NL2032449B1

NL2032449B1 - High temperature-resistant and universal metal-dependent protease and use thereof

Info

Publication number: NL2032449B1
Application number: NL2032449A
Authority: NL
Inventors: Cai Jianling; Pan Chaoming; Hua Yuejin; Wang Liangyan
Original assignee: Univ Zhejiang
Priority date: 2021-07-13
Filing date: 2022-07-12
Publication date: 2024-03-01
Also published as: CN113637662A; NL2032449A

Abstract

The present disclosure provides a high temperature—resistant and universal metal—dependent protease and use thereof. The protease has a Jabl/MPN (EXnHXHX7SX2D) conserved domain, where X is any one of essential amino acids, and has an activity strictly dependent on metal ions and an extremely broad selectivity for the metal ions; the protease can recognize ubiquitin—like modification sites to conduct cleavage; during an enzyme digestion reaction, the protease has an activity at 4°C to 100°C, and an extremely strong activity at 30°C, 37°C, and 100°C. In the present disclosure, the metalloprotease has a broad selectivity to metal ions and an excellent heat resistance, and is not easily deactivated during production, storage, and transportation. Therefore, the protease has a desirable value of use in basic research and in medicine, chemical industry, and feed industry.

Description

HIGH TEMPERATURE-RESISTANT AND UNIVERSAL METAL-DEPENDENT PROTEASE

AND USE THEREOF

TECHNICAL FIELD

The present disclosure relates to a metal-dependent protease

DRJAMM derived from Deinococcus radiodurans, in particular to structural conservation, an active reaction system, an optimum temperature and a temperature resistance of the protease.

BACKGROUND ART

Protease is a general term for a class of enzymes that hydro- lyze protein peptide chains as well as the most important indus- trial enzyme preparation, which is widely found in animal offals, plant stems, leaves and fruits, and microorganisms. Due to limited animal and plant resources, the industrial production of protease preparations is mainly achieved by fermentation with microorgan- isms such as Bacillus subtilis and Aspergillus terricola. By the beginning of this century, more than 900 microbial proteases have been reported; and with the in-depth study of proteases, increas- ing attention has been paid to industrial uses of these proteases.

Proteases are widely used in the fields of food, brewing and fermentation, as well as textile, medicine, leather, daily chemi- cal, feed, and aquatic products processing. For example, plant proteases have long been used to prevent beer turbidity and im- prove beer abiotic stability in beer production. Plant proteases or microbial neutral proteases can also be used together with o- amylase, glucoamylase, isoamylase, and B-glucanase for beer exter- nal enzymatic saccharification. Depilation and softening of the leather industry have also made extensive use of the proteases, which saves time and improves labor hygiene conditions. During the production of bread, the proteases are added into flour to shorten the dough mixing time and improve dough viscosity. In addition, proteases are widely used in biochemical molecular experiments as a scalpel of proteins, which are indispensable for life science research. Proteases are closely related to human beings and are involved in all aspects of life, such that the importance of in- dustrial utilization of the proteases is becoming increasingly ob- vious.

With the increasing level of industrial production, proteases adapted to special production environments, such as microbial high-temperature proteases and cold-adaptive proteases, have grad- ually attracted the attention of researchers. Compared with ordi- nary proteases, high-temperature proteases, due to high catalytic efficiency and strong thermal stability at high temperatures, are more suitable for high-temperature catalytic processes in indus- trial uses than normal-temperature proteases. Therefore, the de- velopment and utilization of high temperature resistant protease is particularly important. Current studies have found that a cer- tain amount of metal ions can significantly improve protease ac- tivity, and the metal ions also play an important role in protease stability as a part of the protease structure or as an enzyme ac- tivator. However, most proteases have been found to specifically depend on one or several metal ions. Therefore, the discovery of universal metal-dependent proteases is of great significance for the utilization and research of proteases. In the present disclo- sure, a high temperature-resistant protease DRJAMM is a metal ion- dependent protease that is derived from Deinococcus radiodurans and has a Jabl/MPN domain. Although homologous proteins exist widely in nature, extensive adaptability of the high temperature- resistant protease to metal ions has not yet been reported. Moreo- ver, it has been reported that these homologous proteins do not remain active under high temperatures in other organisms in addi- tion to thermophilic bacteria.

SUMMARY

In order to overcome the existing difficulties, an objective of the present disclosure is to provide a high temperature- resistant and universal metal-dependent protease and use thereof.

The present disclosure provides a high temperature-resistant and universal metal-dependent protease DRJAMM, having an amino ac- id sequence shown in SEQ ID NO: 1.

The present disclosure further provides a recombinant vector of the high temperature-resistant and universal metal-dependent protease, where a high temperature-resistant protease gene is in- serted between Ndel and BamHI restriction sites on a plasmid pET28a, such that the gene is located downstream of a T7 promoter and regulated by the promoter to obtain the recombinant vector.

The present disclosure further provides an in vitro protein purification method of the high temperature-resistant and univer- sal metal-dependent protease, including: transforming the recombi- nant vector into E. coli BL21 (DE3) for expression, conducting in- duction with 0.4 mM of isopropyl-p-d-thiogalactoside (IPTG), and treating by a nickel column, a 70°C water bath, and a molecular sieve to obtain a DRJAMM protein with a purity of not less than 95%.

The present disclosure further provides a reaction buffer of the high temperature-resistant and universal metal-dependent pro- tease, including 100 mM to 500 mM of NaCl, 10 mM to 50 mM of Tris-

HCl pH 8.0, 0.2 mM to 1 mM of dithiothreitol (DTT), 0.2 mM to 2 mM of ethylene diamine tetraacetic acid (EDTA), and 0.02 mM to 1 mM of metal ions.

In the reaction buffer, the metal ions may include one or more selected from the group consisting of zZn®*, Mn", Mg?*, Ba’,

Co“, Fe", Ni%*, ca*%, sr%, and cd.

The present disclosure further provides a reaction optimiza- tion method of the high temperature-resistant and universal metal- dependent protease, including conducting a reaction using the re- action buffer with a substrate protein DRMoaD-MoaE having a pro- tein sequence ID of NP 296326 at 4°C to 100°C for 20 min to 60 min.

In the reaction optimization method, an enzyme activity may have a temperature range of 30°C to 100°C.

In the reaction optimization method, a maximum enzyme activi- ty has a temperature range of 70°C to 100°C.

The present disclosure further provides a use method of the high temperature-resistant and universal metal-dependent protease, including ligating a specific amino acid sequence recognized by a

DRJAMM protein with a target protein to conduct high temperature cleavage or purification of the target protein.

In the high temperature-resistant and universal metal-

dependent protease, the protease may have a desirable value of use in basic research and in medicine, chemical industry, and feed in- dustry.

The present disclosure has the following beneficial effects:

In the present disclosure, the protease has strong conserva- tion, excellent heat resistance, and broad metal ion selectivity, providing a desirable tool for basic research and industrial uti- lization of the protease.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure is further described below with refer- ence to the accompanying drawings.

FIG. 1 shows a sequence alignment of a protease DRJAMM;

FIG. 2 shows a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) diagram of a purified protease DRJAMM, indicating purity of not less than 95%;

FIG. 3 shows a comparison of protease activities under the catalysis of different metal ions;

FIG. 4 shows a comparison of reactions of the protease at different temperatures in the presence of the same metal ions;

FIG. 5 shows a schematic diagram of a conventional protease

TEV and the DRJAMM in treating tagged high temperature-resistant proteins.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Example 1 Construction of a Deinococcus radiodurans DRJAMM protein expression strain

According to a 2016 resequencing-annotated Deinococcus radi- odurans Rl genome (ASM163882vl), DRJAMM (Protein Sequence ID:

NP 294125) included 136 amino acids (SEQ ID NO: 1). Alignment of the DRJAMM with homologous proteins from thermophilic archaea (SEQ

ID NO: 2) and halophilic archaea (SEQ ID NO:3) revealed that the

DRJAMM had a conserved domain EXnHXHX 7 SX 2 D (boxed in FIG. 1).

A genome of the Deinococcus radiodurans Rl was extracted us- ing a bacterial genome DNA extraction kit (DP302-02, TIANGEN Bio- tech), and the concentration and purity of the genome were deter- mined with NanoDrop 1000 (Thermo Company).

A pair of homologous recombination primers were designed ac- cording to an amino acid sequence of the protease DRJAMM and a pet28a plasmid; where an upstream primer DRJAMM-F was 5'-

CCGCGCGGCAGC CATATG GTGCTCCTGACCCTGCCTG-3' (SEQ ID NO: 4), an un- 5 derline part was a restriction site NdeI, and a part in front of the Ndel was a homologous fragment on the pet28a vector; and a downstream primer DRJAMM-R was 5'-GAGCTCGAATTC GGATCC TCAATT-

GCTCTCATCGGCG-3 (SEQ ID NO: 5) ', an underline part was a re- striction site BamHI, and the part in front of the Ndel was the homologous fragment on the pET28a vector. A Deinococcus radi- odurans Rl genomic DNA was used as a template, the target fragment was amplified with a TransStart FastPfu DNA Polymerase (TransGen

Biotech}; after a single band was detected by agarose gel electro- phoresis, a PCR product was purified and recovered using a Promega

Wizard SV Gel and PCRClean-Up kit.

A KlenDr gene fragment was recombined into the pet28a vector (an N-terminus including 6x His tag) after Ndel/BamHI double di- gestion and linearization using a ClonExpress II One Step Cloning recombinase system (Vazyme, Nanjing).

A recombinant product was transformed into E. coli DHba com- petent cells (TransGen Biotech), spread on a solid LB medium con- taining 40 pg/mL Kanamycin and incubated overnight at 37°C upsides down.

A few single colonies were selected and cultured in a 5 ml liquid LB medium containing 40 pg/mL Kanamycin for 10 h at 37°C with shaking. A plasmid was extracted with an Axygen plasmid ex- traction kit, and sequenced according to T7/T7ter primers; after the BLAST sequence was correctly aligned, the plasmid was stored at -20°C.

Example 2 Induced expression of a Deinococcus radiodurans

DRJAMM protein

A successfully constructed Pet28a-DRJAMM expression vector was transformed into an E. coli BL21 (DE3) expression strain {TransGen Biotech), and successfully-transformed cloned strains were screened with a solid LB medium containing 40 pg/mL Kanamycin antibiotic.

The successfully transformed single colonies were added into ml of a liquid medium, cultured at 37°C overnight by shaking, and then transferred to 500 ml of a medium, cultured to an ODggs of 0.6 to 0.8, cooled on ice for 10 min, added with 200 ul of 1 M

IPTG, and then cultured at 30°C for 5 h to induce expression of 5 the target protein.

After the induction, the cells were collected by centrifuga- tion at 8,000 rpm for 8 min, resuspended and washed with 1x PBS, and then centrifuged at 8,000 rpm for 5 min; a supernatant was discarded, and the cells were stored at -80°C.

Example 3 Purification of the Deinococcus radiodurans DRJIAMM protein

Cell lysis: cells were resuspended according to 1 g of the cells (wet weight) added with 15 mL of a lysis buffer (300 mM

NaCl, 20 mM Tris HCl pH 8.0, 5% Glycerol, and 10 mM imidazole).

The cells were disrupted with an ultrasonic cell disruptor (Ningbo xinzhi, JY92-IIN) in an ice-water bath until a suspension was transparent, where the disruption was conducted at an alternating rod of @®6 and a power of 60%, by ultrasonic treatment for 2 sec with an interval of 9.9 sec, for 60 min to 90 min in total. A dis- rupted cell suspension was centrifuged at 15,000 rpm for 35 min to remove cell debris, and supernatant was retained and filtered with a 0.22 uM or 0.45 pM filter.

Nickel column affinity purification: a nickel column (HisTrap

HP 1 mL) was purchased from GE HealthCare Company. The nickel col- umn was equilibrated with a Ni-buffer A (500 mM NaCl, 20 mM Tris

HCl pH 8.0, and 5% Glycerol}, the sample was loaded at 1 mL/min slowly onto the nickel column and fully bound to the nickel col- umn. The impurity proteins were eluted with low concentration gra- dients (1%, 3%, 5%, and 9%) of a Ni-buffer B (500 mM NaCl, 20 mM

Tris HCl pH 8.0, 5% Glycerol, and 250 mM imidazole), and the tar- get protein was eluted with a high concentration gradient (100%) of the Ni-buffer B solution. The target protein was collected, and purity was verified by agarose gel electrophoresis.

The target protein was treated in a 70°C water bath for 10 min, centrifuged at 15,000 rpm for 35 min, a supernatant and a precipitate were separated, and then detected by SDS-PAGE, the target protein was found in the supernatant.

Molecular sieve purification: superdex 75 Increase 10/300 GL was purchased from GE HealthCare. The target protein was concen- trated to about 500 ul with an ultrafiltration tube (10 kDa), and a molecular sieve buffer included 20 mM Tris-HCl pH 8.0 and 100 mM

KCl; the sample was loaded and eluted at 0.4 mL/min, and the tar- get protein peaked at about a 13 mL elution volume. After the tar- get protein was electrophoresed by SDS-PAGE, and obtained protein had a molecular weight of about 15.7 kDa and purity of not less than 95% (FIG. 2). After the target protein was concentrated, a protein concentration was measured with a NanoDrop instrument, snap-frozen with liquid nitrogen, and stored at -80°C.

Example 4 Deinococcus radiodurans DRJAMM protein capable of functioning dependent on different types of divalent metal ions (1) The purified DRJAMM protein was reacted with a substrate protein MoaD-MoaE (protein sequence ID: NP 296326) thereof in a reaction buffer (100 mM KCl, 20 mM Tris-HCl 8.0, 1 mM DTT, and 1 mM EDTA) at 37°C for 30 min; where in each reaction, metal ions with a final concentration of 0.4 mM were added as follows, re- spectively: zn", Mn, cu®’, Mg“, Ba“’, Co%, Fe, Ni“, ca’, sr, and cd**; after the reaction, a product was heated in a metal bath at 100°C for 15 min to fully denature the product, and then de- tected by SDS-PAGE electrophoresis. (2) The experimental results were shown in FIG. 3, indicating that the DRJAMM protease could cut the substrate enzyme into two segments in the reactions catalyzed by other metal ions except for cu** which was toxic to the protein.

Example 5 Deinococcus radiodurans DRJAMM protein capable of functioning at high temperature (1) To further confirm the effect of temperature on the pro- tease activity of DRJAMM, the purified DRJAMM protein and the sub- strate protein MoaD-MoaE thereof were incubated in the reaction buffer (100 mM KCl, 20 mM Tris-HCl 8.0, 1 mM DTT, and 1 mM EDTA), and added with Ca? according to Example 4 and then reacted for 30 min at 4°C, 16°C, 30°C, 37°C, 70°C, and 100°C. After the reaction, the product was heated in a metal bath at 100°C for 15 min to ful- ly denature the product and then detected by SDS-PAGE electropho- resis.

(2) The experimental results were shown in FIG. 4, indicating that the enzyme digestion reaction between the protease and the substrate was conducted at an optimum temperature of 30°C to 100°C; and the protease had an extremely stable activity to con- duct the reaction sufficiently even at 100°C.

Example 6 Use of the DRJAMM protease in the purification of a tagged high temperature-resistant protein

Based on the wide use of specific proteases during protein purification, principles of use were as follows: before the ex- pression and purification of some difficult-to-express or insolu- ble high temperature-resistant proteins, a fragment helpful for expression or soluble was ligated in front of the target protein; the fragment and the target protein were ligated by a specific cleavage site, and the tag and the target protein were isolated using a specific protease cleavage site during the purification, to purify the proteins. As shown in FIG. 5, in the present disclo- sure, the DRJAMM could efficiently and specifically cleave the recognition sites at greater than 70°C; and under this tempera- ture, the impurity proteins and tags were denatured and precipi- tated. Finally, not only the tag and the target protein were iso- lated, but also the protein purification was achieved during the cleavage, thereby simplifying the purification of such high tem- perature-resistant proteins.

In the examples of the present disclosure, the strain is De- inococcus radiodurans (ATCC No. 13939), and the enzyme is Dein- ococcus radiodurans JAMM protease. The above are only preferred examples of the present disclosure, but the present disclosure is not limited to the above-detailed methods. According to the teach- ing and inspiration of the present disclosure, anyone who is fa- miliar with this technology, using the high temperature-resistant protease DRJAMM and derivatives thereof provided in the present disclosure, are all within the protection scope of the present disclosure.

Sequence Listing Information:

DTD Version: V1 3

File Name: JYXP20220700111-sequence listing.xml

Software Name: WIPO Sequence

Software Version: 2.1.2

Production Date: 2022-08-23

General Information:

Current application / Applicant file reference:

JYXP20220700111

Applicant name: Zhejiang University

Applicant name / Language: en

Invention title: HIGH TEMPERATURE-RESISTANT AND UNIVERSAL MET-

AL-DEPENDENT PROTEASE AND USE THEREOF { en )

Sequence Total Quantity: 5

Sequences:

Sequence Number (ID): 1

Length: 136

Molecule Type: AA

Features Location/Qualifiers: - source, 1..136 > mol type, protein > note, Amino acid sequence of the protease DRJAMM > organism, Deinococcus radiodurans

Residues:

MLLTLPAPLR RALWAQVRRE LPRECVGALG GWVRGEQVQA HALYPLPNVA ADPEREYLAD 60

PGDLLRVVRA MOREGLDLVA LYHSHPHGPA APSASDRRLA AYPVPYLIAD PAAEVLRAYL 120

LPGGEEVEVR SADESN 136

Sequence Number (ID): 2

Length: 122

Molecule Type: AA

Features Location/Qualifiers: - source, 1..122 > mol type, protein > note, a DRJAMM-homologous protein from thermophilic archaea

> organism, unidentified

Residues:

MRVKVRKELF EYLLDLAASF HPKEFAGLLR EKNGVFEEVL FLPKGFFGTK SVYFDLNLLP 60

HDETIKGTVH SHPSPYPYPS KADLDFFSKF GGVHIIIAFP YTKESTKAFR SDGTELDIEI 120

VP 122

Sequence Number (ID): 3

Length: 161

Molecule Type: AA

Features Location/Qualifiers: - source, 1..161 > mol type, protein > note, a DRJAMM-homologous protein from halophilic archaea > organism, unidentified

Residues:

MRLFRSREVV GIAADALDFA LEASAETHPN EYMGLLRGEE ARRVGVDRDG YVVTDVLIIP 60

GTVSDPYSAT VRNDLVPNDF RAVGSIHSHP NGVLRPSDAD LDTFGSGRVH IIIGSPYGPD 120

DWEAFDQSGE VRDLDVLDVD LPDPESFEDE TODDIDAELD R 161

Sequence Number (ID): 4

Length: 37

Molecule Type: DNA

Features Location/Qualifiers: - source, 1..37 > mol type, other DNA > note, Primer DRJAMM-F > organism, synthetic construct

Residues: ccgegcggca gccatatggt gctcctgacc ctgcctg 37

Sequence Number (ID): 5

Length: 37

Molecule Type: DNA

Features Location/Qualifiers: - source, 1..37 > mol type, other DNA

> note, Primer DRJAMM-R > organism, synthetic construct

Residues: gagctcgaat tcggatcctc aattgctctc atcggcg 37

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At <INSDOualifier> 47 <INSDOQualifier name>mol type</INSDQualifier name>

CINSDQualifisr valuesprotein</INSDQualifier value» 4% </INSDQualifiers> an <INSDOualifier id="q5"> 51 <IN3DQualifier name>note</INSDQualifier name> 52 <INSDQualifier valuera DRJAMM-homologous protein from thermophilic archaea</INSDQualifisr valus> 53 </INSDQualifier>

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Ha </IN3DFeature guala> 58 </THSDFeatura> oo a0 </INSDSegy featurs-table>

EL

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LITPGTVSDPYSATVRNDLVPNDFRAVGSIHSHPNGVLRPSDADLDTFGSGRVHIIIGSPYGPDDWEAFDQSGE

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Lid <INSDQuaiifier id="gidx>

LiL <INSDOQualifier namerorganism</INSDQualifier name>

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LLS </INSDSeg>

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LAG <Hedgquencelata segusnceliNumec=NS"> 121 <INSDSeqg> 1322 <IN3DSeq length»37</INSDSeq length» 123 ZINSDSegq molitypes>DNAC/INSDSeq moltyper> 124 <IN3DSeq divisior>PAT</INSDIeqg division»

Lan <INSDSeq feature-table> ze <INSDFeature>

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La

Claims

CONCLUSIESCONCLUSIONS

1. Recombinante vector van een hoge temperatuur-resistente en uni- versele metaalafhankelijke protease, waarbij een hoge temperatuur- resistente protease-gen is ingevoegd tussen Ndel en BamHI re- strictieplaatsen op een plasmide pET28a, zodanig dat het gen stroomafwaarts van een T7-promoter wordt gelokaliseerd en wordt gereguleerd door de promotor om de recombinante vector te verkrij- gen; en het hoge temperatuur-resistente en universele metaalafhankelijke protease DRJAMM een aminozuursequentie heeft die wordt weergegeven in SEQ ID NR: 1.1. Recombinant vector of a high-temperature-resistant and universal metal-dependent protease, wherein a high-temperature-resistant protease gene is inserted between NdeI and BamHI restriction sites on a plasmid pET28a, such that the gene is downstream of a T7 promoter is located and regulated by the promoter to obtain the recombinant vector; and the high temperature-resistant and universal metal-dependent protease DRJAMM has an amino acid sequence shown in SEQ ID NO: 1.

2. Werkwijze voor het in vitro zuiveren van eiwitten van de recom- binante vector van het hoge temperatuur-resistente en universele metaalafhankelijke protease volgens conclusie 1, omvattende: het transformeren van de recombinante vector in E. coli BL21{DE3) voor expressie, het uitvoeren van inductie met 0,4 mM isopropyl-B-d- thiogalactoside (IPTG), en behandeling door een nikkelkolom, een waterbad van 70 °C, en een moleculaire zeef om een DRJAMM-eiwit te verkrijgen met een zuiverheid van niet minder dan 95%.A method for in vitro purification of proteins from the recombinant vector of the high temperature-resistant and universal metal-dependent protease according to claim 1, comprising: transforming the recombinant vector into E. coli BL21{DE3) for expression, performing induction with 0.4 mM isopropyl-β-d-thiogalactoside (IPTG), and treatment through a nickel column, a 70 °C water bath, and a molecular sieve to obtain a DRJAMM protein with a purity of not less than 95% .

3. Reactiebuffer van de recombinante vector van de hoge tempera- tuur-resistente en universele metaalafhankelijke protease volgens conclusie 1, omvattende 100 mM tot 500 mM NaCl, 10 mM tot 50 mM Tris-HCl pH 8,0, 0,2 mM tot 1 mM dithiothreitol (DTT), 0,2 mM tot 2 mM ethyleendiaminetetra-azijnzuur (EDTA), en 0,02 mM tot 1 mM metaalionen.3. Reaction buffer of the recombinant vector of the high temperature-resistant and universal metal-dependent protease according to claim 1, comprising 100 mM to 500 mM NaCl, 10 mM to 50 mM Tris-HCl pH 8.0, 0.2 mM to 1 mM dithiothreitol (DTT), 0.2 mM to 2 mM ethylenediaminetetraacetic acid (EDTA), and 0.02 mM to 1 mM metal ions.

4. Reactiebuffer volgens conclusie 3, waarbij de metaalionen een of meer metaalionen omvatten, gekozen uit de groep bestaande uit zn, Mn", Mg*, Ba*%, Co, Fe%, Ni‘, ca*%, sr” en cd“.4. Reaction buffer according to claim 3, wherein the metal ions comprise one or more metal ions selected from the group consisting of zn, Mn", Mg*, Ba*%, Co, Fe%, Ni', ca*%, sr" and cd “.

5. Werkwijze voor het optimaliseren van de reactie van de recombi- nante vector van het hoge temperatuur-resistente en universele me- taalafhankelijke protease volgens conclusie 1, omvattende: het uitvoeren van een reactie met behulp van de reactiebuffer volgens conclusie 3 met een substraateiwit DRMoaD-MoaE met een eiwit se- quentie-ID van NP 296326 bij 4 °C tot 100 °C gedurende 20 min tot 60 min.A method for optimizing the reaction of the recombinant vector of the high temperature-resistant and universal metal-dependent protease according to claim 1, comprising: carrying out a reaction using the reaction buffer according to claim 3 with a substrate protein DRMoaD -MoaE with a protein sequence ID of NP 296326 at 4°C to 100°C for 20 min to 60 min.

6. Werkwijze voor het optimaliseren van de reactie volgens conclu- sie 5, waarbij een enzymactiviteit een temperatuurbereik van 30 °C tot 100 °C heeft.A method for optimizing the reaction according to claim 5, wherein an enzyme activity has a temperature range of 30°C to 100°C.

7. Werkwijze voor het optimaliseren van de reactie volgens conclu- sie 5, waarbij een maximale enzymactiviteit een temperatuurbereik van 70 °C tot 100 °C heeft.A method for optimizing the reaction according to claim 5, wherein maximum enzyme activity has a temperature range of 70°C to 100°C.

8. Werkwijze voor het gebruik van de recombinante vector van de hoge temperatuur-resistente en universele metaalafhankelijke pro- tease volgens conclusie 1, omvattende: het ligeren van een speci- fieke aminozuursequentie die wordt herkend door een DRJAMM-eiwit met een doeleiwit om splitsing of zuivering van het doeleiwit bij hoge temperatuur uit te voeren.A method of using the recombinant vector of the high temperature-resistant and universal metal-dependent protease according to claim 1, comprising: ligating a specific amino acid sequence recognized by a DRJAMM protein with a target protein to achieve cleavage or purification of the target protein at high temperature.

9. Recombinante vector van de hoge temperatuur-resistente en uni- versele metaalafhankelijke protease volgens conclusie 1, waarbij de recombinante vector een gewenste waarde heeft voor gebruik in fundamenteel onderzoek en in de geneeskunde, de chemische indu- strie en de voedingsindustrie.The recombinant vector of the high temperature-resistant and universal metal-dependent protease according to claim 1, wherein the recombinant vector has a desirable value for use in basic research and in medicine, chemical industry and food industry.