800

caccaccacc accaccactg a 1821

<210> 2

<211> 606

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser

1 5 10 15

Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp

20 25 30

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

35 40 45

Gly Ser Glu Phe Met Ser Pro Thr Val His Thr Ile Asn Gly Thr Tyr

50 55 60

Ser Gly Leu Asn Ile Pro Ser Phe Lys Gln Glu Ala Phe Tyr Gly Ile

65 70 75 80

Pro Tyr Ala Gln Ala Pro Val Gly Asp Leu Arg Leu Arg Lys Ser Leu

85 90 95

Pro Tyr Asn Gln Ser Trp Ser Gly Val Arg Asn Ala Thr Val Arg Ser

100 105 110

Asp Ser Cys Pro Gly Tyr Asn Gly Phe Pro Leu Thr Leu Val Gly Gly

115 120 125

Ser Leu Ile Asp Gly Leu Thr Leu Gly Glu Asp Cys Leu Thr Ile Asp

130 135 140

Ile Val Arg Pro Ala Asn Val Asn Pro Leu Asp Lys Leu Pro Val Phe

145 150 155 160

Val Trp Phe Tyr Gly Gly Gly Phe Ile Ala Gly Gly Ser Ala Asp Pro

165 170 175

Lys Tyr Asn Met Ser Tyr Ile Val Gln Asn Ser Val Glu Met Asn Lys

180 185 190

Pro Ile Met Gly Ala Ile Ile Asn Tyr Arg Thr Met Cys Phe Gly Phe

195 200 205

Leu Ala Ser Lys Glu Val Leu Asp Ala Asn Val Gly Asn Ile Gly Leu

210 215 220

Phe Asp Gln Arg Arg Gly Leu Lys Trp Ile Gln Glu Asn Ile Lys Gly

225 230 235 240

Phe Gly Gly Asp Pro Glu Lys Val Thr Ile Ala Gly Glu Ser Ala Gly

245 250 255

Gly Ser Ser Thr Gly Tyr His Leu Ile Gly Phe Lys Gly Asn Asn Asp

260 265 270

Gly Leu Phe Arg Gly Ala Ile Met Glu Ser Ala Ser Leu Leu Gly Ala

275 280 285

Pro Ile Asn Thr Pro Glu Gln Leu Gln Arg Ser Tyr Gln Gly Met Tyr

290 295 300

Asp Asn Ile Thr Glu Thr Val Gly Cys Ser Thr Ser Asn Asp Ser Leu

305 310 315 320

Ala Cys Leu Arg Ser Val Pro Tyr Asp Thr Leu Tyr Asn Ala Cys Ile

325 330 335

Gly Phe Arg Gln Thr Pro Ile Met Asp Gly Glu Phe Ile Ser Gln Leu

340 345 350

Pro Ser Gln Ser Ile Gln Lys Gly Glu Ile Ala Asp Val Ser Ile Ile

355 360 365

Met Gly Thr Asn Thr Asp Glu Gly Thr Ala Ile Phe Leu Gly Pro Arg

370 375 380

Ala Asn Pro Leu Asn Thr Asp Glu Asp Val Phe Lys Tyr Val Gln Ala

385 390 395 400

Leu Gly Ser Gly Leu Asp Asn Lys Thr Val Glu Thr Val Met Lys Leu

405 410 415

Tyr Pro Asp Asp Pro Thr Trp Gly Cys Pro Phe Gly Thr Gly Pro Glu

420 425 430

Arg Phe Ala Asp Gln Gly Phe Gln Tyr Lys Arg Gly Ala Ala Ile Ala

435 440 445

Gly Asp Tyr Phe Met His Ala Gly Arg Arg Phe Tyr Ala Asn Ser His

450 455 460

Ser Thr Arg Ser His Lys Pro Ile Tyr Thr Tyr Arg Phe Asp Gln Ala

465 470 475 480

Pro Trp Asp Met Arg Glu Pro Ser Ile Met Ile Val Pro Pro Val Tyr

485 490 495

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

500 505 510

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

515 520 525

Phe Met Ser Arg Ser Trp Ala Ser Phe Val His Asp Leu Asn Pro Asn

530 535 540

Asn His Gly Leu Gln Asp Pro Asn Leu Pro Lys Trp Pro Glu Tyr Asp

545 550 555 560

Pro Lys Gln Pro Gln Asn Ile Val Phe Arg Glu Gly Gly Ser Phe Leu

565 570 575

Glu Asn Asp Asp Tyr Arg Lys Pro Gln Leu Ala Phe Trp Gly Thr Ile

580 585 590

Trp Pro Glu Leu Gln Thr Leu Glu His His His His His His

595 600 605

<210> 3

<211> 1821

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgcaccatc atcatcatca ttcttctggt ctggtgccac gcggttctgg tatgaaagaa 60

accgctgctg ctaaattcga acgccagcac atggacagcc cagatctggg taccgacgac 120

gacgacaagg ccatggctga tatcggatcc gaattcatgt cgcctacagt ccataccatc 180

aatggaacat attcaggact gaacatccca tcttttaaac aagaagcatt ttacggcatt 240

ccctatgctc aagcacctgt cggagacctt cgtctccgaa agtctctgcc atacaaccaa 300

tcttggtctg gcgtacggaa tgcaacagtc cgatctgatt cgtgtccagg gtataatgga 360

tttccactca ctcttgttgg tggtagtctc atcgacggac ttactctagg agaagactgc 420

ttgactattg atattgtccg accagccaac gtcaaccctt tggacaaatt acctgtcttt 480

gtctggttct atggtggagg ttttattgct gggggatccg ctgatcccaa gtacaatatg 540

tcatatatcg tccaaaactc ggtggagatg aataagccca ttatgggtgc catcattaac 600

tatcgaacaa tgtgttttgg ctttttggca tctaaagaag ttttggatgc taatgttggc 660

aatattgggc tctttgatca gagacgaggc cttaaatgga tccaagaaaa catcaagggc 720

tttggtggtg atcctgaaaa ggtcacaatc gcgggagaaa gtgccggtgg ttcaagcaca 780

ggctatcacc taattggctt caagggcaat aatgacgggc ttttccgtgg agccattatg 840

gagagcgcaa gtttgctagg agctccaatt aatacccctg aacaacttca gagaagctat 900

caaggcatgt acgacaacat tacagaaact gtgggttgca gtacctcgaa cgatagcttg 960

gcttgtctac gaagtgttcc atatgacact ttatataacg cgtgtattgg gttccgacag 1020

actccaatca tggatggcga gttcatctca caattaccgt ctcagtctat ccaaaaagga 1080

gaaatcgccg atgtttccat catcatgggt accaatactg atgaaggaac tgctattttc 1140

ttagggcctc gtgcaaatcc tttgaatacc gatgaggatg tattcaaata cgttcaggct 1200

cttggaagtg gacttgacaa taaaaccgta gagacggtga tgaagctata tccagatgac 1260

cctacctggg gttgcccatt tggaacaggg cctgaacggt tcgcagacca gggattccag 1320

tacaagcgtg gcgctgctat tgctggcgac tattttatgc acgctgggcg aagattttac 1380

gcaaactcgc acagcactcg aagccacaaa cccatatata cttataggtt tgaccaagca 1440

ccttgggata tgagggagcc ctctatcatg attgtccctc cggtctatgt gacacacttc 1500

tccgagattg tgtatgtgtt tgacaacccc aataacaaca gtaacttcat tggcccttac 1560

ccaagctatg cgaggctgca gtccttcatg tcacgatcct gggcttcatt tgttcatgat 1620

ctcaatccta ataaccatgg actccaagat ccgaatctcc caaaatggcc agagtatgat 1680

ccgaagcagc ctcaaaacat tgtattccgt gaaggaggga gttttcttga gaacgacgat 1740

taccggaaac cacagttggc attttggggc actatttggc cagaacttca gaccctcgag 1800

caccaccacc accaccactg a 1821

Claims

1. A protein characterized by: the protein is any one of A1) -A3):

A1) a protein having the amino acid sequence of SEQ ID No. 2;

A3) the amino acid sequence is the protein at positions 54-598 of SEQ ID No. 2.

2. The protein of claim 1, wherein: the optimum temperature for the lipase reaction of the protein is 80 ℃.

3. The protein of claim 1 or 2, wherein: the optimum pH for the lipase reaction of the protein was 9.5.

4. The biomaterial related to the protein of any one of claims 1 to 3, which is at least one of the following B1) -B4):

B1) a nucleic acid molecule encoding the protein of any one of claims 1-3;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector.

5. The biomaterial of claim 4, wherein: B1) the nucleic acid molecule is a coding gene of the protein shown in any one of the following B11) -B13):

B11) the coding sequence is a DNA molecule of SEQ ID No. 1;

6. Use of a protein according to any one of claims 1 to 3 as a lipase.

7. Use of the biomaterial of claim 4 or 5 in the preparation of a lipase.

8. A method of making a lipase, comprising: expressing a gene encoding the protein of claim 1 in an organism to obtain a lipase; the organism is a microorganism, a plant or a non-human animal.

9. The method of claim 8, wherein: the microorganism is a prokaryotic microorganism.

10. The method of claim 9, wherein: the prokaryotic microorganism is a bacterium of the family Enterobacteriaceae.

11. The method of claim 10, wherein: the bacterium of the family Enterobacteriaceae is a bacterium of the genus Escherichia.

12. The method of claim 11, wherein: the Escherichia bacterium is Escherichia coli.