毕赤酵母表达重组红林蚁AChE高密度发酵工艺研究

【目的】优化毕赤酵母高密度生产重组红林蚁乙酰胆碱酯酶(AChE)的发酵条件,为AChE在农药残留检测中应用提供酶源。【方法】采用毕赤酵母表达系统,选用高表达AChE的重组子X33/pPICZαAAChE,对毕赤酵母高密度生产重组红林蚁AChE的发酵条件进行优化,确定最佳发酵pH值、温度、溶解氧、补料流加速率等因素。【结果】毕赤酵母表达重组红林蚁AChE高密度发酵工艺的最佳参数:pH5.0、溶解氧30%、温度29℃、转速500 r/min、罐压0.06 MPa,甲醇诱导72~96 h时发酵液上清AChE活性增长最快。从发酵结果看,发酵结束菌体湿重可达280 mg/mL,发酵罐内甲醇诱导96 h酶活性最高,达6 000 U/mL,是摇瓶诱导表达的8倍。【结论】毕赤酵母表达系统最佳发酵工艺能实现红林蚁AChE的高密度发酵生产,降低AChE提取成本。

Correlation of cognitive function with acetylcholinesterase activity and P300 event-related potential of patients with type 2 diabetes mellitus

BACKGROUND： At present, central cholinergic neuron system is regarded the most major structural basis of cognitive function. Changes in structure of cholinergic neuron system of brain and receptor expression after brain injury can cause cognitive impairment. OBJECTIVE＂ To comparatively observe the intelligence quotient （IQ）, latent period and wave amplitude of P300 event-related potential and the difference of activity of acetylcholinesterase （ACHE） in blood and cerebrospinal fluid between patients with type 2 diabetes mellitus and with non-diabetes mellitus, and analyze the correlation of IQ of cognitive impairment patients with diabetes mellitus with AChE activity, latent period and wave amplitude of P300 event-related potential in cerebrospinal fluid. DESIGN： Correlation analysis of contrast observation SETTING： Department of Endocrinology, Affiliated Hospital of Binzhou Medical College PARTICIPANTS： Totally 32 patients with type 2 diabetes mellitus who received the treatment in the Department of Endocrinology, Affiliated Hospital of Binzhou Medical College between April 2004 and April 2005 were recruited, serving as diabetes mellitus group. They, including 19 male and 13 female, aged 49 to 73 years, with disease course of 4 to 11 years, all met the diagnostic criteria of diabetes mellitus revised by World Health Organization in 1999. Another 30 patients with non-diabetes mellitus who homeochronously underwent lumbar anesthesia in the Department of Surgery and Department of Gynecology were recruited, serving as non-diabetes mellitus group. The 30 patients included 18 male and 12 female, and their age ranged from 46 to 71 years. Informed consents of detected items were obtained from the involved patients. METHODS： ① Evaluation,on IQ： The IQ of involved subjects was evaluated with Chinese Version of the Wechsler Adult Intelligence Scale revised by Gong Yao-xian （WAIS-RC）. WAIS-RC included 6 verbal subscales and 5 performance subscales. The test scores of the 11 subscales integrated into the scores of the whole scale, and the scores on the WAIS-RC included verbal IQ （VlQ）, performance IQ （PIQ） and full scale IQ （FIQ）. FIQ ≤79 scores indicated low IQ and FIQ≤69 indicated intelligence impairment. ② Detection of P300 wave： P300 wave was detected with evoked potential instrument （MYTOPRO, Italian）, and data of latent period and amplitude of P300 event-related potential were automatically shown by computer. ③ Detection of AChE activity in blood and cerebrospinal fluid： Activity of AChE of blood and cerebrospinal fluid was measured with biochemical methods by using CORNING-560 autoanalyzer.④Correlation analysis： Correlation of FIQ with AChE of cerebrospinal fluid and P300 wave of patients with type 2 diabetes mellitus was analyzed, t test was used in intergroup comparison and linear correlation analysis for relevant treatment. MAIN OUTCOME MEASURES： ① Comparison of IQ, latent period and wave amplitude of P300 wave as well as the activity of AChE between two groups. ② Analysis on the correlation of FIQ of patients with type 2 diabetes mellitus with AChE of cerebrospinal fluid and P300 wave. RESULTS： Thirty-two patients with diabetes mellitus and 30 non-diabetes mellitus participated in the result analysis. ①Comparison of IQ, latent period and wave amplitude of P300 wave as well as the activity of AChE between two groups： The scores of VIP, PIQ and FIQ of patients with type 2 diabetes mellitus were （97.4±10.4）. （92.6±8.4） and （95.2±9.7） scores, respectively; and those of patients with non-diabetes mellitus were （104.7±9.6）, （102.5±8.5）and（102.7±8.9） scores, respectively, and P 〈 0.05-0.01 was set in intergroup comparison. The latent period of P300 wave at points Fz , Cz and Pz of patients with type 2 diabetes mellitus was （370.8±41.8）.（371.5±39.1）and （375.1±43.1） ms, respectively, and that of patients with non-diabetes mellitus was （ 332.1 ±28.3 ）, （335.7 ±29.4）and （339.7 ±27.3） ms, respectively, and P 〈 0.01 was set in intergroup comparison; Wave amplitude of P300 of patients with type 2 diabetes mellitus was （8.6±4.1）,（8.6±4.0） and （7.7±4.0） μV, respectively and that of patients with non-diabetes mellitus was （11.9±4.1）,（11.5±4.4） and （10.9±5.0） μV, respectively , and P 〈 0.05-0.01 was set in intergroup comparison; The level of AChE in blood and cerebrospinal fluid of patients with type 2 diabetes mellitus was （235.61 ±50.34）and （17.89±4.46） μkat/L, respectively, which was significantly higher than that of patients with non-diabetes mellitus [（205.03±44.15）and （14.63±0.48） μkat /L, respectively], and P 〈 0.05-0.01 was set in the intergroup comparison. ② Correlation of FIQ value of patients with type 2 diabetes mellitus with AChE of cerebrospinal fluid and P300 wave： The value of FIQ was significantly negatively correlated with the AChE activity of cerebrospinal fluid （r=-0.588 1, P 〈 0.01 ）, significantly negatively correlated with the latent period at points Fz. C and Pz of P300 wave （r= -0.700 5, -0.689 4, -0.688 5, P 〈 0.01 ）, and significantly positively correlated with the amplitude at points Fz . Cz and Pz of P300 wave（r= 0.607 4,0.616 1,0.592 0,P 〈 0.01 ）. CONCLUSION： ① Cognitive impairment of patients with type 2 diabetes mellitus might be related to the increase of activity of AChE in cerebrospinal fluid. ②Combined application of examination of P300 wave and evaluation of IQ is more useful in deciding the state of cognitive function of patients with type 2 diabetes mellitus.

Effect of Purified Paper Wasp Ropalidia marginata Venom Toxin Enzyme Activity in Blood Serum, Liver, and Gastrocnemius Muscle of Albino Mice

In the present, investigation effects of sub-lethal dose of purified paper wasp Ropalidia marginata venom toxins were evaluated on important metabolic enzymes i.e. ALP ACP, GPT, GOT, LDH, and AchE enzyme activity in serum, liver, and gastrocnemius muscles of albino mice. Alkaline phosphatase was found to be increased up to 119.9% at the 6th hr of the toxin injection in comparison to control. This elevation may be due to cytolysis. Maximum increase i.e., 153.33% level of glutamate pyruvate transaminase (GPT) was found at 6 hrs of 40% of 24-h LD50 treatment while it was found to be 151.1% at 6 hrs of 24 hr 80% of LD50, venom injection. A significant elevation was observed in LDH activity in serum, liver, and muscles, while the activity of AchE was decreased in serum, liver, and gastrocnemius muscles of albino mice after injecting the sub-lethal dose of Ropalidia marginata venom. This increase in the activity of LDH produces liver damage, massive disintegration and necrosis of hepatic cells. This elevation in LDH level led to a significant increase in the glucose catabolism and elevated oxidative stress in muscle and liver cells. It also displays insufficient oxygen supply and consequently leads to cell death. In experimental animals, venom toxin treatment decreased AchE level, and animals showed muscular paralysis. When mice were treated with 40% and 80% of 24-h LD50 of purified venom caused a significant (p < 0.05) elevation in the level of ACP, GOT, GPT, and LDH while the reduction in ALP and AChE level. Present study will be useful in the development of prototypes for study of pharmacological and therapeutic effects of various venom toxins. For this purpose structure activity relationship of enzyme and venom toxin, its due interaction to various metabolic enzymes and receptors must be explored.

Actions of Sodium Fluoride on Acetylcholinesterase Activities in Rats

This study was carried out to observe the effects of sodium fluoride on acetylcholinesterase (AChE) activities in the cerebral synaptic membranes (SPM) and the peripheral red blood cells (RBC) of rats by in vivo and in vitro experiments. In the in vivo study,pregnant rats ingested ad libitum fluorinated drinking water (5, 15, 50 ppm F ) during their gestation and lactation. It was shown that the AChE activities of the SPM and peripheral RBCs in maternal rats exposed 5 ~ 50 ppm F for 60 days were elevated significantly by 30. 0 ~ 67. 6 % and 12. 5 ~ 31. 9 % in a dose-dependent manner, respectively. The AChE activities of their offspring 80 days after birth were also increaased (8. 7 ~ 28. 7 % for SPM and20. 6 ~ 32. 4 % for RBC). In contrast, the AChE activities of SPM in vitro were inhibited by 5. 0~ 50 .0 mmol F /L treatment in a time-and dose dependent manner. Analaysis with the Hanes plots suggested that the enzymesubstrate kinetics are consistent with a mixed type ofinhibition.

毒死蜱和三唑磷对斑马鱼头部AChE活性影响及在鱼体内的富集

采用半静态法，测定了在96h—Lc50的1／10、1／20、1／40及1／80作用剂量下慢性暴露，毒死蜱和三唑磷对斑马鱼头部乙酰胆碱酯酶（AChE）活性的抑制及两种农药在鱼体的富集作用，并研究解除30d后酶活性的恢复。结果表明，随暴露剂量增大、暴露时间延长，农药对AChE活性抑制越强。在96h—LC50的1／10作用剂量下连续暴露30d，毒死蜱和三唑磷处理组斑马鱼头部AChE活性分别为对照的42．25％和31．80％。毒死蜱不同剂量处理组酶活性在解除药剂后30d均可恢复到对照的90％以上，而三唑磷高剂量处理组（96h—LC50的1/10和1／20）分别只有对照的74．91％和71．46％。毒死蜱极容易在鱼体富集，慢性暴露30d富集系数达260．91～1320．03L·kg^-1，而三唑磷仅为8．23-26．84L·kg^-1。

麦穗鱼脑乙酰胆碱酯酶(AChE)的纯化及其比较性研究

用 PEG2 0 0 0双水相萃取、DEAE- Sephadex A- 5 0和 Sephadex G- 2 0 0方法分离纯化麦穗鱼Pseudorasbora parva脑中的乙酰胆碱酯酶 (ACh E) ,然后比较分析纯酶液与粗酶液中鱼脑 ACh E的动力学特性和抗抑制性 ,以便更直接地了解底物与酶以及毒剂与酶的反应关系。研究结果表明 ,经过一系列步骤的纯化 ,最后所得的 ACh E是纯度较高的酶液 ;通过对 ACh E的动力学研究发现 ,纯化后的麦穗鱼脑 ACh E与底物之间的亲和力和其在粗酶状态时没有显著的差别 ,而且纯化后的鱼脑 ACh E对底物抑制作用更敏感 ;抗抑制性研究发现 ,纯化后的麦穗鱼脑 ACh E对经溴水氧化的马拉硫磷 (malathion)和三唑磷 (triazophos)的敏感性显著高于粗酶状态的 ACh E。

对有机磷农药敏感的海鱼脑AChE筛选研究

以10种海洋鱼类：鲅鱼、黄花鱼、鲈鱼、黄鱼、美国红鱼、真鲷、黑鲷、面包鱼、黑头鱼和鳗鲡为实验材料，采用半抑制浓度（IC50）和双分子速率常数（Ki）为指标，通过体外抑制作用比较了这10种海鱼脑AChE对马拉硫磷和甲基对硫磷的敏感性，并分析了海鱼脑AChE作为监测海水中有机磷农药指示酶的可行性，由此筛选出适于制备海水有机磷农药酶传感器的指示酶．实验表明：10种海鱼脑AChE的基础活性在4．62～35．65μmol／（min·g）之间，面包鱼的酶活性最高，黄花鱼的酶活性最低．对马拉硫磷，由IC50所得到的AChE敏感性为：鲅鱼、黄鱼、鲈鱼、真鲷〉黑鲷〉黑头鱼、鳗鲡〉美国红鱼、黄花鱼〉面包鱼；由Ki所得到的AChE敏感性为：鲅鱼〉黄鱼、鲈鱼、真鲷〉黑鲷、黑头鱼、鳗鲡〉美国红鱼、黄花鱼〉面包鱼．对甲基对硫磷，由IC50所得到的AChE敏感性为：鲅鱼〉鲈鱼〉黄鱼、黄花鱼、真鲷、黑鲷、黑头鱼、鳗鲡〉美国红鱼〉面包鱼；由Ki所得到的AChE敏感性为：鲅鱼〉鲈鱼、黄鱼、黄花鱼、黑鲷、黑头鱼、鳗鲡〉真鲷〉美国红鱼〉面包鱼．鲅鱼脑AChE对2种有机磷农药都较为敏感．影响鱼脑AChE对有机磷农药敏感性的主要因素为生物来源、农药种类和抑制方式，而与酶的基础活性无关．海鱼脑AChE活力抑制作用与水体中的有机磷农药具有良好的剂量-效应关系，在1～50mg／L浓度范围内，两者间呈现较好的线性相关性（r^2≥0．93）．根据AChE的敏感性及其对有机磷农药的线性响应程度，鲅鱼脑AChE适于作为监测马拉硫磷和甲基对硫磷酶传感器的指示酶．

海鱼AChE监测海水有机磷农药的可行性研究

有机磷农药 (OPs)对乙酰胆碱酯酶 (AChEEC 3.1 .1 .7)具有专一性抑制作用 ,因此AChE常作为淡水和土壤OPs污染的特异性标识物 (biomarker)。本文在海水背景下 ,通过测定两种有机磷农药 (马拉硫磷和甲基对硫磷 )对海洋鱼类—鲈鱼 [Lateolabraxjaponicus(cuvier) ]脑组织AChE的抑制效应 ,分析了海鱼AChE监测海水OPs的可行性。结果显示 ,抑制时间为 2 0min时 ,抑制程度达到相对稳定 ;在低浓度OPs抑制下 ,纯酶液比粗酶液表现出更好的敏感性 ,对马拉硫磷和甲基对硫磷的灵敏度分别达到 0 .2 8μg·L 1和 0 .2 μg·L 1;在一定浓度范围内 ,AChE活力对OPs具有显著的线性响应。研究表明 ,利用鲈鱼AChE指示和监测海水OPs的微污染是完全可行的。

黄鱼(Hexagrammosotakii)脑组织AChE的亲和层析

采用亲和层析法对黄鱼脑组织的乙酰胆碱酯酶进行分离纯化。纯化酶最大比活力为6 3.1 36 U /mg蛋白 ,最适 p H为 8,最适反应温度为 35℃。此酶经过 SDS- PAGE呈一条带 ,表明其纯度达电泳纯。对纯化酶进行底物特异性、过量底物抑制效应和抑制剂选择性检验 ,结果证实该酶为乙酰胆碱酯酶。

有机磷农药对海洋动物乙酰胆碱酯酶(AChE)的毒性效应研究进展

近几十年来，农业和海水养殖业所使用的有机磷农药 (OPs) 造成沿岸海水的严重污染，威胁到海洋动物的生存。有机磷农药的海洋生态毒理学研究已经成为海洋环境科学中的热门领域之一。作为动物神经传导过程中的关键酶，乙酰胆碱酯酶 (AChE) 能够被有机磷农药专一性抑制，因此，有机磷农药对海洋动物 AChE 的影响受到更多的关注。本文以近年来该领域的研究成果为基础，综述了有机磷农药对海洋动物乙酰胆碱酯酶的毒性效应。

溴氰菊酯对草鱼鱼种脑AchE及ATP酶活性的影响

本试验研究了溴氰菊酯对草鱼鱼种的急性毒性效应以及溴氰菊酯对草鱼鱼种脑ＡｃｈＥ及ＡＴＰ酶活性的影响。结果表明，溴氰菊酯对规格Ⅰ（平均体重８．２１ｇ）、规格Ⅱ（平均体重６６．６ｇ）草鱼鱼种９６ｈ半数致死浓度（９６ｈＬＣ５０）分别为２．１６μｇ／Ｌ、４．２６μｇ／Ｌ，９５％置信区间分别为１．９３～２．４２μｇ／Ｌ、３．８２～４．７５μｇ／Ｌ，离体条件下对草鱼种脑Ｍｇ２＋－ＡＴＰ酶活性抑制显著，呈一定的浓度－效应关系，回归方程ｙ＝５．８９＋０．３２ｘ（ｒ＝０．９２）；对离体、活体脑ＡｃｈＥ、离体脑Ｎａ＋Ｋ＋－ＡＴＰ酶活性无显著影响。

基于固定化AChE的流动注射型酶传感器研究

以固定化鲅鱼脑组织乙酰胆碱酯酶[acetylcholinesterase，ACHE（EC3．1．1．7）]为识别元件，以pH电极为换能器，构建流动注射型AChE酶传感器．该传感器在以磷酸盐缓冲液为载液的条件下具有良好的重现性（RSD=1．427%，n=10）和敏感性，可实现对有机磷化合物的在线监测；对甲基对硫磷具有线性响应的浓度范围为4.29×10^-10～4.29×10^-8mol·L^-1，最低检测限为1．3×10^-10mol·L^-1。在以清洁海水为载液条件下，该传感器对海水中甲基对硫磷的敏感性较差，但是采用NaCIO溶液对海水中甲基对硫磷进行预氧化后，传感器对其的最低检测限可达到2．16×10^-7mol·L^-1。

鲅鱼脑组织AChE的分离纯化及某些生化性质

采用亲和层析法对鲅鱼[Scomberomorus niphonius （Cuvier）]脑组织的胆碱酯酶[acetylcholinesterase （ACHE），EC3．1．1．7]进行分离纯化，然后对其进行底物特异性、过量底物抑制效应和抑制剂选择性检验。结果证实：该酶为乙酰胆碱酯酶（ACHE），其最大比活力为297．82μmol／（min·mg），最佳pH范围在7．0～8．0之间，最佳温度为30℃。此酶经过SDS—PAGE呈一条带，表明其纯度达电泳纯。

麦穗鱼脑AChE间接非竞争ELISA定量分析法的建立

用PEG2000双水相萃取、DEAE-SephadexA-50和SephadexG-200方法分离纯化麦穗鱼Pseudorasboraparva脑中的乙酰胆碱酯酶(AChE),并制备了兔抗麦穗鱼脑AChE抗血清。用兔抗麦穗鱼脑AChE抗体连接抗原,建立了定量分析麦穗鱼脑AChE的间接非竞争酶联免疫吸附法(Indirectandnon-competitiveELISA),此法操作简便、灵敏度高,适用于定量检测。该方法的建立有利于AChE在环境科学和农药学研究领域的进一步研究和应用。

重金属污染条件下花背蟾蜍跳跃能力与骨骼肌ATP酶和AChE活性变化研究

以相对无污染的刘家峡地区和重金属污染较重的白银地区为研究样点,以黄河流域广泛分布的花背蟾蜍Bufo raddei为研究对象,对比调查研究了两地花背蟾蜍成体的单次跳跃能力以及后肢骨骼肌组织中与运动密切相关的总ATP酶、Ca2+-ATP酶、AChE活性的变化情况。结果显示,与相对无污染的刘家峡地区相比,白银地区花背蟾蜍成体的跳跃能力极显著降低,说明重金属污染对蟾蜍运动能力有一定的影响,使其减弱。白银地区蟾蜍骨骼肌中总ATP酶、Ca2+-ATP酶、AChE活性均呈升高趋势,分别达刘家峡地区的1.91、2.64和1.37倍,酶活性的升高可能与低浓度重金属诱导有关。导致花背蟾蜍跳跃能力下降的途径及机理还需要进一步深入研究。

家蝇AChE检测毒死蜱残留的最佳条件研究

以纯化的家蝇(Museadomestica)乙酰胆碱酯酶(AChE)为试验材料,研究了缓冲液pH值、底物(ATCh)浓度、温度三个因素对酶活性的影响。结果表明:在pH值为8.5￣9.0,温度为30℃,底物浓度为2.5mmol/L的条件下,该酶具有最大活性。在此基础上,测定了不同pH值的缓冲反应体系和不同抑制时间条件下毒死蜱(Chiorpyifos)对AChE活性的抑制,发现pH值为8.0时AChE对毒死蜱最为敏感,而且对毒死蜱具有良好的线性响应关系(R2=0.9973),半抑制浓度仅为39.1μg/(L抑制时间为20min),检测下限为4.4μg/L。随着抑制时间的延长,毒死蜱对AChE的抑制程度逐渐增强,检测限可进一步降低。可见家蝇AChE是指示环境中毒死蜱污染程度的良好材料。

3种农药对红裸须摇蚊体内蛋白质质量分数和AChE活性的影响

为评价农药对水生生物的毒理效应,测定了氧化乐果、毒死蜱和三氟氯氰菊酯对红裸须摇蚊(Propsi-locerus akamusi)4龄幼虫的毒力、蛋白质质量分数和AChE活性的影响。结果表明,氧化乐果、毒死蜱和三氟氯氰菊酯24 h LC50分别为9.055、1.087、0.040μg.L-1,表现出高杀虫活性。这3种药剂的高质量浓度(2、4μg.L-1)处理3 h组,红裸须摇蚊体内蛋白质质量分数均低于对照,而0.125μg.L-1处理24、48 h其体内蛋白质质量分数均高于对照。红裸须摇蚊幼虫体内AChE对3种农药处理表现不同的时间—剂量效应。氧化乐果处理随着质量浓度和时间的增加,红裸须摇蚊幼虫体内的AChE活性抑制逐渐减少;而0.125μg.L-1毒死蜱对其体内AChE活性表现为诱导增加,其余处理随着时间的增加对AChE活性抑制逐渐减少;三氟氯氰菊酯处理3 h,其体内AChE活性随着处理质量浓度的增加而增大,但24、48 h处理组AChE均表现为抑制减少。因此,红裸须摇蚊AChE可作为监测有机磷和菊酯类农药污染的生物指示物。

落叶松毛虫AChE最佳反应体系建立及其对3种杀虫剂敏感性比较

通过正交试验和酶活性测定法研究了落叶松毛虫6龄幼虫乙酰胆碱酯酶(AChE)最佳反应体系及对3种杀虫剂的敏感性。结果表明:落叶松毛虫6龄幼虫AChE最佳反应条件为酶液质量浓度0.2g/mL、底物浓度0.8mmol/L、反应体系pH7.5、水浴温度35℃和反应时间5min。敌敌畏、高效氯氟氰菊酯和阿维菌素对落叶松毛虫6龄幼虫体外AChE活性产生不同的抑制作用,且抑制效应与杀虫剂呈现明显的剂量--效应关系;其中敌敌畏抑制效果最显著(IC50=3.018×10-4mg/mL),其次是高效氯氟氰菊酯(IC50=1.436×10-2mg/mL),而阿维菌素对AChE活性抑制效果最弱。

大鼠脑缺血再灌注后海马组织AchE活力的动态观察

目的观察大鼠脑缺血再灌注后海马组织AchE活力的动态变化.方法阻断大鼠双侧颈总动脉进行脑缺血再灌注,于术后第1,7,15 d用比色法测海马组织AchE活力.结果第1,7 d模型大鼠AchE活力分别是(170.95±10.44)mmol·L-1,(168.34±12.02)mmol·L-1,较正常组明显降低(P＜0.01),且7 d组AchE活力低于1 d组,随着缺血再灌注损伤的修复,15 d组AchE活力明显回升.结论 AchE活力降低在缺血再灌注脑损伤中起重要作用.

鲫鱼肌肉AChE的提取、纯化及活性研究

以鲫鱼肌肉为原料提取乙酰胆碱酯酶(Acetylcholinesterase,AChE),然后采用改进的Ellman方法按照正交试验设计分别测定不同环境条件(pH值,温度,时间)下粗酶液活性,得到测定AChE活性的最佳条件组合。再将粗酶液依次通过DEAE-Sephadex A-50和Sephadex G-200柱层析纯化。结果表明,3个因素对AChE活性影响的顺序为温度>pH值>时间;最佳酶活性测定条件组合为环境温度35℃,体系pH值8.0,反应时间30 min;经Sephadex A-50柱层析后的纯化倍数是17.73,产率为40.06%;经Sephadex G-200柱层析后的纯化倍数是47.29,产率为20.65%。