Methods of inactivating a proteinase or an enzymatically active fragment thereof

Abstract

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 ≤about 80 μM; or ii) the concentration of monovalent salt in said composition is ≥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 ≤about 80 μM; or ii) the concentration of monovalent salt in said sample is ≥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 ≤about 80 μM; or ii) the concentration of monovalent salt in said sample is ≥about 20 mM.

Claims

1. A method of inactivating a proteinase or an enzymatically active fragment thereof in a sample, said proteinase comprising the amino acid sequence of SEQ ID NO: 1 or comprising an amino acid sequence which is at least about 90% identical to SEQ ID NO: 1, wherein said method comprises the step of heating the sample at a temperature of from about 53° C. to about 67° C. to inactivate said proteinase or enzymatically active fragment, and wherein i) the concentration of free calcium in said sample is ≤about 80 μM; or ii) the concentration of monovalent salt in said sample is ≥about 20 mM.

2. The method of claim 1, wherein said heating step comprises heating the sample at a temperature of from about 53° C. to about 58° C.

3. The method of claim 2, wherein the concentration of free calcium in the sample is ≤about 10 μM, or the concentration of monovalent salt in the sample is at least about 75 mM.

4. The method of claim 1, wherein said heating step comprises heating the sample at a temperature of from about 63° C. to about 67° C.

5. The method of claim 4, wherein the concentration of free calcium in the sample is ≤about 65 μM, or the concentration of monovalent salt in the sample is at least about 25 mM.

6. The method of claim 1, wherein said heating step comprises heating the sample at a temperature of from about 58° C. to about 63° C.

7. The method of claim 6, wherein said heating step comprises heating the sample for i) about 5 to 15 minutes, wherein the concentration of free calcium in the sample is ≤about 10 μM, or the concentration of monovalent salt in the sample is at least about 75 mM; or ii) about 10 to 20 minutes, wherein the concentration of free calcium in the sample is ≤about 35 μM, or the concentration of monovalent salt in the sample is at least about 50 mM; or i) about 20 to 40 minutes, wherein the concentration of free calcium in the sample is ≤about 65 μM, or the concentration of monovalent salt in the sample is at least about 25 mM.

8. The method of claim 1, wherein any remaining proteinase activity of the proteinase or enzymatically active fragment thereof after said heating step is ≤about 25% as compared to a control, wherein said remaining activity is determined by the following assay steps: i) incubating in a 1000 μl or 250 μl cuvette: 10 to 50 mU/mL heat-treated proteinase, 1 mM substrate Suc-Ala-Ala-Pro-Phe-pNA, ≤15 mM NaCl, 0.1 mM Tris-HCl of a pH of 8, 10 mM CaCl.sub.2), and optionally 1% DMSO; ii) assaying cleavage of the substrate to 4-nitroalinine by measuring the increase in absorbance at 410 nm (ε=8800 M.sup.−1.Math.cm.sup.−1) over 2 minutes via a spectrophotometer at a temperature ≤40° C., wherein one Unit is defined as an amount of enzyme that produces one μmol 4-nitroaniline per minute at the chosen temperature; and iii) comparing the activity observed in step ii) to the activity observed with the same amount of the proteinase that has not been heat-treated but has been otherwise kept under the same conditions as the heat-treated proteinase, by the same assay.

9. The method of claim 1, wherein said sample comprises: a) one or more further enzymes selected from the group consisting of: a nuclease, a DNA or RNA polymerase, a reverse transcriptase, a DNA ligase, an RNA ligase, a methylase, a transferase, a topoisomerase, a guanylyl transferase, a phosphatase, a transposase, a kinase, a helicase, a restriction enzyme, and a glycosylase; b) one or more nucleic acid molecules, and wherein the method comprises, subsequent to said step of heating the sample to inactivate said proteinase, a step of: i) nuclease-mediated digestion of the one or more nucleic acid molecules; ii) phosphorylation or de-phosphorylation of the one or more nucleic acid molecules; or iii) ligation of the one or more nucleic acid molecules; without prior removal or dilution of the proteinase or enzymatically active fragment thereof; c) one or more RNA molecules, and wherein the method comprises, subsequent to said step of heating the sample to inactivate said proteinase, a step of reverse transcription without prior removal or dilution of the proteinase or enzymatically active fragment thereof; d) one or more DNA molecules, and wherein the method comprises, subsequent to said step of heating the sample to inactivate said proteinase, a step of nucleic acid polymerisation, preferably amplification, without prior removal or dilution of the proteinase or enzymatically active fragment thereof; or e) one or more virus particles or cells, and the method comprises, subsequent to said step of heating the sample to inactivate said proteinase, a step of cell lysis without prior removal or dilution of the proteinase or enzymatically active fragment thereof.

Description

(1) The invention will now be described by way of non-limiting Examples with reference to the following figures in which:

(2) FIG. 1 shows the proteinase activity of Proteinase X and K at different temperatures. Activity is presented as % activity relative to maximum activity observed at 65° C. under standard assay conditions with 10 mM free Calcium.

(3) FIG. 2 shows the activity of Proteinase X at different temperatures with either 0 μM, 5 μM or 10 mM free calcium in the assay buffer. The results are presented relative (%) to the standard assay conditions at each temperature, which is 10 mM free calcium.

(4) FIG. 3 shows the activity of Proteinase K at different temperatures with either 0 μM, 5 μM or 10 mM free calcium in the assay buffer. The results are presented relative (%) to the standard assay conditions at each temperature, which is 10 mM free calcium.

(5) FIG. 4 shows the degree of inactivation of Proteinase X after heating at 60° C. for 15 and 30 minutes in the presence of different free calcium concentrations. Activity is presented as % remaining activity, i.e. relative to control (kept on ice, no heating step).

(6) FIG. 5 shows the degree of inactivation of Proteinase K after heating at 60° C. for 15 and 30 minutes in the presence of different free calcium concentrations. Activity is presented as % remaining activity, i.e. relative to control (kept on ice, no heating step).

(7) FIG. 6 shows the degree of inactivation of Proteinase X and Proteinase K after heating at 60° C. for 15 and 30 minutes in presence of varying free calcium concentrations. Activity is presented as % activity relative to maximum activity observed with the same proteinase in the same buffer (comprising 10 mM CaCl.sub.2) kept on ice without heat-treatment.

(8) FIG. 7 shows the effect of NaCl concentration on the thermolability profiles of Proteinase X and Proteinase K. Incubation for 30 minutes at indicated temperatures in the absence of free calcium and in the presence of 50 mM or 300 mM NaCl. Activity is presented as % activity relative to maximum activity observed with the same proteinase in the same buffer (comprising 10 mM CaCl.sub.2) kept on ice without heat-treatment.

(9) FIG. 8 shows the inactivation of Proteinase X at 50 and 60° C. in the presence of various concentrations of NaCl. Activity is presented as % activity relative to maximum activity observed with Proteinase X in the same buffer (comprising 0 M NaCl, 0.03 mM CaCl.sub.2) kept on ice without heat-treatment.

(10) FIG. 9 shows the inactivation of Proteinase X at 60° C. in the presence of various concentrations of NaCl. Activity is presented as % activity relative to maximum activity observed with Proteinase X in the same buffer (comprising 0 M NaCl, 30 μM CaCl.sub.2) kept on ice without heat-treatment.

EXAMPLES

Example 1: Proteinase Specific Activities

(11) In all Examples, Proteinase K was purchased from Thermo Fischer (prod. No. EO0491, 28.9 kDa) and Proteinase X was produced recombinantly in Pichia pastoris at ArcticZymes (Batch 1602-1, SEQ ID NO: 1).

(12) To determine the specific activity of the two proteinases (U per mg proteinase), proteinase concentrations in the solutions were first determined by using NanoDrop.

(13) Nanodrop is a spectrophotometric approach to quantifying protein concentration through measuring absorbance at a wavelength of 280 nm.

(14) Proteinase K was determined to be present at a concentration of 14.3 mg/ml. 10,000 fold stock dilutions (1.43 μg/ml) were used subsequently. Proteinase X was determined to be present at a concentration of 9.2 mg/ml. 1,000 fold stock dilutions (9.2 μg/ml) were used subsequently.

(15) The activity of Proteinase X and K (in U/mL) was determined using a standard kinetic peptide-based assay. In a 1000 μl cuvette was provided 0.4 μg/ml Proteinase X or 0.06 μg/ml Proteinase K 1 mM substrate Suc-Ala-Ala-Pro-Phe-pNA 12 mM NaCl, 0.1 M Tris-HCl pH 8, 10 mM CaCl.sub.2, 1% DMSO Total volume 1000 μl

(16) Enzymatic cleavage of the substrate Suc-Ala-Ala-Pro-Phe-NA to 4-nitrolaniline was assayed by measuring the increase in absorbance at 410 nm (EM 8.8) over two minutes using a UV-spectrophotometer (Ultrospec 2000, Pharmacia Biotec, Sweden) at 25° C. One Unit is defined as the amount of enzyme that produces one μmol 4-nitroaniline at 25° C. per minute.

(17) The following specific activities were determined (Table 1).

(18) TABLE-US-00008 TABLE 1 Specific activity of Proteinase X and K U/ml mg/ml Specific activity Proteinase X ~600 9.2  ~65 (U/mg) Proteinase K ~5700 14.3 ~400 (U/mg)

(19) The results were verified using a vial of Proteinase K from a different vendor (Sigma, 04850, measured to 400 U/mg).

Example 2: Effect of Free Calcium Concentration on Proteinase Activity

(20) The activity of Proteinase X and K was determined at various temperatures using a standard peptide-based assay: In a 1.5 ml cuvette was provided 0.37 μg/ml Proteinase X or 0.06 μg/ml Proteinase K (equivalent to 24 mU/mL proteinase) 1 mM substrate Suc-Ala-Ala-Pro-Phe-pNA 12 mM NaCl, 0.1 M Tris-HCl, pH 8.0, 10 mM CaCl.sub.2, 1% DMSO Total volume 1000 μl

(21) The cuvette was incubated at the indicated temperature for the duration of the assay. Enzymatic cleavage of the substrate Suc-Ala-Ala-Pro-Phe-NA to 4-nitrolaniline was assayed by measuring the increase in absorbance at 410 nm (EM 8.8) over time for 30 seconds in 1.5 mL Semi-micro cuvettes (Brand, Germany) using a UV-spectrophotometer (Ultrospec 2000, Pharmacia Biotech, Sweden).

(22) Activity was calculated as % relative activity compared to the maximum activity, which was observed at 65° C. Measurements above 65° C. were technically not possible. Prior art teaches that the temperature optima of both Proteinase X and K are located between 65 and 70° C.

(23) As shown in FIG. 1, the two proteinases have similar temperature-activity profiles in the presence of 10 mM calcium.

(24) Temperature-activity profiles of Proteinase X and K were also determined under low calcium (5 μM) and calcium free (0 μM) conditions. Again, 24 mU/mL proteinase (equivalent to 0.37 μg/ml Proteinase X or 0.06 μg/ml Proteinase K) was incubated with 1 mM substrate Suc-Ala-Ala-Pro-Phe-pNA at the indicated temperatures in buffer (0.1 M Tris-HCl, pH 8.0, 0 mM/0.005 mM/10 mM CaCl.sub.2, 1% DMSO, 12 mM NaCl); total volume 1000 μl.

(25) Enzymatic cleavage of the substrate Suc-Ala-Ala-Pro-Phe-NA to 4-nitrolaniline was assayed by measuring the increase in absorbance at 410 nm (EM 8.8) over time for 30 seconds using a UV-spectrophotometer.

(26) Activity was calculated as % relative activity compared to the activity observed at 65° C. in the presence of 10 mM CaCl.sub.2 (Table 2), as this was the highest activity observed.

(27) TABLE-US-00009 TABLE 2 Activity of Proteinase X and K relative to activity at 10 mM calcium and 65° C. (%) Prot X Prot K Temp (° C.) 25 35 45 55 65 25 35 45 55 65    0 mM Ca 29.7 47.0 65.1 81.8 82.6 29.4 29.4 64.1 78.0 90.1 0.005 mM Ca 29.1 43.8 64.6 80.3 88.7 30.8 30.8 66.8 82.3 94.2   10 mM Ca 30.2 46.9 72.0 86.3 100.0 31.5 31.5 67.9 85.9 100.0

(28) At each calcium concentration, the temperature profiles for both proteinases are similar, with the maximum activity being observed at 65° C. Reduction of calcium concentration appeared to result in only a small reduction in proteinase activity at some temperatures. The activity profiles of the two proteinases differed when activity was considered relative to the activity achieved using 10 mM calcium at each specific temperature. The results are shown in Tables 3 and 4 and FIGS. 2 and 3, which highlight any calcium dependent effect on proteinase activity at different temperatures.

(29) TABLE-US-00010 TABLE 3 Proteinase X activity relative to activity using 10 mM calcium at stated temperature. 25° C. 35° C. 45° C. 55° C. 65° C.    0 mM Ca 98.2 100.0 90.4 94.8 82.6 0.005 mM Ca 96.2 93.4 89.7 93.1 88.7   10 mM Ca 100.0 100.0 100.0 100.0 100.0

(30) TABLE-US-00011 TABLE 4 Proteinase K activity relative to activity using 10 mM calcium at stated temperature. 25° C. 35° C. 45° C. 55° C. 65° C.    0 mM Ca 93.4 93.4 94.4 90.7 90.1 0.005 mM Ca 97.6 97.6 98.4 95.8 94.2   10 mM Ca 100.0 100.0 100.0 100.0 100.0

(31) FIG. 2 and Table 3 show that for Proteinase X, a low calcium concentration or the absence of calcium results in some loss of activity (maximum decrease of <10%) at 55° C. and 45° C., with no discernible drop in activity at 25° C. or 35° C. A decrease in activity of approximately 20% was observed at 65° C. in the absence of calcium, and a decrease of approximately 10% was observed at 65° C. in low calcium (0.005 mM).

(32) In contrast, FIG. 3 and Table 4, show that the activity of Proteinase K at each temperature was largely unaffected by the calcium conditions, i.e. whether in the presence of high calcium concentrations, low calcium concentrations or in the absence of calcium, only a minimal degree of change in activity was observed with increasing temperature.

(33) Together, the data suggest that low calcium concentrations could induce heat-inactivation of Proteinase X to a larger extent than Proteinase K.

Example 3: Ca.SUP.2+ .Dependent Inducible Thermolability of Proteinases

(34) The calcium-dependent thermolability of Proteinases X and K was investigated further.

(35) Heat-Treatment Steps:

(36) The proteinases were incubated at 60° C. for 15 or 30 minutes in a PCR thermo cycler (Veriti, Applied Biosystems) in buffers comprising various different concentrations of free calcium. Buffers comprised 0.1 mg/ml proteinase X (equivalent to 6.5 U/ml Proteinase X initial activity) or 0.014 mg/ml Proteinase K (equivalent to 5.6 U/ml Proteinase K initial activity) 0.025 M Tris-HCl, pH8, 300 mM NaCl, CaCl.sub.2 (1 mM/0.25 mM/0.125 mM/0.063 mM/0.031 mM/0.016 mM/0.008 mM/0 mM) volume: 50 μl. After inactivation the samples were placed back on ice. Samples used as controls were kept on ice throughout.

(37) Assay of Remaining Activity

(38) After the heat-treatment steps, samples were diluted 1:10 in 0.025 M Tris-HCl, pH8, 300 mM NaCl. The dilution step was performed to reduce enzyme activity U/ml in the samples to bring them within the range that is detectable in a reaction assay.

(39) The remaining activity of the proteinases was assessed as follows: 0.4 μg/ml Proteinase X or 0.06 μg/ml Proteinase K (equivalent to 26 and 24 mU/ml initial activity, respectively) was incubated with 1 mM substrate Suc-Ala-Ala-Pro-Phe-pNA at 37° C. in standard reaction buffer (0.1 M Tris-HCl, pH 8.0, 10 mM CaCl.sub.2, 1% DMSO, 12 mM NaCl); total volume 250 μl.

(40) Enzymatic cleavage of the substrate Suc-Ala-Ala-Pro-Phe-NA to 4-nitrolaniline was assayed by measuring the increase in absorbance at 405 nm (EM 8.8) over time for 10 minutes with signal detection every 11 seconds using a multi-mode platereader (Synergy H1, BioTek, USA).

(41) The results are shown in FIGS. 4 and 5. Activity is displayed as percentage remaining activity as compared to control sample (same proteinase and buffer, kept on ice not exposed to heating step).

(42) As shown in FIG. 5, decreasing free calcium concentration does not affect heat-inactivation of Proteinase K, which loses approximately 40% activity in all samples incubated at 60° C. independent of the free Ca2.sup.+-concentration. Substantial inactivation (≥75%) of Proteinase K was not achieved under any conditions.

(43) In contrast, as shown in FIG. 4, decreasing the free calcium concentration led to increased thermolability of Proteinase X. Substantial inactivation (≥75%, i.e. less than 25% remaining activity) was achieved by heating at 60° C. for 30 minutes at free calcium concentrations ≤0.063 mM, and by heating at 60° C. for 15 minutes at free calcium concentrations ≤0.016 mM. ≥90% inactivation is preferred, and this was achieved with Proteinase X by heating at 60° C. for 30 minutes at free calcium concentrations ≤0.031 mM, or for 15 in the absence of free calcium.

(44) Thus, proteinase X is inducibly thermolabile in the presence of low calcium concentrations, whereas the thermolability of proteinase K is unaffected by calcium concentration.

(45) This difference is demonstrated further in FIG. 6, which shows the inactivation profiles of the two proteinases after heating at 60° C. for 15/30 minutes at various calcium concentrations.

Example 4: Free Calcium Dependent Inactivation Profile of Proteinases

(46) The effect of differing heat-treatment steps on the inactivation of Proteinases X and K was assessed. In the following experiment an upper free calcium limit of 5 μM was used and the heat-inactivation step was performed over a range of temperatures and heating times.

(47) Heat-Treatment Steps:

(48) The proteinases were incubated at various temperatures (45° C., 50° C., 55° C., 60° C., 65° c. and 70° C.) for various times (2, 5, 10, 15, 30 60 minutes) in buffers comprising various different concentrations of free calcium (CaCl.sub.2, 0 μM, 2.5 μM or 5 μM). Buffers further comprised 0.1 mg/ml (6.5 U/ml initial activity) Proteinase X or 0.016 mg/ml (6.4 U/ml initial activity) Proteinase K, 25 mM HEPES, pH 8, 100 mM NaCl, total volume: 50 μl.

(49) Proteinase K was dialysed against a Ca-free storage buffer before use to remove free calcium therein. After inactivation the samples were placed back on ice. Samples used as controls were kept on ice throughout.

(50) Assay of Remaining Activity

(51) After the heat-treatment steps, samples were diluted 1:20 in 50 mM HEPES, pH8, 100 mM NaCl. The dilution step was performed to reduce enzyme activity U/ml in the samples to bring them within the range that is detectable in a reaction assay.

(52) The remaining activity of the proteinases was assessed as follows:

(53) 0.2 μg/ml Proteinase X or 0.03 μg/ml Proteinase K (equivalent to 13 mU and 12 mU initial activity, respectively), was incubated with 1 mM substrate Suc-Ala-Ala-Pro-Phe-pNA at 37° C. in standard reaction buffer (0.1 M Tris-HCl, pH 8.0, 10 mM CaCl.sub.2, 1% DMSO, 4 mM NaCl); total volume 250 μl.

(54) Enzymatic cleavage of the substrate Suc-Ala-Ala-Pro-Phe-NA to 4-nitrolaniline was assayed by measuring the increase in absorbance at 405 nm (EM 8.8) over time for 10 minutes with signal detection every 11 seconds using a multi-mode platereader (Synergy H1, BioTek, USA). Remaining activity is as compared to the control sample kept on ice but otherwise identical to the tested samples.

(55) The results are shown in Tables 5 to 7.

(56) TABLE-US-00012 TABLE 5 Remaining Activity (Percent): Proteinase X, 0 μM CaCl.sub.2 Inacti- vation Temper- 45° 50° 55° 60° 65° 70° ature C. C. C. C. C. C. Inacti-  2 min 110.7 107.6 98.4 83.8 42.1    6.7 vation  5 min 101.6 83.8 80.4 46.3 8.3   1.6 Time 10 min 95.9 76.2 63.6 23.3 1.8   1.5 15 min 94.0 78.2 49.7 11.3 1.2   1.0 30 min 88.2 70.8 29.7  2.4 1.0   1.0 60 min 71.1 49.3 11.2  0.7 1.1   0.9 Ice 100.0 Half life [min] — 60 15   5  <2     <2   75% inactivation <60     <10     <5     <2   [min] 90% inactivation 60   15   <5     <2   [min]

(57) The above data demonstrates that in the absence of free calcium, ≥approximately 75% inactivation of Proteinase X is achieved:

(58) within 2 minutes when heating at at least 70° C.;

(59) within 5 minutes when heating at at least 65° C.;

(60) within 10 minutes when heating at at least 60° C.; and

(61) within 60 minutes when heating at at least 55° C.

(62) And that ≥approximately 90% inactivation of Proteinase X is achieved

(63) within 2 minutes when heating at at least 70° C.;

(64) within 5 minutes when heating at at least 65° C.;

(65) within 15 minutes when heating at at least 60° C.; and

(66) within 60 minutes when heating at at least 55° C.

(67) TABLE-US-00013 TABLE 6 Remaining Activity: Proteinase X, 2.5 μM CaCl.sub.2 Inacti- vation Temper- 45° 50° 55° 60° 65° 70° ature C. C. C. C. C. C. Inacti-  2 min Absorbance Detection Problems vation  5 min 117.0 107.8 83.3 55.7 14.1    1.5 Time 10 min 97.5 94.2 58.5 29.3 2.3   1.2 15 min 108.4 83.8 59.8 17.9 1.3   1.2 30 min 98.7 76.5 37.6  4.1 0.8   0.9 60 min 81.2 62.9 15.9  1.6 0.4   0.8 Ice 100.0 Half life [min] — >60 <30     5  <5     <5   75% inactivation — — <60     <15     <5     <5   [min] 90% inactivation 60   <30     <10     <5   [min]

(68) The above data demonstrates that at free calcium concentrations below 2.5 μM, ≥approximately 75% inactivation of Proteinase X is achieved:

(69) within 5 minutes when heating at at least 70° C.;

(70) within 5 minutes when heating at at least 65° C.;

(71) within 15 minutes when heating at at least 60° C.; and

(72) within 60 minutes when heating at at least 55° C.

(73) And that ≥approximately 90% inactivation of Proteinase X is achieved:

(74) within 5 minutes when heating at at least 70° C.;

(75) within 10 minutes when heating at at least 65° C.; and

(76) within 30 minutes when heating at at least 60° C.

(77) TABLE-US-00014 TABLE 7 Remaining Activity: Proteinase X, 5 μM CaCl.sub.2 Inacti- vation Temper- 45° 50° 55° 60° 65° 70° ature C. C. C. C. C. C. Inacti-  2 min Absorbance Detection Problems vation  5 min 119.3 100.7 93.2 59.9 17.0    1.2 Time 10 min 104.7 86.3 90.8 37.1 3.2   0.6 15 min 90.5 81.3 71.8 21.9 1.3   0.9 30 min 104.1 66.1 46.5  5.6 1.0   0.2 60 min 96.3 60.9 21.8  0.9 1.0   1.1 Ice 100.0 Half life [min] — >60 30   <10     <2     <5   75% inactivation — — <60     <15     <5     <5   [min] 90% inactivation >60     <30     <10     <5   [min]

(78) The above data demonstrates that at free calcium concentrations below 5 μM, approximately 75% inactivation of Proteinase X is achieved:

(79) within 5 minutes when heating at at least 70° C.;

(80) within 5 minutes when heating at at least 65° C.;

(81) within 15 minutes when heating at at least 60° C.; and

(82) within 60 minutes when heating at at least 55° C.

(83) And that ≥approximately 90% inactivation of Proteinase X is achieved:

(84) within 5 minutes when heating at at least 70° C.;

(85) within 10 minutes when heating at at least 65° C.; and

(86) within 30 minutes when heating at at least 60° C.

(87) Thus, at free calcium concentrations ≤5 μM, incubation at 70° C. achieved ≥95% inactivation of Proteinase X within 5 minutes, and incubation at 65° C. achieved ≥95% inactivation of Proteinase X within 10 minutes (within 5 minutes in the absence of free calcium).

(88) In presence of both 2.5 and 5 μM free calcium, incubation at 60° C. achieved ≥90% inactivation of Proteinase X within 30 minutes (within 15 in the absence of free calcium).

(89) Incubation at ≥55° C. for 60 minutes achieved 80% inactivation of Proteinase X in the presence of 5 μM free calcium, 85% inactivation in the presence of ≤2.5 μM free calcium and about 90% inactivation in the absence of free calcium.

(90) The same studies were performed using Proteinase K for comparison. The results are shown in Tables 8 to 10 below.

(91) TABLE-US-00015 TABLE 8 Remaining Activity: Proteinase K, 0 μM CaCl.sub.2 Inacti- vation Temper- 45° 50° 55° 60° 65° 70° ature C. C. C. C. C. C. Inacti-  2 min 77.8 61.0 108.5  86.3 47.6    8.4 vation  5 min 92.6 76.1 92.1 68.4 31.0    0.2 Time 10 min 96.8 86.1 87.7 55.6 8.5   0.1 15 min 83.1 88.1 80.8 42.9 2.7 −0.1 30 min 85.9 90.0 61.0 15.9 0.3 −0.1 60 min 84.0 61.4 19.6  1.7 0.2   0.1 Ice 100 Half life [min] — >60 <60     10   2   <2   75% inactivation — — <60     <30     <10     <2   [min] 90% inactivation >60     <60     <10     <2   [min]

(92) The above data demonstrates that in the absence of free calcium, ≥approximately 75% inactivation of Proteinase K is achieved:

(93) within 2 minutes when heating at at least 70° C.;

(94) within 10 minutes when heating at at least 65° C. (within 5 minutes for Prot X);

(95) within 30 minutes when heating at at least 60° C. (within 10 minutes for Prot X); and

(96) within 60 minutes when heating at at least 55° C. (less inactivation than with Prot X).

(97) And that ≥approximately 90% inactivation of Proteinase K is achieved:

(98) within 2 minutes when heating at at least 70° C.;

(99) within 10 minutes when heating at at least 65° C. (within 5 minutes for Prot X); and

(100) within 60 minutes when heating at at least 60° C. (within 15 minutes for Prot X).

(101) TABLE-US-00016 TABLE 9 Remaining Activity: Proteinase K, 2.5 μM CaCl.sub.2 Inacti- vation Temper- 45° 50° 55° 60° 65° 70° ature C. C. C. C. C. C. Inacti-  5 min 102.1 101.7 100.0  76.3 40.1   0.4 vation 10 min 96.2 97.3 86.2 60.3 14.3   3.3 Time 15 min 100.9 94.3 78.4 45.3  4.7   0.2 30 min 94.5 80.9 53.7 16.9  0.6   0.3 60 min 79.8 61.5 18.4  1.9  0.3   0.3 Ice 100 Half life [min] — >60 30   <15     <5    <5   75% inactivation — — <60     <30     <10     <5   [min] 90% inactivation >60     <60     <15     <5   [min]

(102) The above data demonstrates that at free calcium concentrations below 2.5 μM, ≥approximately 75% inactivation of Proteinase K is achieved:

(103) within 5 minutes when heating at at least 70° C.;

(104) within 10 minutes when heating at at least 65° C. (within 5 minutes for Prot X);

(105) within 30 minutes when heating at at least 60° C. (within 15 minutes for Prot X); and

(106) within 60 minutes when heating at at least 55° C. (less inactivation than with Prot X).

(107) And that ≥approximately 90% inactivation of Proteinase K is achieved:

(108) within 5 minutes when heating at at least 70° C.;

(109) within 15 minutes when heating at at least 65° C. (within 10 minutes for Prot X); and

(110) within 60 minutes when heating at at least 60° C. (within 30 minutes for Prot X).

(111) TABLE-US-00017 TABLE 10 Remaining Activity: Proteinase K, 5 μM CaCl.sub.2 Inacti- vation Temper- 45° 50° 55° 60° 65° 70° ature C. C. C. C. C. C. Inacti-  5 min 112.4 102.7 106.4  95.9 55.9   0.7 vation 10 min 110.5 99.9 99.5 65.4 13.2   0.5 Time 15 min 106.1 103.5 97.6 60.0  8.9   0.0 30 min 94.5 92.4 65.3 22.3  1.0   0.2 60 min 84.8 58.5 19.8  2.5  0.0 −0.2 Ice 100 Half life [min] — >60 <60     <30     5  <5   75% inactivation — — <60     <30     <10     <5   [min] 90% inactivation >60     <60     <15     <5   [min]

(112) The above data demonstrates that at free calcium concentrations below 5 μM, ≥approximately 75% inactivation of Proteinase K is achieved:

(113) within 5 minutes when heating at at least 70° C.;

(114) within 10 minutes when heating at at least 65° C. (within 5 minutes for Prot X);

(115) within 30 minutes when heating at at least 60° C. (within 15 minutes for Prot X); and

(116) within 60 minutes when heating at at least 55° C.

(117) And that ≥approximately 90% inactivation of Proteinase K is achieved with the following heating steps:

(118) within 5 minutes when heating at at least 70° C.;

(119) within 15 minutes when heating at at least 65° C. (within 10 minutes for Prot X); and

(120) within 60 minutes when heating at at least 60° C. (within 30 minutes for Prot X).

(121) Thus, the results show that at all free calcium concentrations, the required heating times to achieve the same degree of inactivation at a given temperature are substantially lower for Proteinase X than Proteinase K. Alternatively viewed, at the vast majority of heating times and temperatures tested, greater inactivation of Proteinase X than Proteinase K is achieved. This is due to the observed thermolability of Proteinase X being induced at low calcium concentrations, which is not observed for Proteinase K.

(122) Thus, the effect of calcium on the ability of proteinase K to be inactivated by heat treatment is significantly less marked than the effect on Proteinase X.

Example 5: Monovalent Salt-Dependent Inducible Thermolability of Proteinases

(123) To determine the effect of NaCl on the thermolability of Proteinases X and K, the inactivation profiles of both proteinases were determined at various temperatures in solutions comprising i) 50 mM (low salt conditions) or ii) 300 mM NaCl (high salt conditions).

(124) Heat-Treatment Steps:

(125) The proteinases were incubated at various temperatures (45° C., 50° C., 55° C., 60° C., 65° c. and 70° C.) for 30 minutes in buffers comprising 50 mM or 300 mM NaCl. Buffers comprised 0.1 mg/ml proteinase (equivalent to 6.5 U/ml Proteinase X or 40 U/ml Proteinase K), 25 mM HEPES, pH 8, 0 μM CaCl.sub.2; volume: 50 μl.

(126) After inactivation the samples were placed back on ice. Samples used as controls were kept on ice throughout.

(127) Assay of Remaining Activity

(128) After the heat-treatment steps, samples were diluted 1:10 or 1:100 for Proteinase X or Proteinase K, respectively, in 50 mM HEPES, pH8, 300 mM NaCl. The remaining activity of the proteinases was assessed as follows:

(129) 0.4 μg/ml Proteinase X (equivalent to initial activity of 26 mU/ml) or 0.04 μg/ml Proteinase K (equivalent to initial activity of 16 mU/ml) was incubated with 1 mM substrate Suc-Ala-Ala-Pro-Phe-pNA at 37° C. in standard reaction buffer (0.1 M Tris-HCl, pH 8.0, 10 mM CaCl.sub.2, 1% DMSO, 12 mM NaCl); total volume 250 μl.

(130) Enzymatic cleavage of the substrate Suc-Ala-Ala-Pro-Phe-NA to 4-nitrolaniline was assayed by measuring the increase in absorbance at 405 nm (EM 8.8) over time for 10 minutes with signal detection every 11 seconds using a multi-mode platereader (Synergy H1, BioTek, USA). Remaining activity is as compared to the control sample kept on ice but otherwise identical to the tested samples.

(131) As shown in FIG. 7, increasing NaCl concentration has an opposite effect on the thermolability of ProtX as compared to ProtK. High NaCl concentrations stabilizes Proteinase K at high temperatures, whereas high NaCl concentrations induces thermolability of Proteinase X.

(132) This result is particularly surprising. The proteinase X of SEQ ID NO: 1 was obtained from a salt water organism, and so would ordinarily be expected to tolerate high salt conditions. In contrast, Prot K is obtained from a non-marine source, the fungus Engyodontium album (formerly Tritirachium album) and would not be expected to be stabilised by high salt conditions.

Example 6: Monovalent Salt-Dependent Thermolability Profiles of Proteinases

(133) To investigate further the effect of NaCl on the thermolability of Proteinase X, a wider range of NaCl concentrations to that used in Example 5 was assessed.

(134) Heat-Treatment Steps:

(135) Proteinase X was incubated at 50° C. or 60° C. for 15 or 30 minutes in buffers comprising 0, 50, 150, 300 or 600 mM NaCl. Buffers comprised 0.1 mg/ml proteinase X (equivalent to 6.5 U/ml), 25 mM HEPES, pH 8, 0.03 mM CaCl.sub.2, volume: 50 μl.

(136) After inactivation the samples were placed back on ice. Samples used as controls were kept on ice throughout.

(137) Assay of Remaining Activity

(138) After the heat-treatment steps, samples were diluted 1:10 in 50 mM HEPES, pH 8, 300 mM NaCl. The remaining activity of the proteinases was assessed as follows:

(139) 0.4 μg/ml Proteinase X (equivalent to 26 mU/ml initial activity), was incubated with 1 mM substrate Suc-Ala-Ala-Pro-Phe-pNA at 37° C. in standard reaction buffer (0.1 M Tris-HCl, pH 8.0, 10 mM CaCl.sub.2, 1% DMSO, 12 mM NaCl); total volume 250 μl.

(140) Enzymatic cleavage of the substrate Suc-Ala-Ala-Pro-Phe-NA to 4-nitrolaniline was assayed by measuring the increase in absorbance at 405 nm (EM 8.8) over time for 10 minutes with signal detection every 11 seconds using a multi-mode platereader (Synergy H1, BioTek, USA). Remaining activity is as compared to the control sample kept on ice but otherwise identical to the tested samples.

(141) The results are shown in FIG. 8 and Table 11.

(142) TABLE-US-00018 TABLE 11 NaCl-dependent effect on thermolability of Proteinase X Inactivation Temp 50° C. 60° C. Ice Inactivation Time 15 min 30 min 15 min 30 min 30 min NaCl conc. 600 mM 79.5 66.1  2.9  0.4 82.3 300 mM 94.2 67.8 17.6  2.9 84.0 150 mM 97.0 72.3 32.9 15.9 86.5  50 mM 101.0 64.2 50.3 38.7 81.7  0 mM 115.7 94.9 86.2 67.9 100

(143) The above data demonstrates that at NaCl concentrations 150 mM, ≥approximately 80% inactivation of Proteinase X is achieved by heating at 60° C. within 30 minutes.

(144) The above data also demonstrates that at NaCl concentrations ≥300 mM, ≥approximately 95% inactivation of Proteinase X is achieved by heating at 60° C. within 30 minutes and ≥approximately 80% inactivation of Proteinase X is achieved by heating at 60° C. within 150 minutes.

(145) A plot of NaCl conc. (x-axis) vs. remaining activity after heating at 60° C. for 30/15 minutes (y-axis), FIG. 9, demonstrates that substantial inactivation (≥75%) is achieved by heating at 60° C. for 15 minutes with an NaCl concentration of at least about 210 mM, and heating for 30 minutes with an NaCl concentration of at least about 100 mM.

Example 7: Combined Effect of Free Calcium and Monovalent Salt Concentrations on Thermolability of Proteinases

(146) The above studies demonstrate that i) lowering the concentration of free calcium or ii) raising the concentration of NaCl induces thermolability of Proteinase X to a greater extent than Proteinase K. The combined effect of these conditions was subsequently investigated.

(147) Heat-Treatment Steps:

(148) Proteinases X and K were incubated at 60° C. for 30 minutes in buffers comprising varying concentrations of NaCl (0, 25, 50, 75, 100, 125 mM) and free calcium (0, 5, 10, 20 and 20 μM CaCl.sub.2). Buffers comprised 6.5 U/ml Proteinase X or 6.4 U/ml Proteinase K (equivalent to 0.1 mg/ml Proteinase X or 0.016 mg/ml Proteinase K), 25 mM HEPES, pH 8 volume: 50 μl.

(149) Proteinase K was dialysed against a Ca-free storage buffer before use to remove free calcium therein

(150) After inactivation the samples were placed back on ice. Samples used as controls were kept on ice throughout.

(151) Assay of Remaining Activity

(152) After the heat-treatment steps, samples were diluted 1:20 in 50 mM HEPES, pH 8, 100 mM NaCl. The remaining activity of the proteinases was assessed as follows:

(153) 0.2 μg/ml Proteinase X (equivalent to 13 mU/ml initial activity) or 0.03 μg/ml Proteinase K (equivalent to 12 mU/ml initial activity), was incubated with 1 mM substrate Suc-Ala-Ala-Pro-Phe-pNA at 37° C. in standard reaction buffer (0.1 M Tris-HCl, pH 8.0, 10 mM CaCl.sub.2, 1% DMSO, 4 mM NaCl); total volume 250 μl.

(154) Enzymatic cleavage of the substrate Suc-Ala-Ala-Pro-Phe-NA to 4-nitrolaniline was assayed by measuring the increase in absorbance at 405 nm (EM 8.8) over time for 10 minutes with signal detection every 11 seconds using a multi-mode platereader (Synergy H1, BioTek, USA). Remaining activity is as compared to the control sample kept on ice but otherwise identical to the tested samples.

(155) The results are shown in Tables 12 and 13

(156) TABLE-US-00019 TABLE 12 Combined effect of CaCl.sub.2 and NaCl on Proteinase X thermolability. CaCl.sub.2 NaCl 0 μM 5 μM 10 μM 20 μM 30 μM  0 mM 35.0 51.3 43.4 59.0 54.9  25 mM 20.0 31.3 35.0 42.2 44.2  50 mM 12.2 20.5 22.4 32.3 39.7  75 mM  4.8 12.6 15.5 25.0 32.6 100 mM  2.8  8.0 10.8 19.2 22.8 125 mM  1.9  4.5  6.6 13.3 16.8

(157) Substantial inactivation (75%) was achieved at all free calcium concentrations in the presence of ≥100 mM NaCl.

(158) Additionally, substantial inactivation (≥about 75%) was achieved with a maximum free calcium concentration of 20 μM in the presence of at least 75 mM NaCl.

(159) Additionally, substantial inactivation (≥about 75%) was achieved with a maximum free calcium concentration of 10 μM in the presence of at least 50 mM NaCl.

(160) Additionally, substantial inactivation (≥about 75%) was achieved with a maximum free calcium concentration of 5 μM in the presence of at least 50 mM NaCl.

(161) Additionally, substantial inactivation (≥about 75%) was achieved in the absence of free calcium and in the presence of at least 25 mM NaCl.

(162) Superior inactivation (≥about 90%) was achieved with a maximum free calcium concentration of 20 μM in the presence of at least 125 mM NaCl.

(163) Superior inactivation (≥about 90%) was achieved with a maximum free calcium concentration of 10 μM in the presence of at least 100 mM NaCl.

(164) Superior inactivation (≥about 90%) was achieved with a maximum free calcium concentration of 5 μM in the presence of at least 75 mM NaCl.

(165) In the absence of free calcium, superior inactivation (≥about 90%) was achieved in the presence of at least 50 mM NaCl.

(166) TABLE-US-00020 TABLE 13 Combined effect of CaCl.sub.2 and NaCl on Proteinase K thermolability. CaCl.sub.2 NaCl 0 μM 5 μM 10 μM 20 μM 30 μM  0 mM  2.3  5.7  6.4  8.9  8.4  25 mM  4.8  8.4  9.4 11.0 10.7  50 mm 10.4 13.6 15.5 20.6 15.5  75 mm 14.1 22.9 23.7 28.3 27.2 100 mM 20.2 24.8 31.3 36.9 34.0 125 mM 31.6 36.5 42.6 44.8 43.7

(167) The results demonstrate that Proteinase K has a very different thermolability profile to Proteinase X. Proteinase K becomes increasingly thermolabile under low salt conditions, is stabilised under high salt conditions and is largely unaffected by calcium concentration. Proteinase X on the other hand becomes increasingly thermolabile under high salt conditions and low free calcium conditions.