Chitin deacetylase (CDA;EC 3. 5. 1. 41) catalyzes the deacetylation of chitin. In this study, we successfully cloned and sequenced a chitin deacetylase gene from the red snow crab Chionoecetes japonicas. By using reve...Chitin deacetylase (CDA;EC 3. 5. 1. 41) catalyzes the deacetylation of chitin. In this study, we successfully cloned and sequenced a chitin deacetylase gene from the red snow crab Chionoecetes japonicas. By using reverse transcription-polymerase chain reaction (RT-PCR) and 5' and 3' rapid amplification of cDNA ends, we obtained a 2141-bp amplicon containing a chitin deacetylase gene (CjCDA) from the epidermis of C. japonicas. The amplicon contains a 1575-bp open reading frame that is predicted to encode a 525-amino acid protein. The structure predicted from the deduced amino acid sequence included an N-terminal signal peptide, chitin-binding domain (CBD), low-density lipoprotein receptor class A domain (LDL-A), and catalytic domain. Comparative analysis of the deduced amino acid sequence of CjCDA revealed the highest homology (74%) to gastrolith protein 59 of Cherax quadricarinatus. We used RT-PCR to evaluate the expression of CjCDA in various tissues of C. japonicas, and we observed that CjCDA was expressed only in the epidermis. A phylogenetic analysis, using the amino acid sequences of CjCDA and other known chitin deacetylases, showed that CjCDA belonged to a group of crustacean chitin deacetylases. To our knowledge, this is the first study reporting the cDNA cloning of a chitin deacetylase from a crab.展开更多
几丁质脱乙酰基酶是几丁质修饰关键酶,在昆虫几丁质组装过程中发挥重要作用,是潜在的绿色农药靶标。课题组近期研究发现家蚕中肠3个几丁质脱乙酰基酶中,BmCDA7对围食膜几丁质具有最高活性,在进食期高表达,可能参与该时期围食膜形成。本...几丁质脱乙酰基酶是几丁质修饰关键酶,在昆虫几丁质组装过程中发挥重要作用,是潜在的绿色农药靶标。课题组近期研究发现家蚕中肠3个几丁质脱乙酰基酶中,BmCDA7对围食膜几丁质具有最高活性,在进食期高表达,可能参与该时期围食膜形成。本研究通过进一步优化分离纯化条件,建立了金属螯合层析和阴离子交换层析两步分离纯化方法,获得了高纯度的BmCDA7重组蛋白。采用SDS-PAGE结合Calcofluor White M2R显色定性确定了BmCDA7对底物乙二醇几丁质的催化活性。通过测定催化反应过程中释放的乙酸量,确定了BmCDA7的最适反应条件、稳定性及底物选择性。BmCDA7催化反应最适温度为60℃,最适pH为8.0,在温度低于60℃以及中性、偏碱性条件下较为稳定。此外,BmCDA7对乙二醇几丁质、胶体几丁质的酶活力分别为2.9926和0.4270μmol/(min·μmol),而对高结晶度的α-几丁质和β-几丁质无活力。这些结果丰富了昆虫几丁质脱乙酰基酶的生物化学基础研究。展开更多
Chitosan and chitooligosaccharides(COS),as derivatives of chitin through deacetylation reaction,have broad applications due to their good biodegradability,biocompatibility,and solubility.In addition,chitosan and COS a...Chitosan and chitooligosaccharides(COS),as derivatives of chitin through deacetylation reaction,have broad applications due to their good biodegradability,biocompatibility,and solubility.In addition,chitosan and COS are involved in cell wall morphogenesis and host–pathogen interactions in vivo.Chitin deacetylases(CDAs)are enzymes that can catalyze the deN-acetylation of chitin.They are widely distributed in protozoa,algae,bacteria,fungi,and insects with important physiological functions.Compared with the traditional chemical method,enzymatic catalysis by CDAs provides an enzymatic catalysis method to produce chitosan and COS with controllable deacetylation site and environmental friendliness.These characteristics attract researchers to produce CDAs by fungicides or pesticides.However,researches on heterologous expression and directed evolution of CDAs are still lacking.In this review,we summarize the latest knowledge of CDAs,especially for heterologous expression systems and directed evolution strategies,which may contribute to the industrial production and future application of CDAs.展开更多
文摘Chitin deacetylase (CDA;EC 3. 5. 1. 41) catalyzes the deacetylation of chitin. In this study, we successfully cloned and sequenced a chitin deacetylase gene from the red snow crab Chionoecetes japonicas. By using reverse transcription-polymerase chain reaction (RT-PCR) and 5' and 3' rapid amplification of cDNA ends, we obtained a 2141-bp amplicon containing a chitin deacetylase gene (CjCDA) from the epidermis of C. japonicas. The amplicon contains a 1575-bp open reading frame that is predicted to encode a 525-amino acid protein. The structure predicted from the deduced amino acid sequence included an N-terminal signal peptide, chitin-binding domain (CBD), low-density lipoprotein receptor class A domain (LDL-A), and catalytic domain. Comparative analysis of the deduced amino acid sequence of CjCDA revealed the highest homology (74%) to gastrolith protein 59 of Cherax quadricarinatus. We used RT-PCR to evaluate the expression of CjCDA in various tissues of C. japonicas, and we observed that CjCDA was expressed only in the epidermis. A phylogenetic analysis, using the amino acid sequences of CjCDA and other known chitin deacetylases, showed that CjCDA belonged to a group of crustacean chitin deacetylases. To our knowledge, this is the first study reporting the cDNA cloning of a chitin deacetylase from a crab.
文摘几丁质脱乙酰基酶是几丁质修饰关键酶,在昆虫几丁质组装过程中发挥重要作用,是潜在的绿色农药靶标。课题组近期研究发现家蚕中肠3个几丁质脱乙酰基酶中,BmCDA7对围食膜几丁质具有最高活性,在进食期高表达,可能参与该时期围食膜形成。本研究通过进一步优化分离纯化条件,建立了金属螯合层析和阴离子交换层析两步分离纯化方法,获得了高纯度的BmCDA7重组蛋白。采用SDS-PAGE结合Calcofluor White M2R显色定性确定了BmCDA7对底物乙二醇几丁质的催化活性。通过测定催化反应过程中释放的乙酸量,确定了BmCDA7的最适反应条件、稳定性及底物选择性。BmCDA7催化反应最适温度为60℃,最适pH为8.0,在温度低于60℃以及中性、偏碱性条件下较为稳定。此外,BmCDA7对乙二醇几丁质、胶体几丁质的酶活力分别为2.9926和0.4270μmol/(min·μmol),而对高结晶度的α-几丁质和β-几丁质无活力。这些结果丰富了昆虫几丁质脱乙酰基酶的生物化学基础研究。
基金This work was financially supported by the Key Research and Development Program of China(2018YFA0900300)the National Natural Science Foundation of China(32021005,31870069)+1 种基金the Fundamental Research Funds for the Central Universities(JUSRP52019A,JUSRP121010,JUSRP221013)Shandong Province Key R&D Program(Major Science and Technology Innovation Project)Project(2019JZZY011002).
文摘Chitosan and chitooligosaccharides(COS),as derivatives of chitin through deacetylation reaction,have broad applications due to their good biodegradability,biocompatibility,and solubility.In addition,chitosan and COS are involved in cell wall morphogenesis and host–pathogen interactions in vivo.Chitin deacetylases(CDAs)are enzymes that can catalyze the deN-acetylation of chitin.They are widely distributed in protozoa,algae,bacteria,fungi,and insects with important physiological functions.Compared with the traditional chemical method,enzymatic catalysis by CDAs provides an enzymatic catalysis method to produce chitosan and COS with controllable deacetylation site and environmental friendliness.These characteristics attract researchers to produce CDAs by fungicides or pesticides.However,researches on heterologous expression and directed evolution of CDAs are still lacking.In this review,we summarize the latest knowledge of CDAs,especially for heterologous expression systems and directed evolution strategies,which may contribute to the industrial production and future application of CDAs.
