CN112280816B - 一种海洋源i型胶原亚基的规模化制备方法 - Google Patents

一种海洋源i型胶原亚基的规模化制备方法 Download PDF

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CN112280816B
CN112280816B CN202010991917.8A CN202010991917A CN112280816B CN 112280816 B CN112280816 B CN 112280816B CN 202010991917 A CN202010991917 A CN 202010991917A CN 112280816 B CN112280816 B CN 112280816B
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陈俊德
高然
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Abstract

本发明公开了一种海洋源I型胶原亚基的规模化制备方法，以海洋生物加工副产物为原材料，利用醋酸提取、盐析电渗析纯化集成技术制备I型胶原；以I型胶原为原料，利用胶原酶酶解协同多级膜分离联动技术制备I型胶原亚基α1和α2混合溶液；在此基础上，利用盐析协同电渗析技术制备I型胶原亚基α1和α2。针对目前胶原亚基的制备工艺，只能制备毫克级、微克级样品，无法实现规模化生产的技术瓶颈，本发明构建胶原酶酶解协同多级膜分离联动技术、盐析协同电渗析技术集成的技术体系，规模化制备I型胶原亚基α1和α2。

Description

一种海洋源I型胶原亚基的规模化制备方法
技术领域
本发明属于生物提取技术领域，具体涉及一种海洋源I型胶原亚基的规模化制备方法。
背景技术
胶原是脊椎动物中含量最丰富的蛋白质，是动物皮肤和骨***中的最主要结构蛋白质，约占总蛋白质的30％。目前确定的胶原的类型有29种，包括胶原I-XXIX型，每种类型的胶原都具有独特的氨基酸序列、结构和功能。在所有胶原类型中，I型胶原具有良好的生物相容性、生物降解性和弱抗原性，能为食品、药品、生物功能材料和组织工程等产业提供良好的原料来源。
近年来由于由于禽流感、疯牛病、***等传染性疾病的发生，导致陆源生物来源I型胶原的全球需求量逐年下降。以此同时，海洋生物来源I型胶原特别是鱼类副产物(主要是鱼鳞、鱼皮、鱼鳔)I型胶原的需求量迅速上升，已成为全球胶原产品最主要的原料来源之一。
I型胶原分子具有由三条胶原α亚基肽链通过右手螺旋形成的稳定的三螺旋四级结构，该结构导致胶原无法被人体吸收，无法开发成药品、保健品、食品。食品科学家利用酶法水解I型胶原，获得I型胶原蛋白肽，可以应用到食品中，但这类型产品含有大量的多肽，现有技术无法确定所有肽的氨基酸序列，产品质量不稳定，无法成为高附加值的胶原药品、保健品。因此，开发氨基酸序列明确的海洋源I型胶原亚基成为研究的热点。
目前I型胶原亚基制备方法有：(1)色谱法：如：Weng等以金鲳鱼鱼皮为原料制备鱼皮胶原，该技术方案将所制备的鱼皮胶原溶解在硼酸盐缓冲液中，45℃加热30min后，利用CM-52羧甲基纤维素色谱柱分离纯化，得到胶原亚基α1和α2；(2)电泳法：如：Deyl等提出将胶原溶解在pH为9.2的硼酸缓冲液中降解15min后，利用毛细管电泳分离纯化，得到胶原亚基。然而这些制备方法只能得到毫克级、微克级的胶原亚基样品，不能实现工业化生产。
发明内容
本发明的目的在于克服现有技术缺陷，提供一种海洋源I型胶原亚基的规模化制备方法。
本发明的技术方案如下：
一种海洋源I型胶原亚基的规模化制备方法，包括如下步骤：
(1)将预处理后的海洋生物加工副产物用醋酸溶液提取，再经离心去除固体杂质后，将所获得的清液经氯化钠盐析和电渗析纯化，得到I型胶原溶液；
(2)在上述I型胶原溶液中加入胶原酶进行常温酶解，再经离心除杂；将所获得的清液用截留相对分子质量200kD的纳滤膜去除未酶解的I型胶原，得到透过液；将该透过液用截留相对分子质量50kD的纳滤膜去除无机盐，获得I型胶原亚基α1和α2混合溶液；
(3)将上述I型胶原亚基α1和α2混合溶液经氯化钠盐析后离心获得清液和沉淀；将该清液经电渗析脱盐后进行冷冻干燥，获得如SEQ ID NO.01所示的I型胶原亚基α1；将该沉淀用醋酸溶液溶解后，再依次经电渗析脱盐和冷冻干燥，获得如SEQ ID NO.02所示的I型胶原亚基α2。
在本发明的一个优选实施方案中，所述海洋生物加工副产物包括鱼鳞、鱼皮和鱼鳔。
进一步优选的，所述预处理为：将所述海洋生物加工副产物投入清洗罐中，加入质量体积浓度为2-4％碳酸氢钠溶液，室温搅拌4-6h，然后进行水洗，碳酸氢钠溶液与海洋生物加工副产物的比例为8-12L∶1kg。
在本发明的一个优选实施方案中，所述步骤(1)中的醋酸溶液的浓度为0.4-0.6M。
在本发明的一个优选实施方案中，所述步骤(1)中的盐析包括在所述清液中加入质量体积百分比为2-4％的氯化钠。
在本发明的一个优选实施方案中，所述步骤(1)中的电渗析的极水为质量体积百分比为4-6％的氯化钠溶液。
在本发明的一个优选实施方案中，所述步骤(1)中的电渗析的流量≤1m3/h。
在本发明的一个优选实施方案中，所述步骤(3)中的电渗析的极水为质量体积百分比为1.5-2.5％的氯化钠溶液。
在本发明的一个优选实施方案中，所述步骤(3)中的电渗析的流量≤1m3/h。
在本发明的一个优选实施方案中，所述步骤(1)和(3)中的电渗析所用的电渗析膜为聚乙烯异相离子交换膜，且步骤(1)中的电渗析的电流为14-16A，步骤(3)中的电渗析的电流为10-12A。
本发明的有益效果是：
1、针对目前胶原亚基的制备工艺，只能制备毫克级、微克级样品，无法实现规模化生产的技术瓶颈，本发明构建胶原酶酶解协同多级膜分离联动技术、盐析协同电渗析技术集成的技术体系，规模化制备I型胶原亚基α1和α2。
2、本发明以低值海洋生物加工副产物为原料，生产成本低，工艺易于控制，适合规模化生产。
3、本发明生产的I型胶原亚基α1和α2，氨基酸序列明确，纯度大于90％，可应用在药品、保健食品领域。
附图说明
图1为本发明的工艺流程图。
图2为本发明实施例1的标样的高效凝胶色谱图。
图3为本发明实施例1的标样高效凝胶色谱的标准曲线。
图4为本发明实施例1的I型胶原亚基α1凝胶色谱图。
图5为本发明实施例1的I型胶原亚基α2凝胶色谱图。
图6为本发明实施例1的I型胶原亚基α1的肽指纹图谱。
图7为本发明实施例1的I型胶原亚基α2的肽指纹图谱。
具体实施方式
以下通过具体实施方式对本发明的技术方案进行进一步的说明和描述。
实施例1
如图1所示，一种海洋源I型胶原亚基的规模化制备方法，，包括以下步骤：
(1)将100kg罗非鱼鱼鳞/鱼皮/鱼鳔投入清洗罐中，加入1000L的碳酸氢钠溶液，溶液的质量体积浓度为3％，室温搅拌5h，水洗净后，加入反应釜中，加入0.5M的醋酸溶液搅拌提取，提取液利用10000rpm的离心机除杂后，所得清液即为I型胶原粗提物溶液。在I型胶原粗提物溶液加入3％(W/V)氯化钠盐析，转速为10000rpm离心机离心后，得到沉淀物。加入0.5M的醋酸溶液溶解沉淀物后，利用电渗析技术去除酸和无机盐，电渗析膜为聚乙烯异相离子交换膜，电流为15A，极水箱为5％(W/V)的氯化钠溶液，电渗析流量≤1m3/h，，得到I型胶原溶液。
(2)将I型胶原溶液加入反应釜中，加入100g胶原酶，常温下酶解4h。酶解液10000rpm离心机除杂，清液利用截留相对分子质量为200kD的纳滤膜去除未酶解的I型胶原，得到透过液。透过液利用截留相对分子质量为50kD的纳滤膜，去除溶液中的无机盐，压力为2Mpa，温度为25℃，得到I型胶原亚基α1和α2混合溶液。
(3)往I型胶原亚基α1和α2混合溶液中加入5％(W/V)氯化钠盐析。10000rpm离心机离心后，清液利用电渗析技术脱盐，电渗析膜为聚乙烯异相离子交换膜，电流为11A，极水箱为2％(W/V)的氯化钠溶液，电渗析流量≤1m3/h，冷冻干燥电渗析后的物料溶液，得到I型胶原亚基α1冻干样。此外，利用0.1M醋酸溶液溶解离心机离心后的沉淀物，利用电渗析技术脱盐，电渗析膜为聚乙烯异相离子交换膜，电流为13A，极水箱为3％的氯化钠溶液，电渗析流量≤1m3/h，冷冻干燥电渗析后的物料溶液，得到I型胶原亚基α2冻干样。
利用高效凝胶色谱法检测本发明得到的I型胶原亚基α1和I型胶原亚基α2的纯度和分子量。检测条件为：SuperdexTM Peptide 10/300GL色谱柱，流动相：50mmol/L磷酸盐、0.15mpl/L氯化钠、pH7.0缓冲液；流速为0.5ml/min，检测波长为210nm，进样量：50uL。标样Mark分别为：①甲状腺球蛋白，分子量669000Da，保留时间8.04；②铁蛋白，分子量440000Da，保留时间14.30；③牛血清蛋白，分子量67000Da，保留时间16.02；④β-乳球蛋白，分子量35000Da，保留时间16.02；⑤细胞色素，分子量13600Da，保留时间18.06；⑥抑肽酶，分子量6512Da，保留时间19.37(图2)。得到标样的标准曲线为lgMw＝-0.1726x+7.2402(R2＝0.9951)(图3)。由图4、图5可见，本实施例制备的胶原亚基α1和α2。α1纯度为93.2％，α2纯度为90.3％。经标准曲线(图2)对照计算，胶原亚基α1的分子量为131KD，胶原亚基α2的分子量为125KD。
利用高效液相色谱-质谱联用技术鉴定本发明得到的I型胶原亚基α1和I型胶原亚基α2。胶原亚基α1的肽指纹图谱鉴定(图6)结果表明，胶原亚基α1的MASCOT分数为1137，覆盖率为32％。胶原亚基α1为collagen，type I，alpha 1 OS＝Oreochromis niloticus GN＝COL1A1 PE＝2 SV＝1，蛋白编码为tr|G9M6I5|G9M6I5_ORENI，分子量为137283Da，等电点(PI)值为5.64。胶原胶原亚基α1的氨基酸序列如SEQ ID NO.01所示。胶原亚基α2的肽指纹图谱鉴定(图7)结果表明，胶原亚基α2的MASCOT分数为1344，覆盖率为36％。胶原亚基α2为Collagen type I alpha 2 OS＝Oreochromis niloticus GN＝COL1A2PE＝2 SV＝1，蛋白编码为tr|G9M616|G9M6I6_ORENI，分子量为126477Da，等电点(PI)值为9.18。胶原胶原亚基α2的氨基酸序列如SEQ ID NO.02所示。
以上所述，仅为本发明的较佳实施例而已，故不能依此限定本发明实施的范围，即依本发明专利范围及说明书内容所作的等效变化与修饰，皆应仍属本发明涵盖的范围内。
序列表
<110> 自然资源部第三海洋研究所
<120> 一种海洋源Ⅰ型胶原亚基的规模化制备方法
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Ser Ala Gly Ile Pro Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Ile
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Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly Phe Pro Gly Leu Ala
945 950 955 960
Gly Pro Val Gly Glu Pro Gly Lys Gln Gly Pro Ser Gly Pro Ser Gly
965 970 975
Glu Arg Gly Pro Pro Gly Pro Met Gly Pro Pro Gly Leu Ala Gly Ala
980 985 990
Pro Gly Glu Pro Gly Arg Glu Gly Thr Pro Gly Asn Glu Gly Ala Ala
995 1000 1005
Gly Arg Asp Gly Ala Pro Gly Pro Lys Gly Asp Arg Gly Glu Ser Gly
1010 1015 1020
Pro Ala Gly Ala Pro Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly Pro
1025 1030 1035 1040
Val Gly Pro Ala Gly Lys Thr Gly Asp Arg Gly Glu Thr Gly Pro Ala
1045 1050 1055
Gly Pro Ala Gly Ala Ala Gly Pro Ala Gly Pro Arg Gly Pro Ala Gly
1060 1065 1070
Ala Pro Gly Leu Arg Gly Asp Lys Gly Glu Thr Gly Glu Ala Gly Glu
1075 1080 1085
Arg Gly Met Lys Gly His Arg Gly Phe Thr Gly Met Gln Gly Pro Pro
1090 1095 1100
Gly Pro Pro Gly Thr Ser Gly Glu Ser Gly Pro Ala Gly Ala Ala Gly
1105 1110 1115 1120
Pro Ala Gly Pro Arg Gly Pro Ser Gly Ala Ala Gly Ala Pro Gly Lys
1125 1130 1135
Asp Gly Val Ser Gly Leu Pro Gly Pro Thr Gly Pro Pro Gly Pro Arg
1140 1145 1150
Gly Arg Ser Gly Glu Met Gly Pro Ala Gly Pro Pro Gly Pro Pro Gly
1155 1160 1165
Pro Pro Gly Ala Pro Gly Ala Pro Gly Gly Gly Phe Asp Leu Gly Phe
1170 1175 1180
Met Val Gln Pro Gln Glu Lys Ala Pro Asp Pro Phe Arg Met Tyr Arg
1185 1190 1195 1200
Ala Asp Asp Ala Asn Val Leu Arg Asp Arg Asp Leu Glu Val Asp Ser
1205 1210 1215
Thr Leu Lys Ser Leu Ser Gln Gln Ile Glu Gln Ile Arg Ser Pro Asp
1220 1225 1230
Gly Thr Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met Cys
1235 1240 1245
His Pro Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asp Gln Gly
1250 1255 1260
Cys Thr Gln Asp Ala Ile Lys Val Tyr Cys Asn Met Glu Thr Gly Glu
1265 1270 1275 1280
Thr Cys Val Ser Pro Thr Gln Arg Glu Val Ala Lys Lys Asn Trp Tyr
1285 1290 1295
Ile Ser Lys Asn Ile Lys Glu Lys Lys His Val Trp Phe Gly Glu Ala
1300 1305 1310
Met Asn Glu Gly Phe Gln Phe Glu Tyr Gly Ser Glu Gly Ser Leu Pro
1315 1320 1325
Glu Asp Val Asn Ile Gln Met Thr Phe Leu Arg Leu Met Ser Thr Glu
1330 1335 1340
Ala Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met
1345 1350 1355 1360
Asp Ala Ala Ala Gly Asn Leu Lys Lys Ala Leu Leu Leu Gln Gly Ser
1365 1370 1375
Asn Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser
1380 1385 1390
Val Leu Glu Asp Gly Cys Thr Ser His Thr Gly Thr Trp Gly Lys Thr
1395 1400 1405
Val Ile Asp Tyr Lys Thr Ser Lys Thr Ser Arg Leu Pro Ile Ile Asp
1410 1415 1420
Ile Ala Pro Met Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Glu
1425 1430 1435 1440
Val Gly Pro Val Cys Phe Leu
1445
<210> 2
<211> 1350
<212> PRT
<213> 罗非鱼(Oreochroms mossambcus)
<400> 2
Met Leu Ser Phe Val Asp Thr Arg Ile Leu Leu Leu Leu Ala Val Thr
1 5 10 15
Ser Tyr Leu Ala Ser Cys Gln Phe Ala Gly Pro Lys Gly Pro Arg Gly
20 25 30
Asp Arg Gly Pro Pro Gly Pro Asn Gly Lys Asp Gly Leu Pro Gly Pro
35 40 45
Pro Gly Pro Ala Gly Pro Pro Gly Pro Pro Gly Leu Gly Gly Asn Phe
50 55 60
Ala Ala Gln Tyr Asp Gly Val Lys Ala Pro Asp Pro Gly Pro Gly Pro
65 70 75 80
Met Gly Leu Met Gly Pro Arg Gly Pro Pro Gly Pro Pro Gly Ala Ser
85 90 95
Gly Pro Gln Gly His Thr Gly His Ala Gly Glu Pro Gly Glu Pro Gly
100 105 110
Gln Ala Gly Ala Val Gly Pro Arg Gly Pro Pro Gly Pro Pro Gly Lys
115 120 125
Ala Gly Glu Asp Gly Asn Asn Gly Arg Pro Gly Lys Pro Gly Asp Arg
130 135 140
Gly Ala Pro Gly Pro Gln Gly Ala Arg Gly Phe Pro Gly Thr Pro Gly
145 150 155 160
Leu Pro Gly Met Lys Gly His Arg Gly Tyr Thr Gly Leu Asp Gly Arg
165 170 175
Lys Gly Glu Pro Gly Ala Ala Gly Pro Lys Gly Glu Pro Gly Ala His
180 185 190
Gly Ala Ala Gly Ser Pro Gly Leu Ala Gly Ala Arg Gly Leu Pro Gly
195 200 205
Glu Arg Gly Arg Pro Gly Pro Ala Gly Pro Ala Gly Ala Arg Gly Ala
210 215 220
Asp Gly Asn Ala Gly Pro Thr Gly Pro Ala Gly Pro Leu Gly Ala Ala
225 230 235 240
Gly Pro Pro Gly Phe Pro Gly Gly Pro Gly Pro Lys Gly Glu Thr Gly
245 250 255
Pro Val Gly Ala Thr Gly Pro Ser Gly Pro Gln Gly Ser Arg Gly Glu
260 265 270
Pro Gly Pro Asn Gly Ala Val Gly Pro Val Gly Pro Ser Gly Asn Pro
275 280 285
Gly Ala Asn Gly Leu Asn Gly Ala Lys Gly Ala Ala Gly Thr Pro Gly
290 295 300
Val Ala Gly Ala Pro Gly Phe Pro Gly Pro Arg Gly Gly Pro Gly Pro
305 310 315 320
Gln Gly Pro Gln Gly Ala Ala Gly Pro Arg Gly Leu Ala Gly Asp Pro
325 330 335
Gly Ile Gln Gly Val Lys Gly Asp Ser Gly Pro Lys Gly Glu Pro Gly
340 345 350
His Ser Gly Pro Gln Gly Pro Pro Gly Pro Gln Gly Glu Glu Gly Lys
355 360 365
Arg Gly Pro Thr Gly Glu Ile Gly Ala Thr Gly Leu Ala Gly Ala Arg
370 375 380
Gly Ala Arg Gly Ala Pro Gly Ser Arg Gly Met Pro Gly Ala Glu Gly
385 390 395 400
Arg Thr Gly Pro Val Gly Met Pro Gly Ala Arg Gly Ala Thr Gly Ala
405 410 415
Ala Gly Pro Arg Gly Pro Pro Gly Asp Ala Gly Arg Ala Gly Glu Pro
420 425 430
Gly Ala Ala Gly Leu Arg Gly Leu Pro Gly Ser Pro Gly Ser Ser Gly
435 440 445
Pro Pro Gly Lys Glu Gly Pro Ala Gly Pro Ser Gly Gln Asp Gly Arg
450 455 460
Ser Gly Pro Pro Gly Pro Ser Gly Pro Arg Gly Leu Ser Gly Asn Ile
465 470 475 480
Gly Phe Pro Gly Pro Lys Gly Pro Ser Gly Glu Pro Gly Lys Pro Gly
485 490 495
Glu Arg Gly Ala Thr Gly Pro Thr Gly Leu Arg Gly Pro Pro Gly Pro
500 505 510
Asp Gly Asn Asn Gly Ala Thr Gly Ala Thr Gly Val Ala Gly Gly Pro
515 520 525
Gly Glu Lys Gly Glu Gln Gly Pro Ser Gly Ser Pro Gly Phe Gln Gly
530 535 540
Leu Pro Gly Pro Ala Gly Pro Thr Gly Glu Ala Gly Lys Pro Gly Asp
545 550 555 560
Arg Gly Ile Pro Gly Glu Pro Gly Ala Ala Gly Asn Ala Gly Ala Lys
565 570 575
Gly Glu Arg Gly Asn Pro Gly Ala Ala Gly Ser Ala Gly Pro Gln Gly
580 585 590
Pro Ile Gly Pro Arg Gly Pro Ala Gly Ala Pro Gly Pro Asp Gly Gly
595 600 605
Lys Gly Glu Pro Gly Pro Ala Gly Val Ala Gly Ala Pro Gly His Gln
610 615 620
Gly Ala Gly Gly Met Pro Gly Glu Arg Gly Gly Ala Gly Thr Pro Gly
625 630 635 640
Pro Lys Gly Glu Lys Gly Glu Pro Gly His Lys Gly Pro Asp Gly Asn
645 650 655
Pro Gly Arg Asp Gly Pro Arg Gly Leu Ala Gly Pro Ala Gly Pro Pro
660 665 670
Gly Pro Thr Gly Ala Asn Gly Asp Lys Gly Glu Gly Gly Ser Phe Gly
675 680 685
Pro Ala Gly Pro Ala Gly Pro Arg Gly Pro Ser Gly Glu Arg Gly Glu
690 695 700
Val Gly Pro Ala Gly Ala Pro Gly Phe Ala Gly Pro Pro Gly Ala Asp
705 710 715 720
Gly Gln Ala Gly Ala Arg Gly Glu Arg Gly Pro Ser Gly Ala Lys Gly
725 730 735
Glu Val Gly Pro Ser Gly Leu Ala Gly Pro Ala Gly Gln Ser Gly Pro
740 745 750
Ala Gly Pro Ala Gly Pro Gly Gly Pro Pro Gly Ala Arg Gly Asp Asn
755 760 765
Gly Pro Pro Gly Leu Thr Gly Phe Pro Gly Ala Ala Gly Arg Val Gly
770 775 780
Ala Ala Gly Pro Ala Gly Ile Val Gly Pro Pro Gly Pro Ala Gly Pro
785 790 795 800
Ser Gly Lys Asp Gly Pro Arg Gly Pro Arg Gly Asp Pro Gly Pro Ser
805 810 815
Gly Pro Ser Gly Glu Pro Gly Ile Ile Gly Pro Pro Gly Leu Ala Gly
820 825 830
Glu Lys Gly Pro Ser Gly Glu Ser Gly Pro Pro Gly Ser Pro Gly Ala
835 840 845
Pro Gly Thr Ser Gly Pro Leu Gly Leu Gln Gly Phe Val Gly Leu Pro
850 855 860
Gly Ser Arg Gly Asp Arg Gly Ala Pro Gly Gly Ala Gly Gly Val Gly
865 870 875 880
Glu Pro Gly Arg Leu Gly Pro Ala Gly Pro Pro Gly Ala Arg Gly Ala
885 890 895
Pro Gly Asn Ile Gly Leu Pro Gly Met Thr Gly Pro Gln Gly Glu Ala
900 905 910
Gly Arg Glu Gly Ser Pro Gly Asn Asp Gly Pro Pro Gly Arg Pro Gly
915 920 925
Ala Ala Gly Leu Lys Gly Asp Arg Gly Glu Pro Gly Ser Ala Gly Thr
930 935 940
Thr Gly Leu Ala Gly Ala Pro Gly Pro Ala Gly Pro Thr Gly Ala Ala
945 950 955 960
Gly Arg Pro Gly Asn Arg Gly Glu Ala Gly Pro Ser Gly Pro Ser Gly
965 970 975
Ala Val Gly Pro Ala Gly Ala Arg Gly Ala Ser Gly Pro Ala Gly Pro
980 985 990
Arg Gly Glu Lys Gly Val Ala Gly Asp Lys Gly Glu Arg Gly Met Lys
995 1000 1005
Gly Leu Arg Gly His Pro Gly Leu Gln Gly Met Pro Gly Pro Ser Gly
1010 1015 1020
Pro Pro Gly Asp Thr Gly Ala Ala Gly Ala His Gly Pro Ser Gly Pro
1025 1030 1035 1040
Arg Gly Pro Ala Gly Pro His Gly Pro Val Gly Lys Asp Gly Arg Pro
1045 1050 1055
Gly Ala His Gly Thr Met Gly Ala Pro Gly Ala Arg Gly Pro Asn Gly
1060 1065 1070
Tyr Ser Gly Pro Val Gly Pro Pro Gly Pro Pro Gly Leu Pro Gly Pro
1075 1080 1085
Pro Gly Pro Ala Gly Gly Gly Tyr Asp Val Ser Gly Gly Tyr Asp Glu
1090 1095 1100
Tyr Arg Ala Asp Gln Pro Ala Leu Arg Ala Lys Asp Tyr Glu Val Asp
1105 1110 1115 1120
Ala Thr Ile Lys Ser Leu Asn Thr Gln Ile Glu Asn Leu Leu Thr Pro
1125 1130 1135
Glu Gly Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Ile Lys Leu
1140 1145 1150
Ser His Pro Asp Trp Ser Ser Gly Phe Tyr Trp Ile Asp Pro Asn Gln
1155 1160 1165
Gly Cys Thr Asn Asp Ala Ile Lys Val Phe Cys Asp Phe Thr Thr Arg
1170 1175 1180
Glu Thr Cys Ile Tyr Ala His Pro Glu Ser Ile Ala Arg Lys Asn Trp
1185 1190 1195 1200
Tyr Arg Ser Thr Glu Asn Lys Lys His Val Trp Phe Gly Glu Thr Ile
1205 1210 1215
Asn Gly Gly Thr Glu Phe Thr Tyr Asn Asp Glu Thr Leu Ser Pro Gln
1220 1225 1230
Ser Met Ala Thr Gln Leu Ala Phe Met Arg Leu Leu Ser Asn Gln Ala
1235 1240 1245
Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met Asp
1250 1255 1260
Gly Glu Ser Gly Ser Leu Lys Lys Ala Val Val Leu Gln Gly Ser Asn
1265 1270 1275 1280
Asp Val Glu Leu Arg Ala Glu Gly Asn Ser Arg Phe Thr Phe Ser Val
1285 1290 1295
Leu Glu Asp Gly Cys Thr Thr His Thr Gly Glu Trp Ser Lys Thr Val
1300 1305 1310
Ile Glu Tyr Arg Thr Asn Lys Pro Ser Arg Leu Pro Ile Leu Asp Ile
1315 1320 1325
Ala Pro Leu Asp Ile Gly Gly Ala Asp Gln Glu Phe Gly Leu Asp Ile
1330 1335 1340
Gly Pro Val Cys Phe Lys
1345 1350

Claims (6)

1.一种海洋源Ⅰ型胶原亚基的规模化制备方法，其特征在于：包括如下步骤：
（1）将预处理后的海洋生物加工副产物用浓度为0.4-0.6M的醋酸溶液提取，再经离心去除固体杂质后，将所获得的清液经氯化钠盐析和电渗析纯化，得到Ⅰ型胶原溶液；
（2）在上述Ⅰ型胶原溶液中加入胶原酶进行常温酶解，再经离心除杂；将所获得的清液用截留相对分子质量200kD的纳滤膜去除未酶解的I型胶原，得到透过液；将该透过液用截留相对分子质量50kD的纳滤膜去除无机盐，获得Ⅰ型胶原亚基α1和α2混合溶液；
（3）将上述Ⅰ型胶原亚基α1和α2混合溶液经氯化钠盐析后离心获得清液和沉淀；将该清液经电渗析脱盐后进行冷冻干燥，获得如SEQ ID NO.01所示的Ⅰ型胶原亚基α1；将该沉淀用醋酸溶液溶解后，再依次经电渗析脱盐和冷冻干燥，获得如SEQ ID NO.02所示的Ⅰ型胶原亚基α2；该步骤中的电渗析的极水为质量体积百分比为1.5-2.5%的氯化钠溶液，电渗析的流量≤1m3/h；
上述步骤（1）和（3）中的电渗析所用的电渗析膜为聚乙烯异相离子交换膜，且步骤（1）中的电渗析的电流为14-16A，步骤（3）中的电渗析的电流为10-12A。
2.如权利要求1所述的规模化制备方法，其特征在于：所述海洋生物加工副产物包括鱼鳞、鱼皮和鱼鳔。
3. 如权利要求2所述的规模化制备方法，其特征在于：所述预处理为：将所述海洋生物加工副产物投入清洗罐中，加入质量体积浓度为2-4%碳酸氢钠溶液，室温搅拌4-6 h，然后进行水洗，碳酸氢钠溶液与海洋生物加工副产物的比例为8-12L: 1kg。
4.如权利要求1所述的规模化制备方法，其特征在于：所述步骤（1）中的盐析包括在所述清液中加入质量体积百分比为2-4%的氯化钠。
5.如权利要求1所述的规模化制备方法，其特征在于：所述步骤（1）中的电渗析的极水为质量体积百分比为4-6%的氯化钠溶液。
6.如权利要求1所述的规模化制备方法，其特征在于：所述步骤（1）中的电渗析的流量≤1m3/h。
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