WO2015196829A1 - 蛋白酶k在皮肤保健和化妆品领域中的应用 - Google Patents

蛋白酶k在皮肤保健和化妆品领域中的应用 Download PDF

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WO2015196829A1
WO2015196829A1 PCT/CN2015/073796 CN2015073796W WO2015196829A1 WO 2015196829 A1 WO2015196829 A1 WO 2015196829A1 CN 2015073796 W CN2015073796 W CN 2015073796W WO 2015196829 A1 WO2015196829 A1 WO 2015196829A1
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proteinase
mutation
skin
cosmetic composition
protease
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French (fr)
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安海谦
鲁刚伟
冉波
任玉珍
张贵财
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金普诺安蛋白质工程技术（北京）有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q9/00—Preparations for removing hair or for aiding hair removal

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  • the present invention relates to the use of native proteinase K and proteinase K mutants in the cosmetic and cosmetic industries. More specifically, the present invention relates to the use of native proteinase K and genetically engineered mutant recombinant proteinase K in the cosmetic and cosmetic industries for the removal of aged keratinocytes.
  • proteases are safe and free of toxins, they have strong hydrolysis ability to animal and vegetable proteins and peptides.
  • papain which has been applied to skin beauty, acts on the stratum corneum of human skin to promote its decomposition, degradation, and removal, and promotes cell growth.
  • Papain can also directly hydrolyze dark brown casein to pale or Colorless tyrosine, a hydrolyzate that forms a thin film of amino acid derivatives on the surface of the skin, keeping the skin moist and smooth.
  • US6846485B2 discloses the use of serine proteases derived from fish in pharmaceuticals and cosmetics.
  • the document discloses the treatment of various diseases and disorders (such as inflammatory diseases, by viruses, using serine proteases extracted from fish, including trypsin and chymotrypsin extracted from salmon (such as Atlantic salmon). Infectious diseases caused by bacteria and fungi, etc.) and pharmaceutical compositions containing these proteases.
  • Protease K in the protease family is a serine protease capable of digesting disulfide-rich natural keratin derived from Tritirachium album limber (Ebeling W. et al., Eur J Biochem. 1974 Aug 15; 47(1): 91-7).
  • the biggest feature of proteinase K is its broad-spectrum and high-efficiency cleavage ability for natural proteins, which is the most active of the existing proteases.
  • the enzyme prefers to cleave fatty amino acids and aromatic amino acids, particularly the carboxy terminal peptide bonds of alanine.
  • proteinase K is very stable, active at pH 4 to pH 12, temperature range 20 ° C ⁇ 65 ° C, in a variety of chemical agents that can denature proteins (such as SDS, urea, chelating agents (such as EDTA) and sulfhydryl reagents (such as trypsin inhibitors or chymotrypsin inhibitors) also have the ability to cleave proteins. That is, proteinase K is still active under complex solution buffer conditions in which other protease species have been inactivated.
  • denature proteins such as SDS, urea, chelating agents (such as EDTA) and sulfhydryl reagents (such as trypsin inhibitors or chymotrypsin inhibitors)
  • sulfhydryl reagents such as trypsin inhibitors or chymotrypsin inhibitors
  • Proteinase K has the potential to replace existing proteases in the washing, tanning and food feed industries to achieve more efficient and low cost.
  • the application of proteinase K has not only been limited to medical diagnosis and scientific research.
  • proteinase K can also replace other proteases with narrow scope of action, and is used in industrial tanning, papermaking, feed and other industries.
  • proteinase K can also replace raw materials that may be contaminated with conventional chemical methods to remove unwanted protein impurities.
  • the skin is divided into epidermis, dermis and subcutaneous tissue.
  • the epidermis of the skin is further divided into five layers, from bottom to top, the basal layer, the spinous cell layer, the granular layer, the transparent layer and the stratum corneum.
  • the basal layer of skin is the "origin of the skin".
  • the nascent skin cells gradually leave the basal layer, move to the epidermis of the outer layer, become a spine cell, and then become a granule.
  • the cells, finally completely keratinized into keratinocytes then gradually move to the outermost layer of the stratum corneum until they fall off the surface of the skin.
  • the stratum corneum is composed of multiple layers of flat keratinocytes.
  • the cells are completely keratinized, dry and hard, without nucleus and organelles, and the cell outline is unclear.
  • the skin has a thick cuticle, and the skin loses its luster, gray, peeling, wrinkles, and acne.
  • proteinase K has a similar effect to papain, and can effectively remove the skin aging cuticle, thereby restoring the skin to tenderness and luster; in addition, proteinase K is more stable at room temperature, and its action is extreme. pH, salt ions, detergents and the like are still very effective in the case of other proteases. According to reports in the literature, proteinase K is the most active of all proteases, so proteinase K can achieve the same effect as other proteases in a small amount. Less dosage means less possible allergic reactions and increased stability of liquid and semi-solid cosmetics. In addition, Proteinase K is highly safe, selectively removing the aged stratum corneum as needed, retaining part of the stratum corneum and all of the basal layer without damaging the remaining skin epidermis and dermis.
  • the present inventors have found that, in addition to the natural proteinase K, as long as the activity of the native proteinase K is retained, the gene mutant type recombinant proteinase K, particularly the gene mutant recombinant proteinase K having a higher activity than the native proteinase K, is keratinized in the skin. Layer removal can also show excellent results.
  • the present invention provides the use of the natural proteinase K and its mutant in the preparation of a cosmetic composition for removing aged keratinocytes, thereby completing the present invention.
  • mutant proteinase K as used herein has the same meaning as “wild type protease K” and is used to describe proteinase K which is naturally occurring in nature. Furthermore, the terms “gene mutant recombinant protein K” and “proteinase K mutant” are used interchangeably herein.
  • proteinase K As used herein, when referring to the term “proteinase K”, it is intended to encompass both native proteinase K and proteinase K mutants.
  • the native proteinase K precursor consists of 384 amino acids (SEQ ID NO. 1, UniProtKB/Swiss-Prot: P06873.2), including a signal peptide of 15 amino acids in length, a propeptide of 90 amino acids in length, and a length of 279 amino acids. Mature proteinase K.
  • native proteinase K can be used to prepare a cosmetic composition for removing aged keratinocytes.
  • the amino acid sequence of the native proteinase K is represented by SEQ ID NO. 2, the amino acid sequence consisting of 369 amino acids, including a propeptide of 90 amino acids in length and a mature protease of 279 amino acids in length. K.
  • such a protease K mutant can be used to prepare a cosmetic composition for removing aged keratinocytes as long as the protease K mutant retains the activity of the native proteinase K.
  • the proteinase K mutant may be a proteinase K mutant disclosed in CN102839165A having the following mutations based on the native proteinase K:
  • the Y mutation at position 151 of the native proteinase K amino acid sequence (SEQ ID NO. 2) is A, the K mutation at position 208 is H, the S mutation at position 273 is T, and the G mutation at position 293 is For A, the K mutation at position 332 is R, and the S mutation at position 337 is N; optionally, any one or more of the following mutations are also carried out:
  • the absolute position of the N-terminus of the proteinase K is expressed as a relative position compared to the amino acid sequence of SEQ ID NO.
  • the relative position of the amino acid is determined by aligning the amino acid sequence of the protease of interest with the amino acid sequence of SEQ ID NO.
  • mutProK6 mutProK7-1
  • mutProK7-2 mutProK8
  • mutProK8 mutProK8
  • the native proteinase K and the genetically modified recombinant protein K (such as, but not limited to, mutProK6, mutProK7-1, mutProK7-2, and mutProK8) used to prepare the cosmetic compositions of the present invention can be obtained by any method known to those skilled in the art.
  • the preparation method disclosed in CN102839165A can be employed.
  • the cosmetic composition of the present invention in addition to the proteinase K, it may contain other cosmetically acceptable active agents such as, but not limited to, substances having anti-aging activity; decolorizing, whitening or cleaning activity on the skin.
  • active agents such as, but not limited to, substances having anti-aging activity; decolorizing, whitening or cleaning activity on the skin.
  • Substance a substance having a slimming activity; a substance having moisturizing activity; a substance having calming, soothing or relaxing activity; a substance having an activity of stimulating skin microcirculation to improve the luster of a facial complexion; for caring for oily skin, a substance having sebum regulating activity; a substance for washing or purifying skin; having anti-free radical activity Substances; and any mixture of them.
  • the cosmetic composition of the present invention may further comprise at least one cosmetically acceptable adjuvant such as, but not limited to, a pigment, a surfactant, a viscous agent, a fragrance, a pH adjuster, an antioxidant. Preservatives, stabilizers, solubilizers, and any mixtures thereof.
  • the cosmetic composition may be a serum, an emulsion, a cream, a hydrogel, or the like, and may be, for example, a mask, an oil-in-water emulsion, an anhydrous composition, or may be in the form of a strip, In the form of a patch, or in the form of a lipstick, mascara or foundation.
  • the cosmetic composition can be applied by topical application to the skin of an individual.
  • the cosmetic composition can be applied to the skin of an individual over 35 years old, over 40 years old, over 45 years old, over 50 years old, over 55 years old, and even over 60 years old.
  • those skilled in the art can determine the content of proteinase K in the cosmetic composition as needed. For example, when the proteinase K content is too low, it may be difficult to effectively remove the skin-derived stratum corneum; when the proteinase K content is too high, an unnecessary cost increase may be caused.
  • the concentration of proteinase K may be 80 ⁇ g / ml - 100 ⁇ g / ml, 80 ⁇ g / ml - 150 ⁇ g / ml, 80 ⁇ g / ml - 200 ⁇ g / ml; alternatively may be 100 ⁇ g / ml - 150 ⁇ g / ml, 100 ⁇ g / ml - 200 ⁇ g / ml; or may be 150 ⁇ g / ml - 200 ⁇ g / ml.
  • Figure 1 shows the mutation sites in the amino acid sequences of the genetically modified recombinant proteases K mutProK6, mutProK7-1, mutProK7-2 and mutProK8 exemplarily used in the examples of the present invention.
  • 2A-2D show artificial skin of the positive control group (1% Triton-100 treatment), the negative control group (PBS treatment), and the experimental group (200 ⁇ g/ml and 2000 ⁇ g/ml natural proteinase K treatment) in Example 1 respectively.
  • the 3D model processes the results.
  • 3A-3B show the results of artificial skin 3D model treatment of the positive control group (1% Triton-100 treatment) and the negative control group (PBS treatment) in Example 2, respectively.
  • 4A-4G show artificial production of the experimental group (200 ⁇ g/ml, 150 ⁇ g/ml, 100 ⁇ g/ml, 80 ⁇ g/ml, 50 ⁇ g/ml, 20 ⁇ g/ml, and 2 ⁇ g/ml proteinase K mutant) in Example 2, respectively.
  • Skin 3D model processing results 200 ⁇ g/ml, 150 ⁇ g/ml, 100 ⁇ g/ml, 80 ⁇ g/ml, 50 ⁇ g/ml, 20 ⁇ g/ml, and 2 ⁇ g/ml proteinase K mutant
  • Fig. 5 shows a standard curve showing the relationship between the human IL-1 ⁇ concentration and the OD value (A450-A550) in Example 3.
  • Figure 6 shows the human IL-1 ⁇ concentration detected in different samples in Example 3.
  • test sample natural proteinase K, Sigma, prepared by diluting the solution (10 mM PBS + 30% glycerol) into 2 mg/ml and storing in a refrigerator at 4 °C.
  • Negative control phosphate buffered saline (PBS), Corning Cellgro, Cat# R21-040-CV.
  • Culture medium DMEM medium, Corning Cellgro, Cat#R10-014-CV.
  • the positive control group, the negative control group and the experimental group were designed, and two parallel experiments were performed in each group.
  • Positive control group 1% Triton-100, 2 skin tissues.
  • Negative control group PBS, 2 skin tissues.
  • the washed skin tissue was placed in a new 6-well cell culture plate, 0.9 ml of the culture solution was added to each well, placed in a 37 ° C carbon dioxide incubator (5% CO 2 ), and cultured for 24 hours;
  • the artificial skin 3D model was placed in a formalin solution, embedded in paraffin, sectioned, and finally subjected to HE staining.
  • Triton-100 positive control group significantly disrupted the basic structure of skin tissue, not only blurred the stratum corneum, but also reduced the basal cells that are the lowest source of skin cells in the epidermis (Fig. 2A); PBS (negative control group) had no visually visible effect on cells in all layers of skin tissue (Fig. 2B);
  • the skin sample treated for 30 minutes was able to effectively remove the dead cell layer on the surface of the stratum corneum (Fig. 2C); for the high-dose group of proteinase K of 2000 ⁇ g/ml, the skin sample was treated for 30 minutes. No dead cell layer was present on the surface of the stratum corneum, and keratinocytes in the lower part of the stratum corneum also partially disappeared (Fig. 2D). The results of this experiment show that native proteinase K can completely or partially remove aged stratum corneum cells.
  • mutProK6 a protease K mutant developed and produced by Jinpunoan Protein Technology Engineering (Beijing) Co., Ltd. was used, which has the following mutations based on the natural proteinase K:
  • the Y mutation at position 151 is A
  • the K mutation at position 208 is H
  • the S mutation at position 273 is T
  • the G mutation at position 293 is A
  • the K mutation at position 332 is R
  • 337th position The same experiment was performed with the S mutation to N), and consistent results were obtained. This proves that this effect is caused by the enzyme activity of the proteinase K itself. Therefore, as long as it is a protein having stable proteinase K activity, both the native proteinase K and the proteinase K mutant can digest the aged stratum corneum to achieve the effect of removing dead cells without damaging the underlying cells.
  • Test sample Protease K mutant mutProK6, developed and produced by Jinpu Nuoan Protein Technology Engineering (Beijing) Co., Ltd., diluted in solution (10 mM PBS + 30% glycerol) to the corresponding concentration and stored in a refrigerator at 4 ° C .
  • Negative control phosphate buffered saline (PBS), Corning Cellgro, Cat# R21-040-CV.
  • Culture medium DMEM medium, Corning Cellgro, Cat#R10-014-CV.
  • the positive control group, the negative control group and the experimental group were designed, and two parallel experiments were performed in each group.
  • Positive control group 1% Triton-100, 2 skin tissues.
  • Negative control group PBS, 2 skin tissues.
  • the washed skin tissue was placed in a new 6-well cell culture plate, 0.9 ml of the culture solution was added to each well, placed in a 37 ° C carbon dioxide incubator (5% CO 2 ), and cultured for 24 hours;
  • the artificial skin 3D model was placed in a formalin solution, embedded in paraffin, sectioned, and finally subjected to HE staining.
  • Triton-100 positive control group significantly destroys the basic structure of skin tissue, not only blurring the stratum corneum, but also reducing the basal cells that are the lowest source of skin cells in the epidermis.
  • PBS negative control group had no visually visible effect on cells of each layer of skin tissue (Fig. 3B).
  • IL-1 ⁇ is a commonly used model for evaluating whether cells are stimulated or not, if detected by ELISA.
  • An increase in the amount of secretion indicates that the cells are stimulated by the test substance to secrete the inflammatory factor; otherwise, the cells do not respond to the stimulatory response of the test substance.
  • the artificial skin 3D model of Beijing Fulong Kangtai Biotechnology Co., Ltd. can be used to detect the stimulating effect of the test substance: if the skin cells are stimulated, human IL-1 ⁇ is secreted; otherwise, the level of the factor remains normal.
  • the experimental sample was a cell culture supernatant of an artificial skin 3D model, and parallel experiments of two tissues were performed under each experimental condition, for a total of 12 skin tissues. There are 2 duplicate holes in the supernatant of each organization, a total of 24 holes.
  • Skin tissue specimen number Subject name Sample volume ( ⁇ l) 1,2 1% Triton-100 210 3,4 PBS 210 5,6 Proteinase K (150 ⁇ g/ml) 210 7,8 Proteinase K (100 ⁇ g/ml) 210 9,10 Proteinase K (80 ⁇ g/ml) 210 11,12 Proteinase K (50 ⁇ g/ml) 210
  • each reagent and standard according to the instructions of the ELISA kit. Among them, since the reagent cannot be directly dissolved at 37 ° C, the reagents are equilibrated to room temperature before the start of the experiment; when the reagent or sample is diluted, it is necessary to mix, and avoid foaming when mixing. Predict the sample content before the experiment. If the sample concentration is too high (more than 500pg/ml), the sample should be diluted so that the diluted sample meets the detection range of the kit, and then multiplied by the corresponding dilution factor.
  • Biotinylated anti-IL-1 ⁇ antibody 100 ⁇ l of diluted biotinylated anti-IL-1 ⁇ antibody per well, The plate was incubated with the plate and incubated for 60 minutes at room temperature.
  • Termination reaction After the color development was completed, 50 ⁇ l of the stop solution was added to each well.
  • Microplate reader reading The OD values were read at 450 nm and 550 nm, respectively.
  • Proteinase K had no effect on the amount of IL-1 ⁇ secreted by human skin cells at concentrations of 50 ⁇ g/ml, 80 ⁇ g/ml, 100 ⁇ g/ml, 150 ⁇ g/ml, and 200 ⁇ g/ml (results not shown in 200 ⁇ g/ml). It was shown that proteinase K did not induce the secretion of inflammatory factor IL-1 ⁇ under the above detection conditions, indicating that proteinase K had no stimulating effect on living cells in the cultured artificial skin 3D model, but only epidermal dead cells were removed.
  • proteinase K does not stimulate the skin, and the proteinase K concentration between 80-200 ⁇ g/ml can achieve the purpose of partial and total digestion of the stratum corneum of the skin, and does not exhibit irritating effect on the skin. Actually, you need to choose different doses to use.
  • Example 3 the artificial skin 3D model treated with the test product native proteinase K in Example 1 was used to detect the secretion of human IL-1 ⁇ by the action of proteinase K.
  • the natural proteinase K does not induce the secretion of the inflammatory factor IL-1 ⁇ in the experimental dosage range, and does not stimulate the skin, indicating that the natural proteinase K and the mutant proteinase K have a uniform effect on the digestion of the stratum corneum of the skin.
  • both the native proteinase K and the proteinase K mutant can achieve partial and total digestion of the skin stratum corneum in a suitable concentration range (between 80 and 200 ⁇ g/ml), and the skin is not It has a stimulating effect and has high application value and prospect in skin cosmetics.

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Abstract

蛋白酶K在制备用于去除老化角质细胞的化妆品组合物中的用途以及含有蛋白酶K作为活性成分的化妆品组合物。其中蛋白酶K的氨基酸序列如SEQ ID NO.2所示。

Description

蛋白酶K在皮肤保健和化妆品领域中的应用 技术领域
本发明涉及天然蛋白酶K和蛋白酶K突变体在美容、化妆品行业的应用。更具体而言，本发明涉及天然蛋白酶K及经过基因改造的突变型重组蛋白酶K在用于去除老化角质细胞的美容、化妆品行业中的应用。
背景技术
前瞻产业研究院发布的《2013-2017年中国化妆品终端渠道建设与投资前景分析报告》数据显示，2008-2012年，我国化妆品行业的市场规模总体上呈逐年上升态势。2008年，我国化妆品行业的市场规模仅为465.3亿元；到2012年，已上升为近843亿元，5年内增长了80％多。从增长速度来看，受金融危机的影响，2008年和2009年的增速较缓，2010-2012年的增速持续上升，2012年达到20.55％。从这些数据可以看出，化妆品产业的发展创新仍大有可为。
由于蛋白酶安全无毒素，对动植物蛋白、多肽具有较强的水解能力，目前在化妆品行业，已存在关于木瓜蛋白酶、菠萝蛋白酶、弹性蛋白酶、胰蛋白酶和糜蛋白酶的应用报道。
例如，目前已经应用于皮肤美容的木瓜蛋白酶通过作用于人体皮肤老化角质层，促使其分解、退化、去除，达到促进细胞生长的效果；木瓜蛋白酶还可直接将深褐色的酪蛋白水解为淡色或无色的酪氨酸，水解物在皮肤表层形成一层氨基酸衍生物薄膜，使皮肤保持润湿与光滑。
US6846485B2中则公开了来源于鱼类的丝氨酸蛋白酶在药物和化妆品方面的应用。具体而言，该文献公开了用从鱼提取的丝氨酸蛋白酶(包括从鳕鱼(如大西洋鳕鱼)中提取的胰蛋白酶和胰凝乳蛋白酶)治疗各种疾病和紊乱(如炎性疾病，由病毒、细菌和真菌引起的传染性疾病等)以及含有这些蛋白酶的药物组合物。
蛋白酶家族中的蛋白酶K是一种丝氨酸蛋白酶，能够消化富含二硫键的天然角蛋白，来源于林伯氏白色念球菌(Tritirachium album limber)(Ebeling W.等，Eur J Biochem.1974Aug 15；47(1):91-7)。蛋白酶K最大的特点在于对天然蛋白质具有广谱高效的切割能力，是现有蛋白酶中活性最高的一种。该酶偏好于切割脂肪族氨基酸和芳香族氨基酸，特别是丙氨酸的羧基端肽键。此外，蛋白酶K稳定性非常好，在pH 4～pH 12、温度范围20℃～65℃的条件下均有活性，在多种能够使蛋白质变性的化学试剂(如SDS、尿素、螯合剂(如EDTA)及巯基试剂(如胰蛋白酶抑制剂或糜蛋白酶抑制剂))中也具有切割蛋白质的能力。亦即，在其它蛋白酶种类已经失活的复杂溶液缓冲条件下，蛋白酶K仍然具有活性。
鉴于此特性，蛋白酶K在洗涤、制革和食品饲料行业具有替代现有蛋白酶，实现更高效和低成本的潜在能力。近年来，蛋白酶K的应用不仅仅局限在医疗诊断及科研领域。在实现规模化工业生产的前提下，蛋白酶K还可以替代作用范围较窄的其它蛋白酶，应用于工业制革、造纸、饲料等行业中。另外，蛋白酶K也可以替代传统的可能存在污染的化学方法处理原材料，去除不必要的蛋白质杂质。
尽管蛋白酶K具有以上出色活性，但目前国内外未见蛋白酶K应用于美容、化妆品行业的报道。
发明内容
皮肤分为表皮、真皮及皮下组织。皮肤的表皮又分为五层，从下往上依序是基底层、棘状细胞层、颗粒层、透明层及角质层。其中，皮肤基底层是皮肤细胞的“发源地”，新生的皮肤细胞随着其结构和功能的不断成熟、分化，逐步离开基底层，向外层表皮移动，变成棘细胞，再变成粒细胞，最后完全角化成为角质细胞，然后逐渐向角质层最外面移动，直至从皮肤表面脱落。角质层则由多层扁平的角质细胞组成，细胞已完全角化、干硬，无细胞核和细胞器，细胞轮廓不清。皮肤表面角质层厚，皮肤会失去光泽，灰暗、脱皮、产生皱纹、长痘等。
本发明人通过深入研究，发现蛋白酶K具有和木瓜蛋白酶类似的作用，能够有效地去除皮肤老化角质层，从而使皮肤恢复细嫩和光泽；此外，蛋白酶K在室温条件下更加稳定，其作用在极端pH、盐离子、除垢剂等不利于其它蛋白酶的情况下仍然十分有效。据文献报道，蛋白酶K是所有蛋白酶中活性最高的品种，所以蛋白酶K在用量较少的情况下也能达到与其它蛋白酶相同的效果。较少的用量意味着可以减少可能的过敏反应并增加液体和半固体化妆品的稳定性。此外，蛋白酶K还具有很高的安全性，能够根据需要选择性地去除老化角质层，保留部分角质层及全部基底层，不会伤及剩余的皮肤表皮层和真皮层。
进而，本发明人发现，除天然蛋白酶K外，只要保留了天然蛋白酶K的活性，基因突变型重组蛋白酶K、尤其是比天然蛋白酶K的活性更高的基因突变型重组蛋白酶K在皮肤老化角质层去除方面也能够显示出出色的效果。
基于以上研究，本发明提供了天然蛋白酶K及其突变体在制备用于去除老化角质细胞的化妆品组合物中的用途，从而完成了本发明。
具体实施方式
本文中使用的术语“天然蛋白酶K”具有与“野生型蛋白酶K”相同的含义，用于描述自然界中天然存在的蛋白酶K。此外，术语“基因突变型重组蛋白酶K”及“蛋白酶K突变体”在本文中可互换使用。
在本文中，当提及术语“蛋白酶K”时，旨在涵盖天然蛋白酶K和蛋白酶K突变体。
天然蛋白酶K前体由384个氨基酸组成(SEQ ID NO.1，UniProtKB/Swiss-Prot:P06873.2)，包括15个氨基酸长度的信号肽、90个氨基酸长度的前肽及279个氨基酸长度的成熟蛋白酶K。
根据本发明的一个实施方式，可将天然蛋白酶K用于制备去除老化角质细胞的化妆品组合物。根据本发明的一些实施方式，所述天然蛋白酶K的氨基酸序列如SEQ ID NO.2所示，该氨基酸序列由369个氨基酸组成，包括90个氨基酸长度的前肽及279个氨基酸长度的成熟蛋白酶K。
根据本发明的另一个实施方式，只要蛋白酶K突变体保留了天然蛋白酶K的活性，便可将此类蛋白酶K突变体用于制备去除老化角质细胞的化妆品组合物。根据本发明的一些实施方式，所述蛋白酶K突变体可以是CN102839165A中所公开的蛋白酶K突变体，其在天然蛋白酶K的基础之上具有如下突变：
(1)将天然蛋白酶K氨基酸序列(SEQ ID NO.2)中第151位的Y突变为A、第208位的K突变为H、第273位的S突变为T、第293位的G突变为A、第332位的K突变为R、第337位的S突变为N；任选地，还进行下述任意1种以上的突变：
(2)将第123位的S突变为A；
(3)将第180位的L突变为I。
在本发明中，“123位”、“151位”、“180位”、“208位”、“273位”、“293位”、“332位”及“337位”并不是必然表示从所述蛋白酶K的N端起的绝对位置，而是表示与SEQ ID NO.2的氨基酸序列相比的相对位置。例如，在含有SEQ ID NO.2的氨基酸序列的蛋白酶中，当该蛋白酶123位的N端某一位置缺失了一个氨基酸，上述的123位即变为122位。即使在这一情况下，所述从N端残基起计数为122位的氨基酸在本发明中仍为“123位”的氨基酸。通过将感兴趣的蛋白酶的氨基酸序列与SEQ ID NO.2的氨基酸序列进行比对而确定所述氨基酸的相对位置。
下文中，将上述蛋白酶K突变体分别称为mutProK6、mutProK7-1、mutProK7-2及mutProK8(参见图1)。
可通过本领域技术人员已知的任何方法获得用于制备本发明化妆品组合物的天然蛋白酶K以及基因突变型重组蛋白酶K(例如但不限于mutProK6、mutProK7-1、mutProK7-2和mutProK8)。优选地，可采用CN102839165A中所公开的制备方法。
在本发明的化妆品组合物的制备中，除蛋白酶K外，可使其包含化妆品上可接受的其它活性剂，例如但不限于，具有抗老化活性的物质；对皮肤具有脱色、美白或清洁活性的物质；具有减肥活性的物质；具有保湿活性的物质；具有镇定、舒缓或放松活性的物质；具有刺激皮肤微循环以改善特别是面部肤色光泽的活性的物质；用于对油性皮肤进行护理、具有皮脂调节活性的物质；用于清洗或净化皮肤的物质；具有抗自由基活性 的物质；以及它们的任意混合物。
在本发明的化妆品组合物的制备中，还可使其包含至少一种化妆品上可接受的辅剂，例如但不限于，色素、表面活性剂、粘性剂、芳香剂、pH调节剂、抗氧化剂、防腐剂、稳定剂、增溶剂以及它们的任意混合物。
在本发明的实施方式中，所述化妆品组合物可为精华素、乳液、霜、水凝胶等，例如可为面膜、水包油乳剂、无水组合物，或可为条状物形式、贴剂形式，或为唇膏、睫毛膏或粉底型化妆产品的形式。
根据本发明的一些实施方式，该化妆品组合物可通过局部施用到个体皮肤进行施用。根据本发明一个优选的实施方式，该化妆品组合物可施用到超过35岁、超过40岁、超过45岁、超过50岁、超过55岁乃至超过60岁的个体的皮肤上。
在本公开的基础之上，本领域技术人员能够根据需要确定化妆品组合物中蛋白酶K的含量。例如，当蛋白酶K含量过低时，可能难以有效去除皮肤老化角质层；当蛋白酶K含量过高时，可能会导致不必要的成本提高。根据本发明的一些实施方式，在所制备的用于去除老化角质细胞的化妆品组合物中，蛋白酶K的浓度可以为80μg/ml-100μg/ml、80μg/ml-150μg/ml、80μg/ml-200μg/ml；或者可以为100μg/ml-150μg/ml、100μg/ml-200μg/ml；又或者可以为150μg/ml-200μg/ml。
附图说明
从与附图结合的下列详细描述中，将更清楚地理解本发明的上述和其它目的、特征和其它优点，其中：
图1显示了本发明实施例中示例性使用的基因突变型重组蛋白酶K mutProK6、mutProK7-1、mutProK7-2及mutProK8氨基酸序列中的突变位点。
图2A-图2D分别显示了实施例1中阳性对照组(1％Triton-100处理)、阴性对照组(PBS处理)以及实验组(200μg/ml和2000μg/ml天然蛋白酶K处理)的人造皮肤3D模型处理结果。
图3A-图3B分别显示了实施例2中阳性对照组(1％Triton-100处理)以及阴性对照组(PBS处理)的人造皮肤3D模型处理结果。
图4A-图4G分别显示了实施例2中实验组(200μg/ml、150μg/ml、100μg/ml、80μg/ml、50μg/ml、20μg/ml和2μg/ml蛋白酶K突变体处理)的人造皮肤3D模型处理结果。
图5显示了实施例3中的表示人IL-1α浓度与OD值(A450-A550)之间关系的标准曲线。
图6显示了实施例3中的不同样品中检测到的人IL-1α浓度。
实施例
下面通过实施例具体描述本发明，然而应当理解本发明并不限于这些实施例。
实施例1
1.实验目的：
验证蛋白酶K是否对人造皮肤3D模型的角质层具有消化作用。
2.材料与方法：
2.1材料：
1)人造皮肤3D模型：北京富龙康泰生物技术有限公司制备。
2)供试品：天然蛋白酶K，Sigma公司，溶液稀释后(10mM PBS+30％甘油)配制成2mg/ml放置于4℃冰箱保存。
3)对照品：
阳性对照：Triton-100；国药Cat#30188993。
阴性对照：磷酸盐缓冲液(PBS)，Corning Cellgro，Cat#R21-040-CV。
4)培养液：DMEM培养液，Corning Cellgro，Cat#R10-014-CV。
2.2实验设计：
设计阳性对照组、阴性对照组和实验组，每组实验做两个平行实验。
1)阳性对照组：1％Triton-100，2个皮肤组织。
2)阴性对照组：PBS，2个皮肤组织。
3)实验组：2000μg/ml(高剂量组)及200μg/ml(低剂量组)的蛋白酶K溶液，每个处理条件分别用2个皮肤组织。
2.3实验方法：
1)准备2个6孔细胞培养板，每孔加入0.9ml培养液，将人造皮肤3D模型放入其中，37℃二氧化碳孵箱(5％CO2)培养两个小时；
2)在人造皮肤3D模型中央分别滴加对照品(1％Triton-100；PBS)或供试品(蛋白酶K溶液)10μl；
3)放置于37℃二氧化碳孵箱(5％CO2)中，孵育30分钟；
4)用PBS冲洗皮肤组织15遍；
5)将经冲洗的皮肤组织放入新的6孔细胞培养板中，每孔加入0.9ml培养液，放置于37℃二氧化碳孵箱(5％CO2)中，培养24小时；
6)将人造皮肤3D模型放入福尔马林溶液中固定、石蜡包埋、切片、最后进行HE染色。
3.实验结果：见图2(HE染色，10X)。
从图2中的实验结果可以看出：
1％Triton-100(阳性对照组)显著破坏皮肤组织的基本结构，不仅使角质层变得模糊，还使得表皮中最底层的、作为皮肤细胞“发源地”的基底层细胞也有所减少(图2A)；PBS(阴性对照组)则对皮肤组织各层细胞均没有视觉上可见的影响(图2B)；
对于200μg/ml的低剂量组蛋白酶K，经过30分钟处理的皮肤样品能够有效去除角质层表面死细胞层(图2C)；对于2000μg/ml的高剂量组蛋白酶K，经过30分钟处理的皮肤样品未见到角质层表面死细胞层存在，并且角质层下部的角质细胞也部分消失(图2D)。该实验结果显示天然蛋白酶K能够全部或部分去除老化角质层细胞。
除上述购自Sigma公司的天然蛋白酶K外，还利用由金普诺安蛋白质技术工程(北京)有限公司研发和生产的蛋白酶K突变体(mutProK6，在天然蛋白酶K的基础之上具有如下突变：第151位的Y突变为A、第208位的K突变为H、第273位的S突变为T、第293位的G突变为A、第332位的K突变为R、且第337位的S突变为N)进行了同样的实验，得到了一致的结果。由此证明，这一效果是由蛋白酶K本身的酶活引起。因此，只要是具有稳定蛋白酶K活性的蛋白，无论是天然蛋白酶K、还是蛋白酶K突变体，均可以消化老化角质层，达到去除死细胞而不伤及底层细胞的效果。
为了探索更适宜的去除角质层表面死细胞层的条件，在下述实施例中测试了不同浓度蛋白酶K对人造皮肤3D模型的角质层消化作用。
实施例2
1.实验目的：
设计不同的实验组和对照组，检测蛋白酶K对人造皮肤3D模型的角质层消化作用。
2.材料与方法：
2.1材料：
1)人造皮肤3D模型：北京富龙康泰生物技术有限公司制备。
2)供试品：蛋白酶K突变体mutProK6，由金普诺安蛋白质技术工程(北京)有限公司研发和生产，溶液稀释后(10mM PBS+30％甘油)配制成相应浓度放置于4℃冰箱保存。
3)对照品：
阳性对照：Triton-100；国药Cat#30188993。
阴性对照：磷酸盐缓冲液(PBS)，Corning Cellgro，Cat#R21-040-CV。
4)培养液：DMEM培养液，Corning Cellgro，Cat#R10-014-CV。
2.2实验设计：
设计阳性对照组、阴性对照组和实验组，每组实验做两个平行实验。
1)阳性对照组：1％Triton-100，2个皮肤组织。
2)阴性对照组：PBS，2个皮肤组织。
3)实验组：200μg/ml、150μg/ml、100μg/ml、80μg/ml、50μg/ml、20μg/ml及2μg/ml的蛋白酶K溶液，每个处理条件分别用2个皮肤组织。
2.3实验方法：
1)准备2个6孔细胞培养板，每孔加入0.9ml培养液，将人造皮肤3D模型放入其中，37℃二氧化碳孵箱(5％CO2)培养两个小时；
2)在人造皮肤3D模型中央分别滴加对照品(1％Triton-100；PBS)或供试品(蛋白酶K溶液)10μl；
3)放置于37℃二氧化碳孵箱(5％CO2)中，孵育15分钟；
4)用PBS冲洗皮肤组织15遍；
5)将经冲洗的皮肤组织放入新的6孔细胞培养板中，每孔加入0.9ml培养液，放置于37℃二氧化碳孵箱(5％CO2)中，培养24小时；
6)将人造皮肤3D模型放入福尔马林溶液中固定、石蜡包埋、切片、最后进行HE染色。
3.实验结果：见图3-图4(HE染色，200X)。
从图3-图4中的实验结果可以看出：
1)1％Triton-100(阳性对照组)显著破坏皮肤组织的基本结构，不仅使角质层变得模糊，还使得表皮中最底层的、作为皮肤细胞“发源地”的基底层细胞也有所减少(图3A)；PBS(阴性对照组)则对皮肤组织各层细胞均没有视觉上可见的影响(图3B)。
2)200μg/ml的蛋白酶K处理基本去除了皮肤组织中的角质层细胞，对下层细胞没有影响(图4A)。
3)150μg/ml的蛋白酶K处理使皮肤组织中的角质层明显变薄，并且角质层形成明显的网状结构(图4B)。
4)100μg/ml的蛋白酶K处理使皮肤组织中的角质层被消化，并且角质层出现网状结构(图4C)。
4)80μg/ml的蛋白酶K处理使皮肤组织中的角质层略微变薄，但下部的角质细胞没有变化、也无网状结构出现(图4D)。
5)50μg/ml及以下浓度(20μg/ml及2μg/ml)的蛋白酶K处理过的皮肤样品，未见角质层有明显减少或破坏(图4E-图4G)。
上述实验结果证实了，选择合理浓度的蛋白酶K(80μg/ml-200μg/ml)可以消化老化角质层，达到去除死细胞而不伤及底层细胞的效果。
实施例3
IL-1α的产生是常用的评价细胞受刺激与否的模式指标，如果通过ELISA检测显示 其分泌量增加，则表明细胞受到受试物刺激而分泌该炎症因子；反之则说明细胞没有应答该受试物的刺激反应。北京富龙康泰生物技术有限公司的人造皮肤3D模型可用于检测受试物的刺激作用：如果皮肤细胞受到刺激，会分泌人IL-1α；反之，该因子的水平保持正常值。
1.实验目的：
设计不同的实验组和对照组，检测人造皮肤3D模型细胞在蛋白酶K作用下分泌人IL-1α的情况，从而确定蛋白酶K对人皮肤是否具有刺激作用。
2.实验材料及设备：
2.1ELISA检测试剂盒：人IL-1α试剂盒，北京富龙康泰生物技术有限公司：货号：FP-E01。
2.2仪器设备：M3读板机：Molecular Devices，型号：spectraMax M3。
2.3样品信息：
实验样品是人造皮肤3D模型的细胞培养上清，每个实验条件下进行两个组织的平行实验，共计12个皮肤组织。每个组织的上清设2个复孔，共24个孔。
在检测人IL-1α之前，样品储存在4℃冰箱中保存过夜。
表1.样品(上清)信息
皮肤组织标本号 受检物名称 样品体积(μl)
1，2 1％Triton-100 210
3，4 PBS 210
5，6 蛋白酶K(150μg/ml) 210
7，8 蛋白酶K(100μg/ml) 210
9，10 蛋白酶K(80μg/ml) 210
11，12 蛋白酶K(50μg/ml) 210
3.实验方法和步骤
3.1按照ELISA试剂盒的说明书，配制各试剂和标准品。其中，由于试剂不能直接在37℃溶解，在实验开始前将各试剂平衡至室温；试剂或样品稀释时，均需混匀，混匀时尽量避免起泡。实验前预测样品含量，如样品浓度过高(超过500pg/ml)应对样品进行稀释，以使稀释后的样品符合试剂盒的检测范围，计算时再乘以相应的稀释倍数。
3.2各样品和标准品均做复孔，根据样品的数量决定要使用的酶标包被板条。
3.3加样：将标准品、样品和空白对照按100μl/孔加入，用封板膜封好后，室温孵育60分钟。用ELISA试剂盒说明书最后一页的加样记录纸记录各孔加样的顺序。
3.4洗板：室温孵育60分钟后，弃去孔内液体，甩干，洗板5次，每次浸泡3分钟。洗板时每孔注入300μl洗涤液。最后一次洗板时，将孔内的液体彻底甩干，将酶标板在新鲜滤纸或医用纱布上面用力拍几下。
3.5生物素标记的抗IL-1α抗体：每孔加100μl稀释过的生物素标记的抗IL-1α抗体， 酶标板加上封板膜，室温孵育60分钟。
3.6洗板：重复步骤4。
3.7亲和素-HRP：最后一次甩干后，每孔加100μl稀释过的亲和素-HRP。酶标板加上封板膜，室温孵育30分钟。
3.8洗板：重复步骤4。
3.9显色：最后一次甩干后，每孔加100μl TMB底物液。室温避光孵育30分钟显色。
3.10终止反应：显色完全后，每孔加终止液50μl。
3.11读数及分析数据：
酶标仪读数：分别于450nm和550nm处读取OD值。
计算标准曲线：用标准品的浓度与OD值(OD450nm减OD550nm)计算出标准曲线的回归方程式(y＝0.002x+0.016，R2＝0.999，参见图5)。
计算样品浓度：将样品的OD值(OD450nm减OD 550nm)代入上述方程式(y＝OD值；x＝样品浓度)，计算出样品中IL-1α的浓度，再乘以稀释倍数，即为样品中IL-1α的实际浓度。
4.实验结果：
4.1标准曲线：如图5所示。
4.2人IL-1α在样品中检测到的实际浓度：如表2及图6所示。
表2.不同受检物对人IL-1α表达的影响(pg/ml，平均值±标准差)
Figure PCTCN2015073796-appb-000001
5实验结论：
蛋白酶K在浓度为50μg/ml、80μg/ml、100μg/ml、150μg/ml及200μg/ml(200μg/ml的结果未示出)时对人皮肤细胞所分泌的IL-1α的量均没有影响，表明蛋白酶K在上述检测条件下不会诱导炎症因子IL-1α分泌，显示蛋白酶K对培养的人造皮肤3D模型中的活细胞没有刺激作用，而仅去除了表皮死细胞。因而，蛋白酶K对皮肤不呈现刺激作用，蛋白酶K浓度在80-200μg/ml之间可以达到对皮肤角质层部分和全部消化的目的，并且对皮肤不呈现刺激作用，在皮肤化妆品应用上可以根据实际需要选择不同剂量使用。
其中，表2及图5-图6中示例性示出的是蛋白酶K突变体mutProK6的处理结果。
实施例4
采用与实施例3中相同的方法，利用实施例1中经供试品天然蛋白酶K处理的人造皮肤3D模型检测其在蛋白酶K作用下分泌人IL-1α的情况。
结果如下表3所示：
表3.不同受检物对人IL-1α表达的影响(pg/ml，平均值±标准差)
Figure PCTCN2015073796-appb-000002
可见，天然蛋白酶K在实验用量范围内也不会诱导炎症因子IL-1α分泌，对皮肤也不呈现刺激作用，表明天然蛋白酶K和突变型蛋白酶K对皮肤角质层的消化具有一致效果。
由上述结果可知，无论是天然蛋白酶K、还是蛋白酶K突变体，都能够在合适的浓度范围内(80-200μg/ml之间)对皮肤角质层达到部分和全部消化的目的，并且对皮肤不呈现刺激作用，在皮肤化妆品方面具备较高应用价值和前景。
本领域技术人员将会理解，为了实现与本发明相同的目的，可容易地将在以上描述中所公开的概念和具体实施方式用作基础来修改或设计出其它实施方式。本领域技术人员还将理解的是，此类等同实施方式并未背离在所附权利要求中请求保护的本发明的精神和范围。

Claims (10)

  1. 蛋白酶K在制备用于去除老化角质细胞的化妆品组合物中的用途。
  2. 如权利要求1所述的用途，其中，所述蛋白酶K为天然蛋白酶K；
    优选地，所述天然蛋白酶K的氨基酸序列如SEQ ID NO.2所示。
  3. 如权利要求1所述的用途，其中，所述蛋白酶K为蛋白酶K突变体；
    优选地，所述蛋白酶K突变体具有如下突变：
    (1)将如SEQ ID NO.2所示的天然蛋白酶K氨基酸序列中第151位的Y突变为A、第208位的K突变为H、第273位的S突变为T、第293位的G突变为A、第332位的K突变为R、第337位的S突变为N；
    任选地，还进行下述任意1种以上的突变：
    (2)将第123位的S突变为A；
    (3)将第180位的L突变为I。
  4. 如权利要求1-3中任一项所述的用途，其中，所述化妆品组合物中的所述蛋白酶K的浓度为80-200μg/ml。
  5. 如权利要求1-3中任一项所述的用途，其中，所述化妆品组合物中进一步包含至少一种化妆品上可接受的辅剂；
    优选地，所述辅剂选自于色素、表面活性剂、粘性剂、芳香剂、pH调节剂、抗氧化剂、防腐剂、稳定剂、增溶剂，以及它们的任意混合物。
  6. 如权利要求1-3中任一项所述的用途，其中，所述化妆品组合物中进一步包含至少一种化妆品上可接受的其它活性剂；
    优选地，所述其它活性剂选自于具有抗老化活性的物质；对皮肤具有脱色、美白或清洁活性的物质；具有保湿活性的物质；具有镇定、舒缓或放松活性的物质；具有刺激皮肤微循环以改善面部肤色光泽的活性的物质；用于对油性皮肤进行护理、具有皮脂调节活性的物质；具有抗自由基活性的物质；以及它们的任意混合物。
  7. 含有蛋白酶K作为活性成分的化妆品组合物。
  8. 如权利要求7所述的化妆品组合物，其中，所述蛋白酶K为天然蛋白酶K；
    优选地，所述天然蛋白酶K的氨基酸序列如SEQ ID NO.2所示。
  9. 如权利要求7所述的化妆品组合物，其中，所述蛋白酶K为蛋白酶K突变体；
    优选地，所述蛋白酶K突变体具有如下突变：
    (1)将如SEQ ID NO.2所示的天然蛋白酶K氨基酸序列中第151位的Y突变为A、第208位的K突变为H、第273位的S突变为T、第293位的G突变为A、第332位的K突变为R、第337位的S突变为N；
    任选地，还进行下述任意1种以上的突变：
    (2)将第123位的S突变为A；
    (3)将第180位的L突变为I。
  10. 如权利要求7-9中任一项所述的化妆品组合物，其中，所述化妆品组合物中的所述蛋白酶K的浓度为80-200μg/ml。
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1253585A (zh) * 1997-01-10 2000-05-17 诺沃挪第克公司 一种用于皮肤护理的被修饰酶
CN102839165A (zh) * 2012-09-26 2012-12-26 金普诺安生物科技（苏州）有限公司 基因突变型重组蛋白酶k及其工业化生产方法

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IL117350A0 (en) * 1995-03-09 1996-07-23 Procter & Gamble Proteinase k variants having decreased adsorption and increased hydrolysis
DE10105911A1 (de) * 2001-02-09 2002-08-14 Roche Diagnostics Gmbh Expression der rekombinanten Proteinase K aus Tritirachium album in Hefe
US8005620B2 (en) * 2003-08-01 2011-08-23 Dna Twopointo Inc. Systems and methods for biopolymer engineering

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1253585A (zh) * 1997-01-10 2000-05-17 诺沃挪第克公司 一种用于皮肤护理的被修饰酶
CN102839165A (zh) * 2012-09-26 2012-12-26 金普诺安生物科技（苏州）有限公司 基因突变型重组蛋白酶k及其工业化生产方法

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