CN114790450B - 一种阿拉伯糖苷酶和木聚糖酶的同步诱导合成方法 - Google Patents
一种阿拉伯糖苷酶和木聚糖酶的同步诱导合成方法 Download PDFInfo
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Abstract
本发明公开了一种阿拉伯糖苷酶和木聚糖酶的同步诱导合成方法,以里氏木霉作为产酶菌种,利用车前子壳、车前子多糖、或车前子壳与车前子多糖的混合物为碳源诱导物在产酶培养基中进行发酵,实现阿拉伯糖苷酶和木聚糖酶的同步诱导合成。该发明所生产的具备高活性的阿拉伯糖苷酶和木聚糖酶的酶液,可满足以阿拉伯木聚糖为酶水解对象实现阿拉伯低聚木糖高效率制备的需要。
Description
技术领域
本发明属生物化学中微生物培养技术领域,具体涉及一种阿拉伯糖苷酶和木聚糖酶的同步诱导合成方法。
背景技术
半纤维素作为丰度仅次于纤维素的第二大可再生农林资源,而阿拉伯木聚糖作为半纤维素家族的重要成员,主要由木糖单体经β-1,4-糖苷键聚合而成的木聚糖主链和α-1,2/α-1,3-阿拉伯糖苷键随机取代形成阿拉伯糖支链。基于阿拉伯木聚糖的分子结构解析,其水解酶系主要涵括:木聚糖酶、木糖苷酶、阿拉伯糖苷酶等,其中木聚糖酶和阿拉伯糖苷酶作为关键水解酶极为重要。相比于同质木聚糖而言,阿拉伯木聚糖类异质杂多糖因受支链结构空间位阻的影响,酶水解难度和复杂性显著提升,木聚糖酶和阿拉伯糖苷酶间协同作用对于阿拉伯低聚木糖的高效制备极为重要。在阿拉伯木聚糖定向水解过程中,利用阿拉伯糖苷酶有选择去除阿拉伯木聚糖的支链结构,对于木聚糖酶水解效率和阿拉伯低聚木糖得率的提高意义深远,而如何实现木聚糖酶和阿拉伯糖苷酶的高效协同制备是能否实现阿拉伯木聚糖高效水解效率的关键。
目前,生物酶的制备多采用产酶菌株筛选、底物特异性诱导和工程菌的可控改造等技术加以实现,其中以基因工程菌的可控改造技术研究最为广泛。基因工程菌的构建多以大肠杆菌和毕赤酵母为目的基因载体,但大肠杆菌或毕赤酵母次级代谢产物的生物安全性和基因工程菌的传代稳定性,是限制其在食品工业大规模应用的限制因素。里氏木霉作为分泌木聚糖水解酶系的主要原始菌株,其基因组中包含阿拉伯木聚糖所需水解酶系的全部基因,利用底物诱导技术可实现木聚糖水解酶的高效制备,所得酶的四维结构和稳定性相较于基因工程酶优势明显,然而如何筛选合适的诱导底物对于实现阿拉伯糖苷酶和木聚糖酶协同制备的关键。
发明内容
发明目的:针对现有技术存在阿拉伯木聚糖生物酶法制备阿拉伯低聚木糖过程关键水解酶(阿拉伯糖苷酶和木聚糖酶)无法高效协同制备的瓶颈问题,本发明提出一种阿拉伯糖苷酶和木聚糖酶的同步诱导合成方法。
为实现上述目的,本发明公开了一种阿拉伯糖苷酶和木聚糖酶的同步诱导合成方法,以里氏木霉作为产酶菌种,利用车前子壳、车前子多糖、或车前子壳与车前子多糖的混合物为碳源诱导物在产酶培养基中进行发酵,实现阿拉伯糖苷酶和木聚糖酶的同步诱导合成。
其中,所述车前子壳主要为平车前子、大车前子的种皮。
具体地,所述车前子多糖为车前子壳经热水抽提和乙醇分级沉淀法制备得到的车前子多糖。
所述乙醇分级沉淀使用体积浓度依次为15%、30%、45%、和60%的乙醇进行分级沉淀,将沉淀反复洗涤,经冷冻干燥即得到不同分子量区间的车前子多糖。
其中,所述车前子多糖通过如下方法制备得到:车前子壳经机械粉碎,过筛后,按固液比1:5-1:20,于30℃-50℃下提取10-100min后,离心收集上清液,优选地,10000rpm下离心10min收集上清液,控制乙醇浓度进行分级沉淀,得到的沉淀经固液分离、洗涤、干燥,收集固形物,得到车前子多糖。
通过逐步分级乙醇沉淀,制备得到的车前子多糖采用凝胶渗透色进行分子量的标准。其中凝胶渗透色谱法的测定条件为:色谱仪为安捷伦高效液相色谱仪1260;色谱柱为TSK-Gel G3000PWXL和TSK-Gel G5000PWXL双柱串联;检测器为示差检测器;流动相为去离子水;流速为0.6mL/min;柱温65℃;进样体积10.0μL,以不同分子量的普鲁兰多糖为标准样品。
得到的车前子多糖的分子量范围为104~105Da。
本发明的车前子壳和不同分子量的车前子多糖均为具有阿拉伯支链的木聚糖,其中阿拉伯糖与木糖的摩尔质量比0.05:1~0.5:1,其中,两者摩尔比通过如下方法进行测定:采用4%硫酸在121℃下将车前子壳和不同分子量的车前子多糖水解处理60min,利用利用高效液相色谱检测稀硫酸水解液中木糖与阿拉比糖的浓度进行计算。高效液相色谱测定条件为:色谱仪为安捷伦高效液相色谱仪1260;色谱柱为Bio-Rad Aminex HPX-87H;检测器为示差检测器;流动相为5mmol/L的硫酸溶液;流速为0.6mL/min;柱温55℃;进样体积10.0μL。
当碳源诱导物为车前子壳与车前子多糖的混合物时,两者的质量比为1:9-9:1。
优选地,碳源诱导物的添加浓度为5-30g/L。
具体地,发酵条件为:在28-30℃,150-170rpm条件下于产酶培养基中发酵产酶,产酶时间控制在72-120h,离心分离去除菌体,收集上清液。
其中,所述产酶培养基每1000mL中包含葡萄糖1.00g、磷酸二氢钾20.00g、尿素3.00g、无水硫酸镁1.50g、硫酸铵14.00g、氯化钙4.00g、FeSO4·7H2O 5.00mg、MnSO4·7H2O1.60mg、ZnSO4·7H2O 1.40mg和CoCl2 2.00mg。
车前草作为中草药资源在我国应用历史悠远,车前子壳为大粒车前草或平车前草的成熟种皮,细胞壁包含50%~70%亲水性阿拉伯木聚糖,具有杀菌消炎、调节免疫和改善便秘等生理活性。有研究表明,车前子壳中包含62.5%的阿拉伯木聚糖,其化学结构为β-1,4-糖苷键形成的木聚糖主链,在其主链上随机进行阿拉伯糖或木糖取代,阿拉伯糖与木糖(Ara/Xyl)的摩尔质量比为0.41:1.00。因此,本发明提出利用车前子壳、不同分子量车前子多糖和不同质量配比车前子壳/车前子多糖混合物为碳源诱导物,利用里氏木霉液态发酵,实现阿拉伯糖苷酶和木聚糖酶的协同制备,为阿拉伯木聚糖资源的高效生物炼制奠定基础。
有益效果:与现有技术相比,本发明采用里氏木霉以车前子壳、不同分子量车前子多糖和不同质量配比车前子壳/车前子多糖混合物为诱导底物,采用液态发酵方式,实现阿拉伯糖苷酶和木聚糖酶的高效协同制备。该混合酶液可破除阿拉伯木聚糖中阿拉伯糖支链空间位阻的限制,显著提升木聚糖酶水解效率,有助于阿拉伯低聚木糖的高效制备。
附图说明
图1为不同阿拉伯糖苷酶添加量对阿拉伯低聚木糖得率的影响。
具体实施方式
下面结合具体实施例对本发明做进一步详细说明,实施例将有助于理解本发明,但是本发明的保护范围不限于下述的实施例。
以下实施例中,不同分子量范围的车前子多糖的平均相对分子质量采用凝胶渗透色谱法(GPC)进行测定,具体的测试条件如下:色谱仪为安捷伦高效液相色谱仪1260;色谱柱为TSK-Gel G3000PWXL和TSK-Gel G5000PWXL双柱串联;检测器为示差检测器;流动相为去离子水;流速为0.6mL/min;柱温65℃;进样体积10.0μL,以不同分子量的普鲁兰多糖为标准样品。
以下实施例中,车前子壳和不同分子量的车前子多糖中的阿拉伯糖和木糖的摩尔质量比主要采用4%稀硫酸水解法将车前子壳和不同分子量的车前子多糖完全水解为木糖和阿拉伯糖,利用高效液相色谱仪测定稀硫酸水解液中的木糖和阿拉伯糖的浓度,具体的测试条件如下:色谱仪为安捷伦高效液相色谱仪1260;色谱柱为Bio-Rad Aminex HPX-87H;检测器为示差检测器;流动相为5mmol/L的硫酸溶液;流速为0.6mL/min;柱温55℃;进样体积10.0μL。阿拉伯糖与木糖的摩尔质量比计算公式如下:
式中:Ala:Xyl代表阿拉伯与木聚糖的摩尔质量比;CAla为酸水解液中阿拉伯糖的质量浓度,g/L;CXyl代表酸水解液中木糖的质量浓度,g/L;V为稀硫酸水解液的体积,L;150为阿拉伯糖和木糖的摩尔质量,g/mol。
以下实施例中,阿拉伯糖苷酶活力的测定方法:于15mL试管中加入0.1mL适当稀释的产酶上清液和0.9mL 1mmol/L pNPA溶液于50℃下保温10min,立即加入2.0mL 1mol/L的Na2CO3溶液终止反应,加入10mL蒸馏水,充分摇匀,于400nm下测定反应混合物的吸光度,并依据吸光度与对硝基苯酚的相关关系,计算反应生成的对硝基苯酚的浓度。1个阿拉伯糖苷酶活力单位(IU)以每分钟水解pNPA释放1μmol对硝基苯酚所需阿拉伯糖苷酶的酶量进行计算。
以下实施例中,木聚糖酶活力的测定方法:25mL刻度试管中加入0.9mL的1.0%的桦木木聚糖底物溶液,50℃预热5min,加入0.1mL适当稀释的产酶上清液,于50℃下反应30min,立即加入3.0mL DNS试剂终止反应,随后沸水浴中处理5min,冷却后定容到25mL,充分摇匀,于550nm下测定反应混合物的吸光度,并根据吸光度与还原糖的相关关系,计算反应生成的还原糖的浓度。1个木聚糖酶活力单位(IU)以每分钟水解底物产生1μmol木糖所需木聚糖酶的酶量进行计算。
实施例1:
不同分子量车前子多糖的制备方法,包括以下步骤:
(1)车前子壳经机械粉碎,过筛100目筛,按固液比(g/mL)1:20,于50℃的恒温摇床中170rpm下提取12h,取出后置于10000rpm下离心10min收集上清液,上清液即为车前子多糖混合溶液;
(2)取步骤(1)中得到的车前子多糖混合溶液,不断搅拌下加入无水乙醇,控制体系中乙醇浓度(体积浓度)为15%,将混合溶液经固液分离得到上清液Ι和沉淀,将沉淀于15%(体积浓度)的乙醇溶液中反复洗涤3次,冷冻干燥即为车前子多糖组分Ι,采用凝胶渗透色谱法测定车前子多糖组分Ι的平均分子质量,并利用稀硫酸水解和高效液相色谱法测定车前子多糖组分Ι的Ala:Xyl的摩尔质量比。
(3)取步骤(2)中的上清液Ι,在不断搅拌下加入无水乙醇,使体系中乙醇的浓度达到30%,将混合溶液经固液分离得到上清液П和沉淀,将沉淀于30%的乙醇溶液中反复洗涤3次,冷冻干燥即为车前子多糖组分П,采用凝胶渗透色谱法测定车前子多糖组分П的平均分子质量,并利用稀硫酸水解和高效液相色谱法测定车前子多糖组分П的Ala:Xyl的摩尔质量比;
(4)取步骤(3)中的上清液П,在不断搅拌下加入无水乙醇,使体系中乙醇的浓度达到45%,将混合溶液经固液分离得到上清液Ⅲ和沉淀,将沉淀于45%的乙醇溶液中反复洗涤3次,冷冻干燥即为车前子多糖组分Ⅲ,采用凝胶渗透色谱法测定车前子多糖组分Ⅲ的平均分子质量,并利用稀硫酸水解和高效液相色谱法测定车前子多糖组分Ⅲ的Ala:Xyl的摩尔质量比;
(5)取步骤(4)中的上清液Ⅲ,在不断搅拌下加入无水乙醇,是体系中乙醇的浓度达到60%,将混合溶液经固液分离得到上清液Ⅳ和沉淀,将沉淀于60%的乙醇溶液中反复洗涤3次,冷冻干燥即为车前子多糖组分Ⅳ,采用凝胶渗透色谱法测定车前子多糖组分Ⅳ的平均分子质量,并利用稀硫酸水解和高效液相色谱法测定车前子多糖组分Ⅳ的Ala:Xyl的摩尔质量比;
结果表明,车前子壳经热水提取可得到不同分子量范围的车前子多糖,采用乙醇分级沉淀法获得不同分子量范围的车前子多糖组分的平均分子质量为:组分Ι、组分П、组分Ⅲ和组分Ⅳ的平均相对分子质量分别为1.60×105Da、6.65×104Da、4.73×104Da和1.57×104Da;不同分子量范围的车前子多糖的Ala:Xyl的摩尔质量比0.26、0.22、0.19和0.17。
实施例2:
以车前子壳、不同分子量车前子多糖为诱导碳源的发酵产酶法,主要包括以下步骤:
(1)分别称取车前子壳、不同分子量的车前子多糖(实施例1制备)20.00g,葡萄糖1.00g,磷酸二氢钾20.00g,尿素3.00g,无水硫酸镁1.50g,硫酸铵14.00g,氯化钙4.00g,FeSO4·7H2O 5.00mg,MnSO4·7H2O 1.60mg,ZnSO4·7H2O 1.40mg,CoCl2 2.00mg,充分溶解于蒸馏水后,加入1mol/L柠檬酸缓冲液50mL,蒸馏水定容至1000mL,将产酶培养基分装后置于121℃灭菌15min。
(2)将里氏木霉孢子悬浮液(孢子数量为2.0×107cfu/mL)按10%的接种量与产酶培养基混合均匀,于170rpm摇床中培养,产酶温度第一天设定为30℃,第二天将温度调整至28℃。摇床培养5d后,将发酵液置于3000rpm离心30min,收集上清液,测定上清液中的阿拉伯糖苷酶和木聚糖酶活力。
结果见表1。
表1车前子壳和不同分子量车前子多糖为诱导碳源对酶活力的影响
碳源 | 分子量(Da) | Ala:Xyl | 木聚糖酶(IU/mL) | 阿拉伯糖苷酶(IU/mL) |
车前子壳 | / | 0.23 | 10.59 | 8.99 |
组分Ι | 1.60×105 | 0.26 | 4.93 | 10.27 |
组分П | 6.65×104 | 0.22 | 6.75 | 8.43 |
组分Ⅲ | 4.73×104 | 0.19 | 8.47 | 6.66 |
组分Ⅳ | 1.57×104 | 0.17 | 11.45 | 4.85 |
结果表明,里氏木霉分别以车前子壳和不同分子量的车前子多糖为底物诱导产酶,车前子多糖的分子量和Ala:Xyl摩尔质量比均对木聚糖酶和阿拉伯糖苷酶的活力产生影响。随着车前子多糖分子量的降低,产酶液中木聚糖酶活力呈现逐渐增加的趋势,而阿拉伯糖苷酶却呈现逐渐降低的趋势。此结果,表明木聚糖主链上的阿拉伯支链取代情况对于阿拉伯糖苷酶的诱导合成极为重要。
实施例3:
车前子壳与不同分子量车前子多糖混合后为诱导碳源的发酵产酶法,主要包括以下步骤:
(1)将车前子壳分别与实施例(1)中的组分Ι、组分П、组分Ⅲ和组分Ⅳ,按质量比为1:1,于机械粉碎机中充分粉碎均匀,分别记为混合Ι、混合П、混合Ⅲ和混合Ⅳ。
(2)分别称取混合Ι、混合П、混合Ⅲ和混合Ⅳ(步骤(1)中制备)20.00g,葡萄糖1.00g,磷酸二氢钾20.00g,尿素3.00g,无水硫酸镁1.50g,硫酸铵14.00g,氯化钙4.00g,FeSO4·7H2O 5.00mg,MnSO4·7H2O 1.60mg,ZnSO4·7H2O 1.40mg,CoCl2 2.00mg,充分溶解于蒸馏水后,加入1mol/L柠檬酸缓冲液50mL,蒸馏水定容至1000mL,将产酶培养基分装后置于121℃灭菌15min。
(3)将里氏木霉孢子悬浮液(孢子数量为2.0×107cfu/mL)按10%的接种量与步骤(2)中配制的产酶培养基混合均匀,于170rpm摇床中培养,产酶温度第一天设定为30℃,第二天将温度调整至28℃。摇床培养5d后,将发酵液置于3000rpm离心30min,收集上清液,测定上清液中的阿拉伯糖苷酶和木聚糖酶活力。
结果见表2。
表2车前子壳与不同分子量车前子多糖混合对酶活力的影响
碳源 | 木聚糖酶(IU/mL) | 阿拉伯糖苷酶(IU/mL) |
混合Ι | 8.47 | 9.22 |
混合П | 9.41 | 8.24 |
混合Ⅲ | 10.81 | 6.41 |
混合Ⅳ | 12.94 | 5.82 |
比较例1:
不同碳源对里氏木霉产木聚糖酶和阿拉伯糖苷酶的影响,包括以下步骤:
(1)分别称取木聚糖、纤维素、甘露聚糖和车前壳20.00g,葡萄糖1.00g,磷酸二氢钾20.00g,尿素3.00g,无水硫酸镁1.50g,硫酸铵14.00g,氯化钙4.00g,FeSO4·7H2O5.00mg,MnSO4·7H2O 1.60mg,ZnSO4·7H2O 1.40mg,CoCl2 2.00mg,充分溶解于蒸馏水后,加入1mol/L柠檬酸缓冲液50mL,蒸馏水定容至1000mL,将产酶培养基分装后置于121℃灭菌15min。
(2)将里氏木霉孢子悬浮液(孢子数量为2.0×107cfu/mL)按10%的接种量与步骤(1)中配制的产酶培养基混合均匀,于170rpm摇床中培养,产酶温度第一天设定为30℃,第二天将温度调整至28℃。摇床培养5d后,将发酵液置于3000rpm离心30min,收集上清液,测定上清液中的阿拉伯糖苷酶和木聚糖酶活力。
结果见表3。
表3不同结构碳源对里氏木霉产酶的影响
碳源 | 木聚糖酶(IU/mL) | 阿拉伯糖苷酶(IU/mL) |
木聚糖 | 3.40 | 0.45 |
纤维素 | 1.98 | 0.11 |
甘露聚糖 | 0.52 | 0.12 |
车前子壳 | 10.59 | 8.99 |
由比较例1的结果可知,里氏木霉以车前子壳为碳源诱导物,相较于以木聚糖酶、纤维素和甘露聚糖作为碳源诱导物,发酵产酶液中木聚糖酶和阿拉伯糖苷酶的酶活显著提高,尤其是对阿拉伯糖苷酶活力的诱导效果更好。结合实施例2、实施例3和比较例可知,以车前子可及不同分子量车前子多糖作为优质碳源均可实现木聚糖酶和阿拉伯糖苷酶的高效协同诱导合成。
比较例2:
为体现酶水解体系中阿拉伯糖苷酶的存在对阿拉伯低聚木糖的得率的影响,分别以比较例1中木聚糖和车前子壳为诱导碳源所获得的发酵液为产酶液(分别记为木聚糖产酶液和车前子壳产酶液),具体的操作步骤如下:
精确称取0.5000g车前子壳粉末,分别加入木聚糖产酶液(29.41mL)和车前子壳产酶液(9.44mL),控制木聚糖酶的添加量为200IU/g-车前子壳粉,此时木聚糖产酶液中阿拉伯糖苷酶总活力为12.23IU,车前子壳产酶液中阿拉伯糖苷酶总活力为84.87IU,分别加入pH5.0的1.00mol/L的柠檬酸缓冲液2.5mL,再次补加一定量的蒸馏水,使得酶水解体系的总体积为50mL。将上述混合体系置于150rpm摇床上,于50℃下进行酶水解,分别取12、24、36、48、60h时的酶水解混合液,于10000rpm下离心5min,收集上清液进行后续的处理。
分别取不同酶水解时间的上清液15mL,将其置于超滤管中(超滤管的截留分子量为3000Da),于5000rpm下离心30min,记录透过液的体积为V。将上述的透过液按体积比1:1与8%的稀硫酸溶液混合均匀后,置于121℃下酸水解60min,收集酸水解液,利用高效液相色谱法测定酸水解液和透过液中阿拉伯糖和木聚糖的浓度。阿拉伯低聚木糖得率按下式计算:
式中:C1-----酸水解液中阿拉伯糖浓度,g/L;
C2-----酸水解液中木糖浓度,g/L;
C3-----透过液中阿拉伯糖浓度,g/L;
C4-----透过液中木糖浓度,g/L;
V-----透过液体积,L;
0.5-----车前子壳粉末质量,g;
0.05-----酶水解液体积,L;
0.015-----用于超滤处理的酶水解上清液体积,L。
以木聚糖产酶液和车前子壳产酶液为阿拉伯木聚糖水解酶液,控制木聚糖酶添加量为200IU/g-车前子壳粉,探讨阿拉伯糖苷酶添加量对阿拉伯低聚木糖得率的影响,结果如图1所示。当酶水解时间为48h,以木聚糖产酶液和车前子壳产酶液为阿拉伯木聚糖水解酶,体系中阿拉伯低聚木糖得率分别为35.42%和55.21%。此结果表明,酶水解体系中阿拉伯糖苷酶的存在有助于提升阿拉伯低聚糖得率。
本发明提供了一种阿拉伯糖苷酶和木聚糖酶的同步诱导合成的思路及方法,具体实现该技术方案的方法和途径很多,以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。本实施例中未明确的各组成部分均可用现有技术加以实现。
Claims (6)
1.一种阿拉伯糖苷酶和木聚糖酶的同步诱导合成方法,其特征在于,以里氏木霉作为产酶菌种,利用车前子壳、车前子多糖、或车前子壳与车前子多糖的混合物为碳源诱导物在产酶培养基中进行发酵,实现阿拉伯糖苷酶和木聚糖酶的同步诱导合成;其中:
车前子多糖的分子量范围为104~105 Da;车前子壳与车前子多糖的混合物的质量比为1:9-9:1;
所述碳源诱导物的添加浓度为5-30 g/L;
所述产酶培养基,每1000 mL中包含葡萄糖 1.00 g、磷酸二氢钾20.00 g、尿素 3.00g、无水硫酸镁1.50 g、硫酸铵 14.00 g、氯化钙 4.00 g、FeSO4‧7H2O 5.00 mg、MnSO4‧7H2O1.60 mg、ZnSO4‧7H2O 1.40 mg和CoCl2 2.00 mg。
2.根据权利要求1所述的方法,其特征在于,所述车前子壳为平车前子或大车前子的种皮。
3.根据权利要求1所述的方法,其特征在于,所述车前子多糖为车前子壳经热水抽提和乙醇分级沉淀法制备得到的车前子多糖。
4.根据权利要求3所述的方法,其特征在于,所述乙醇分级沉淀使用体积浓度依次为15%、30%、45%、和60%的乙醇进行分级沉淀,将沉淀反复洗涤,经冷冻干燥即得到不同分子量区间的车前子多糖。
5.根据权利要求1所述的方法,其特征在于,所述车前子多糖通过如下方法制备得到:车前子壳经机械粉碎,过筛后,按固液比1:5-1:20,于30 ℃-50 ℃下提取10-100 min后,离心收集上清液,控制乙醇浓度进行分级沉淀,得到的沉淀经固液分离、洗涤、干燥,收集固形物,得到车前子多糖。
6.根据权利要求1所述的方法,其特征在于,发酵条件为:在28-30 ℃,150-170 rpm条件下于产酶培养基中发酵产酶,产酶时间控制在72-120 h,离心分离去除菌体,收集上清液。
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