β-D-半乳糖苷酶殼聚糖固定方法、特性及應(yīng)用研究

1、-D-半乳糖苷酶殼聚糖固定方法、特性及應(yīng)用研究摘 要“乳糖不耐癥”是影響我國乳業(yè)發(fā)展的瓶頸技術(shù)之一，而利用固定化-D-半乳糖苷酶水解奶中的乳糖是解決“乳糖不耐癥”的有效方法。本研究以殼聚糖為載體和戊二醛為交聯(lián)劑，采用共價交聯(lián)和吸附交聯(lián)的方法，優(yōu)選出不同形態(tài)殼聚糖固定化乳糖酶的方法，并進(jìn)一步研究了固定化乳糖酶的特性和應(yīng)用的效果。另外，還比較研究了測定乳糖水解率的方法，優(yōu)選出了簡便、準(zhǔn)確和快捷的測定方法。主要得出以下結(jié)論：1. 對碘量法、蒽酮法、費(fèi)林法、旋光法和酶催法測定乳糖的準(zhǔn)確性和精密性進(jìn)行了比較研究，結(jié)果表明酶催法測定乳糖水解率的標(biāo)準(zhǔn)偏差為0.0102，變異系數(shù)為2.7935%，其準(zhǔn)確性和精

2、密度好，操作簡單快速，是一種準(zhǔn)確、經(jīng)濟(jì)和快捷的乳糖水解率測定方法。2. 對殼聚糖薄膜、殼聚糖凝膠和殼聚糖珠3種形態(tài)固定化乳糖酶的優(yōu)化方法及其活力回收率進(jìn)行了比較研究，結(jié)果表明殼聚糖珠與戊二醛吸附交聯(lián)固定化乳糖酶的活力回收率最高達(dá)到52.9%，殼聚糖凝膠與戊二醛共價交聯(lián)固定化乳糖酶的活力回收率最高達(dá)到71.1%，殼聚糖薄膜與戊二醛共價交聯(lián)固定化乳糖酶的活力回收率達(dá)到77.8%。3. 殼聚糖膜與戊二醛共價交聯(lián)固定化乳糖酶的最佳工藝條件為：戊二醛濃度為2.5%，pH值為6.5，乳糖酶濃度為0.4 mg/mL，交聯(lián)時間12h，4下固定12h。4. 殼聚糖膜與戊二醛共價交聯(lián)法固定乳糖酶的特性發(fā)生了變化。

3、與游離酶相比，固定化后酶最適pH由6.8移至6.5，固定化酶的最適溫度由40升至45, 60保溫1h后仍保留初始活力的50%，而游離酶則幾乎完全失活；固定化乳糖酶米氏常數(shù)Km與游離酶相比差異不大，說明在對底物的親和性上，在乳糖酶固定化后能夠基本上按游離酶一樣進(jìn)行。固定化酶重復(fù)使用8次后其活力僅損失10%；412下儲存25d后固定化酶活力仍保留50%以上。5. 研究了殼聚糖膜固定化乳糖酶在不同條件下水解乳糖的特性，結(jié)果表明： 隨著固定化酶用量的增加，單位時間內(nèi)對牛奶中的乳糖水解率升高，即在4下，達(dá)到70%以上水解率所需的酶量和時間為：酶添加量10.0g/25mL，水解18h；酶添加量14.0g/

4、25mL，水解18h。在45下，達(dá)到70%以上水解率所需的酶量和時間為：酶添加量6.0g/25mL，水解3.5h；酶添加量10.0g/25mL，水解2.5h；酶添加量14.0g/25mL，水解2h； 固定化酶濃度為10.0g/25mL時，在低溫4、中溫20和最適溫度45下，達(dá)到70%乳糖水解率所需時間分別為18h、18h和2.5h； 水解羊乳中乳糖時，達(dá)到70%以上水解率所需的固定化酶量和時間為：在4下，酶添加量為10.0g/25mL，水解24.0h；在45下，酶添加量10.0g/25mL，水解4.0h。 固定化乳糖酶在相同水解條件下，分別對不同介質(zhì)中的乳糖進(jìn)行水解，乳糖水解率從高到低依次為4

5、.8%乳糖牛乳羊乳乳清； 0.01%的鎂離子對固定化乳糖酶活力有較提高作用。關(guān)鍵詞：-D-半乳糖苷酶；固定化；殼聚糖；乳糖水解率 STUDY ON METHODS, CHARACTERS AND APPLICATIONS OF CHITOSAN IMMOBILIZED-D-GALACTOSIDASEABSTRACT“Lactose malabsorption” is the bottleneck of the dairy industry development in our country. To make use of immobilized-D-galactosidase to hydr

6、olyze lactose in milk can settle “l(fā)actose malabsorption” effectively.This study adopted chitosan as carrier, and glutaraldehyde as crosslinked reagent to immobilize-D-galactosidase. We used ways of covalent-crosslinked and absorption- crosslinked to find the conditions to immobilize lactase in diffe

7、rent forms of chitosan in order to pick out an optimal condition for immobilization. Based on above researches, we had studied the characteristics of the immobilized lactase that had been prepared under the optimal conditions. Additional, we had compared five methods which mensurated ratio of hydrol

8、yzed lactose in order to find a method that is simple、nicety and economy. The results were as follows:1. Compared among five methods including Fehling method, the colorimetric method of Anthrone, the Sacchairmetry, iodine number method and enzyme catalyzed method. The result showed that enzyme catal

9、yzed method was a nicety, economy, simple and convenient way to mensurate content of lactose.2. Studied of the methods of three forms of chitosan including membrane、gelatin and bead to immobilize-D-galactosidase as well as activity recycle rate. The results showed that the activity recycle rate of c

10、hitosan bead adsorption-crosslinked with glutaraldehyde immobilized lactase reached to 52.9%, chitosan gel covalent-crosslinked with glutaraldehyde reached immobilized lactase to 71.1%, chitosan membrane covalent-crosslinked with glutaraldehyde immobilized lactase reached to 77.8%.3. The immobilizat

11、ion conditions of enzyme were confirmed. The best efficiency of immobilization could be obtained under the condition that the concentration of glutaraldehyde was 2.5%, the pH of glutaraldehyde was 6.5, the adding amount-D-galactosidase was 0.4mg/mL, crosslink and immobilization time both were 12 hou

12、rs. 4. The characters of chitosan membrane covalent-crosslinked with glutaraldehyde immobilized lactase had changed. Compared with free lactase, the the optimum pH shifted from 6.8 to 6.5, the optimum temperature for immobilized lactase shifted from 40 to 45.Under the condition of 60/1h, it was stil

13、l left 50% avtivity while the free lactase lose acvtivety completely. Km for immobilized lactase had just a little difference from free lactase, It showed immobilized lactase had almost the same appetency to lactose with free lactase. Immobilized lactase had left 90% activity after be reused 8 times

14、;when storaged 412 for 25d, the activity still had 50%.5. Studied the characteristics of immobilized lactase hydrolyzing lactose in different conditions. The result showed that: The lactose hydrolyzing rate was changed with the amount of immobilized enzyme.To reach upwards of 70% lactose hydrolyzing

15、 rate under 45, the enzymes amount and hydrolyzing time were as follows: 6.0g/25mL, 3.5h; 10.0g/25mL, 2.5h; 14.0g/25mL, 2.0 h.And to reach the same hydrolyzing rate under 4, the enzymes amount and hydrolyzing time is 10.0g/25mL and 18.0h respectively. When the concentration of immobilized enzyme is

16、10.0g/25mL, the time of reaching 70% hydrolyzing lactose rate is 18.0h、18.0h、2.5h respectively under 4、20 and 45. The amount of immobilized lactase and time that need to reach 70% lactose hydrolyzing rate were as follows: adding 10.0g/25mL immobilized lactase and hydrolyzing 24.0h under 4; adding 10.0g/25mL immobilized lactase and hydrolyzing 4.0h under 45. Under the same hydrolyzing