Thermolysin (EC 3.4.24.27; TML)   中文名称: 嗜热菌蛋白酶

Peptide bond

嗜热菌蛋白酶（100 μg，1 mL，64°C，4 小时）可选择性去除分离淀粉颗粒中 10 至 27 kD 之间的低分子量蛋白质 [2]。牛肝肌肉血浆蛋白可被嗜热菌蛋白酶水解[3]。

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最佳pH: 8.0。从pH值5到9.5相当稳定。
最佳温度：70℃

在 SHR 大鼠中，嗜热菌蛋白酶（50 mg/kg，口服，一次）可以降低收缩压[4]。急性毒性不是嗜热菌蛋白酶的问题。根据急性毒性评估，小鼠口服LD50超过24,000 mg/kg，大鼠口服LD50超过18,000 mg/kg[5]。

玉米淀粉颗粒（Zea mays）含有一组与淀粉基质紧密相关的多肽。玉米蛋白约占颗粒相关蛋白的50%，在本研究中确定了它们在淀粉颗粒中的空间分布。在亚糊化温度下，使用Thermolysin/嗜热蛋白酶 对淀粉颗粒进行蛋白水解，导致玉米蛋白被选择性去除，而32 kD或以上的颗粒相关蛋白，包括蜡质蛋白、淀粉合成酶I和淀粉分支酶IIb，仍然难以被蛋白水解。因此，来自玉米的颗粒相关蛋白由两个不同的类别组成，10至27 kD的表面定位的玉米蛋白和32 kD或更高的颗粒内在蛋白。通过比较匀浆的全胚乳和淀粉体分离的淀粉颗粒中δ -玉米蛋白的含量，探讨了表面定位的δ -玉米蛋白的来源。来自淀粉体的淀粉颗粒所含的玉米蛋白含量明显低于来自整个胚乳的淀粉颗粒，这表明玉米蛋白在淀粉体被破坏后附着在颗粒表面。交联实验表明，玉米蛋白以聚集体的形式沉积在颗粒表面。相反，颗粒内禀蛋白容易发生共价修饰，但不形成分子间交联。我们得出的结论是，单个颗粒内在蛋白质作为单体存在，而不是以淀粉基质内多聚簇的形式沉积。[2] <人力资源> 用Thermolysin酶在37℃水解2h，从牛肝脏中分离出肌浆蛋白。水解产物经分子量切断膜（MWCO）过滤得到滤液。未水解的肌浆蛋白、完全水解产物、10-kDa和3-kDa滤液的水活度（a(w)）均低于微生物生长所需的极限。采用2,2-二苯基-1-苦味肼基（DPPH）自由基清除活性、铁离子还原抗氧化能力（FRAP）和铁（2+）螯合能力测定对滤液和馏分的抗氧化活性进行了评价。采用反相高效液相色谱法对全水解产物、10-kDa和3-kDa滤液进行纯化。全水解产物、10-kDa和3-kDa滤液的肽含量采用Dumas法测定，各部分的肽含量采用电喷雾四极杆飞行时间（ESI-Q-TOF）质谱法进行表征，所得谱图采用Protein Lynx Global Server 2.4软件进行分析。和TurboSEQUEST。从每个部分中发现特征肽的氨基酸组成与先前报道的抗氧化肽之间存在相似性。本研究表明，利用Thermolysin酶，肉类副产品（如肝脏）可作为生产具有体外抗氧化活性的生物活性肽的原料。这是重要的，因为它为肉类加工商提供了一个潜在的机会，利用他们的废物流来产生生物活性肽，用于潜在的功能性食品用途。［３］

Animal/Disease Models: Spontaneously hypertensive rats (SHR) rats[4]
Doses: 50 mg/kg
Route of Administration: Oral administration (po), once
Experimental Results: Lowered systolic blood pressure in SHR rats, with maximal reduction by 22 mm in systolic blood pressure being observed 6 hrs (hours) after administration.

Thermolysin is a zinc metalloprotease that has potential uses in the food industry. The safety of thermolysin has not been demonstrated before, and therefore a series of standard toxicological tests to assess its potential toxicity was undertaken. The thermolysin used in this study was derived from the thermophilic bacterium Geobacillus stearothermophilus, which had undergone chemical mutagenesis to generate strains with increased thermolysin production. Acute toxicity studies in rats and mice showed that thermolysin powder is not acutely toxic with an oral LD₅₀ of more than 18,000 mg/kg (2520 mg/kg thermolysin protein) in rats and more than 24,000 mg/kg (3360 mg/kg protein) in mice. Subchronic feeding studies in rats for 91 days at doses up to 1000 mg/kg (390 mg/kg protein) revealed no significant differences between treated and non-treated groups and a No Observed Effect Level (NOEL) of 1000 mg/kg (390 mg/kg protein) per day was established. Results from genotoxicity tests such as in vitro chromosomal aberration assay and in vivo mouse micronucleus were negative. Allergenicity sequence analysis revealed no evidence suggesting that thermolysin is an allergen. The data presented in this study support the conclusion that thermolysin is safe for use in food production.[5]

[1]. Bertusvan den Burg, et al. Chapter 111 - Thermolysin and Related Bacillus Metallopeptidases.
[2]. Mu-Forster C, et al. Surface localization of zein storage proteins in starch granules from maize endosperm. Proteolytic removal by thermolysin and in vitro cross-linking of granule-associated polypeptides. Plant Physiol. 1998 Apr;116(4):1563-71.
[3]. Di Bernardini R, et al. Isolation, purification and characterization of antioxidant peptidic fractions from a bovine liver sarcoplasmic protein thermolysin hydrolyzate. Peptides. 2011 Feb;32(2):388-400.
[4]. Fujita H, et al: a prodrug-type ACE-inhibitory peptide derived from fish protein. Immunopharmacology. 1999 Oct 15;44(1-2):123-7.
[5]. Ke Q, et al. Safety evaluation of a thermolysin enzyme produced from Geobacillus stearothermophilus. Food Chem Toxicol. 2013 Sep;59:541-8.

It has been previously documented that the thermolysin-digest of "Katsuo-bushi", a Japanese traditional food processed from dried bonito possesses potent inhibitory activity against angiotensin I-converting enzyme (ACE). The present authors isolated eight kinds of ACE-inhibitory peptides from it. Of these isolated peptides, LKPNM (IC50 = 2.4 microM) was found to be hydrolyzed by ACE to produce LKP (IC50 = 0.32 microM) with 8-fold higher ACE-inhibitory activity relative to the parent peptide or LKPNM, suggesting that LKPNM can be regarded as a prodrug-type ACE-inhibitory peptide. For assessment of relative antihypertensive activities of LKPNM and LKP to that of captopril, they were orally administered to SHR rats to monitor time-course changes of blood pressures, whereby it was evidenced that both LKPNM and captopril showed maximal decrease of blood pressure 4 h after oral administration and their efficacies lasted until 6 h post-administration. In sharp contrast, however, maximal reduction of blood pressure occurred as early as 2 h after administration of LKP. Minimum effective doses of LKPNM, LKP and captopril were 8, 2.25 and 1.25 mg/kg, respectively. When compared on molar basis, antihypertensive activities of LKPNM and LKP accounted for 66% and 91% relative to that of captopril, respectively, whereas in vitro ACE-inhibitory activities of LKPNM and LKP were no more than 0.92% and 7.73% compared with that of captopril (IC50 = 0.022 microM). It is of interest to note that both of these peptides exert remarkably higher antihypertensive activities in vivo despite weaker in vitro ACE-inhibitory effects, which was ascertained by using captopril as the reference drug. [4]

CAH2ZN

107.502875804901

9073-78-3

504687751

Typically exists as White to light yellow solid at room temperature

[CaH2].[Zn]

Thermolysin; Thermophilicbacterial protease; DTXSID801054296; EINECS 232-973-4;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。