CN113817710B - 一种琼胶酶冻干保护剂及琼胶酶的保存方法 - Google Patents
一种琼胶酶冻干保护剂及琼胶酶的保存方法 Download PDFInfo
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- CN113817710B CN113817710B CN202111320964.0A CN202111320964A CN113817710B CN 113817710 B CN113817710 B CN 113817710B CN 202111320964 A CN202111320964 A CN 202111320964A CN 113817710 B CN113817710 B CN 113817710B
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Abstract
本发明属于酶保存技术领域,具体涉及一种琼胶酶冻干保护剂及琼胶酶的保存方法。所述琼胶酶冻干保护剂含有琼胶寡糖1~10w/v%、缓冲溶液0.1~0.5mol/L、非还原性二糖1~5w/v%、可溶性淀粉1~10w/v%、糊精1~10w/v%、甘油5~20w/v%、甘露醇0.5~5w/v%且余量为水。本发明提供的琼胶酶冻干保护剂将琼胶寡糖作为主要组分并配以特定含量的缓冲溶液、非还原性二糖、可溶性淀粉、糊精、甘油和甘露醇一起使用,由此所得酶冻干保护剂可以使得琼胶酶的蛋白结构免遭破坏,能够最大限定地保持琼胶酶的理化组成和生物活性,从而实现对琼胶酶的良好保护。
Description
技术领域
本发明属于酶保存技术领域,具体涉及一种琼胶酶冻干保护剂及琼胶酶的保存方法。
背景技术
琼胶酶是能够催化琼胶的糖苷键发生断裂并产生一系列低聚合度琼胶寡糖的糖苷水解酶,是一类很重要的海藻多糖降解酶。由于琼胶酶具有能够降解琼胶的特性,使其在许多领域均得到了广泛应用,例如,琼胶酶可以作为海藻遗传的工具酶,用于降解红藻的细胞壁来制备原生质体或单细胞;琼胶酶在分子生物学中可以液化琼脂糖凝胶,用于PCR产物的纯化;琼胶酶可以用于海藻多糖的结构研究;琼胶酶可以用于制备琼胶寡糖,酶解法制备琼胶寡糖与传统的酸解法相比具有环保、高效、专一等特点。
琼胶酶冷冻干燥技术又称升华干燥技术,是指在低温下将湿物料或溶液冻结成固态,然后在真空下将物料中的水分进行升华,最终使物料脱水的一种干燥技术。虽然经冷冻干燥之后能够在一定程度上延长琼胶酶的保质期,便于琼胶酶的保存和运输,但由于目前国内的冷冻干燥技术尚不够完善,将琼胶酶直接进行冷冻干燥,容易使得琼胶酶的蛋白结构遭到破坏,失活率较高。
鉴于琼胶酶的应用已越来越广泛,显示出较高的商业化价值,因此,亟需开发一种便于琼胶酶保存及运输的方法,以最大效果地保护琼胶酶,使其后续应用不会受到影响。目前,关于琼胶酶冻干保护剂的研究目前还未见报道。
发明内容
本发明的目的是为了克服现有技术采用冷冻干燥技术对琼胶酶进行保存时会导致其失活率较高的缺陷,而提供一种失活率非常低的琼胶酶冻干保护剂及琼胶酶的保存方法。
具体地,本发明提供了一种琼胶酶冻干保护剂,其中,所述琼胶酶冻干保护剂含有琼胶寡糖1~10w/v%、缓冲溶液0.1~0.5mol/L、非还原性二糖1~5w/v%、可溶性淀粉1~10w/v%、糊精1~10w/v%、甘油5~20w/v%、甘露醇0.5~5w/v%且余量为水。
进一步地,所述琼胶寡糖的聚合度为2~10。
进一步地,所述琼胶寡糖选自新琼二糖、新琼四糖、新琼六糖和新琼八糖中的至少一种。
进一步地,所述琼胶寡糖按照以下方法制得:将琼胶水溶液在β-琼胶酶的存在下进行酶解,所述β-琼胶酶的基因序列为SEQ ID NO:1,之后将所得酶解产物进行纯化和浓缩。
进一步地,所述酶解的条件包括温度为40~50℃,时间为3~8h。
进一步地,以琼胶水溶液的总重量为100g计,所述β-琼胶酶的用量为0.1~1mL。
进一步地,所述缓冲溶液为Tris-HCl缓冲溶液,所述Tris-HCl缓冲溶液的pH值为8.5~9.5。
进一步地,所述非还原性二糖为海藻糖和/或蔗糖。
进一步地,所述糊精选自白糊精、黄糊精、麦芽糊精和环糊精中的至少一种。
本发明还提供了一种琼胶酶的保存方法,该方法包括往琼胶酶溶液中加入上述琼胶酶冻干保护剂混合均匀得到琼胶酶保护液,之后将该琼胶酶保护液进行冷冻干燥。
进一步地,所述琼胶酶冻干保护剂与琼胶酶溶液的体积比为1:(5~15)。
进一步地,所述冷冻干燥的方法包括将琼胶酶保护液于-45℃~-35℃下冻结1~5h,再置于真空冷冻干燥机中并于-10℃~0℃下干燥5~10h,之后以5~10℃/min的速率逐步升温至25℃~35℃并保持2~5h完成冷冻干燥。
本发明针对琼胶酶的特定蛋白结构特点,将琼胶寡糖作为主要组分并配以特定含量的缓冲剂、非还原性二糖、可溶性淀粉、糊精、甘油和甘露醇一起使用,由此所得酶冻干保护剂可以使得琼胶酶的蛋白结构免遭破坏,能够最大限定地保持琼胶酶的理化组成和生物活性,从而实现对琼胶酶良好保护。研究数据表明,往琼胶酶溶液中加入10%的琼胶酶冻干保护剂混合后冷冻干燥,在37℃下保存3个月后失活率保持在16%以内,在4℃下保存2年后失活率保持在20%以内。
具体实施方式
本发明提供的琼胶酶冻干保护剂含有琼胶寡糖1~10w/v%、缓冲溶液0.1~0.5mol/L、非还原性二糖1~5w/v%、可溶性淀粉1~10w/v%、糊精1~10w/v%、甘油5~20w/v%、甘露醇0.5~5w/v%且余量为水。
所述琼胶寡糖是琼胶多糖经水解后所得低聚糖,主要由琼二糖的重复单位连接而成,包括琼寡糖(agaroligo saccharides)和新琼寡糖(neoagarooligo saccharides)两个系列,琼寡糖以3,6-内醚-α-L-半乳糖残基为还原性末端,新琼寡糖以β-D-半乳糖残基为还原性末端。所述琼胶寡糖的聚合度为2~20,具体可以为新琼二糖、新琼四糖、新琼六糖和新琼八糖中的至少一种。
在本发明中,所述琼胶寡糖能在冻结过程中起低温保护剂的功能,又能在干燥脱水的过程中起到脱水保护剂的作用。所述琼胶寡糖的含量为1~10w/v%,具体可以为1w/v%、2w/v%、3w/v%、4w/v%、5w/v%、6w/v%、7w/v%、8w/v%、9w/v%、10w/v%。当琼胶寡糖的浓度过低时,不能有效发挥保护作用;当琼胶寡糖的浓度过高时,会在琼胶酶的实际应用中对检测结果产生干扰。
根据本发明的一种优选实施方式,所述琼胶寡糖按照以下方法制得:将琼胶水溶液在β-琼胶酶的存在下进行酶解,所述β-琼胶酶的基因序列为SEQ ID NO:1,之后将所得酶解产物进行纯化和浓缩,此时所得琼胶寡糖对琼胶酶的保护效果更佳。其中,所述酶解的条件优选包括温度为40~50℃,时间为3~8h。此外,以琼胶水溶液的总重量为100g计,所述β-琼胶酶的用量优选为0.1~1mL,如0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1.0mL。
在上述琼胶寡糖的制备过程中,所述β-琼胶酶通过克隆或化学合成法从相应菌属或菌种中获得酶基因,将该酶基因引入原核表达载体中得到重组载体,之后将所述重组载体转化至大肠杆菌得到重组菌株,所述重组菌株经发酵、分离纯化后得到所述β-琼胶酶。所述β-琼胶酶的基因序列为SEQ ID NO:1,其对应的氨基酸序列为SEQ ID NO:2。其中,通过克隆或化学合成法获得酶基因、将酶基因引入原核表达载体中得到重组载体、将重组载体转化至大肠杆菌得到重组菌株的方式均为本领域技术人员公知,在此不作赘述。所述原核表达载体例如可以为PET系列的表达载体(如PET-14、PET-21、PET-22、PET-25、PET-28)、PGEX系列的表达载体(如PGEX-4T-2、PGEX-6T-1)等。所述大肠杆菌例如可以为大肠杆菌BL21(DE3)。
SEQ ID NO:1:
ATGCATCATCATCATCATCATCGACCTAAGTTCATAAACTTTCACGCAAAGTCCCACGATAAAGACGTGACCGCTTTCTACGAAGAATATGACGTATACCTTGGCCGAGGCTTCTGGGGAATGATTAAAACAGTGCTGCGCAAGGGATTTACAAAACGCTCCACGGTGGGTATACACGTTGACACAGCAGACGTCGGCTCGACTTATCCGAATTCTACTATAATTGATAAATCAACGGACGTCAAGGAGGTCTCTAATCTCATAGGAACAGAACACGAGAAACCGTTTTGGCCGGATGCTGGAGAAATGTCGCTGATGGACGCATCTACCTCGGGGGACTTTATGGGTCAGTTTTTTAACGAGTTTGTCGATGCAGGTGCTACTAACTCGGATGATCGGCCAAAATACGTCGAAATCATCAATGAACCCTTTTGGCATGCTCATGACTTTTATGAGATTACTGCAAAGGAGATGGCGGAACTCTTTGGTACTATCGCAGCGCAAGTTCACGATACTCCGTCGCTCGGAAAGATGAAGGTTGGTGGCTATTGTACCGGTTTCCCAGATTTTGAAATCAATAACTTTGCACATTGGGAAGATAATATGAAAATGTTCATGGATGTGGCGGGAGATGATATGGACTTTTGGTCTATACACCTATACGATTTTCCTTCGGGAATAACCCAGAACAATAATCGCTCGGGATCCAACATGGAAGCGGTTCTAGATCTTATAGAGACGTATTCGATGTGGAAGGGAGGCAAGGTCAAACCTCATGCGATCACACAATACGGGGTTATCACCCATGGTTTCGACAATTACACACCGTATCGGGATTGGCTTCACATCAAATCAACAAATTCTATGCTGATGCAGTTTATGGAGCGCACGGACAATATATGCTATGCAATGCCGTTTGCCATGGATAAATCCACGTGGCACCTAACGGAGAATAATGGGCAGCCTGGTGCACTCTTCATACCTACCAATATCGGTGAAAAGGAAGTAGAGCAGTGGGTGTTCACGGAAATGATCAAATTCTATCAACTTTGGAAGACCGGAGTTTCTGTCAATCCGGCGTCAACGTCCGTAGCAGTTGCGGCTACGCAGAAATTCACAGCAGGCATAACGATGGCTGATGCTGGCGTGGTATGGTCCGTTGCTAATGAATCGGTGGCACTAGTCTCCGCGACTGGGAAGGTAACAGCTGTTAAGAAGGGCAAGCTGACCATCACAGCAACCACTACGGACGAATTTGCGGCAGTACTTATCCAAAAGATAGAAGCGGAAGATTTCGGGGCTTATCTAGATCGCATCAACGAGGGCGTTAATATCGATTCTACAACAGCTGGGCAGGAGACGGGCGAATGGACCTCATACGCAGGTACGGATGTTAATATACCCGAATCAGCAATCTATACGATCTCGGTCAACACATCAACTTCGACAGAGTCGTCCCTTGATCTTATAGAGGATAACAAAATCTGTGAAACTATCGAAGTAAATAACAATGCCCTTACGGAGGTAAAAGTGGAGCTAGCCGGTTCACATGTGCTCGGACACGGGGATGTTAAGATAAATTGGCTTTTCAGTTAA。
SEQ ID NO:2:
MHHHHHHRPKFINFHAKSHDKDVTAFYEEYDVYLGRGFWGMIKTVLRKGFTKRSTVGIHVDTADVGSTYPNSTIIDKSTDVKEVSNLIGTEHEKPFWPDAGEMSLMDASTSGDFMGQFFNEFVDAGATNSDDRPKYVEIINEPFWHAHDFYEITAKEMAELFGTIAAQVHDTPSLGKMKVGGYCTGFPDFEINNFAHWEDNMKMFMDVAGDDMDFWSIHLYDFPSGITQNNNRSGSNMEAVLDLIETYSMWKGGKVKPHAITQYGVITHGFDNYTPYRDWLHIKSTNSMLMQFMERTDNICYAMPFAMDKSTWHLTENNGQPGALFIPTNIGEKEVEQWVFTEMIKFYQLWKTGVSVNPASTSVAVAATQKFTAGITMADAGVVWSVANESVALVSATGKVTAVKKGKLTITATTTDEFAAVLIQKIEAEDFGAYLDRINEGVNIDSTTAGQETGEWTSYAGTDVNIPESAIYTISVNTSTSTESSLDLIEDNKICETIEVNNNALTEVKVELAGSHVLGHGDVKINWLFS
在本发明中,所述缓冲溶液所起的作用为保护溶液pH值的稳定,使酶蛋白始终处于最适pH环境中,冻干后的样品贮藏稳定性好。所述缓冲溶液的含量为0.1~0.5mol/L,具体可以为0.1mol/L、0.2mol/L、0.3mol/L、0.4mol/L、0.5mol/L。当缓冲剂的含量低于0.1mol/L时,缓冲溶液的缓冲容量降低,对蛋白的保护能力减弱;当缓冲剂的含量高于0.5mol/L时,会影响蛋白质的空间构像,使酶活下降。此外,所述缓冲溶液优选为Tris-HCl缓冲溶液。所述Tris-HCl缓冲溶液的pH值优选为8.5~9.5。
在本发明中,所述非还原性二糖可以作为填充剂和贮藏保护剂,有效降低干燥后的酶制剂在长期保存过程中的酶活损失率。所述非还原性二糖的含量为1~5w/v%,具体可以为1w/v%、1.5w/v%、2.0w/v%、2.5w/v%、3.0w/v%、3.5w/v%、4.0w/v%、4.5w/v%、5.0w/v%。所述非还原性二糖优选为海藻糖和/或蔗糖。
所述可溶性淀粉为淀粉经过氧化剂、酸、甘油或酶处理而成的淀粉衍生物,可溶性淀粉为白色或类白色粉末,其不溶于冷水、乙醇和乙醇,在沸水中可溶解为透明溶液,冷却后不结冰。所述可溶性淀粉可以通过商购得到,也可以按照现有的各种方法制备得到。在本发明中,所述可溶性淀粉的含量为1~10w/v%,具体可以为1w/v%、2w/v%、3w/v%、4w/v%、5w/v%、6w/v%、7w/v%、8w/v%、9w/v%、10w/v%。
所述糊精为淀粉在加热、酸或淀粉酶作用下水解成的小分子中间物质。所述糊精的具体实例包括但不限于:白糊精、黄糊精、麦芽糊精和环糊精中的至少一种。在本发明中,所述糊精的含量为1~10w/v%,具体可以为1w/v%、2w/v%、3w/v%、4w/v%、5w/v%、6w/v%、7w/v%、8w/v%、9w/v%、10w/v%。
在本发明中,所述可溶性淀粉和糊精能够促进其他小分子物质发挥保护作用,并能有效地保持样品冻干过程中的形状,减少样品的皱缩,提高冻干效率。
在本发明中,所述甘油所起的作用为防冻剂,在冻干过程中可有效的保护蛋白质分子结构免受冰晶的损伤。所述甘油的含量为5~20w/v%,具体可以为5w/v%、6w/v%、7w/v%、8w/v%、9w/v%、10w/v%、11w/v%、12w/v%、13w/v%、14w/v%、15w/v%、16w/v%、17w/v%、18w/v%、19w/v%、20w/v%。当甘油含量过低时,不能有效达到低温保护的作用;而当甘油含量过高时,则使液体的黏度加大,影响样品的冻干效率及最终样品的外观形态。
在本发明中,所述甘露醇被作为填充剂,在冻结过程中为活性物质提供支撑结构。所述甘露醇的含量为0.5~5w/v%,具体可以为0.5w/v%、1w/v%、1.5w/v%、2w/v%、2.5w/v%、3w/v%、3.5w/v%、4w/v%、4.5w/v%、5w/v%。所述甘露醇对酶的保护作用与其浓度有关,在以上浓度范围内,甘露醇能使酶蛋白分子保持稳定,阻止蛋白质分子的聚集,浓度过高时,甘露醇本身会形成结晶,反而会对酶蛋白造成损伤。
本发明提供的琼胶酶的保存方法包括往琼胶酶溶液中加入所述琼胶酶冻干保护剂混合均匀得到琼胶酶保护液,之后将该琼胶酶保护液进行冷冻干燥。其中,所述琼胶酶为能够催化琼胶的糖苷键发生断裂并产生一系列低聚合度琼胶寡糖的酶制剂。按照作用方式的不同,所述琼胶酶分为α-琼胶酶和β-琼胶酶,其中,α-琼胶酶为裂解琼胶糖的α-1,3糖苷键生成以3,6-内醚-α-L半乳糖为还原性末端的琼寡糖系列,β-琼胶酶为裂解琼胶糖的β-1,4糖苷键生成以β-D-半乳糖为还原性末端的新琼寡糖系列。所述琼胶酶可以从海洋环境中的菌株中分离得到,其中,α-琼胶酶主要来自假单胞菌属、单胞菌属和弧菌属,β-琼胶酶主要来自弧菌属和交替单胞菌属。所述琼胶酶可以通过商购得到,也可以按照现有的方法从海洋环境中的菌株中分离得到,对此本领域技术人员均能知悉,在此不作赘述。所述琼胶酶冻干保护剂与琼胶酶溶液的体积比优选为1:(5~15),具体可以为1:5、1:6、1:7、1:8、1:9、1:10、1:11、1:12、1:13、1:14、1:15等。
在一种优选实施方式中,所述冷冻干燥的方法包括将琼胶酶保护液于-45℃~-35℃下冻结1~5h,再置于真空冷冻干燥机中并于-10℃~0℃下干燥5~10h,之后以5~10℃/min的速率逐步升温至25℃~35℃并保持2~5h完成冷冻干燥。此时,琼胶酶在冷冻干燥过程中失去水分,琼胶酶冻干保护剂中的羟基能够替代蛋白质表面的水的羟基,使蛋白质表面形成一层假定的水化膜,这样可保护氢键的联结位置不直接暴露在周围环境中,稳定蛋白质的高级结构,防止蛋白质因冻干而变性,使其即使在低温冷冻和干燥失水的情况下,仍能够保持蛋白质结构和功能的完整性,保护效果更为优异。
以下将通过实施例对本发明进行详细描述。
以下制备例中,β-琼胶酶通过以下方法获得:按照通过化学合成法从相应菌属或菌种中获得基因序列为SEQ ID NO:1的酶基因,将该酶基因引入PET-14表达载体中得到重组载体,之后将所述重组载体转化至大肠杆菌BL21(DE3)得到重组菌株,所述重组菌株经发酵、分离纯化后得到所述β-琼胶酶。
在没有特别说明的情况下,所述β-琼胶酶中涉及的操作可按照本领域常规技术手段进行,例如,如“《分子克隆实验指南(第四版)》J.萨姆布鲁克、M.R.格林”所公开的。
以下实施例和对比例中:可溶性淀粉购自上海阿拉丁生化科技股份有限公司,CAS号为9005-84-9;糊精购自上海阿拉丁生化科技股份有限公司,CAS号为9004-53-9;海藻糖购自北京索莱宝科技有限公司,CAS号为6138-23-4;蔗糖购自北京索莱宝科技有限公司,CAS号为57-50-1;甘油购自国药集团化学试剂有限公司,CAS号为56-81-5;甘露醇购自国药集团化学试剂有限公司,CAS号为69-65-8。
以下制备例中,酶活单位的定义为:在最适条件下每分钟释放1μmol还原糖(mmol,U)所需要的酶量(mL)。
以下实施例和对比例中,比活力按照以下方法测定:将50μL琼胶酶溶液与50mL溶有0.2%琼脂糖的Tris-HCl溶液(pH值为9.0)混合,之后将所得混合液在40℃下水浴加热10min,随后立即煮沸灭活,取上述反应液1mL与3mL DNS试剂混合,沸水加热10min,充分冷却后在540nm波长下测定产物的吸光值。参照半乳糖标准曲线计算还原糖产量,计算比活力。
制备例1
往100g的琼胶水溶液(琼胶浓度为1wt%)中加入0.1mLβ-琼胶酶(基因序列为SEQID NO:1且氨基酸序列为SEQ ID NO:2,酶活为200U/mL),之后于40℃下酶解8h,再将所得酶解产物经高速冷冻离心机离心以去除未被降解的胶体,之后再将上清液减压浓缩得到琼胶寡糖,记为Q1,其主要产物为新琼四糖和新琼六塘。
制备例2
往100g的琼胶水溶液(琼胶浓度为1.5wt%)中加入0.5mLβ-琼胶酶(基因序列为SEQ ID NO:1且氨基酸序列为SEQ ID NO:2,酶活为200U/mL),之后于45℃下酶解5h,再将所得酶解产物经高速冷冻离心机离心以去除未被降解的胶体,之后再将上清液减压浓缩得到琼胶寡糖,记为Q2。
制备例3
往100g的琼胶水溶液(琼胶浓度为2wt%)中加入1mLβ-琼胶酶(基因序列为SEQ IDNO:1且氨基酸序列为SEQ ID NO:2,酶活为200U/mL),之后于50℃下酶解3h,再将所得酶解产物经高速冷冻离心机离心以去除未被降解的胶体,之后再将上清液减压浓缩得到琼胶寡糖,记为Q3。
制备例4
按照制备例1的方法制备琼胶寡糖,不同的是,将基因序列为SEQ ID NO:1的β-琼胶酶采用CN110438182A中公开的β-琼胶酶(酶活为200U/mL)替代,具体氨基酸序列为SEQID NO:3:
MTFTKSKIATVLSLSLLGIYGCASTTPQNEQAAAGEQVVEDMGGALPDFESDKFFSKLKAEHAKASAVTDTGVTAGSQALKIDFDSVNEANKFKFWPNVKLHPDTGNWNWNAKGSLTLDVTNPTDSTANIILKIADNVGVMGAGDNQLNYALSVPAGETVPVEMIFNGSKRKLDGYWGGEKINLRKLVEFQIFVQGPIDQQSVIVDNFALVDATGDFVEASGAEEVVTGPVPTVLAITDFEKGQDSFISAERSVATTISPVKTDDGAAIDVLFSASNSYPNITFRPDVPWDWSGQGDFNVAFDMVNKSDEPLQLFVRVDDDEHEAFGGTANGVQNSWSGYVTIAPNDEGTYYLSLMPAGDQMVSGMRGEPPKKSYKAQAISYGWGDNNLDLSNIYSMQLYLQNPTADQKLQISSVRLIPNLESDTSRYEGLLDEFGQYTGQDWAQKVKSLEDLQAAGAAELDSLEHPTQLPDRSKFGGWADGPKLEATGFFRAEKVDGKWALVDPEGYLFFVTGLDNIRMDDTVTITGVDFSNKETREGREVASELRNSMFTWLPEYDDVLAESYDYADWIHTGALKKGEVFSFYSANLQRKYQTSREEALKIWKDVTLNRMQDWGFTTLGNWADPKFYDNQQIAYAANGWIFGDHARISTGNDYWGPIHDPFDPEFAVSTRKMAEKVASEVSKDDPWLMGIFVDNEISWGNTKNEANHYGLVVNALSYDIKESPAKAAFTKHLQDKYSSIDALNQSWGTKVTSWADFEVSFDHRSRLSSSMKKDYSEMLQMLSEKYFSTVQAELKKVLPNHMYLGARFADWGVTPEIARGAAPYVDVMSYNLYAEDLNSKGDWSLLPELDKPSIIGEFHFGATDTGLFHGGIVSASNQADRAKKYTHYMQSIVDNPYFVGAHWFQYLDSPTTGRAWDGENYNVGFVSITDTPYQELIDAAKQFNRDLYNLRYKK,其余条件与制备例1相同,得到琼胶寡糖,记为Q4。
实施例1
将琼胶寡糖Q1 6w/v%、Tris-HCl缓冲液(pH值为9)0.5mol/L、α,α-海藻糖2w/v%、可溶性淀粉5w/v%、糊精5w/v%、甘油10w/v%、甘露醇1w/v%溶于超纯水中,得到琼胶酶冻干保护剂,记为M1。
实施例2
将琼胶寡糖Q2 1w/v%、Tris-HCl缓冲液(pH值为8.5)0.1mol/L、蔗糖1w/v%、可溶性淀粉1w/v%、糊精1w/v%、甘油5w/v%、甘露醇0.5w/v%溶于超纯水中,得到琼胶酶冻干保护剂,记为M2。
实施例3
将琼胶寡糖Q3 10w/v%、Tris-HCl缓冲液(pH值为9.5)0.3mol/L、蔗糖3w/v%、可溶性淀粉10w/v%、糊精10w/v%、甘油20w/v%、甘露醇2w/v%溶于超纯水中,得到琼胶酶冻干保护剂,记为M3。
实施例4
按照实施例1的方法制备琼胶酶冻干保护剂,不同的是,将琼胶寡糖Q1采用相同重量份的琼胶寡糖Q4替代,其余条件与实施例1相同,得到琼胶酶冻干保护剂,记为M4。
对比例1
按照实施例1的方法制备琼胶酶冻干保护剂,不同的是,将琼胶寡糖Q1采用相同重量份的α,α-海藻糖替代,其余条件与实施例1相同,得到参比琼胶酶冻干保护剂,记为DM1。
对比例2
按照实施例1的方法制备琼胶酶冻干保护剂,不同的是,将α,α-海藻糖采用相同重量份的葡萄糖替代,其余条件与实施例1相同,得到参比琼胶酶冻干保护剂,记为DM2。
对比例3
按照实施例1的方法制备琼胶酶冻干保护剂,不同的是,将可溶性淀粉采用相同重量份的葡萄糖替代,其余条件与实施例1相同,得到参比琼胶酶冻干保护剂,记为DM3。
对比例4
按照实施例1的方法制备琼胶酶冻干保护剂,不同的是,将糊精采用相同重量份的葡萄糖替代,其余条件与实施例1相同,得到参比琼胶酶冻干保护剂,记为DM4。
对比例5
按照实施例1的方法制备琼胶酶冻干保护剂,不同的是,将甘露醇采用相同重量份的甘油替代,其余条件与实施例1相同,得到参比琼胶酶冻干保护剂,记为DM5。
测试例
(1)冻干保护效果测定:
将以上实施例和对比例所得琼胶酶冻干保护剂分别与琼胶酶溶液按体积比1:9的比例混合,将其置于-40℃下冻结5h,之后将冻结物置于真空冷冻干燥机中并于-10℃下干燥10h,随后以5℃/min的速率逐步升温至30℃并保持2h完成冷冻干燥。将干燥后的粉末复溶于超纯水中,使其体积恢复至原始加入的琼胶酶溶液的体积,测定其酶活力,检测时取三次测量结果计算平均值。琼胶酶溶液的原始比活力、干燥后比活力以及酶活损失率见表1。
表1
从以上结果可以看出,往琼胶酶溶液中分别加入10%的M1、M2、M3后进行冷冻干燥,酶活力均能得到良好的保护,酶活损失率在5%以内;往琼胶酶溶液中分别加入10%的M4后进行冷冻干燥,酶活力均能得到较好的保护,酶活损失率在9%以内;而往琼胶酶溶液中分别加入10%的DM1、DM2、DM3、DM4、DM5后进行冷冻干燥,酶活损失较多,损失率超过12%,最高可达15.8%。这说明本发明提供的琼胶酶冻干保护剂能在琼胶酶的冷冻干燥中对琼胶酶进行有效保护,并且采用基因序列为SEQ ID NO:1的琼胶酶对琼胶水溶液进行酶解所得琼胶寡糖所对应的琼胶酶冻干保护剂的保护效果更好。
(2)琼胶酶冻干粉稳定性测试:
将按照以上实施例和对比例所述的琼胶酶冻干保护剂与琼胶酶溶液按体积比1:9的比例混合,将其置于-40℃下冻结5h,之后将冻结物置于真空冷冻干燥机中并于-10℃下干燥10h,随后以5℃/min的速率逐步升温至30℃并保持2h完成冷冻干燥,得到琼胶酶冻干粉。所得琼胶酶冻干粉置于37℃、相对湿度(RH)为60%的恒温恒湿箱中,连续放置3个月后检测其比活力,计算酶活损失率。另将按以上方法所得琼胶酶冻干粉置于4℃冰箱中,连续冷藏2年后检测其比活力并计算酶活损失率,结果见表2。
表2
从以上结果可以看出,实施例1~4所得琼胶酶冻干保护剂M1、M2、M3和M4可以有效延长琼胶酶的贮藏期限,与对比例所得琼胶酶冻干保护剂相比表现出更优异的保护效果。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在不脱离本发明的原理和宗旨的情况下在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
SEQUENCE LISTING
<110> 蓝脑科技(厦门)有限公司
<120> 一种琼胶酶冻干保护剂及琼胶酶的保存方法
<130> NNKE-21001-NUI
<160> 3
<170> PatentIn version 3.3
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gagggcgtta atatcgattc tacaacagct gggcaggaga cgggcgaatg gacctcatac 1380
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cacggggatg ttaagataaa ttggcttttc agttaa 1596
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530
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<213> β-琼胶酶
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Trp Ile Phe Gly Asp His Ala Arg Ile Ser Thr Gly Asn Asp Tyr Trp
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Gly Pro Ile His Asp Pro Phe Asp Pro Glu Phe Ala Val Ser Thr Arg
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Claims (11)
1.一种琼胶酶冻干保护剂,其特征在于,所述琼胶酶冻干保护剂含有琼胶寡糖1~10w/v%、缓冲溶液0.1~0.5mol/L、非还原性二糖1~5w/v%、可溶性淀粉1~10w/v%、糊精1~10w/v%、甘油5~20w/v%、甘露醇0.5~5w/v%且余量为水;所述缓冲溶液为Tris-HCl缓冲溶液,所述Tris-HCl缓冲溶液的pH值为8.5~9.5。
2.根据权利要求1所述的琼胶酶冻干保护剂,其特征在于,所述琼胶寡糖的聚合度为2~10。
3.根据权利要求1所述的琼胶酶冻干保护剂,其特征在于,所述琼胶寡糖选自新琼二糖、新琼四糖、新琼六糖和新琼八糖中的至少一种。
4.根据权利要求1所述的琼胶酶冻干保护剂,其特征在于,所述琼胶寡糖按照以下方法制得:将琼胶水溶液在β-琼胶酶的存在下进行酶解,所述β-琼胶酶的基因序列为SEQ IDNO:1,之后将所得酶解产物进行纯化和浓缩。
5.根据权利要求4所述的琼胶酶冻干保护剂,其特征在于,所述酶解的条件包括温度为40~50℃,时间为3~8h。
6.根据权利要求4所述的琼胶酶冻干保护剂,其特征在于,所述琼胶水溶液与β-琼胶酶的用量比为100g:(0.1~1)mL;所述β-琼胶酶的酶活为200U/mL。
7.根据权利要求1所述的琼胶酶冻干保护剂,其特征在于,所述非还原性二糖为海藻糖和/或蔗糖。
8.根据权利要求1所述的琼胶酶冻干保护剂,其特征在于,所述糊精选自白糊精、黄糊精、麦芽糊精和环糊精中的至少一种。
9.一种琼胶酶的保存方法,其特征在于,该方法包括往琼胶酶溶液中加入权利要求1~8中任意一项所述的琼胶酶冻干保护剂混合均匀得到琼胶酶保护液,之后将该琼胶酶保护液进行冷冻干燥。
10.根据权利要求9所述的保存方法,其特征在于,所述琼胶酶冻干保护剂与琼胶酶溶液的体积比为1:(5~15)。
11.根据权利要求9所述的保存方法,其特征在于,所述冷冻干燥的方法包括将琼胶酶保护液于-45℃~-35℃下冻结1~5h,再置于真空冷冻干燥机中并于-10℃~0℃下干燥5~10h,之后以5~10℃/min的速率逐步升温至25℃~35℃并保持2~5h完成冷冻干燥。
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