本文選題：磁性殼聚糖微球 + 固定化酶��； 參考：《河北大學(xué)》2014年碩士論文

【摘要】：哌嗪-2-羧酸作為非蛋白組成成分的氨基酸，是重要的手性藥物中間體，其不同的單一對映體（S型和R型）衍生物具有不同的藥理活性。本實驗室篩選得到的副球菌(Paracoccous sp.) W10173產(chǎn)生的(S)-酰胺酶能夠立體選擇性水解(S)-哌嗪-2-甲酰胺生成重要醫(yī)藥中間體(S)-哌嗪-2-羧酸的單一對映體。由于固定化酶與游離酶相比具有便于回收、穩(wěn)定性高、操作連續(xù)可控、工藝簡便等一系列的優(yōu)點，故對(S)-酰胺酶進行固定化研究對其應(yīng)用于工業(yè)生產(chǎn)有重要意義。 本研究首先對實驗室保藏的副球菌W10173經(jīng)超聲波破壁和分級鹽析得到實驗用粗酶。超聲波破壁條件為功率400W，工作時間3S，間歇時間6S，循環(huán)240次；并確定分級鹽析的最佳硫酸銨飽和度為20%-50%，且重復(fù)三次。 以化學(xué)共沉淀法制備的Fe3O4磁性粒子為磁核，液體石蠟為分散介質(zhì)，Span-80為乳化劑，，戊二醛為交聯(lián)劑，采用反相懸浮交聯(lián)法合成磁性殼聚糖微球。以此為載體，通過吸附-交聯(lián)聯(lián)合固定化方法制備固定化(S)-酰胺酶，并探討了酶給予量、吸附時間、交聯(lián)時間、戊二醛濃度等因素對固定化效果的影響。最終確定(S)-酰胺酶的最佳固定條件為：10mL0.95mg/mL粗酶液/50mg載體吸附2h、戊二醛濃度1%交聯(lián)4.5h。 對固定化(S)-酰胺酶酶學(xué)性質(zhì)進行研究結(jié)果表明，固定化(S)-酰胺酶的最佳溫度為32℃；最佳pH為11，比游離酶最佳pH向堿性移動2個單位。同時還發(fā)現(xiàn)該固定化酶具有良好的熱穩(wěn)定性，60℃處理4h后仍能保留約88%活性。獲得的固定化(S)-酰胺酶的活力為2.3U/mg載體，酶活回收率為36.9%。固定化酶連續(xù)反復(fù)使用10批次后，酶活仍保留71%以上，具良好的操作穩(wěn)定性。(S)-酰胺酶被固定化后，與游離酶相比，其貯存穩(wěn)定性也有所提高。
[Abstract]:Piperazine-2-carboxylic acid is an important chiral drug intermediate as an amino acid of non-protein component. Its different single enantiomers S-type and R type) derivatives have different pharmacological activities. Paracoccous sp.) W10173 can stereoselective hydrolyze the single enantiomer of the important pharmaceutical intermediates, namely, Schi-piperazine-2-carboxylic acid. The monozyme can be stereoselective hydrolyzed by the thiosamine-piperazine-2-formamide. Because the immobilized enzyme has a series of advantages such as easy recovery, high stability, continuous and controllable operation, simple process and so on, it is of great significance for the application of immobilized enzyme to industrial production. In this study, the experimental crude enzymes were obtained by ultrasonic wave breaking and salting out. The conditions of ultrasonic wall breaking are power 400W, working time 3s, intermittent time 6S, cycle 240, and the optimum saturation of ammonium sulfate for grading salting out is determined to be 20 ~ 50 and repeated three times. Magnetic chitosan microspheres were synthesized by reverse phase suspension crosslinking method using Fe _ 3O _ 4 magnetic particles prepared by chemical coprecipitation as magnetic nucleus, liquid paraffin as dispersing medium Span-80 as emulsifier and glutaraldehyde as crosslinking agent. In this paper, the immobilized Scu-actamases were prepared by the method of adsorption-crosslinking and immobilization, and the effects of enzyme dosage, adsorption time, crosslinking time and glutaraldehyde concentration on the immobilization effect were discussed. The optimal immobilization conditions were determined as follows: 1 10mL0.95mg / mL crude enzyme solution / 50mg carrier adsorbed for 2 h, glutaraldehyde 1% crosslinked for 4.5h. The results showed that the optimum temperature and pH were 32 鈩

本文編號：2038978