臭腹腺蝗肠道中共存的淀粉和纤维素消化酶的纯化、动力学和抑制研究(英文)

Purification,kinetics and inhibition studies on co-existing starch and cellulose-digesting enzymes from the gut of the variegated grasshopper( Zonocerus variegatus)

【目的】本研究旨在了解臭腹腺蝗Zonocerus variegatus中碳水化合物的消化。【方法】依次通过CM-Sepharose CL-6B阳离子交换、QAE Sephadex A-50阴离子交换以及Sephadex G-100凝胶过滤,对臭腹腺蝗中肠的酶粗提取液(其中α-淀粉酶和纤维素酶的比活性分别为283±1.72 U/mg和17±0.83 U/mg)进行了纯化。最后通过层析将共存的酶分离成α-淀粉酶和纤维素酶。【结果】这两种酶都是单体,α-淀粉酶分子量为38 k Da,纤维素酶分子量为21 k Da。α-淀粉酶对淀粉的Km值为13.8±1.29 mg/m L,Vmax为3 883±52.25 U/mg pro;而纤维素酶对羧甲基纤维素底物的Km值为5.03±0.57 mg/m L,Vmax为433.00±5.24 U/mg pro。臭腹腺蝗α-淀粉酶能以不同的效率水解原淀粉,纤维素酶也能不同程度水解各种纤维材料。α-淀粉酶的水解活性在p H 6和40℃下最高,而纤维素酶的水解高活性在p H 9和45℃下最高。【结论】从番木瓜Carica papaya种子中分离到蛋白类抑制剂,发现它们是这两种酶的有效抑制剂,说明这些蛋白抑制剂(Cp AI和Cp CI)如果在害虫取食的作物上过量表达,则可能有利于害虫防治。尽管纤维类和淀粉类物质可能都显著提供该害虫的能量需求,但是动力学分析表明臭腹腺蝗更偏向于淀粉类食物。

[ Aim ] This study aims to gain information on carbohydrate digestion in the variegated grasshopper Zonocerus variegatus. [ Methods ] Crude enzyme extracts of the midgut with specific activity of 283 ± 1.72 U/mg for α-amylase and 17 ± 0. 83 U/mg for cellulase, were subsequently purified by a combination of cation-exchange on CM-Sepharose CL-6B, anion-exchange on QAE Sephadex A-50 and gel filtration on Sephadex G-100. The co-existing enzymes were separated into alpha amylase and eellulase by the final step of chromatography. [ Results] Both enzymes are monomers with molecular weights of 38 kDa for α-amylase and 21 kDa for cellulase. Alpha amylase has a Km of 13.8 ± 1.29 mg/mL for starch and a Vmax of 3 883 ± 52.25 U/mg protein, whereas the values of 5.03 ± 0.57 mg/mL and 433.00 ± 5.24 U/mg protein were respectively obtained for cellulase with carboxymethyl cellulose （ CMC ） as substrate. Z. variegatus a-amylase could hydrolyse raw starches with varying efficiency while the cellulase could also hydrolyze various cellulosic materials to varying degrees. Hydrolytic activity was maximal for α-amylase at pH 6 and 40℃ and cellulase at pH 9 and 45℃. [ Conclusion] The proteinaceous inhibitors were isolated from Carica papaya seeds and found to be effective inhibitors to both enzymes, suggesting that these protein inhibitors （ CpAI and CpCI） may be useful in the pest control if overexpressed in crop plants that the pests feed on. Though both cellulosic and starchy materials may contribute significantly to the pests＇ energy requirements, kinetic analysis suggests that Z. variegatus prefer starchy foods.