Identification of a novel endochitinase from a marine bacterium Vibrio proteolyticus strain No. 442.
Identification of a novel endochitinase from a marine bacterium Vibrio proteolyticus strain No. 442.
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DOI：
10.1016/j.bbapap.2007.06.003
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发表时间：
2007-09
期刊：
影响因子：
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通讯作者：
S. Itoi;Y. Kanomata;Yuki Koyama;K. Kadokura;S. Uchida;T. Nishio;T. Oku;H. Sugita
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文献类型：
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作者：
S. Itoi;Y. Kanomata;Yuki Koyama;K. Kadokura;S. Uchida;T. Nishio;T. Oku;H. Sugita
从溶蛋白弧菌中提取几丁质结合蛋白，经SDS-PAGE分析，其N-末端110-kDa的氨基酸序列与其它弧菌几丁质酶A前体蛋白N-末端22 - 41位氨基酸序列有85-90%的同源性。我们克隆了相应的基因，该基因编码一个含有26个残基的信号序列的850个氨基酸的推定蛋白。解蛋白弧菌的几丁质酶前体与副溶血弧菌、溶藻弧菌和弧菌的几丁质酶前体有78-80%的同源性。然而，在其他弧菌的几丁质酶前体中的R597和K598之间的C-末端加工的蛋白水解切割位点在解蛋白弧菌的氨基酸序列中没有观察到，而是具有序列R600和A601。随后，在大肠杆菌中产生全长和截短的几丁质酶。在大肠杆菌中表达的全长几丁质酶比活为1.25 × 10 - 6。大肠杆菌中的胶体和α-几丁质（不溶性底物）分别比C-末端截短的酶高17倍和20倍。然而，这两种重组体显示出相似的水解模式的六-N-乙酰基-壳六糖（可溶性底物），产生二-N-乙酰基-壳二糖作为主要产物的TLC分析。我们发现，解蛋白弧菌几丁质酶A的C-末端对于表达针对不溶性几丁质的高比活性是重要的。
Chitin binding proteins prepared from Vibrio proteolyticus were purified and the N-terminal amino-acid sequence of a protein from a 110-kDa band on SDS-PAGE was found to be 85–90% identical to the 22nd–41st residues of the N-termini of chitinase A precursor proteins from other vibrios. We cloned the corresponding gene, which encodes a putative protein of 850 amino acids containing a 26-residue signal sequence. The chitinase precursor from V. proteolyticus was 78–80% identical to those from Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio carchariae. However, the proteolytic cleavage site for C-terminal processing between R597and K598in the chitinase precursor of other vibrios was not observed in the amino acid sequence of V. proteolyticus, which instead had the sequence R600and A601. Subsequently, full-length and truncated chitinases were generated in Escherichia coli. The specific activity of full-length chitinase expressed in E. coli was 17- and 20-folds higher for colloidal and α-chitins (insoluble substrate), respectively, than that of the C-terminal truncated enzyme. However, both recombinants showed similar hydrolysis patterns of hexa-N-acetyl-chitohexaose (soluble substrate), producing di-N-acetyl-chitobiose as major product on TLC analysis. We showed that the C-terminus of the V. proteolyticus chitinase A was important for expression of high specific activity against insoluble chitins.