Proteinase K NGS (powder)

NGS Proteinase K NGS (powder) is a stable serine protease with broad substrate specificity. Free of DNase, RNase, Nickase, transported in room temperature with shelf life 3 years. Enzymes ativity is ≥40U/mg. DNA residue ≤5 pg/mg, Bioburden: ≤ 50 CFU/g.

Cat No: HC4507A

Package: 1g/10g/100g/500g

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Product Description

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Product Tags

NGS Protease K is a stable serine protease with high enzyme activity and wide substrate specificity.The enzyme preferentially decomposes ester bonds and peptide bonds adjacent to the C-terminal of hydrophobic amino acids, sulfur-containing amino acids and aromatic amino acids. So, it is often used to degrade proteins into short peptides. NGS Protease K is a typical serine protease with the Asp₃₉-His₆₉-Ser₂₂₄ catalytic triad which is unique to serine proteases, and the catalytic center is surrounded by tow Ca²⁺ binding sites for stabilization, maintaining high enzyme activity under a wider range of conditions.

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Specification

Appearance	White to off-white amorphous powder, lyophilized
Specific activity	≥40U/mg solid
DNase	None detected
RNase	None detected
Bioburden	≤50CFU/g solid
Nucleic acid residue	<5pg/mg solid

Properties

Source	Tritirachium album
EC number	3.4.21.64 (Recombinant from Tritirachium album)
Molecular weight	29kDa (SDS-PAGE)
Isoelectric point	7.81 Fig.1
Optimum pH	7.0-12.0 (All perform high activity) Fig.2
Optimum temperature	65℃ Fig.3
pH Stability	pH 4.5-12.5 (25℃,16h) Fig.4
Thermal stability	Below 50℃ (pH 8.0, 30min) Fig.5
Storage stability	Stored at 25℃ for 12months Fig.6
Activators	SDS, urea
Inhibitors	Diisopropyl fluorophosphate; benzylsulfonyl fluoride

Storage Conditions

Store the lyophilized powder at -25~-15 ℃ for a long time away from light; After dissolution, aliquot into appropriate volume for short-term storage at 2-8℃ away from light or long-term storage at -25~-15 ℃ away from light.

Precautions

Wear protective gloves and goggles when using or weighing, and keep well ventilated after use. This product may cause skin allergic reaction and serious eye irritation. If inhaled, it may cause allergy or asthma symptoms or dyspnea. May cause respiratory irritation.

Unit definition

One unit of NGS Protease K is defined as the amount of enzyme required to hydrolyze casein into 1 μmol L-tyrosine under standard determination conditions.

Reagents preparation

Reagent	Manufacturer	Catalog
Casein technical from bovine milk	Sigma Aldrich	C7078
NaOH	Sinopharm Chemical Reagent Co., Ltd.	10019762
NaH₂PO₄ ·2H₂O	Sinopharm Chemical Reagent Co., Ltd.	20040718
Na2HPO4	Sinopharm Chemical Reagent Co., Ltd.	20040618
Trichloroacetic acid	Sinopharm Chemical Reagent Co., Ltd.	80132618
Sodium acetate	Sinopharm Chemical Reagent Co., Ltd.	10018818
Acetic acid	Sinopharm Chemical Reagent Co., Ltd.	10000218
HCl	Sinopharm Chemical Reagent Co., Ltd.	10011018
Sodium carbonate	Sinopharm Chemical Reagent Co., Ltd.	10019260
Foline-phenol	Sangon Biotech (Shanghai) Co., Ltd.	A500467-0100
L-tyrosine	Sigma	93829

Reagent I:

Substrate: 1% Casein from bovine milk solution: dissolve 1g bovine milk casein in 50ml of 0.1M sodium phosphate solution, pH 8.0, heat in water bath at 65-70 °C for 15mins, stir and dissolve, cool with water, adjusted by sodium hydroxide to pH 8.0, and dilute in to 100ml.

Reagent II:

TCA solution: 0.1M trichloroacetic acid, 0.2M sodium acetate and 0.3M acetic acid (weigh 1.64g trichloroacetic acid + 1.64g sodium acetate + 1.724mL acetic acid successively, add 50mL deionized water, adjust with HCl to pH 4.03, and dilute into 100ml).

Reagent III:

0.4m sodium carbonate solution (weigh 4.24g anhydrous sodium carbonate and dissolve in 100mL water)

Reagent IV:

Folin phenol reagent: dilute 5 times with deionized water.

Reagent V:

Enzyme diluent: 0.1 M sodium phosphate solution, pH 8.0.

Reagent VI:

L-tyrosine standard solution:0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol/ml L-tyrosine dissolved with 0.2M HCl.

Procedure

1. Turn on the UV-Vis spectrophotometer and select photometric measurement.

2. Set the wavelength as 660nm.

3. Turn on the water bath, set the temperature to 37℃, ensure the temperature unchanged for 3-5mins.

4. Preheat 0.5mL substrate in a 2mL centrifuge tube at 37℃ water bath for 10mins.

5. Extract 0.5mL diluted enzyme solution into the preheated centrifuge tube for 10 minutes. Set enzyme diluent as blank group.

6. Add 1.0 mL TCA reagent immediately after the reaction. Mix well and incubate in water bath for 30 minutes.

7. Centrifugate reaction solution.

8. Add the following components in the order specified.

Reagent	Volume
Supernatant	0.5 mL
0.4M Sodium carbonate	2.5 mL
Folin phenol reagent	0.5 mL

9. Mix well before incubating in water bath a 37℃ for 30 mins.

10. OD₆₆₀ was determined as OD₁; blank control group: Enzyme diluent is used to replace enzyme solution to determine OD₆₆₀ as OD₂, ΔOD=OD₁-OD₂.

11. L-tyrosine standard curve: 0.5mL different concentration L-tyrosine solution, 2.5mL 0.4M Sodium carbonate, 0.5mL Folin phenol reagent in 5mL centrifuge tube, incubate in 37℃ for 30mins, detect for OD₆₆₀ for different concentration of L-tyrosine, then obtained the standard curve Y=kX+b,where Y is the L-tyrosine concentration, X is OD₆₀₀.

Calculation

2: Total volume of reaction solution (mL)

0.5: Volume of enzyme solution (mL)

0.5: Reaction liquid volume used in chromogenic determination (mL)

10: Reaction time (min)

Df: Dilution multiple

C: Enzyme concentration (mg/mL)

Figures

Fig.1 DNA residue

Sample	Ave C4	Nucleic Acid Recovery(pg/mg)	Recovery(%)	Total Nucleic Acid ( pg/mg)
PRK	24.66	2.23	83%	2.687
PRK+STD2	18.723	126.728	—	—
STD1	12.955	—	—	—
STD2	16
STD3	19.125
STD4	23.135
STD5	26.625
RNA-Free H2O	Undetermined	—	—	—