脂肪酶(triacylglycerol acyl hydrolases,EC 3.1.1.3)是一类丝氨酸水解酶,在微水和非水相中能够催化酯化、转酯、酯解等多种反应[1-2],被认为是目前生物领域中最为重要的工业酶之一[3],在食品和饲料等领域具有较大的商业价值[4]。米根霉源脂肪酶(Rhizopus oryzae lipase,ROL)作为常用的商业酶之一,能够改变甘油骨架连接的脂肪酸类型,在油脂加工、风味酯类物质合成和天然等同酯香料制备方面具有重要的应用[5-8]。然而,游离的ROL存在化学稳定性差、回收困难和无法连续化操作等问题[9-10],限制了其在工业中的实际应用。寻找提升ROL稳定性及可重复利用率,同时在最大程度上保持其催化活性的方法,一直是研究者面对的艰巨任务与挑战[11]。

固定化酶技术是解决生物酶稳定性差、回收困难等问题的有效方法之一[12-14]。目前,研究人员已成功将ROL固定在 NKA-9树脂、HPMC-PVA膜、磁性纳米颗粒、苯乙烯-二乙烯基苯等多种基质上[10, 15-17]。尽管基于这些载体制备的固定化ROL展现了稳定性提高及可重复利用等优点,但由于其大多采用吸附法制备,载体对酶分子的束缚较弱,在一定条件下容易出现酶渗漏、浸出等问题[11]。沸石-咪唑框架-8(zeolitic imidazolate framework-8,ZIF-8)是以Zn2+和咪唑连接体通过配位键自组装形成的一种金属有机骨架材料[18-19]。ZIF-8材料具有较大的比表面积、可调控的孔道尺寸及较强的化学和热稳定性等优点,在酶固定化领域备受关注[20-21]。以ZIF-8材料为载体制备固定化酶的方法主要包括吸附法、共价交联法、孔隙封装法和原位自封装法[11,18]。原位自封装法是将酶加入ZIF-8材料的前驱体溶液中,在自组装形成ZIF-8材料的同时,直接把酶固定在ZIF-8内部[18]。与吸附法制备的固定化酶相比,原位自封装法制备的固定化酶减少了酶渗漏、浸出等问题,其稳定性和可重复利用性提高。目前,基于ZIF-8材料利用原位自封装法已实现了产碱杆菌源脂肪酶、黑曲霉源脂肪酶和皱褶假丝酵母源脂肪酶的固定化[22-24]。

尽管基于ZIF-8材料采用原位自封装法制备的固定化脂肪酶已展现了较好的应用前景,但是由于ZIF-8材料较小的孔径,酶的底物或产物在进出ZIF-8材料孔隙时会产生较大的传质阻力,降低固定化酶的催化效率[22-25]。为了解决该问题,研究人员开发了多种具有更大孔径的ZIF-8材料。Feng 等[26]在无水甲醇体系下合成了具有微孔(孔径<2 nm)和介孔(孔径为2～50 nm)的多级孔ZIF-8材料,并基于此材料利用原位自封装法制备了包含细胞色素C和葡萄糖氧化酶的多酶催化纳米反应器。然而,由于ROL的甲醇耐受性较差,在固定化过程中ROL可能发生变性失活[27]。Cui等[28]研究发现,在一定的前驱体初始物质的量比例条件下可以制备出具有介孔的十字花形状 ZIF-8材料,利用此材料原位封装过氧化氢酶制备的固定化酶活性,比采用传统ZIF-8材料制备的固定化酶活性提高了4倍。Wu等[29]通过缺陷法制备出具有微孔及介孔的多级孔ZIF-8材料,应用该材料通过原位自封装法制备了固定化葡萄糖氧化酶,其显示出比基于仅具有微孔的ZIF-8材料制备的固定化酶高5～20倍的表观活性。采用多级孔ZIF-8材料固定化酶不仅提高了酶的稳定性和可重复利用性,而且保持甚至提高了酶的催化活性。

在原位自封装过程中,酶分子表面的电荷分布、酶分子与ZIF-8材料间形成的氢键、疏水相互作用等显著影响ZIF-8材料的成核、生长及组装,进而影响固定化酶的形貌及催化活性 [11, 28, 30]。Liang等[19]基于ZIF-8材料采用原位自封装法固定脂肪酶、尿素酶、葡萄糖脱氢酶等不同酶时发现,固定化酶因酶的结构差异呈现不同的形貌特征。Chen等[31]比较了利用ZIF-8材料原位自封装葡萄糖氧化酶、细胞色素C、辣根过氧化物酶、过氧化氢酶、尿酸氧化酶和乙醇脱氢酶制备的固定化酶的催化性能,发现酶的表面净电荷对固定化酶的固载率和酶活力具有较大影响。目前,利用多级孔ZIF-8材料采用原位自封装法固定ROL的效果仍不清楚。本研究拟通过调节前驱体初始物质的量的比例,制备无定形ZIF-8(aZIF-8)材料,希望获得一种具有更大孔径的aZIF-8材料。采用原位自封装法固定米根霉源脂肪酶,制备固定化脂肪酶ROL@aZIF-8, 研究ZIF-8、aZIF-8、ROL@aZIF-8及采用ZIF-8材料固定化ROL(ROL@ZIF-8)的形貌及结构差异,比较游离ROL与ROL@aZIF-8的热稳定性、酸碱耐受性、贮存稳定性及对邻苯二甲酸二丁酯(dibutyl phthalate,DBP)和邻苯二甲酸二异丁酯(diisobutyl phthalate,DIBP)的水解能力,以期为基于多级孔ZIF-8材料制备固定化脂肪酶提供更多的思路。

1 材料与方法

1.1 材料与试剂

ROL、2-甲基咪唑、醋酸锌、异丙醇和对硝基苯酚丁酸酯(p-nitrophenyl butyrate,p-NPB),美国Sigma-Aldrich公司;乙二胺四乙酸(ethylene diamine tetraacetic Acid,EDTA),西陇科学股份有限公司;DBP和DIBP,国药集团化学试剂有限公司。实验所用试剂均为分析纯。

1.2 仪器与设备

CR 22N型多用途冷冻离心机、jsm7610f型扫描电子显微镜(scanning electron microscopy,SEM)、Jem2100p型透射电子显微镜(transmission electron microscopy,TEM)、扫描透射电镜-能量色散X射线光谱仪(scanning transmission electron microscopy-energy dispersive X-ray spectrometry,STEM-EDX),日本日立公司;Free zone 4.5 plus型真空冷冻干燥机,美国Labconco公司;Nicolet IS50型傅里叶变换红外光谱仪(Fourier transform infrared spectroscopy,FT-IR),赛默飞世尔科技有限公司;D8advance 型X-射线衍射仪(X-ray diffraction,XRD),布鲁克公司;N2吸附解吸仪,美国康塔公司;Theta Flex型水接触角仪,瑞典百欧林公司;电感耦合等离子体发射光谱仪(inductively coupled plasma optical emission spectrometer,ICP-OES)、7890B型气相色谱仪,美国安捷伦科技有限公司。

1.3 实验方法

1.3.1 aZIF-8、ROL@aZIF-8、ZIF-8及ROL@ZIF-8的制备

1.3.1.1 aZIF-8及ROL@aZIF-8的制备

参考Wu等[29]的方法并适当修改。将1 mL 20 mmol/L的醋酸锌溶液逐滴加至1 mL 33.8 mmol/L的2-甲基咪唑溶液中,立刻加入80 μL、 5 mg/mL的ROL溶液,在300 r/min下反应30 min。反应结束后用去离子水洗涤沉淀物3次,收集沉淀,冷冻干燥12 h,得到ROL@aZIF-8。在相同条件下不添加ROL溶液制备aZIF-8材料。

1.3.1.2 ZIF-8及ROL@ZIF-8的制备

参考梁鑫等[24]的方法并适当修改。将20 mL 1 mol/L的2-甲基咪唑溶液与80 mg的ROL混合均匀,加入2 mL 0.5 mol/L的醋酸锌溶液,在300 r/min下反应30 min。反应结束后用去离子水洗涤沉淀物3次,收集沉淀,冷冻干燥12 h,得到ROL@ZIF-8。在相同条件下不添加ROL溶液制备ZIF-8材料。

1.3.2 aZIF-8、ROL@aZIF-8、ZIF-8及ROL@ZIF-8的形貌及结构观察

取一定量的aZIF-8、ROL@aZIF-8、ZIF-8以及ROL@ZIF-8样品,研磨至均匀粉末。采用XRD、SEM和TEM观察样品形貌和晶体结构;采用FT-IR、STEM-EDX及ICP-OES分析样品中元素组成、分布及比例;采用N2吸附解吸仪测定温度为77 K时样品的N2吸附解吸等温线,计算Brunauer-Emmett-Teller (BET)面积、孔径分布和孔隙体积;采用水接触角仪分析样品的亲疏水性。

1.3.3 ROL和ROL@aZIF-8酶活力的测定

ROL和ROL@aZIF-8酶活力的测定方法参考Qi等[32]的方法并稍加修改。将一定量的ROL和ROL@aZIF-8加入pH值为7.5的1 mL 0.01 mol/L的磷酸盐缓冲液中,与1 mL 2 mg/mL溶于异丙醇中的p-NPB溶液混匀后,在室温条件下反应5 min。反应结束后,加入1 mL终止液(40 g NaOH和93.5 g EDTA溶于1 L去离子水中)终止反应,在4 ℃条件下离心收集上清液,在405 nm波长处测定吸光值。酶活力单位定义为:每分钟释放1 μmol对硝基苯酚(p-nitrophenol, p-NP)所需的ROL和ROL@aZIF-8的质量,以U/mg表示。

1.3.4 不同质量浓度的ROL与ROL@aZIF-8酶活力的测定

采用不同质量浓度(3、4、5、6、7 mg/mL)的ROL溶液分别制备ROL@aZIF-8,分析ROL@aZIF-8与相同质量浓度ROL的酶活力。

1.3.5 不同条件下ROL与ROL@aZIF-8酶活力的测定

1.3.5.1 不同pH值条件下ROL与ROL@aZIF-8酶活力的测定

将ROL以及ROL@aZIF-8置于不同pH 值(7.5、8、9、10)的 0.01 mol/L PBS 缓冲液中,在室温下保温60 min,测定其酶活力。

1.3.5.2 不同温度条件下ROL与ROL@aZIF-8酶活力的测定

将ROL以及ROL@aZIF-8置于pH值为7.5的0.01 mol/L PBS缓冲液中,在不同温度(30、40、50、60、70 ℃)下保温60 min,测定其酶活力。

1.3.5.3 不同储藏时间条件下ROL与ROL@aZIF-8酶活力的测定

将ROL以及ROL@aZIF-8置于pH值为7.5的0.01 mol/L PBS缓冲液中,在4 ℃下储藏不同时间 (0、2、4、6、8、10、15、20、25 d),测定其酶活力。

1.3.6 ROL 及ROL@aZIF-8水解DBP和DIBP能力的测定

以pH值为7.4的0.05 mol/L Tris-HCl 溶液为缓冲体系,将0.78 mL Tris-HCl 缓冲液、200 μL 50 mg/mL的ROL溶液或50 mg ROL@aZIF-8,以及20 μL 10 g/L的DBP和DIBP混合溶 液充分混合,在150 r/min、37 ℃条件下反应2 h。反应结束后,使用正己烷(含内标油酸乙酯)萃取,进行气相色谱分析。分析条件:安捷伦HP-INNO WAX色谱柱,80 ℃保持 3 min,以 20 ℃/min 的速率升至 250 ℃,保持22.5 min。以未加入ROL溶液或ROL@aZIF-8的催化反应体系中的 DBP和DIBP残余量为100%,计算ROL以及ROL@aZIF-8对DBP和DIBP的分解率。

1.4 数据处理

数据以平均值±标准偏差表示。采用SPSS 25统计软件经单因素方差分析进行数据比较,P<0.05为差异显著。

2 结果与分析

2.1 ZIF-8、aZIF-8、ROL@ZIF-8和ROL@aZIF-8的形貌比较

在原位自封装过程中,前驱体的初始物质的量比例不同,会形成具有不同形貌及结构的ZIF-8材料[28-29]。在本研究中,ZIF-8材料是在2-甲基咪唑和醋酸锌(前驱体)初始物质的量比例为20∶1的条件下制备获得,而aZIF-8材料是在2-甲基咪唑和醋酸锌初始物质的量比例为1.69∶1.00的条件下制备获得。ROL@ZIF-8和ROL@aZIF-8分别是在ZIF-8和aZIF-8材料组装过程中加入ROL制备获得。对ZIF-8、ROL@ZIF-8、aZIF-8和ROL@aZIF-8进行了晶体结构和形貌分析,分析结果见图1至图3。由图1可见,ZIF-8材料和ROL@ZIF-8在2θ为7.40°、10.41°、12.78°、14.74°、16.50°和18.07°处出现强峰,其衍射峰分布规律与基于理论数据模拟的ZIF-8材料一致,且相对强度相似[33-34];而aZIF-8材料和ROL@aZIF-8在2θ为11.05°、12.14°、17.25°和18.43°处出现强峰,这与ZIF-8材料和ROL@ZIF-8衍射峰分布规律具有明显差异,表明相较于ZIF-8材料和ROL@ZIF-8,aZIF-8材料和ROL@aZIF-8的晶体结构发生了明显改变[35]。由图2(a)和(c)可见,ZIF-8材料呈现出规则的多面体形貌,而aZIF-8材料呈现出包含十字花形、正方形、球形等多种形态的无定形形貌。微观结构分析结果与形貌分析结果一致[图3(a)和(c)]。研究发现,在2-甲基咪唑和醋酸锌的初始物质的量比例为4∶1以及更高的条件下,ZIF-8材料呈现出规则的菱形十二面体结构,这与我们的研究结果相似[23, 29]。Cui等[28]研究发现,在2-甲基咪唑和硝酸锌的初始物质的量比例为1.6∶1.0 的条件下,catalase@ZIF-8呈现十字花形的聚合物形貌,而在2-甲基咪唑和硝酸锌的初始物质的量比例为1.8∶1.0的条件下,catalase@ZIF-8呈现多面体形貌。这表明, aZIF-8材料的无定形形貌是因为在相对低的前驱体初始物质的量比例条件下制备获得的。

尽管ROL@ZIF-8也呈现出多面体形貌,但相较于ZIF-8材料,ROL@ZIF-8的边界变得凹凸不平,并且面积有轻微的减小[图2(b)、图3(b)]。ROL@aZIF-8呈现出与aZIF-8材料相似的无定形形貌,但其形态更加多样[图2(d)、图3(d)]。为了确证ROL@ZIF-8和ROL@aZIF-8中是否含有ROL,对ROL、ZIF-8、ROL@ZIF-8、aZIF-8和ROL@aZIF-8进行了红外光谱分析(图4)。由图4可见,ZIF-8、ROL@ZIF-8、aZIF-8和ROL@aZIF-8均在波数为560 cm-1和1 308 cm-1处出现特异吸收峰。位于560 cm-1处的吸收峰是Zn-N拉伸振动产生的,而位于1 350～1 500 cm-1处的吸收峰是2-甲基咪唑振动产生的。这些结果说明,ZIF-8、ROL@ZIF-8、aZIF-8和ROL@aZIF-8结构中存在Zn2+与2-甲基咪唑[36-37]。ROL@ZIF-8和ROL@aZIF-8均在1 600～1 700 cm-1处出现蛋白质酰胺键的特异吸收峰,而ZIF-8以及aZIF-8未观察到相应的吸收峰,表明ROL已成功固定在ZIF-8和aZIF-8材料上[35, 38-39]。这些结果表明,在固定化ROL后,与ZIF-8和aZIF-8材料相比,ROL@ZIF-8和ROL@aZIF-8的形貌发生了改变[19, 40-41]。

2.2 ZIF-8、aZIF-8、ROL@ZIF-8和ROL@aZIF-8的结构比较

ZIF-8材料形貌的改变往往伴随着其结构的变化[28-29]。利用STEM-EDX及ICP-OES对ZIF-8、ROL@ZIF-8、aZIF-8和ROL@aZIF-8 中Zn和N元素分布及比例进行了研究,分析结果见图5和表1。由图5可见,ZIF-8、ROL@ZIF-8、aZIF-8和ROL@aZIF-8中Zn和N元素分布均匀[40]。由表1可见,ZIF-8材料和ROL@ZIF-8中N与Zn的物质的量比为4.58和4.35,而aZIF-8材料和ROL@aZIF-8中N与Zn的物质的量比则降至1.53和1.94。这表明在ZIF-8材料和ROL@ZIF-8中,平均一个锌离子与4.3～4.5个氮原子配位,而在aZIF-8材料和ROL@aZIF-8中,平均一个锌离子与1.5～1.9个氮原子配位。Wu等[29]研究发现,与仅具有微孔的ZIF-8材料相比,具有微孔及介孔的多级孔ZIF-8材料的N与Zn的物质的量比例降低,这与本研究结果相似。当锌离子与相对较少的氮原子配位时,ZIF-8材料会出现结构缺陷,导致其孔径变大甚至出现介孔[29]。为了探究ZIF-8、ROL@ZIF-8、aZIF-8和ROL@aZIF-8的孔径大小,利用N2吸附法测定了其N2吸附-解吸等温线、孔径分布及比表面积,分析结果见图6、图7和表2。由图6可见,随相对压力的增加,不同材料及固定化ROL的N2吸附量呈现不同的变化趋势。通过与6种类型N2吸附-解吸等温线的标准形状比较发现,ZIF-8材料和ROL@ZIF-8的N2吸附-解吸等温线呈现出Ⅰ型吸附等温线特征,表明ZIF-8材料和ROL@ZIF-8的微孔性质;而aZIF-8和ROL@aZIF-8的N2吸附-解吸等温线呈现出Ⅱ型吸附等温线特征,表明aZIF-8材料和ROL@aZIF-8存在微孔和介孔[42-43]。由图7可见,ZIF-8材料和ROL@ZIF-8的孔径分布在1～2 nm,表明ZIF-8材料和ROL@ZIF-8形成了微孔;而aZIF-8材料和ROL@aZIF-8的孔径分布在1～20 nm,表明aZIF-8材料和ROL@aZIF-8中既存在微孔又存在介孔。由表2可见,相较于ZIF-8材料和ROL@ZIF-8,aZIF-8材料和ROL@aZIF-8的BET比表面积和孔隙体积均出现显著减小,这可能是因为,在相对低的前驱体初始物质的量比例条件下,aZIF-8材料出现结构缺陷,造成BET比表面积减小和孔容下降[29]。本研究结果表明,通过调节前驱体的物质的量比例制备得到的aZIF-8材料是一种具有微孔和介孔的多级孔材料。

2.3 酶质量浓度对ROL@aZIF-8酶活力的影响

为探究利用aZIF-8材料采用原位自封装法固定ROL的效果,本研究比较了在不同ROL质量浓度条件下制备的ROL@aZIF-8与相同质量浓度的ROL的酶活力差异,分析结果见图8。由图8可见,当ROL的质量浓度从3 mg/mL增加到4 mg/mL时,ROL@aZIF-8催化p-NPB水解的能力逐渐增强;当ROL的质量浓度从4 mg/mL增加到7 mg/mL时,ROL@aZIF-8的酶活力逐渐减弱。在ROL的质量浓度为4 mg/mL的条件下制备的ROL@aZIF-8呈现最大酶活力,为5.69 U/mg。通过与文献报道的固定化ROL的酶活力相比较,本研究中制备的ROL@aZIF-8的酶活力处于最高水平[10, 13, 15-17, 44-48]。在相同质量浓度条件下,ROL@aZIF-8的酶活力比ROL高,表明利用aZIF-8材料采用原位自封装法固定ROL可以增强酶的催化活性。

ROL@aZIF-8催化活性增强的效果可能与ROL“盖子”结构的构象变化有关[49-51]。ROL具有由两亲性氨基酸组成的“盖子”结构[52]。“盖子”结构具有独特的打开-闭合构象变化现象[52]。当“盖子”结构处于闭合构象时,ROL的催化中心会被“盖子”盖住,阻止了底物进入催化中心,表现为酶的催化活性较低;当“盖子”结构处于打开构象时,底物和产物可以较容易地进出催化中心,表现为酶的催化活性大幅提高[53-54]。研究发现,当使用疏水性的载体固定化脂肪酶时,脂肪酶的“盖子”结构可以处于稳定的打开构象,固定化脂肪酶表现出较高的催化活性[53-56]。为探究ROL@aZIF-8催化活性增强效果的可能原因,本研究比较了ZIF-8、ROL@ZIF-8、aZIF-8和ROL@aZIF-8的亲疏水性差异,分析结果见图9。由图9可见,ZIF-8材料和aZIF-8材料均具有较强的疏水性,aZIF-8材料的疏水性相较于ZIF-8材料减弱。这表明利用aZIF-8材料固定化ROL可能诱导ROL的“盖子”结构处于打开构象,从而使ROL@aZIF-8的催化活性提高[49-50]。疏水性的载体可能在固定化过程中诱导酶的聚集,导致固定化酶催化活性的下降[57]。aZIF-8材料的疏水性相较于ZIF-8材料减弱可能有助于减少在固定化过程中ROL的聚集,促使固定化酶的催化活性保持甚至提高[57]。由图9可知,ROL@ZIF-8和ROL@aZIF-8均呈现出亲水性,这可能是由于在固定化ROL后,ROL增强了材料的亲水性[29]。ROL@aZIF-8中因结构缺陷形成的多级孔结构可能造成部分的ROL暴露于固定化酶表面,导致ROL@aZIF-8的亲水性相较于ROL@ZIF-8增强,这也同时促使了底物更容易与ROL的催化活性中心结合[29]。本研究结果表明,aZIF-8材料的疏水性及多级孔特性对ROL@aZIF-8催化活性的增强有重要的促进作用。

2.4 不同条件下ROL和ROL@aZIF-8酶活力的比较

2.4.1 pH值对ROL和ROL@aZIF-8酶活力的影响

pH值对ROL和ROL@aZIF-8酶活力的影响,实验结果见图10。由图10可见,在pH值为7.5 ～ 10.0时,固定化酶ROL@aZIF-8的酶活力均高于游离脂肪酶ROL,表明固定化酶比游离酶具有更好的碱耐受性。这可能是由于aZIF-8材料和ROL间通过疏水相互作用、范德华相互作用和氢键等创造了一个保护酶分子的微环境从而改善了酶的碱稳定性[30, 39]。

2.4.2 温度对ROL和ROL@aZIF-8酶活力的影响

温度对ROL和ROL@aZIF-8酶活力的影响,实验结果见图11。由图11可见,在30～70 ℃下保温1 h后, ROL@aZIF-8比ROL具有更高的酶活力,这表明固定化酶比游离酶具有更好的温度耐受性,这可能是因为aZIF-8材料对ROL产生的保护作用[28-29]。

2.4.3 储藏时间对ROL和ROL@aZIF-8酶活力的影响

储藏时间对ROL和ROL@aZIF-8酶活力的影响,实验结果见图12。由图12可见,随储藏时间的延长, ROL@aZIF-8和ROL的酶活力均下降,但ROL@aZIF-8的酶活力降低幅度相较于ROL较小。在经过25 d储藏后,ROL@aZIF-8的酶活力降至其初始酶活力的30.5%,而ROL的酶活力降至其初始酶活力的20.4%,表明ROL@aZIF-8比ROL具有更好的储藏稳定性。储藏稳定性的提高可能是因为aZIF-8材料包裹在ROL周围,防止ROL水解和聚合以及环境对酶活性位点的破坏,从而使酶活力降低减缓[2, 51]。Cheng等[58]研究发现,利用Cu-BTC金属有机骨架材料采用原位自封装法制备的固定化脂肪酶在经过20d的储藏后仍保持了97.1%的酶活力。Nadar等[23]研究发现,利用ZIF-8材料采用原位自封装法制备的固定化脂肪酶在经过25 d的储藏后酶活力降低了10%。与这些固定化脂肪酶相比,ROL@aZIF-8的储藏稳定性较弱,原因可能是由于aZIF-8材料的结构缺陷降低了ROL@aZIF-8在苛刻条件下的稳定性[10]。

2.5 ROL和ROL@aZIF-8对DBP和DIBP水解能力的比较

DBP和DIBP是塑化剂的典型代表,具有较强的生殖毒性,即使微量摄入也会造成脂质代谢异常,诱发肥胖病,干扰免疫和过敏反应等[59-60]。利用生物酶法水解DBP和DIBP具有条件温和、效率高和不产生二次污染等优势[59-60]。为了进一步探究ROL@aZIF-8催化活性增强的效果,本研究比较了ROL和ROL@aZIF-8对DBP和DIBP的水解能力,分析结果见图13。由图13可见,ROL@aZIF-8催化DBP和DIBP水解2h后,DBP和DIBP的残留量分别为83.7%和83.1%,而ROL催化水解2 h后,DBP和DIBP的残留量分别90.8%和91.2%。这表明与ROL相比,ROL@aZIF-8对DBP和DIBP的水解能力有一定程度提高。

3 结 论

本研究通过调节2-甲基咪唑和醋酸锌的初始物质的量比例,制备了一种aZIF-8材料,采用原位自封装法将ROL固定在aZIF-8材料中,制备出ROL@aZIF-8。研究结果表明,aZIF-8材料是一种具有微孔和介孔的多级孔材料。aZIF-8材料和ROL@aZIF-8均呈现出包含十字花形、正方形、球形等多种形态的无定形形貌。在ROL的质量浓度为4 mg/mL的条件下制备的ROL@aZIF-8具有最大酶活力,为5.69 U/mg。在相同的ROL质量浓度条件下,ROL@aZIF-8的酶活力比ROL高,其原因与aZIF-8材料的疏水性及多级孔特性密切相关。与ROL相比,ROL@aZIF-8具有更好的碱稳定性、温度耐受性、储藏稳定性以及对DBP和DIBP更高的水解能力。本研究旨在为基于多级孔ZIF-8材料制备固定化脂肪酶提供理论依据。

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Immobilization of Lipase Derived from Rhizopus oryzae Based on Amorphous Zeolitic Imidazolate Framework-8 Material

LU Yuhan1, ZHANG Chengnan1,*, LI Xiuting1,2, XU Youqiang1, LI Weiwei1

(1.School of Food and Health/Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University, Beijing 100048, China;2.Beijing Engineering and Technology Research Center of Food Additives, Beijing 100048, China)

Abstract：Hierarchical porous zeolitic imidazolate framework-8 (ZIF-8) materials have larger pore sizes compared with conventional ZIF-8 materials, and show better application prospects in the field of immobilized enzymes. At present, the effect of immobilizing Rhizopus oryzae lipase (ROL) using hierarchical porous ZIF-8 material remains unclear. In this study, an amorphous zeolitic imidazolate framework-8 (aZIF-8) material was prepared by adjusting the initial molar ratio of the precursor of ZIF-8 material, and the immobilized lipase ROL@ZIF-8 and ROL@aZIF-8 were prepared by in situ self-encapsulation based on the ZIF-8 and aZIF-8 materials, respectively. The morphological and structural differences of ZIF-8, aZIF-8, ROL@ZIF-8 and ROL@aZIF-8 were investigated by using infrared spectroscopy, X-ray diffractometer and scanning electron microscope, etc. The enzyme activities of ROL@aZIF-8 prepared under different ROL mass concentrations were compared with those of the same mass concentration of ROL. The acid-alkali stability, temperature tolerance, storage stability, and hydrolysis ability of ROL and ROL@aZIF-8 against dibutyl phthalate (DBP) and diisobutyl phthalate (DIBP) were investigated. The results showed that the aZIF-8 material presented an amorphous morphology containing various forms such as cruciate flower-like, square, and spherical, and was a hierarchical porous material with micropores and mesopores. ROL@aZIF-8 prepared at ROL mass concentration of 4 mg/mL showed the maximum enzyme activity of 5.69 U/mg. The hydrophobicity and hierarchical porous properties of the aZIF-8 material contributed to the higher enzyme activity of ROL@aZIF-8 than that of ROL at the same mass concentration of ROL. ROL@aZIF-8 showed better alkali stability, temperature tolerance, storage stability, and higher hydrolytic capacity for DBP and DIBP than ROL.

Keywords：lipase; hierarchical porous zeolitic imidazolate framework-8 material; immobilized enzyme; in situ encapsulation method; morphology

doi：10.12301/spxb202200982

文章编号：2095-6002(2023)06-0039-13

引用格式：芦瑜涵, 张成楠, 李秀婷, 等. 基于无定形沸石-咪唑框架-8材料固定米根霉源脂肪酶研究[J]. 食品科学技术学报,2023,41(6):39-51.

LU Yuhan, ZHANG Chengnan, LI Xiuting, et al. Immobilization of lipase derived from Rhizopus oryzae based on amorphous zeolitic imidazolate framework-8 material [J]. Journal of Food Science and Technology, 2023,41(6):39-51.

中图分类号：TS202.3

文献标志码：A

收稿日期：2022-10-27

基金项目：国家自然科学基金资助项目(32001638; 31830069)。

Foundation: National Natural Science Foundation of China (32001638; 31830069).

第一作者：芦瑜涵,女,硕士研究生,研究方向为食品酶学。

*通信作者：张成楠,男,副教授,博士,主要从事食品生物技术方面的研究。

(责任编辑：叶红波)