Laboratory of Molecular Genetics of Microorganisms, Kazan (Volga Region) Federal University, Kazan 420008, Russia.
Department of Biophysics and Biotechnology, Voronezh State University, Voronezh 394018, Russia.
Mar Drugs. 2021 Mar 31;19(4):197. doi: 10.3390/md19040197.
Chitosan, the product of chitin deacetylation, is an excellent candidate for enzyme immobilization purposes. Here we demonstrate that papain, an endolytic cysteine protease (EC: 3.4.22.2) from latex immobilized on the matrixes of medium molecular (200 kDa) and high molecular (350 kDa) weight chitosans exhibits anti-biofilm activity and increases the antimicrobials efficiency against biofilm-embedded bacteria. Immobilization in glycine buffer (pH 9.0) allowed adsorption up to 30% of the total protein (mg g chitosan) and specific activity (U mg protein), leading to the preservation of more than 90% of the initial total activity (U mL). While optimal pH and temperature of the immobilized papain did not change, the immobilized enzyme exhibited elevated thermal stability and 6-7-fold longer half-life time in comparison with the soluble papain. While one-half of the total enzyme dissociates from both carriers in 24 h, this property could be used for wound-dressing materials design with dosed release of the enzyme to overcome the relatively high cytotoxicity of soluble papain. Our results indicate that both soluble and immobilized papain efficiently destroy biofilms formed by and . As a consequence, papain, both soluble and immobilized on medium molecular weight chitosan, is capable of potentiating the efficacy of antimicrobials against biofilm-embedded . Thus, papain immobilized on medium molecular weight chitosan appears a presumably beneficial agent for outer wound treatment for biofilms destruction, increasing antimicrobial treatment effectiveness.
壳聚糖是甲壳素脱乙酰化的产物，是酶固定化的理想候选物。在这里，我们证明了木瓜蛋白酶（一种内切半胱氨酸蛋白酶，EC：3.4.22.2）从乳胶中固定在中分子量（200 kDa）和高分子量（350 kDa）壳聚糖基质上，具有抗生物膜活性，并提高了抗生物膜嵌入细菌的抗菌剂效率。在 pH 值为 9.0 的甘氨酸缓冲液中固定化允许吸附高达 30%的总蛋白（mg g 壳聚糖）和比活度（U mg 蛋白），导致初始总活性（U mL）的保留率超过 90%。虽然固定化木瓜蛋白酶的最适 pH 值和温度没有改变，但固定化酶表现出更高的热稳定性和比游离木瓜蛋白酶长 6-7 倍的半衰期。虽然两种载体中有一半的总酶在 24 小时内解离，但这种性质可用于设计伤口敷料材料，以控制酶的剂量释放，从而克服游离木瓜蛋白酶相对较高的细胞毒性。我们的结果表明，游离和固定化的木瓜蛋白酶都能有效地破坏 和 形成的生物膜。因此，游离和固定在中分子量壳聚糖上的木瓜蛋白酶都能够增强抗菌剂对生物膜嵌入 的功效。因此，固定在中分子量壳聚糖上的木瓜蛋白酶似乎是一种用于破坏生物膜的外伤口处理的有益剂，提高了抗菌治疗的效果。
