CN110499348B - 一种酶法改性淀粉制备dp值15-30的直链糊精的方法 - Google Patents
一种酶法改性淀粉制备dp值15-30的直链糊精的方法 Download PDFInfo
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- CN110499348B CN110499348B CN201910893818.3A CN201910893818A CN110499348B CN 110499348 B CN110499348 B CN 110499348B CN 201910893818 A CN201910893818 A CN 201910893818A CN 110499348 B CN110499348 B CN 110499348B
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Abstract
本发明公开了一种酶法改性淀粉制备DP值15‑30的直链糊精的方法,属于淀粉改性技术领域。本发明提供了一种改性淀粉从而提高聚合度在15‑30的直链糊精产率的方法,该方法通过从热变形菌(Thermoproteus uzoniensis)中克隆到一株4‑α‑糖基转移酶基因(TuαGT),通过大肠杆菌异源表达,酶学性质表征,确定其为4αGT,并且该酶具有较好的温度稳定性和底物耐受性,使其能够在高温下催化高浓度的淀粉改性,延长其支链淀粉的侧链。
Description
技术领域
本发明涉及一种酶法改性淀粉制备DP值15-30的直链糊精的方法,属于淀粉改性技术领域。
背景技术
淀粉是许多植物主要的能量储存方式,淀粉在人类生产生活中扮演重要角色,不仅是人类能量的重要来源之一,而且因其低廉的价格,还是许多工业生产商非常重要的原材料,例如食品业,纺织业等。不同淀粉的性质特征各有不同,因此可以生产出多种多样的淀粉基食品。然而,原淀粉不溶于冷水、分散性差、糊化后易回生导致产品出现明显的粉感等缺点,在实际生产应用中受到了很大的限制。
在淀粉众多的衍生物中,糊精的应用相当广泛。直链糊精是糊精中较为特殊的一种,是由数个或数十个葡萄糖单元通过α-1,4-糖苷键连接而成,其分子量小,因而具有较好的水溶性。与直链淀粉结构相似,直链糊精具有螺旋空间结构,具有良好的包埋特性,可包埋不同的客体,对包埋的客体起到保护或缓释的效果,可以添加到一些具有香味的食品或饮料中,使香味更长久。此外,直链糊精可作为食品添加物应用于低脂低热量食品中,添加直链糊精的食品具有良好的流变性、稳定性和口感。
据研究表明,直链糊精的聚合度大小及分布是影响其性质与功能的关键因素。当聚合度过低,小于10个糖基单元,直链糊精溶解性较好,但是形成的螺旋结构较短,对一些较长的客体物质的包埋效果较差。但是当聚合度过长,大于30个糖基单元,容易出现直链糊精间分子链的重排,形成氢键,反而影响其包埋效果。因此,聚合度在10~30的直链糊精,可以在维持较好溶解性的情况下,又保留了其包埋特性,是良好的工业原料。
目前,常见的直链糊精生产方式主要有两种:
(1)水解法:使用淀粉为底物,将淀粉经普鲁兰酶脱支后,通过包括色谱法、膜滤法和醇逐步沉淀法在内的分级方法来实现糊精的分级以得到直链糊精。
(2)合成法:以腺苷二磷酸葡萄糖(ADPGlc)或尿苷二磷酸葡萄糖(ADPGlc)为底物,利用淀粉合成酶或者蔗糖磷酸化酶等酶法合成直链糊精。
目前经上述方法制备得到的直链糊精存在两大问题:(1)聚合度分布广,并无法获得较窄聚合度分布的直链糊精;(2)聚合度分布窄,但是分离效率低且分离成本较高。因此,提高聚合度分布窄的直链糊精的产率具有重要的意义。
发明内容
为了解决上述存在的技术问题,本发明提供了一种改性淀粉从而提高聚合度在15~30的直链糊精产率的方法,该方法通过从热变形菌(Thermoproteus uzoniensis)中克隆到一株4-α-糖基转移酶基因(TuαGT),通过大肠杆菌异源表达,酶学性质表征,确定其为4αGT,并且该酶具有较好的温度稳定性和底物耐受性,使其能够在高温下催化高浓度的淀粉改性,延长其支链淀粉的侧链。本发明主要采取了三个关键方面的控制来实现高强度抗回生淀粉基凝胶的制备:1)淀粉种类的筛选;2)优化4-α-糖基转移酶的添加量;3)优化4-α-糖基转移酶的作用时间;4)优化醇逐步沉淀法的醇水比。
本发明的第一个目的是提供一种改性淀粉制备DP值15-30的直链糊精的方法,包括如下步骤:
(1)将淀粉溶于缓冲液中进行调浆,在70-120℃条件下完全糊化;
(2)将糊化温度降至40-90℃,pH调至4.0-13.0,添加氨基酸序列如SEQ ID NO.1所示的4-α-糖基转移酶,反应1-12h,灭酶;
(3)将灭酶温度降至40-90℃,pH调至4.0-13.0,添加普鲁兰酶,反应1-12h,灭酶,干燥;
(4)采用醇逐步沉淀法对直链糊精进行分级。
在本发明一种实施方式中,酶解作用时间为1-12h。
在本发明一种实施方式中,步骤(1)中所述淀粉的质量浓度是10%~40%。
在本发明一种实施方式中,步骤(1)中所述淀粉主要包括马铃薯淀粉、玉米淀粉、木薯淀粉、小麦淀粉、豌豆淀粉和大米淀粉。
在本发明一种实施方式中,优选地,所述淀粉为木薯淀粉。
在本发明一种实施方式中,步骤(2)中所述4-α-糖基转移酶添加量为0.5~10U/g淀粉。
在本发明一种实施方式中,优选地,4-α-糖基转移酶添加量为2~10U/g淀粉。
在本发明一种实施方式中,优选地,4-α-糖基转移酶作用时间为4-12h。
在本发明一种实施方式中,步骤(3)中所述灭酶方法包括高温灭酶,酸碱灭酶,乙醇灭酶。
在本发明一种实施方式中,步骤(3)中所述普鲁兰酶的添加量为50~200U/g。
在本发明一种实施方式中,步骤(3)中所述普鲁兰酶的最适反应温度为40~70℃,最适反应pH为3.0~8.0。
在本发明一种实施方式中,步骤(3)中所述灭酶方法包括高温灭酶,酸碱灭酶,乙醇灭酶。
在本发明一种实施方式中,步骤(3)中所述的干燥方法包括冷冻干燥、常压干燥、喷雾干燥、滚筒干燥、微波干燥。
在本发明一种实施方式中,步骤(4)中所述醇逐步沉淀法为乙醇沉淀。
在本发明一种实施方式中,步骤(4)中所述醇逐步沉淀法的醇水比为(1-3):(1-5)。
在本发明一种实施方式中,优选地,醇水比为1:(1-5)。
在本发明一种实施方式中,步骤(4)中所述醇逐步沉淀法的干燥方法包括烘干和真空冷冻干燥;
在本发明一种实施方式中,步骤(4)中所述醇逐步沉淀法的pH为4.0~8.0。
本发明的第二个目的是提供一种应用上述方法制备得到的直链糊精。
本发明第三个目的是提供一种上述直链糊精作为食品添加剂中的应用。
本发明的有益效果:
(1)4-α-糖基转移酶的来源为Thermoproteus uzoniensis。通过序列比对发现该酶序列与其他4-α-糖基转移酶序列最高相似度仅为70%;4-α-糖基转移酶的最适反应温度为50~80℃,最适反应pH为5.0~8.0;4-α-糖基转移酶的最小作用底物单元为麦芽糖,对葡萄糖等单糖并无活力。
(2)本发明的4-α-糖基转移酶,在65℃-80℃之间,该酶的相对酶活均保持在90%以上,具有较好的温度稳定性;可以在高温60-90℃下催化淀粉改性,淀粉转化率高达80%以上;可以催化10~30%(w/v)高浓度的淀粉改性,淀粉转化率高达90%以上。
(3)与现有技术相比,本发明得到的直链糊精中DP值在15-30的产率最高可达到63%。
附图说明
图1:4-α-糖基转移酶镍柱纯化结果。
图2:酶的最适反应温度。
图3:酶的最适反应pH。
图4:4-α-糖基转移酶改性淀粉侧链分布。其中,黑色为原木薯淀粉,灰色为改性木薯淀粉。
具体实施方式
以下对本发明的优选实施例进行说明,应当理解实施例是为了更好地解释本发明,不用于限制本发明。
(1)4αGTase酶活测定方法
取100μL适当稀释的酶液加入1%麦芽三糖(G3)溶液(50mM柠檬酸钠缓冲液配制,pH6.0),75℃反应6h。沸水浴10min停止反应。采用葡萄糖氧化酶试剂盒法测定反应液中的葡萄糖含量。歧化活力定义为每分钟产生1μmol葡萄糖所需的酶的量。
酶活单位定义为:每分钟产生1μmol葡萄糖所需的酶的量。
(2)淀粉转化率的计算公式:
式中:X为淀粉转化率;c0为原淀粉中直链淀粉含量;c1为酶改性后淀粉中直链淀粉含量。
(3)聚合度的测定方法:
使用阴离子色谱仪测定淀粉的聚合度分布。配制2mg/ml的淀粉溶液(50mM醋酸钠缓冲液,pH4.5),沸水浴30min。降温至42℃,加入2U的异淀粉酶,反应过夜(12h)。待反应结束后,沸水浴5min,5000rpm离心2min,取上清液4℃保存备用。流动相NaOH/NaAC;流速0.5mL/min;柱温30℃;色谱柱型号PA-200;上样量25μL。
实施例1:酶的制备
人工合成核苷酸序列如SEQ ID NO.2所示(氨基酸序列如SEQ ID NO.1所示)的基因片段,选用pET-28a质粒构建重组质粒,利用BL21表达菌株表达,经摇瓶发酵,离心沉淀,超声破壁得到粗酶,经过镍柱纯化最终获得4αGTase纯酶(见图1)。
MLRGAGILLPLFSLPGPHGIGDMGPAAYRFVEFLRDAGQTYWQLLPLNPVLPEYDNSPYSSTSSFAGEPAYISLELLAEQGVLRRSASAEFGAGRADYEAARAYKVKIIAEAERPKDLDEFVGRNEWLEDYALYSALREYFGRPWVEWPRELRDRDPAALRQWREKLRRRIELEYLLQYFFWTQWRELKRHANSLGIFLIGDLPIYLSLDSADVWAHRELFKLDAEGRPLYVAGVPPDYFSPTGQLWGNPVYDWDAMRKEGFRWWLDRLRYTLSVFDYVRLDHFRGYAAYWEVPAGEKTAVNGRWVPAPGGELLERARDELGGLKIIAEDLGYITPDVVELRDRFGLPGMRVLQFAWDGNPANEHKPHNHVKNSVVYTGTHDNNTIVGWFFREASPKARREALEYMGRRSAKDIHWAFIRLAYFSVADVAVVPMQDFMGLGPEARINRPGTRGGNWVWRMTELPGRALARRIRRLARLYGR
实施例2:酶的性质
(1)酶的最适反应温度
在不同温度下(50℃-90℃)测定4αGTase歧化活力以确定最适温度。4-α-糖基转移酶的最适反应温度为50~80℃。
表1 4αGTase最适反应温度
(2)酶的最适反应pH
用不同pH值(3.0-10.0)的缓冲液代替4αGTase歧化测定方法中的缓冲液,在75℃下测定4αGTase活力,考察酶的最适pH。最适反应pH为5.0~8.0。
表2 4αGTase最适pH的测定
(3)酶的温度稳定性
在不同温度(50℃-90℃)保温,每1h将部分酶液取出,迅速冷却,测定残留总酶活。结果表明:温度在65℃-80℃之间,该酶的相对酶活均保持在90%以上。
表3温度稳定性测定
实施例3:淀粉种类对直链糊精产率的影响
(1)将淀粉溶于缓冲液中进行调浆,在120℃条件下完全糊化。
(2)将温度降至70℃,pH调至6.0,添加4-α-糖基转移酶,反应6h,灭酶。
(3)将温度降至70℃,pH调至6.0,添加普鲁兰酶,反应6h,灭酶,干燥。
(4)采用醇逐步沉淀法对直链糊精进行分级;所述淀粉主要包括马铃薯淀粉、玉米淀粉、木薯淀粉、小麦淀粉、豌豆淀粉和大米淀粉。
通过测定改性淀粉中直链淀粉的含量确定改性淀粉的转化率,采用阴离子色谱仪对直链糊精聚合度进行测定,结果见表4。其中,木薯淀粉的DP值15~30的直链糊精产率最高,为39.5%。
表4淀粉种类对直链糊精产率的影响
实施例4:淀粉浓度对直链糊精产率的影响
(1)将淀粉溶于缓冲液中进行调浆,在120℃条件下完全糊化。
(2)将温度降至70℃,pH调至6.0,添加4-α-糖基转移酶6U/g淀粉,反应6h,灭酶。
(3)将温度降至70℃,pH调至6.0,添加普鲁兰酶,反应6h,灭酶,干燥。
(4)采用醇逐步沉淀法对直链糊精进行分级;所述淀粉浓度为10%~30%。
通过测定改性淀粉中直链淀粉的含量确定改性淀粉的转化率,采用阴离子色谱仪对直链糊精聚合度进行测定,结果见表5。
表5淀粉浓度对直链糊精产率的影响
底物浓度(%) | 10% | 20% | 30% |
转化率(%) | 95.4% | 93.7% | 92.9% |
DP值15~30的直链糊精产率(%) | 38.71 | 34.92 | 35.26 |
实施例5:4-α-糖基转移酶的添加量对直链糊精产率的影响
(1)将淀粉溶于缓冲液中进行调浆,在120℃条件下完全糊化。
(2)将温度降至70℃,pH调至6.0,添加4-α-糖基转移酶,反应6h,灭酶。
(3)将温度降至70℃,pH调至6.0,添加普鲁兰酶,反应6h,灭酶,干燥。
(4)采用醇逐步沉淀法对直链糊精进行分级;4-α-糖基转移酶添加量分别为0.5~10U/g淀粉。
通过测定改性淀粉中直链淀粉的含量确定改性淀粉的转化率,采用阴离子色谱仪对直链糊精聚合度进行测定,结果见表6。优选地,4-α-糖基转移酶添加量为2~10U/g淀粉。
表6 4-α-糖基转移酶添加量对直链糊精产率的影响
4-α-糖基转移酶添加量(U/g) | 0.5 | 1 | 2 | 4 | 6 | 8 | 10 |
转化率(%) | 40.68 | 60.81 | 84.36 | 92.61 | 92.79 | 92.98 | 93.15 |
DP值15~30的直链糊精产率(%) | 16.16 | 23.91 | 33.32 | 36.58 | 36.65 | 36.72 | 36.79 |
实施例6:4-α-糖基转移酶的作用时间对直链糊精产率的影响
(1)将淀粉溶于缓冲液中进行调浆,在120℃条件下完全糊化。
(2)将温度降至70℃,pH调至6.0,添加4-α-糖基转移酶6U/g淀粉,灭酶。
(3)将温度降至70℃,pH调至6.0,添加普鲁兰酶,反应6h,灭酶,干燥。
(4)采用醇逐步沉淀法对直链糊精进行分级;4-α-糖基转移酶作用时间分别为0.5-12h。
通过测定改性淀粉中直链淀粉的含量确定改性淀粉的转化率,采用阴离子色谱仪对直链糊精聚合度进行测定,结果见表7。优选地,4-α-糖基转移酶作用时间为4-12h。
表7 4-α-糖基转移酶作用时间对直链糊精产率的影响
4-α-糖基转移酶作用时间(h) | 0.5 | 1 | 2 | 4 | 6 | 8 | 12 |
转化率(%) | 54.48 | 63.12 | 74.54 | 92.21 | 93.51 | 93.62 | 93.88 |
DP值15~30的直链糊精产率(%) | 21.52 | 24.93 | 29.44 | 36.43 | 36.94 | 36.98 | 37.08 |
实施例7:醇水比对淀粉基凝胶性能的影响
(1)将淀粉溶于缓冲液中进行调浆,在70-120℃条件下完全糊化。
(2)将温度降至40-90℃,pH调至4.0-13.0,添加4-α-糖基转移酶,反应1-12h,灭酶。
(3)将温度降至40-90℃,pH调至4.0-13.0,添加普鲁兰酶,反应1-12h,灭酶,干燥。
(4)采用醇逐步沉淀法对直链糊精进行分级;醇水比分别为1:5、1:2、1:1、2:1、3:1。
通过测定改性淀粉中直链淀粉的含量确定改性淀粉的转化率,采用阴离子色谱仪对直链糊精聚合度进行测定,结果见表8。优选地,醇水比为1:(1-5)。
表8醇水比对直链糊精产率的影响
醇水比 | 1:5 | 1:2 | 1:1 | 2:1 | 3:1 |
DP值15~30的直链糊精产率(%) | 63.24 | 62.77 | 39.98 | 14.85 | 12.71 |
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 江南大学
<120> 一种酶法改性淀粉制备DP值15-30的直链糊精的方法
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<170> PatentIn version 3.3
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Claims (5)
1.一种改性淀粉制备DP值15-30的直链糊精的方法,其特征在于,包括如下步骤:
(1)将淀粉溶于缓冲液中进行调浆,在70-120℃条件下完全糊化;
(2)将糊化温度降至50-80℃,pH调至5.0-8.0,添加氨基酸序列如SEQ ID NO.1所示的4-α-糖基转移酶,灭酶;
(3)将步骤(2)温度降至40-90℃,pH调至4.0-13.0,添加普鲁兰酶,反应1-12h,灭酶,干燥;
(4)采用醇逐步沉淀法对直链糊精进行分级;
所述淀粉为木薯淀粉;
步骤(2)中4-α-糖基转移酶添加量为2~10U/g淀粉,反应4-12h;
步骤(4)中所述醇逐步沉淀法为乙醇沉淀;所述醇逐步沉淀法的pH为4.0~8.0;所述醇逐步沉淀法的醇水比为1:(1-5)。。
2.根据权利要求1所述的方法,其特征在于,步骤(1)中所述淀粉的质量浓度是10%~40%。
3.根据权利要求1所述的方法,其特征在于,步骤(2)和步骤(3)中所述灭酶方法包括高温灭酶,酸碱灭酶,乙醇灭酶。
4.根据权利要求1所述的方法,其特征在于,步骤(3)中所述普鲁兰酶的添加量为50~200U/g;所述普鲁兰酶的反应温度为40~70℃,反应pH为3.0~8.0。
5.根据权利要求1所述的方法,其特征在于,步骤(3)中所述的干燥方法包括冷冻干燥、常压干燥、喷雾干燥、滚筒干燥、微波干燥。
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