几丁质酶抑制剂的研究
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摘要
几丁质酶在许多真菌和昆虫的生理和发育上具有许多功能，以其为靶标的几丁质酶抑制剂可以作为抗真菌剂、生物杀虫剂和抗疟药。
论文首先筛选得到一株产几丁质酶菌株，并对其所产的几丁质酶酶学性质做了初步研究，考察了最适反应温度、最适pH及金属离子对酶活的影响。
通过建立的模型，从浙江东海的海水中筛选得到能分泌几丁质酶抑制剂的细菌，经鉴定属于Acinetobacter lwoffii ChI-06(洛菲不动杆菌)；并考察了ChI-06的生长曲线及遗传稳定性。
比较系统地研究了ChI-06发酵工艺的优化，以提高其酶抑制剂的产率。经优化确定的较佳培养基为：蛋白胨10.0g／L，酵母膏2.0g／L，葡萄糖5.0g／L，NaCl 5.0g／L，Al(NO_3)_3 2mmol／L，海水晶30g／L；较佳培养条件为：培养基初始pH为7.0，摇瓶装量为50mL培养基／250mL三角瓶，接种量为6％，在28℃，180r／min摇床培养6d。按优化工艺进行发酵，酶抑制剂的活性达到65.42％，比优化前提高了86.91％。在3L发酵罐的研究中，采用发酵前、后期改变搅拌转速，发酵周期比摇瓶缩短了一半多，酶抑制剂的活性达到81.73％，比摇瓶发酵结果提高了24.93％。
对该酶抑制剂理化性质的初步研究表明，它可能是一种(多)糖类酶抑制剂。以果蝇为昆虫模型，Allosamidin和抑太保作对照的杀虫实验表明，当酶抑制剂的添加量为0.75mL时，在第三天对果蝇的杀虫率达到91.67％。
The importance of chitinases in the physiological and developmental processes of fungus and insects makes their inhibitors important targets for potential antifungal and insecticidal agents, as well as antimalarial agents.
First of all, this study screened a bacterium producing chitinase. The characteristic of enzyme, such as optimum temperature and pH as well as the effects of metal irons on the enzyme, was studied.
Based on the screening model, a marine bacterium producing chitinase inhibitor was screened from the East Sea ZheJiang. This strain was identified as Acinetobacter lwoffii ChI-06. The growth curve and stability of genetics were preliminarily studied.
In order to enhance the productivity of the inhibitor, the optimization of the fermentation technology of ChI-06 was systematically carried out in flasks. Under optimum conditions, a maximum inhibitor activity of 65.42% was observed after 6 days cultivation at 28℃on a 180 r/min shaker in 50 mL of fermentation medium containing peptone 10.0 g/L, yeast 2.0 g/L, glucose 5.0 g/L, NaC15.0 g/L, Al(NO_3)_3 2 mmol/L, marine water extraction 30 g/L with 6% inoculum's sizes. In the study in a 3 L fermentor, using the strategy of decreasing the agitation rate during fermentation, the time course was cut off half and the inhibitor activity was achieved 81.73%, which is 24.93% higher compared to the result in flasks.
Preliminary studies show chitinase inhibitor could be saccharide or polysaccharide. The Drosophila-killing trials were conducted with the control of Allosamidin and Chlorfluazuron. With the dose of 0.75 mL, chitinase inhibitor can kill 91.67% flies after 3 days cultivation.
论文首先筛选得到一株产几丁质酶菌株，并对其所产的几丁质酶酶学性质做了初步研究，考察了最适反应温度、最适pH及金属离子对酶活的影响。
通过建立的模型，从浙江东海的海水中筛选得到能分泌几丁质酶抑制剂的细菌，经鉴定属于Acinetobacter lwoffii ChI-06(洛菲不动杆菌)；并考察了ChI-06的生长曲线及遗传稳定性。
比较系统地研究了ChI-06发酵工艺的优化，以提高其酶抑制剂的产率。经优化确定的较佳培养基为：蛋白胨10.0g／L，酵母膏2.0g／L，葡萄糖5.0g／L，NaCl 5.0g／L，Al(NO_3)_3 2mmol／L，海水晶30g／L；较佳培养条件为：培养基初始pH为7.0，摇瓶装量为50mL培养基／250mL三角瓶，接种量为6％，在28℃，180r／min摇床培养6d。按优化工艺进行发酵，酶抑制剂的活性达到65.42％，比优化前提高了86.91％。在3L发酵罐的研究中，采用发酵前、后期改变搅拌转速，发酵周期比摇瓶缩短了一半多，酶抑制剂的活性达到81.73％，比摇瓶发酵结果提高了24.93％。
对该酶抑制剂理化性质的初步研究表明，它可能是一种(多)糖类酶抑制剂。以果蝇为昆虫模型，Allosamidin和抑太保作对照的杀虫实验表明，当酶抑制剂的添加量为0.75mL时，在第三天对果蝇的杀虫率达到91.67％。
The importance of chitinases in the physiological and developmental processes of fungus and insects makes their inhibitors important targets for potential antifungal and insecticidal agents, as well as antimalarial agents.
First of all, this study screened a bacterium producing chitinase. The characteristic of enzyme, such as optimum temperature and pH as well as the effects of metal irons on the enzyme, was studied.
Based on the screening model, a marine bacterium producing chitinase inhibitor was screened from the East Sea ZheJiang. This strain was identified as Acinetobacter lwoffii ChI-06. The growth curve and stability of genetics were preliminarily studied.
In order to enhance the productivity of the inhibitor, the optimization of the fermentation technology of ChI-06 was systematically carried out in flasks. Under optimum conditions, a maximum inhibitor activity of 65.42% was observed after 6 days cultivation at 28℃on a 180 r/min shaker in 50 mL of fermentation medium containing peptone 10.0 g/L, yeast 2.0 g/L, glucose 5.0 g/L, NaC15.0 g/L, Al(NO_3)_3 2 mmol/L, marine water extraction 30 g/L with 6% inoculum's sizes. In the study in a 3 L fermentor, using the strategy of decreasing the agitation rate during fermentation, the time course was cut off half and the inhibitor activity was achieved 81.73%, which is 24.93% higher compared to the result in flasks.
Preliminary studies show chitinase inhibitor could be saccharide or polysaccharide. The Drosophila-killing trials were conducted with the control of Allosamidin and Chlorfluazuron. With the dose of 0.75 mL, chitinase inhibitor can kill 91.67% flies after 3 days cultivation.
引文
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[5] Zhu Q, Maher EA, Masoud S, et al. Enhanced protection against fungal attack by constitutive co-expressing of chitinase and glucanase genes in transgenic tobacco[J]. Bio/Technology, 1994, 12: 807-812.
[6] Schlumbaum A, Mauch F, Vogeli U, et al. Plant chitinase are potent inhibitors of fungal growth[J]. Nature, 1986, 324(27): 355-356.
[7] Mauch F, Mauch-Mani B, Boiler T. Antifungal hydrolyses in pea tissue: Inhibition of fungal growth by combination of chitinase and β-1,3-glucanase[J]. Plant Physiol, 1988, 88: 936-942.
[8] Verburg JG, Huynh QK. Purification. and Characterization of an Antifungal Chitinase from Arabidopsis thaliana. Plant Physiol[J], 1991, 95(2): 450-455.
[9] Haki GD, Rakshit SK. Development in industrially important thermoStable enzymes[J]. Bioresource Technology, 2003, 89(1): 17-34.
[10] Carlini CR, Grossi DS, Maria F. Plant toxic proteins with insecticidal properties[J]. Toxicon, 2002, 40(11): 1519-1539.
[11] 郑志成，周美英，姚丙新．海洋链霉菌几丁质酶的研究[J]．厦门大学学报(自然科学版)，31(5)：543-547．
[12] 陈三凤，李季伦．几丁质酶研究历史和发展前景[J]．微生物学通报，1993，20(3)：156-160．
[13] 彭仁旺，黄秀梨．球饱白僵菌饱内几丁质酶的分离纯化及性质[J]．微生物学报，1995，35(6)：427-432．
[14] Vyas P and Deshpande M. Purification and characterization of an endochitinase from Myrotheci- um Verrucaria[J]. J Gen Appl Microbiol, 1993, 39: 91-99.
[15] 陈亚成，陈红歌，王石磊等．产碱性几丁质酶菌种的筛选与发酵条件的研究．河南农业大学学报．1998，32(4)：389-393．
[16] Hara S, Yamamura Y, Mega T and Ikenaka T. Purification and characterization of chitinase produced by Sreptomyces erythraeus[J]. J Biochem, 1989, 105: 484-489.
[17] Fuchs R, Mcpherson S and Drahos D. Cloning a Serratia marcescens gene ineoding chitinase [J]. Appl Environ Microbiol, 1986, 51(3): 504-509.
[18] Fuchs R L. Purification and Properties of Chitinases from Sarratia Marcescens[J]. Environ Microbiol, 1987, 53(7): 1718-1720.
[19] Phillips W R, Charles A, Barbara B. Cloning and Expression of a Streptomyces plicatus Clitinase in E. coli[J]. Biol Chem 1988, 263(1): 443-447.
[20] Saburo H, Yoshitaka Y, Yoko F., et al. Purification and Characterization of Chitinase Produced by Streptomyces erythraeus[J]. Biochem, 1989, 105: 484-489.
[21] Takeshi W, Wataru O, KazushiS, et al. Chitinase System of Bacillus eirculans W L-12 and importance of Chitinase Al in Chtin Degradation[J]. Bacteriol, 1990, 172(7): 4017-4022.
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