Purification and characterization of chitin deacetylase from Scopulariopsis brevicaulis
Purification and characterization of chitin deacetylase from Scopulariopsis brevicaulis
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DOI：
10.1016/j.carbpol.2006.01.003
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发表时间：
2006-07-25
影响因子：
11.2
通讯作者：
Kennedy, John F.
中科院分区：
文献类型：
--
作者：
Cai, Jun;Yang, Jianhong;Kennedy, John F.
使用甲壳素作为培养基的唯一碳源，从短柄帚霉中产生了细胞外甲壳素脱乙酰酶。菌株29℃、200rpm振荡96h后，培养上清酶活力为10～11单位ml(-1)。通过硫酸铵沉淀、Sephadex G-25 和 G-100 柱层析，将该酶纯化 74 倍，产率 38%。通过 SDS-PAGE 和凝胶过滤色谱测定，表观分子量为 55 kDa，表明该酶作为单一组分存在。该酶对具有至少两个 N-乙酰基-葡糖胺残基的壳寡糖有活性，但活性随着 N-乙酰基-葡糖胺残基的数量而增加。以六-N-乙酰壳六糖为底物，确定酶活的最适pH为7.5，最适温度为55℃。在此条件下，对水溶性壳聚糖、黑曲霉甲壳素和虾结晶甲壳素进行酶活研究。通过FT-IR、XRD和电位滴定对产物结构进行了表征。结果表明，底物结晶度对酶活性有重要影响。该酶对来自黑曲霉菌丝体的无定形几丁质（脱酰率37%）和水溶性壳聚糖（33%）具有较高的脱乙酰活性，但较低。对虾结晶甲壳素的活性（3.7%）。 (c) 2006 Elsevier Ltd. 保留所有权利。
An extracellular chitin deacetylase from Scopulariopsis brevicaulis has been produced using chitin as sole carbon resource of culture medium. The enzyme activity was 10-11 units ml(-1) culture supernatant after the strain was shaken at 200 rpm and 29 degrees C for 96 h. The enzyme was purified 74-fold at 38% yield through ammonium sulfate precipitation, and Sephadex G-25, and G-100 column chromatography. The apparent molecular weight of 55 kDa, as determined by SDS-PAGE and gel filtration chromatography, suggested that the enzyme exists as a single component. The enzyme was active on chitooligosacchari des with at least two N-acetyl-glucosamine residues, but the activity increased with the number of Nacetyl-glucosamine residues. When hexa-N-acetylchitohexaose was used as substrate, the optimum pH for enzyme activity was determined to be 7.5, and the optimum temperature was 55 degrees C. Under these conditions, the activity of enzyme was studied on water-soluble chitosan, chitin from Aspergillus niger and shrimp crystalline chitin. The structures of products were characterized by FT-IR, XRD and potentiometric fitration. The results indicated that degree of substrate crystallinity had an important effect on enzyme activity. The enzyme had high deacetylating activity on amorphous chitin from A. niger mycelium (37% deacylation) and water-soluble chitosan (33%) but low. activity on shrimp crystalline chitin (3.7%). (c) 2006 Elsevier Ltd. All rights reserved.