CN116479073A - 一种半酶法催化唾液酸合成唾液酸酯的方法和用途 - Google Patents
一种半酶法催化唾液酸合成唾液酸酯的方法和用途 Download PDFInfo
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- CN116479073A CN116479073A CN202310460510.6A CN202310460510A CN116479073A CN 116479073 A CN116479073 A CN 116479073A CN 202310460510 A CN202310460510 A CN 202310460510A CN 116479073 A CN116479073 A CN 116479073A
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- Prior art keywords
- sialic acid
- reaction
- acid
- acid ester
- lipase
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Abstract
本发明公开一种半酶法催化唾液酸合成唾液酸酯的方法和用途,属于生物合成领域,以唾液酸和C1‑C4醇为反应底物,以脂肪酶和占底物唾液酸质量0.05‑5‰的酸联合作为催化剂,反应速率大大加快,远优于单一的脂肪酶或酸催化,所述反应底物C1‑C4醇的选择取决于目标唾液酸酯类,加入分子筛吸水剂,在温度为30‑60℃、振荡转速为200‑300r/min的条件下进行酯化反应,反应结束后,反应液经分离纯化获得唾液酸酯。本发明方法具有酶的区域选择性强、反应快速、反应转化率高等优点,且下游分离简单,能耗低,反应溶介回收套用,对环境友好,适合工业化生产,产率高。
Description
技术领域
本发明属于生物合成领域,特别涉及一种半酶法催化唾液酸合成唾液酸酯的方法和用途。
背景技术
唾液酸(SA)是指一系列含9个碳原子的羧基化单糖酰化衍生物的统称,发现于动物及某些细菌中,通常以低聚糖、糖脂或者糖蛋白的形式存在。唾液酸最早从颌下腺的粘蛋白中分离而出,因而得名。唾液酸的种类繁多,目前已知的有50多种。哺乳动物中,最常见的唾液酸衍生物是N-乙酰神经氨酸(Neu5Ac)。人体中,脑的唾液酸含量最高,其中,脑灰质中的唾液酸含量是肝、肺等内脏器官的15倍。唾液酸的主要食物来源是母乳,也存在于牛奶、鸡蛋和奶酪中,其化学结构式如下所示:
唾液酸类化合物具有重要的生物学功能,在生命体许多重要的生理、生化、病理过程中直接参与细胞与细胞、细胞与微生物、细胞与毒素、细胞与抗体间的相互作用,在抗炎、抗肿瘤、抗病毒、抗老年痴呆等领域具有重要的应用价值。
唾液酸已被欧盟(2018年)和我国(2019年)批准为食品添加剂,主要是婴儿食品配料和营养增补剂,2021年又被我国批准为化妆品原料。唾液酸的来源有化学合成、从天然乳品中提取纯化、微生物发酵和生物酶法催化合成等几种,各有优缺点。作为食品添加剂或化妆品原料用途的唾液酸的制备要求较高,不能残留有害物质,因此,化学合成法来源的唾液酸不适合作为食品添加剂或化妆品原料。
唾液酸因为含有羧基,在体内产生负电荷极易溶于水,溶于甲醇,微溶于乙醇,不溶于乙醚、丙酮和氯仿。唾液酸羧基被酯化形成唾液酸酯,极性降低,在甲醇或乙醇中溶解度增大,同时因为多个羟基的存在,易与水形成氢键,所以在水中溶解度也较好,唾液酸酯的化学结构式如下所示。
唾液酸在细胞间的相互作用中起到了重要的作用,例如病原体-宿主的识别、肿瘤的转移、毒素-受体的相互作用、细胞的分化和增殖。唾液酸极性较大,作为药物或营养物质不利于细胞吸收,生物利用率较低。通过将唾液酸酯化,作为前药,可降低极性,有利于细胞迅速吸收利用唾液酸。传统唾液酸酯的合成都是化学合成,如唾液酸甲酯的合成,以甲醇为溶剂,加入三氟乙酸(TFA)催化,产率较高,但是化学合成法制备的唾液酸酯反应耗时须3-7d,生产的时间成本较大,且需要应用的三氟乙酸用量多,终产物可能残留微量的三氟乙酸盐,副产物多,不适合用于食品添加剂、药品或化妆品原料。
传统的唾液酸酯均采用化学合成法,因为唾液酸溶解性等问题,加入催化量(1%-10%)的三氟乙酸(TFA),无水溶剂室温反应至少3d以上,产率90%以上,如果单一应用脂肪酶40℃反应至少7d以上。用传统已知方法进行唾液酸酯的工业化生产,生产耗时长,生产效率低,生产成本高,迫切需要一种新的催化合成唾液酸酯的方法解决前述问题。
发明内容
本发明的目的是为了解决上述化学合成方法的不足,利用脂肪酶和极少量的0.05-5‰的三氟乙酸或盐酸或硫酸等(TFAor HCl or H2SO4 etc)联合作为催化剂,采用半酶法合成唾液酸酯,且利用脂肪酶催化唾液酸合成唾液酸酯亦未见现有文献或专利报道,本发明具有生产工艺条件温和、步骤简单、产品收率高、环境友好、节能环保等优点。本发明首次应用半酶法,使用脂肪酶和极少量的酸催化唾液酸合成唾液酸酯,安全高效,反应速率大大加快,催化剂可循环多次使用(本实验已重复50次以上验证催化率仍保持98%以上),溶剂回收套用,对环境友好、绿色无污染,无有毒物质残留,适合批量等工业化生产,适用于食品添加剂、药品或化妆品原料等用途。
为达到发明目的本发明采用的技术方案是:
一种半酶法催化唾液酸合成唾液酸酯的方法:以唾液酸和C1-C4醇为反应底物,以脂肪酶和酸联合作为催化剂,且酸的用量为底物唾液酸对应质量的0.05-5‰,所述反应底物C1-C4醇的选择取决于目标唾液酸酯类,同时,C1-C4醇还兼作有机溶剂介质使用;将唾液酸溶于C1-C4醇中,再加入脂肪酶、占底物唾液酸质量0.05-5‰的酸、分子筛吸水剂,在温度为30-60℃、振荡转速为200-300r/min的条件下进行酯化反应,反应结束后,反应液分离纯化,获得唾液酸酯。
优选的,所述唾液酸为N-乙酰神经氨酸、N-羟乙酰神经氨酸、聚唾液酸(PolysialicAcid)中的一种。
优选的,所述脂肪酶为固定化脂肪酶,具体选自丹麦诺维信公司Novozym435,来源于CandidaAntarctica B的固定化脂肪酶,其由一种经过基因改性的米曲霉(Aspergillusoryzae)微生物进行深层发酵并吸附在大孔丙烯酸树脂上而制成。该商业化酶易得,催化效率高,稳定性好,其粒径为0.3-0.9mm,堆积密度为430kg/m3,含水率为1-2%,具有较宽的底物范围,最适催化温度为40-60℃,酶活为10000U/g。
优选的,所述C1-C4醇为甲醇、乙醇、丙醇、异丙醇、正丁醇、仲丁醇、叔丁醇、异丁醇中的一种。
优选的,所述的酸为可电离出H+的有机酸或无机酸,包括三氟乙酸、盐酸、硫酸中的一种。
优选的,所述脂肪酶、唾液酸、C1-C4醇、分子筛吸水剂的用量比例为(0.5-1)g:1g:(100-125)mL:10g,且三氟乙酸或盐酸或硫酸等(TFAor HCl or H2SO4etc)的用量比例为底物唾液酸质量的0.05-5‰。
优选的,所述酯化反应的时间为6-20h。
优选的,所述反应液分离纯化的方法为:反应结束后,过滤反应液,去除脂肪酶和吸水剂,并将滤液减压蒸馏至无馏出物,得到所述白色固体唾液酸酯。
本发明还进一步提供上述方法合成的唾液酸酯在制备营养补充剂、食品、药品、饲料、食品添加剂、饲料添加剂、化妆品、护肤品中的用途。
本发明还进一步提供上述方法合成的唾液酸酯在制备防治病毒性疾病、预防和治疗瘢痕疙瘩、治疗自身免疫病、治疗肿瘤、治疗鼻炎、治疗哮喘、治疗神经系统疾病、治疗炎症性疾病、治疗带状疱疹、新冠后遗症、抗衰老药物中的用途。
本发明的有益效果:
1.本发明首次利用已商业化的固定化脂肪酶Novozyme 435和占反应底物唾液酸质量0.05-5‰的酸联合做为催化剂的半酶法催化唾液酸合成唾液酸酯。发明人通过创新性研究发现,在催化合成过程中,在脂肪酶、唾液酸、C1-C4醇、吸水剂的用量比例为(0.5-1)g:1g:(100-125)mL:10g的反应体系中,仅滴加占反应底物唾液酸质量0.05-5‰的三氟乙酸(或盐酸或硫酸等可电离出H+的有机酸或无机酸),即可使反应时间缩短至6-20h。本发明的半酶法合成唾液酸酯相比于传统的单一的三氟乙酸催化法(需要5d反应时间)或单一的脂肪酶酶促法(需要8d的反应时间),三氟乙酸等酸和脂肪酶产生了协同催化的效应,0.05-5‰的三氟乙酸等酸可以显著提升脂肪酶的酶促反应速率,大大加速了整个反应体系的反应效率,极大缩短了反应时间。本发明升级优化了唾液酸酯的生产工艺,极大提高了唾液酸酯的生产率。此外,相比于传统化学合成法合成唾液酸酯的反应(传统化学合成法需1-10wt%左右的三氟乙酸等酸),三氟乙酸等酸的用量被极大降低,终产物中三氟乙酸等酸的残留痕量或无,副产物含量极低,终产品适合用于食品添加剂、药品或化妆品原料等用途。
2.本发明首次利用已商业化的固定化脂肪酶Novozyme 435和占反应底物唾液酸质量0.05-5‰的酸联合做为催化剂的半酶法催化唾液酸合成唾液酸酯,酶的区域选择性强,反应转化率高,安全高效,催化剂可循环多次使用,溶剂介质可回收套用,对环境友好,绿色无污染,无有毒物质残留,适合批量生产。
3.本发明以反应底物C1-C4醇作为有机溶剂反应介质,反应底物C1-C4醇的选择取决于目标唾液酸酯类,下游分离简单,固定化脂肪酶和分子筛经过滤即可去除且回收套用,/>分子筛高温除水活化,能耗低,环境污染小,无有害物质残留和三废,适合工业化生产,唾液酸转化率超过95%。
附图说明
图1为实施例1所得唾液酸甲酯制备成的胶囊产品图;
图2为实施例2制出的产物唾液酸乙酯的高效液相色谱图(HPLC);
图3为实施例7中患者1的带状疱疹症状图;
图4为实施例7中患者2的带状疱疹症状图;
图5为实施例7中患者3的带状疱疹症状图;
图6为实施例9中试用者使用唾液酸甲酯溶液护肤前的面部皮肤图;
图7为实施例9中试用者使用唾液酸甲酯溶液护肤后的面部皮肤图。
具体实施方式
下面结合实施例对本发明做进一步的详细说明,以令本领域技术人员参照说明书文字能够据以实施。
本发明提供一实施例的半酶法催化唾液酸合成唾液酸酯的方法:以唾液酸和C1-C4醇为反应底物,以脂肪酶为催化剂,所述反应底物C1-C4醇的选择取决于目标唾液酸酯类,同时,C1-C4醇还兼作有机溶剂介质使用;将唾液酸溶于C1-C4醇中,再加入脂肪酶、占底物唾液酸质量0.05-5‰的三氟乙酸(TFA)或盐酸(HCl)或硫酸(H2SO4)等、分子筛吸水剂,在温度为30-60℃、振荡转速为200-300r/min的条件下进行酯化反应8-15h,反应结束后,反应液分离纯化,获得唾液酸酯;
所述唾液酸为N-乙酰神经氨酸、N-羟乙酰神经氨酸、聚唾液酸(Polysialic Acid)中的一种;所述脂肪酶为固定化脂肪酶,具体选自固定化南极假丝酵母(Candidaantarctica)脂肪酶B(CAL-B),商品名为Novozyme 435,该商业化酶价廉易得,催化效率高,稳定性好,其粒径为0.3-0.9mm,堆积密度为430kg/m3,含水率为1-2%,具有较宽的底物范围,最适催化温度为40-60℃,酶活为10000U/g。
所述C1-C4醇为甲醇、乙醇、丙醇、异丙醇、正丁醇、仲丁醇、叔丁醇、异丁醇中的一种;所述脂肪酶、唾液酸、C1-C4醇、分子筛吸水剂、三氟乙酸或盐酸或硫酸的用量比例为(0.5-1)g:1g:(100-125)mL:10g:(0.00005-0.005)g;
所述反应液分离纯化的方法为:反应结束后,过滤反应液,去除固定化脂肪酶和吸水剂,并将滤液减压蒸馏至无馏出物,得到所述唾液酸酯。
该实施例方法合成的唾液酸酯在制备营养补充剂、食品、药品、饲料、食品添加剂、饲料添加剂、化妆品、护肤品中的用途。
该实施例方法合成的唾液酸酯在制备防治病毒性疾病、预防和治疗瘢痕疙瘩、治疗自身免疫病、治疗肿瘤、治疗鼻炎、治疗哮喘、治疗神经系统疾病、治疗炎症性疾病、治疗带状疱疹、新冠后遗症、抗衰老药物中的用途。
下列实施例1-5选用的脂肪酶为固定化脂肪酶,具体为固定化南极假丝酵母(Candida antarctica)脂肪酶B(CAL-B),商品名Novozyme 435,生产厂家丹麦诺维信公司,酶活10000U/g。
下列实施例1~5中极少量的三氟乙酸(TFA)或盐酸(HCl)或硫酸(H2SO4)等用量为底物唾液酸质量的0.05-5‰。
下列实施例1-5及对比例1-3中的唾液酸和唾液酸酯的液相色谱仪(HPLC)测定条件:ZORBAX SB C18250×4.6mm色谱柱;采用流动相:pH3.0的硫酸水溶液;流动相流速:1.0mL/min;检测器:UV 210nm;进样量:5.0μL。
实施例1
一种半酶法催化唾液酸合成唾液酸酯的方法:将4.0gN-乙酰神经氨酸、400mL无水甲醇加入500mL的三角瓶中,搅拌溶解后,加入2.0g脂肪酶Novozyme 435、40.0g 分子筛和0.01mL三氟乙酸(根据三氟乙酸分子量,通过质量容积换算确定),30℃恒温振荡反应15h,振荡速度为200r/min,反应结束后,过滤反应液,去除脂肪酶Novozyme 435和/>分子筛,并将滤液减压蒸馏至无馏出物,再取样进行液相色谱分析,测得唾液酸甲酯产率为95.6%。
实施例2
一种半酶法催化唾液酸合成唾液酸酯的方法:将4.0gN-乙酰神经氨酸、400.0mL无水乙醇加入1000mL的三角瓶中,搅拌溶解后,加入4.0g脂肪酶Novozyme 435、40.0g 分子筛和0.005mL的1M盐酸(根据盐酸分子量,通过质量容积换算确定),40℃恒温振荡反应10h,振荡速度为200r/min,反应结束后,过滤反应液,去除脂肪酶Novozyme 435和/>分子筛,并将滤液减压蒸馏至无馏出物,再取样进行液相色谱分析,测得唾液酸乙酯产率为94.3%。
实施例3
一种半酶法催化唾液酸合成唾液酸酯的方法:将4.0gN-羟乙酰神经氨酸、400.0mL无水乙醇加入1000mL的三角瓶中,搅拌溶解后,加入4.0g脂肪酶Novozyme 435、40.0g分子筛和0.0025mL的1M硫酸(根据硫酸分子量,通过质量容积换算确定),50℃恒温振荡反应12h,振荡速度为300r/min,反应结束后,过滤反应液,去除脂肪酶Novozyme 435和/>分子筛,并将滤液减压蒸馏至无馏出物,再取样进行液相色谱分析,测得唾液酸乙酯产率为95.9%。
实施例4
一种半酶法催化唾液酸合成唾液酸酯的方法:将4.0gN-羟乙酰神经氨酸、500.0mL无水丙醇加入1000mL的三角瓶中,搅拌溶解后,加入2.0g脂肪酶Novozyme 435、40.0g 分子筛和0.002mL三氟乙酸(根据三氟乙酸分子量,通过质量容积换算确定),60℃恒温振荡反应8h,振荡速度为200r/min,反应结束后,过滤反应液,去除脂肪酶Novozyme 435和/>分子筛,并将滤液减压蒸馏至无馏出物,再取样进行液相色谱分析,测得唾液酸丙酯产率为92.8%。
实施例5
一种半酶法催化唾液酸合成唾液酸酯的方法:将4.0g聚唾液酸、500.0mL无水丁醇加入1000mL的三角瓶中,搅拌溶解后,加入2.0g脂肪酶Novozyme 435、40.0g 分子筛和0.001mL三氟乙酸(根据三氟乙酸分子量,通过质量容积换算确定),50℃恒温振荡反应12h,振荡速度为200r/min,反应结束后,过滤反应液,去除脂肪酶Novozyme 435和/>分子筛,并将滤液减压蒸馏至无馏出物,再取样进行液相色谱分析,测得唾液酸丁酯产率为91.5%。
对比例1-5
对比例1-5与实施例1相同,区别在于:所选用的具体脂肪酶不同,并根据100%×(初始唾液酸质量-反应后唾液酸质量)/初始唾液酸质量计算唾液酸转化率,具体如表1所示:
由表1可知,用不同脂肪酶作为催化剂,其他条件不变,对唾液酸进行半酶法酯化,实施例1的脂肪酶Novozyme 435的催化效果优于其他脂肪酶。
对比例6
对比例6与实施例1相同,区别在于:在反应过程中不滴加三氟乙酸,待对比例6反应体系的唾液酸甲酯产率达到95.6%,记录所需反应时间。
试验结果及分析:对比例6的纯酶法达到与实施例1相同的95.6%唾液酸甲酯产率时,需要耗费8d的反应时长,证明了实施例1中极少量的三氟乙酸对脂肪酶的酶促催化产生了巨大的协同效应。
对比例7
对比例7与实施例1相同,区别在于:在反应过程中不添加2.0g脂肪酶Novozyme435,反应时间为6d。
试验结果及分析:对比例7在反应过程中未添加脂肪酶Novozyme 435,振荡反应6d后,测得唾液酸甲酯产率仅为0.4%,说明极少量的三氟乙酸对唾液酸酯合成的催化效应微乎其微。
对比例8
一种三氟乙酸催化唾液酸合成唾液酸酯的方法:将4.0gN-乙酰神经氨酸、400.0mL无水甲醇加入1000mL的三角瓶中,搅拌溶解后,加入40.0g 分子筛和0.26mL三氟乙酸(根据三氟乙酸分子量,通过质量容积换算确定),30℃恒温振荡反应5d,振荡速度为200r/min,反应结束后,过滤反应液,去除/>分子筛,并将滤液减压蒸馏至无馏出物,再取样进行液相色谱分析,测得唾液酸甲酯产率为88.4%。
试验结果及分析:相比于对比例8传统三氟乙酸催化合成唾液酸酯的反应,实施例1的半酶法催化合成方法,使反应时间压缩至15h内,且实施例1的目标产物产率也得到较明显地提升,证明了实施例1结合脂肪酶和三氟乙酸的半酶法催化合成方法,有效提高了反应速率和目标产物唾液酸酯的产出率;同时,三氟乙酸用量被极大降低(对比例8化学合成法中三氟乙酸用量占反应底物N-乙酰神经氨酸质量的10%左右,而实施例1仅需3.375‰左右),终产物中三氟乙酸残留痕量或无,副产物含量极低。
综合实施例1-5及对比例1-8可知,实施例1-5首次创造性利用已商业化的固定化脂肪酶Novozyme 435和占反应底物N-乙酰神经氨酸质量0.05-5‰的三氟乙酸(或盐酸或硫酸等可电离出H+的有机酸或无机酸)联合做为催化剂的半酶法催化唾液酸合成唾液酸酯。发明人通过创新性研究发现,在催化合成过程中,在脂肪酶、唾液酸、C1-C4醇、吸水剂的用量比例为(0.5-1)g:1g:(100-125)mL:10g的反应体系中,仅滴加占反应底物N-乙酰神经氨酸质量0.05-5‰的三氟乙酸(或盐酸或硫酸等可电离出H+的有机酸或无机酸),即可使反应时间缩短至6-20h,本发明的半酶法相比于对比例8中传统的单一的三氟乙酸催化法(需要5d反应时间)或对比例6中单一的脂肪酶酶促法(需要8d的反应时间),显著提升了整个反应体系的反应效率,极大缩短了反应时间,证明了脂肪酶和三氟乙酸产生了协同催化的效应。究其原因可能是,三氟乙酸(或盐酸或硫酸等可电离出H+的有机酸或无机酸)作为质子酸催化剂,在反应过程中作为质子供体,脂肪酶的羧基接受质子,形成离子化脂肪酶,离子化脂肪酶可能是催化效率提升的关键因素,一方面,离子化脂肪酶使酶促合成唾液酸酯反应过程中的活化能被进一步降低,另一方面,三氟乙酸(或盐酸或硫酸等可电离出H+的有机酸或无机酸)与唾液酸及C1-C4醇形成的离子中间体和离子化脂肪酶更易结合,提高了脂肪酶与反应底物的有效接触面积,进而实现脂肪酶与三氟乙酸(或盐酸或硫酸等可电离出H+的有机酸或无机酸)协同快速催化合成唾液酸酯的效果,从而使唾液酸酯的生产工艺升级优化,极大提高了唾液酸酯的生产率。
实施例6
以实施例1所得唾液酸甲酯为原料,制备成唾液酸甲酯含量为70mg/粒的胶囊,如图1所示,用于治疗带状疱疹、炎症性疾病等。
实施例7
采用实施例6制备的唾液酸甲酯胶囊治疗带状疱疹患者:
治疗方案一:患者1,男,60岁,带状疱疹1周余(症状图如图3所示),患处神经痛,剧烈难忍,伴烦躁不安。口服及外敷多种药物无效。空腹口服实施例6所制备的唾液酸甲酯胶囊,140mg/次,1次/日,连服3日。
治疗效果:首次口服唾液酸甲酯胶囊后3.5h疼痛明显缓解,电话随访疼痛及皮损逐渐减轻,停药后症状未加重。
治疗方案二:患者2,女,48岁,带状疱疹初起(症状图如图4所示),患处皮肤灼热、疼痛明显。尚未用药治疗。空腹口服实施例6所制备的唾液酸甲酯胶囊,140mg/次,1次/日,连服3日。
治疗效果:首次口服唾液酸甲酯胶囊后4.5h疼痛缓解,电话随访疼痛及皮损逐渐减轻,停药后症状未加重。
治疗方案三:患者3,女,75岁,带状疱疹初起(症状图如图5所示),患处及周边皮肤灼热、感觉过敏,疼痛尚可忍受。口服对乙酰氨基酚无效。空腹口服实施例6所制备的唾液酸甲酯胶囊,140mg/次,1次/日,连服3日。
治疗效果:首次口服唾液酸甲酯胶囊后5h疼痛缓解,电话随访疼痛及皮损逐渐减轻,5日后痊愈。
实施例8
采用实施例6制备的唾液酸甲酯胶囊治疗炎症性疾病:
患者4,女,51岁,右侧大腿根部的淋巴结区肿胀,局部皮温无发热,伴轻中度疼痛不适,走路、久坐或劳累时局部疼痛不适感加强,不适感放射到右侧小腹对应的背部,自服多种抗生素治疗无效。
治疗方案:睡前空腹口服唾液酸甲酯胶囊70mg/次,1次/日,连服10日。
治疗效果:右侧大腿根部的淋巴结区肿胀现象明显减轻,疼痛感基本消失,走路、久坐或劳累后局部不适感不明显。
实施例9
采用实施例1制出的唾液酸甲酯用于皮肤滋润保湿养护:
使用方法:以实施例1所得唾液酸甲酯为原料,制备成唾液酸甲酯含量为3%的生理盐水溶液,喷雾涂敷于面部皮肤,轻柔按摩,用于面部皮肤滋润保湿养护。
护肤效果:面部喷雾涂敷3%唾液酸甲酯生理盐水溶液后30min,相较于使用前(如图6所示),皮肤明显湿润滑嫩,细小皱纹明显减轻(皮肤滋润保湿效果如图7所示)。
尽管本发明的实施方案已公开如上,但其并不仅仅限于说明书和实施方式中所列运用,它完全可以被适用于各种适合本发明的领域,对于熟悉本领域的人员而言,可容易地实现另外的修改,因此在不背离权利要求及等同范围所限定的一般概念下,本发明并不限于特定的细节。
Claims (10)
1.一种半酶法催化唾液酸合成唾液酸酯的方法,其特征在于:以唾液酸和C1-C4醇为反应底物,以脂肪酶和酸联合作为催化剂,且酸的用量为底物唾液酸对应质量的0.05-5‰,其中,C1-C4醇的选择取决于目标唾液酸酯类;将唾液酸溶于C1-C4醇中,再加入脂肪酶、酸、分子筛吸水剂,在温度为30-60℃、振荡转速为200-300r/min的条件下进行酯化反应,反应结束后,反应液经分离纯化获得纯品唾液酸酯。
2.如权利要求1所述一种半酶法催化唾液酸合成唾液酸酯的方法,其特征在于,所述唾液酸为N-乙酰神经氨酸、N-羟乙酰神经氨酸、聚唾液酸中的一种。
3.如权利要求1所述一种半酶法催化唾液酸合成唾液酸酯的方法,其特征在于,所述脂肪酶为固定化南极假丝酵母脂肪酶B。
4.如权利要求1所述一种半酶法催化唾液酸合成唾液酸酯的方法,其特征在于,所述C1-C4醇为甲醇、乙醇、丙醇、异丙醇、正丁醇、仲丁醇、叔丁醇、异丁醇中的一种。
5.如权利要求1所述一种半酶法催化唾液酸合成唾液酸酯的方法,其特征在于,所述的酸为可电离出H+的有机酸或无机酸,包括三氟乙酸、盐酸、硫酸中的一种。
6.如权利要求1~5任一项所述一种半酶法催化唾液酸合成唾液酸酯的方法,其特征在于,所述脂肪酶、唾液酸、C1-C4醇、分子筛吸水剂、酸的用量比例为(0.5-1)g:1g:(100-125)mL:10g:(0.00005-0.005)g。
7.如权利要求1所述一种半酶法催化唾液酸合成唾液酸酯的方法,其特征在于,所述酯化反应的时间为6-20h。
8.如权利要求1所述一种半酶法催化唾液酸合成唾液酸酯的方法,其特征在于,所述反应液分离纯化的方法为:反应结束后,过滤反应液,去除脂肪酶和吸水剂,并将滤液减压蒸馏至无馏出物,得到白色固体为所述唾液酸酯。
9.一种如权利要求1~5、7、8任一项所述的方法合成的唾液酸酯在制备营养补充剂、食品、药品、饲料、食品添加剂、饲料添加剂、化妆品、护肤品中的用途。
10.一种如权利要求1~5、7、8任一项所述的方法合成的唾液酸酯在制备预防和治疗病毒性疾病、瘢痕疙瘩、自身免疫病、肿瘤、鼻炎、哮喘、神经系统疾病、炎症性疾病、带状疱疹、新冠后遗症以及抗衰老药物中的用途。
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