CN115247169A - 一种利用双MOFs固定化脂肪酶制备生物柴油的方法 - Google Patents
一种利用双MOFs固定化脂肪酶制备生物柴油的方法 Download PDFInfo
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Abstract
本发明公开一种利用双MOFs固定化脂肪酶制备生物柴油的方法,其包括以下步骤:制备ZIF‑8;利用ZIF‑8制备ANL/ZIF‑8;制备三维有序聚苯乙烯模板;利用三维有序聚苯乙烯模板制备M‑ZIF‑8;利用M‑ZIF‑8制备ANL@M‑ZIF‑8;将油脂、短链醇、ANL/ZIF‑8、及ANL@M‑ZIF‑8混合反应,制得生物柴油。本发明同时采用ANL/ZIF‑8和ANL@M‑ZIF‑8作为双MOFs用以制备生物柴油,不但解决了采用单一MOFs介孔结构的孔径不足而抑制催化合成效果的问题,而且集成两种MOFs的介孔结构的物理化学性能,形成多层次的孔吸附结构,进而极大地提高生物柴油的得率。
Description
技术领域
本发明涉及生物化工领域,具体涉及一种利用双MOFs固定化脂肪酶制备生物柴油的方法。
背景技术
金属有机骨架材料(MOFs)作为一种新兴材料,主要用于脂肪酶的固定化,对油脂高值化转化具有重要开发应用前景。MOFs对酶的固定化一般通过孔吸附法来实现,酶分子固定化在MOFs孔内后,受到MOFs孔结构的限域效应,分子构象不易发生变化,因而大大地提高其稳定性。但通常MOFs结晶性孔孔径<5nm,理论上无法实现孔吸附对脂肪酶进行固定化。一些学者提出通过软模板法合成含有介孔结构的多级孔MOF进而用于脂肪酶的固定化,但由于受其介孔结构(20~30nm)传质限制,固定化的脂肪酶仍然只能用来催化小分子底物月桂酸和苯甲醇的酯化反应,而无法催化动植物油脂中的甘油三酯底物进行生物柴油的制备。以三油酸甘油酯底物制备生物柴油为例,平均分子尺寸在3.5nm左右,而不同脂肪酶分子平均尺寸在5~10nm以上,在实际催化体系中,脂肪酶往往与水合分子结合形成酶-水合分子,其分子尺寸往往在数十纳米,使得这种微介孔载体通过孔吸附法固定化脂肪酶,综合考虑酶及底物分子尺寸、以及传质要求,此种酶-水合分子的尺寸难以满足脂肪酶对生物柴油的催化合成需求。现阶段主要趋向于需要大孔结构(孔径>50nm)才能在吸附脂肪酶的同时,还能催化大分子长链油脂底物制备生物柴油。
发明内容
为了克服上述技术问题,本发明公开了一种利用双MOFs固定化脂肪酶制备生物柴油的方法。
本发明为实现上述目的所采用的技术方案是:
本发明采用的金属有机骨架材料MOFs包括但不限于ZIF-8(ZIF-8是由Zn2+和2-甲基咪唑配体组成的稳定的类沸石结构的有机金属骨架材料),本发明选用ZIF-8合成ZIF-8表面固定化脂肪酶、及通过孔吸附法合成大孔ZIF-8固定化脂肪酶,实现对油脂的高值化、资源化利用,达到更高的酶活回收率和产品得率。
为此,本发明提供一种利用双MOFs固定化脂肪酶制备生物柴油的方法,其包括以下步骤:
步骤1,制备ZIF-8;
步骤2,利用所述ZIF-8制备ANL/ZIF-8;
步骤3,制备三维有序聚苯乙烯模板;
步骤4,利用所述三维有序聚苯乙烯模板制备M-ZIF-8;
步骤5,利用所述M-ZIF-8制备ANL@M-ZIF-8;
步骤6,将油脂、短链醇、所述ANL/ZIF-8、及所述ANL@M-ZIF-8混合反应,制得生物柴油。
上述的利用双MOFs固定化脂肪酶制备生物柴油的方法,其中所述步骤1进一步包括以下步骤:
步骤1-1,将Zn(NO3)2·6H2O溶于甲醇中,制得溶液A;
步骤1-2,将2-甲基咪唑溶于甲醇中,制得溶液B;
步骤1-3,将所述溶液A和溶液B混合,并于室温下震摇或磁力搅拌24h后离心分离;其中,离心分离转速为10000rpm;
步骤1-4,取离心后的沉淀,利用甲醇洗涤数次后浸泡于甲醇中活化1天后,经过离心、甲醇洗涤、真空干燥,得到白色粉末状的所述ZIF-8。
上述的利用双MOFs固定化脂肪酶制备生物柴油的方法,其中所述步骤2进一步包括以下步骤:
步骤2-1,定量称取已干燥的所述ZIF-8,加入超纯水和液体脂肪酶ANL,混匀、震摇、离心,取上清液测定蛋白浓度;其中,震摇条件为:震摇温度45℃,回旋频率200rpm,震荡时间>4小时;
步骤2-2,取离心后的沉淀水洗、离心,利用冻干机冻干>12小时,得到所述ANL/ZIF-8,即为ZIF-8表面固定化脂肪酶。
上述的利用双MOFs固定化脂肪酶制备生物柴油的方法,其中所述步骤3进一步包括以下步骤:
步骤3-1,取足量苯乙烯,依次用10wt%NaOH溶液和去离子水分别洗涤数次,以去除苯乙烯中的阻聚剂;
步骤3-2,将步骤3-1的苯乙烯和聚乙烯吡咯烷酮溶液,用氮气鼓泡15分钟后,在持续转速450rpm机械搅拌和于75℃下加热的条件下,加入K2S2O8溶液引发反应24小时,待反应完成后冷却、抽滤、用去离子水和乙醇依次洗涤、60℃干燥,得到滤饼状的所述三维有序聚苯乙烯模板。
上述的利用双MOFs固定化脂肪酶制备生物柴油的方法,其中所述步骤4进一步包括以下步骤:
步骤4-1,按照比例取Zn(NO3)2·6H2O、2-甲基咪唑和甲醇,配制成足量的ZIF-8前体甲醇溶液;
步骤4-2,取步骤3制得的所述三维有序聚苯乙烯模板浸泡于所述ZIF-8前体甲醇溶液中1小时,抽真空除气泡10分钟、于50℃下干燥12小时,得到滤饼A;
步骤4-3,将所述滤饼A在室温下浸入体积比为1:1的CH3OH/NH3·H2O的混合溶液中,抽真空除气泡10分钟后常温常压下放置反应24小时,在此过程中所述滤饼A因ZIF-8的生长压力渐大而逐步破碎成小块,待反应完成后抽滤,用足量水和乙醇依次洗涤,充分洗去残余的氨水,于50℃下干燥,得到滤饼B;
步骤4-4,将所述滤饼B浸入足量四氢呋喃中24小时,多次刻蚀并于100℃下干燥,得到白色粉末状的所述M-ZIF-8,即为大孔ZIF-8;其中,所述M-ZIF-8的孔径大于100nm,每次刻蚀操作后离心分离出固体,重复刻蚀操作大于5次,以确保聚苯乙烯模板已被充分刻蚀。
上述的利用双MOFs固定化脂肪酶制备生物柴油的方法,其中所述步骤5进一步包括以下步骤:
步骤5-1,称取所述M-ZIF-8,加入超纯水超声分散至均匀分散,加入液体脂肪酶ANL,混匀、震摇、离心(离心后固体实际悬浮于溶液上方),取上清液采用BCA法测定蛋白浓度;其中,震摇条件为:震摇温度45℃,回旋频率200rpm,震荡时间>4小时;
步骤5-2,取离心后的沉淀水洗、离心,利用冻干机冻干>12小时,得到所述ANL@M-ZIF-8,即为大孔ZIF-8固定化脂肪酶。
上述的利用双MOFs固定化脂肪酶制备生物柴油的方法,其中在所述步骤6中,所述短链醇的用量为油脂摩尔比的4.5~6倍,所述短链醇包括甲醇、乙醇、丙醇、丁醇等,所述ANL/ZIF-8用量为油脂质量的600~1000个标准酶活单位,所述ANL@M-ZIF-8用量为油脂质量的600~1000个标准酶活单位。
上述的利用双MOFs固定化脂肪酶制备生物柴油的方法,其中所述步骤6于一级或多级酶反应器中进行,反应温度控制在40~55℃,反应8~20小时,其中短链醇在2个小时内匀速加入;优选地,一级或多级酶反应器与在线脱水设备相偶联,以在线除去反应体系中的水分,其中,在线脱水是指利用膜、分子筛或气提。在线脱水所用的膜为有机膜、无机膜或陶瓷膜等;在线脱水所用的分子筛为或分子筛等;通常情况下,所述ANL/ZIF-8的反应转化率为74%以上,所述ANL@M-ZIF-8的反应转化率为82%以上。
本发明中所述液体脂肪酶包括来源于酵母、霉菌、细菌或其它微生物的脂肪酶;所述液体脂肪酶为单种脂肪酶或多种脂肪酶的组合,例如但不限于来源于南极假丝酵母(Candida antarctica)、嗜热丝孢菌(Thermomyces lanuginosus)、黑曲霉(Aspergillusniger)、米曲霉(Aspergillus oryzae)、米黑根毛霉(Rhizomucor miehei)和米根霉(Rhizopus oryzae)等中的至少一种。
本发明中所述油脂为可再生生物油脂,包括植物油脂、动物油脂、废食用油、酸化油、油脂精练下脚料和微生物油脂等;其中,所述植物油脂为蓖麻油、棕榈油、菜籽油、大豆油、花生油、玉米油、棉子油、米糠油、麻风树油、文冠果油或小桐子油等;所述动物油脂为鱼油、牛油、猪油或羊油等;所述微生物油脂为酵母油脂或微藻类油脂等;所述废食用油为潲水油或地沟油等;所述油脂精炼下脚料为酸化油等。
本发明的有益效果为:
(1)本发明同时采用ANL/ZIF-8和ANL@M-ZIF-8作为双MOFs用以制备生物柴油,不但解决了采用单一MOFs介孔结构的孔径不足而抑制催化合成效果的问题,而且集成了两种MOFs的介孔结构的物理化学性能,形成多层次的孔吸附结构,进而极大地提高生物柴油的得率;其中ZIF-8表面固定化脂肪酶作为常规的微孔表面固定化脂肪酶,可满足基本的反应转化需求,大孔ZIF-8固定化脂肪酶通过孔吸附法将脂肪酶固定化在大孔MOFs的孔内部,极大地提高比表面积,表现出优于微孔表面固定化脂肪酶更高的催化效率、酶活回收率、比酶活,在后续催化油脂制备生物柴油的过程中,也表现出更高的短链醇耐受性,在催化油脂高值化转化制备生物能源和生物基化学品中具有重要开发应用前景;
(2)本发明采用温和的方法合成ANL@M-ZIF-8,同时控制原材料的配比和制备条件,利于对脂肪酶有效吸附固定于大孔介孔结构内部,实现脂肪酶的稳定性和循环实用性;
(3)选择合成M-ZIF-8,其大孔介孔结构的比标面积大、孔隙率高,利于脂肪酶的固定和保护,防止脂肪酶在制备过程中流失或丧失酶活,方便产物和脂肪酶的分离,便于脂肪酶的重复利用。
附图说明
下面结合附图和实施例对本发明进一步说明。
图1为本发明中工艺方法的主要流程示意图。
具体实施方式
下面通过具体实施例对本发明作进一步说明,以使本发明技术方案更易于理解、掌握,而非对本发明进行限制。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段,所用原料均为市售商品。
实施例1:本实施例提供一种利用双MOFs固定化脂肪酶制备生物柴油的方法,其包括以下步骤:
步骤1,将1.68gZn(NO3)2·6H2O(5.65mmol)溶于80mL甲醇中,制得溶液A;将3.70g2-甲基咪唑(45mmol)溶于80mL甲醇中,制得溶液B;将所述溶液A和溶液B混合,并于室温下磁力搅拌24h后,以10000rpm的转速离心分离;取离心后的沉淀,利用甲醇洗涤数次后浸泡于甲醇中活化1天后,经过离心、甲醇洗涤、真空干燥,得到白色粉末状的所述ZIF-8;
步骤2,利用分析天平定量称取30mg已干燥的所述ZIF-8,加入980μL超纯水和20μL液体脂肪酶ANL,混匀后于45℃、频率为200rpm下震摇5小时、离心,取上清液测定蛋白浓度;取离心后的沉淀水洗、离心,利用冻干机冻干14小时,得到所述ANL/ZIF-8;
步骤3,取足量苯乙烯加入至分液漏斗中,依次用10wt%NaOH溶液和去离子水分别洗涤数次,以去除苯乙烯中的阻聚剂;将65mL苯乙烯和500mL聚乙烯吡咯烷酮溶液(K-30,2.50g)添加至圆底三口烧瓶中,用氮气鼓泡15分钟后,在持续转速450rpm机械搅拌下,于75℃下加热30分钟,随后快速加入50mL的K2S2O8(1.00g)溶液引发反应,于75℃下持续转速450rpm机械搅拌24小时,待反应完成后冷却,并于布氏漏斗上叠放两张常规滤纸后水泵抽滤,抽滤24小时后,用去离子水和乙醇依次洗涤形成的滤饼,并置于60℃烘箱中干燥过夜,得到滤饼状的所述三维有序聚苯乙烯模板;
步骤4,取8.15g Zn(NO3)2·6H2O、6.75g 2-甲基咪唑和45mL甲醇,配制成足量的ZIF-8前体甲醇溶液;将所述三维有序聚苯乙烯模板浸泡于所述ZIF-8前体甲醇溶液中1小时,抽真空除气泡10分钟后,将充分浸润的滤饼放入烘箱中于50℃下干燥12小时,得到滤饼A;将所述滤饼A在室温下浸入体积比为1:1的CH3OH/NH3·H2O的混合溶液中,抽真空除气泡10分钟后常温常压下放置反应24小时,在此过程中所述滤饼A因ZIF-8的生长压力渐大而逐步破碎成小块,待反应完成后抽滤,用足量水和乙醇依次洗涤,充分洗去残余的氨水,于50℃烘箱中干燥,得到滤饼B;将所述滤饼B浸入足量四氢呋喃中24小时,多次刻蚀并于100℃下干燥过夜,得到白色粉末状的所述M-ZIF-8;
步骤5,称取60mg所述M-ZIF-8,加入800μL超纯水超声分散至均匀分散,加入200μL液体脂肪酶ANL,混匀并于45℃、频率为200rpm下震摇5小时后离心(离心后固体实际悬浮于溶液上方),取上清液采用BCA法测定蛋白浓度;取离心后的沉淀水洗、离心,利用冻干机冻干14小时,得到所述ANL@M-ZIF-8;
步骤6,将200g菜籽油和基于油脂摩尔比为4.5:1的甲醇置于一级或多级酶反应器中,分别加入基于油脂质量600个标准酶活单位的ANL/ZIF-8和ANL@M-ZIF-8,反应温度控制在40℃,反应8小时,获得生物柴油,经测定,ANL/ZIF-8的反应转化率为80%,ANL@M-ZIF-8的反应转化率为92%;其中,所述步骤2和步骤5中的液体脂肪酶ANL均来源于南极假丝酵母(Candida antarctica),甲醇在2个小时内匀速加入。
实施例2:本实施例提供了一种利用双MOFs固定化脂肪酶制备生物柴油的方法,其中步骤1~5与实施例1中的步骤1~5相同,其不同之处在于:
步骤6,将500g大豆油和基于油脂摩尔比为6:1的乙醇置于一级或多级酶反应器中,分别加入基于油脂质量600个标准酶活单位的ANL/ZIF-8和ANL@M-ZIF-8,反应温度控制在50℃,反应10小时,获得生物柴油,经测定,ANL/ZIF-8的反应转化率为84%,ANL@M-ZIF-8的反应转化率为90%;其中,所述步骤2和步骤5中的液体脂肪酶ANL均来源于米曲霉(Aspergillus oryzae),乙醇在2个小时内匀速加入。
实施例3:本实施例提供了一种利用双MOFs固定化脂肪酶制备生物柴油的方法,其中步骤1~5与实施例1中的步骤1~5相同,其不同之处在于:
步骤6,将200g酸化油和基于油脂摩尔比为6:1的丙醇置于一级或多级酶反应器中,分别加入基于油脂质量1000个标准酶活单位的ANL/ZIF-8和ANL@M-ZIF-8,反应温度控制在55℃,反应12小时,获得生物柴油,经测定,ANL/ZIF-8的反应转化率为78%,ANL@M-ZIF-8的反应转化率为85%;其中,所述步骤2和步骤5中的液体脂肪酶ANL均来源于米黑根毛霉(Rhizomucor miehei),丙醇在2个小时内匀速加入。
实施例4:本实施例提供了一种利用双MOFs固定化脂肪酶制备生物柴油的方法,其中步骤1~5与实施例1中的步骤1~5相同,其不同之处在于:
步骤6,将400g酸化油和基于油脂摩尔比为6:1的丁醇置于一级或多级酶反应器中,分别加入基于油脂质量800个标准酶活单位的ANL/ZIF-8和ANL@M-ZIF-8,通过图1所示的在线脱水,反应温度控制在55℃,反应20小时,获得生物柴油,经测定,ANL/ZIF-8的反应转化率为74%,ANL@M-ZIF-8的反应转化率为82%;其中,所述步骤2和步骤5中的液体脂肪酶ANL均来源于南极假丝酵母(Candida antarctica),丁醇在2个小时内匀速加入。
以上所述,仅是本发明的较佳实施例而已,并非对本发明作任何形式上的限制。任何熟悉本领域的技术人员,在不脱离本发明技术方案范围情况下,都可利用上述揭示的技术手段和技术内容对本发明技术方案做出许多可能的变动和修饰,或修改为等同变化的等效实施例。故凡是未脱离本发明技术方案的内容,依据本发明之形状、构造及原理所作的等效变化,均应涵盖于本发明的保护范围。
Claims (10)
1.一种利用双MOFs固定化脂肪酶制备生物柴油的方法,其特征在于,其包括以下步骤:
步骤1,制备ZIF-8;
步骤2,利用所述ZIF-8制备ANL/ZIF-8;
步骤3,制备三维有序聚苯乙烯模板;
步骤4,利用所述三维有序聚苯乙烯模板制备M-ZIF-8;
步骤5,利用所述M-ZIF-8制备ANL@M-ZIF-8;
步骤6,将油脂、短链醇、所述ANL/ZIF-8、及所述ANL@M-ZIF-8混合反应,制得生物柴油。
2.根据权利要求1所述的利用双MOFs固定化脂肪酶制备生物柴油的方法,其特征在于,所述步骤1进一步包括以下步骤:
步骤1-1,将Zn(NO3)2·6H2O溶于甲醇中,制得溶液A;
步骤1-2,将2-甲基咪唑溶于甲醇中,制得溶液B;
步骤1-3,将所述溶液A和溶液B混合,并于室温下震摇或磁力搅拌24h后离心分离;
步骤1-4,取离心后的沉淀,利用甲醇洗涤数次后浸泡于甲醇中活化1天后,经过离心、甲醇洗涤、真空干燥,得到所述ZIF-8。
3.根据权利要求2所述的利用双MOFs固定化脂肪酶制备生物柴油的方法,其特征在于,所述步骤2进一步包括以下步骤:
步骤2-1,定量称取已干燥的所述ZIF-8,加入超纯水和液体脂肪酶ANL,混匀、震摇、离心,取上清液测定蛋白浓度;
步骤2-2,取离心后的沉淀水洗、离心,利用冻干机冻干,得到所述ANL/ZIF-8。
4.根据权利要求3所述的利用双MOFs固定化脂肪酶制备生物柴油的方法,其特征在于,所述步骤3进一步包括以下步骤:
步骤3-1,取足量苯乙烯,依次用10wt%NaOH溶液和去离子水分别洗涤数次,以去除苯乙烯中的阻聚剂;
步骤3-2,将步骤3-1的苯乙烯和聚乙烯吡咯烷酮溶液,用氮气鼓泡后,在持续机械搅拌和于75℃下加热的条件下,加入K2S2O8溶液引发反应,待反应完成后冷却、抽滤、洗涤、干燥,得到滤饼状的所述三维有序聚苯乙烯模板。
5.根据权利要求4所述的利用双MOFs固定化脂肪酶制备生物柴油的方法,其特征在于,所述步骤4进一步包括以下步骤:
步骤4-1,按照比例取Zn(NO3)2·6H2O、2-甲基咪唑和甲醇,配制成足量的ZIF-8前体甲醇溶液;
步骤4-2,取步骤3制得的所述三维有序聚苯乙烯模板浸泡于所述ZIF-8前体甲醇溶液中,抽真空除气泡、干燥,得到滤饼A;
步骤4-3,将所述滤饼A在室温下浸入体积比为1:1的CH3OH/NH3·H2O的混合溶液中,抽真空除气泡后常温常压下放置反应,待反应完成后抽滤,用足量水和乙醇洗涤、干燥,得到滤饼B;
步骤4-4,将所述滤饼B浸入足量四氢呋喃中,多次刻蚀、干燥,得到所述M-ZIF-8。
6.根据权利要求5所述的利用双MOFs固定化脂肪酶制备生物柴油的方法,其特征在于,所述步骤5进一步包括以下步骤:
步骤5-1,称取所述M-ZIF-8,加入超纯水超声分散,加入液体脂肪酶ANL,混匀、震摇、离心,取上清液测定蛋白浓度;
步骤5-2,取离心后的沉淀水洗、离心,利用冻干机冻干,得到所述ANL@M-ZIF-8。
7.根据权利要求6所述的利用双MOFs固定化脂肪酶制备生物柴油的方法,其特征在于,在所述步骤6中,所述短链醇的用量为油脂摩尔比的4.5~6倍,所述ANL/ZIF-8用量为油脂质量的600~1000个标准酶活单位,所述ANL@M-ZIF-8用量为油脂质量的600~1000个标准酶活单位。
8.根据权利要求7所述的利用双MOFs固定化脂肪酶制备生物柴油的方法,其特征在于,所述步骤6于一级或多级酶反应器中进行,反应温度控制在40~55℃,反应8~20小时,其中短链醇在2个小时内匀速加入。
9.根据权利要求8所述的利用双MOFs固定化脂肪酶制备生物柴油的方法,其特征在于,所述液体脂肪酶包括来源于酵母、霉菌、细菌或其它微生物的脂肪酶。
10.根据权利要求9所述的利用双MOFs固定化脂肪酶制备生物柴油的方法,其特征在于,所述油脂为可再生生物油脂,包括植物油脂、动物油脂、废食用油、酸化油、油脂精练下脚料或微生物油脂。
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