Arcticzymes as

Universitetet I Tromsø—norges Arktiske Universitet

Biotec Pharmacon Asa

Marit Sjo Lorentzen

Olav Lanes

Terese Solstad

Atle Noralf Larsen

Yvonne Piotrowski

Morten Elde

Nils Peder Willassen

Elisabeth Lill Andreassen

Bjørn Altermark

Linda Havdalen

Ingar Leiros

Ronny Helland

Bernd Ketelsen Striberny

Cathrine Pedersen

Jørn Remi Henriksen

Bjorn Altermark

The invention provides a composition comprising a proteinase or an enzymatically active fragment thereof, said proteinase comprising the amino acid sequence of SEQ ID NO: 1 or comprising an amino acid sequence which is at least about 70% identical to SEQ ID NO: 1, wherein i) the concentration of free calcium in said composition is &#x2264;about 80 &#x3bc;M; or ii) the concentration of monovalent salt in said composition is &#x2265;about 20 mM. Under such conditions, the proteinases and enzymatically active fragments thereof are inducibly thermolabile. The invention further provides samples comprising one or more polypeptides and a proteinase or an enzymatically active fragment thereof, said proteinase comprising the amino acid sequence of SEQ ID NO: 1 or comprising an amino acid sequence which is at least about 70% identical to SEQ ID NO: 1, wherein i) the concentration of free calcium in said sample is &#x2264;about 80 &#x3bc;M; or ii) the concentration of monovalent salt in said sample is &#x2265;about 20 mM. The invention further provides methods comprising the inactivation of such proteinases or enzymatically active fragments thereof, wherein said method comprises the step of heating the sample to inactivate said proteinase or enzymatically active fragment, and wherein i) the concentration of free calcium in said sample is &#x2264;about 80 &#x3bc;M; or ii) the concentration of monovalent salt in said sample is &#x2265;about 20 mM.

Methods of inactivating a proteinase or an enzymatically active fragment thereof

The invention provides an exonuclease or an enzymatically active fragment thereof, said exonuclease having the amino acid sequence of SEQ ID NO:1 or an amino acid sequence which is at least about 50% identical thereto, wherein said exonuclease or enzymatically active fragment thereof (i) is substantially irreversibly inactivated by heating at a temperature of about 55&#xb0; C. for 10 minutes in a buffer consisting of 10 mM Tris-HCl, pH 8.5 at 25&#xb0; C., 50 mM KCl and 5 mM MgCl; (ii) is substantially specific for single stranded DNA; and (iii) has a 3'-5&#x2032; exonuclease activity. The invention further provides a method of removing single stranded DNA from a sample, a method of nucleic acid amplification, a method of reverse transcription and a method of nucleic acid sequence analysis in which the exonuclease or enzymatically active fragment thereof is used. The invention still further provides nucleic acids encoding said exonuclease or an enzymatically active fragment thereof and kits or compositions comprising the same.

Thermolabile exonucleases

The invention provides an exonuclease or an enzymatically active fragment thereof, said exonuclease having the amino acid sequence of SEQ ID No. 1 or an amino acid sequence which is at least about 50% identical thereto, wherein said exonuclease or enzymatically active fragment thereof (i) is substantially irreversibly inactivated by heating at a temperature of about 55&#xb0; C. for 10 minutes in a buffer consisting of 10 mM Tris-HCI, pH 8.5 at 25&#xb0; C., 50 mM KCI and 5 mM MgCI; (ii) is substantially specific for single stranded DNA; and (iii) has a 3'-5&#x2032; exonuclease activity. The invention further provides a method of removing single stranded DNA from a sample, a method of nucleic acid amplification, a method of reverse transcription and a method of nucleic acid sequence analysis in which the exonuclease or enzymatically active fragment thereof is used. The invention still further provides nucleic acids encoding said exonuclease or an enzymatically active fragment thereof and kits or compositions comprising the same.

The present invention provides an endonuclease I or enzymatically active fragment thereof wherein said endonuclease I has the sequence of SEQ ID No. 4 or a sequence which is at least 70% identical thereto and wherein the amino acid residue which is immediately N-terminal of the FYCGC pentapeptide motif has been substituted with a residue which is negatively charged as well as nucleic acid molecules encoding these enzymes and methods of removing contaminating polynucleotides from a sample using these enzyme.

Endonucleases

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Krokelvdalen, Norway

Marit Sjo Lorentzen