Sinitsyna Olga A, Volkov Pavel V, Zorov Ivan N, Rozhkova Alexandra M, Emshanov Oleg V, Romanova Yulia M, Komarova Bozhena S, Novikova Natalia S, Nifantiev Nikolay E, Sinitsyn Arkady P
Department of Chemistry, M.V. Lomonosov Moscow State University, Moscow, Russia.
Federal Research Centre «Fundamentals of Biotechnology» of the Russian Academy of Sciences, Moscow, Russia.
Appl Environ Microbiol. 2025 Feb 19;91(2):e0022624. doi: 10.1128/aem.00226-24. Epub 2025 Jan 23.
The gene encoding fungus mutanase (MutA, GH71 family, α-1,3-glucanase, EC 3.2.1.59) was cloned and heterologously expressed by the highly productive fungus. strain secreted crude enzyme preparations with the recombinant MutA content of 40% of the total secreted protein, and the specific activity increased 150 folds compared to that of enzyme preparation obtained by the host strain. Homogeneous MutA had molecular mass of 70 kDa and displayed maximum of the activity on mutan at pH 5.0 and 50°C, with and being 1.0 g/L and 30 s, respectively. At 40-50°C, the MutA was stable for at least 3 h. Glucose was the main product of long-term mutan hydrolysis. HPLC analysis of hydrolysis product of oligo-α-(1→3)-D-glucosides bearing UV-detectable --cinnamoyl residue in the aglycon clearly indicated that MutA has an endo-processive hydrolytic mode of action. It was demonstrated that MutA can destroy the polysaccharide matrix of both gram-positive and gram-negative pathogenic bacteria biofilms.
The manuscript describes the properties of a novel recombinant GH71 mutanase Mut A from . Gene encoding mutanase was heterologously expressed in the host strain B1-537 (ΔniaD). The recipient strain has a high secretory ability and allowed to obtain preparations containing the target recombinant enzyme up to 80% of the total protein pool. MutA exhibited a high activity against mutan and negligible or zero activity toward other types of glucans including α-(1→4)-, β-(1→3)-, β-(1→4)-, and β-(1→6)-glucans. By using a series of synthetic oligo-α-(1→3)-D-glucosides, we demonstrated that MutA is an endo-processive enzyme, which hydrolyzes the internal glucosidic bonds and releases glucose from the reducing end sliding into the non-reducing end. MutA recognizes tetrasaccharide as a minimal substrate and hydrolyzes it to trisaccharide and glucose. The effectiveness of the use of MutA for the destruction of clinical isolates of gram-positive and gram-negative bacteria is also described.
编码真菌变聚糖酶（MutA，GH71家族，α-1,3-葡聚糖酶，EC 3.2.1.59）的基因被高产真菌克隆并进行了异源表达。该菌株分泌的粗酶制剂中重组MutA含量占总分泌蛋白的40%，与宿主菌株获得的酶制剂相比，比活性提高了150倍。纯的MutA分子量为70 kDa，在pH 5.0和50°C时对变聚糖表现出最大活性，Km和kcat分别为1.0 g/L和30 s。在40 - 五十°C时，MutA至少稳定3小时。葡萄糖是变聚糖长期水解的主要产物。对糖苷配基中带有可紫外检测的肉桂酰残基的寡聚-α-(1→3)-D-葡萄糖苷水解产物的HPLC分析清楚地表明，MutA具有内切渐进性水解作用模式。已证明MutA可破坏革兰氏阳性和革兰氏阴性病原菌生物膜的多糖基质。
该手稿描述了一种来自的新型重组GH71变聚糖酶Mut A的特性。编码变聚糖酶的基因在宿主菌株B1-537（ΔniaD）中进行了异源表达。受体菌株具有高分泌能力，能够获得目标重组酶含量高达总蛋白池80%的制剂。MutA对变聚糖表现出高活性，而对其他类型的葡聚糖，包括α-(1→4)-、β-(1→3)-、β-(1→4)-和β-(1→6)-葡聚糖的活性可忽略不计或为零。通过使用一系列合成的寡聚-α-(1→3)-D-葡萄糖苷，我们证明MutA是一种内切渐进性酶，它水解内部糖苷键并从还原端向非还原端滑动释放葡萄糖。MutA将四糖识别为最小底物并将其水解为三糖和葡萄糖。还描述了使用MutA破坏革兰氏阳性和革兰氏阴性细菌临床分离株的有效性。
