CN112391425A - 一种协同水解槐豆胶的方法 - Google Patents

一种协同水解槐豆胶的方法 Download PDF

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CN112391425A
CN112391425A CN202011139582.3A CN202011139582A CN112391425A CN 112391425 A CN112391425 A CN 112391425A CN 202011139582 A CN202011139582 A CN 202011139582A CN 112391425 A CN112391425 A CN 112391425A
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宋亚囝
潘炳菊
申会涛
罗学刚
李中媛
张同存
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Tianjin University of Science and Technology
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Abstract

本发明涉及一种协同水解槐豆胶的方法，属于生物技术领域。本发明通过两种β‑甘露聚糖酶和一种α‑半乳糖苷酶协同水解半乳甘露聚糖底物槐豆胶制备甘露糖，半乳糖及甘露二糖，酶解过程条件温和，无污染物产生，且无毒、耗能低、产物安全性高，最终降解产物中甘露糖的转化生成率为19.4％、半乳糖的转化生成率为22.3％、甘露二糖的转化生成率为58.2％；且未检测到其他聚合度甘露寡糖。

Description

一种协同水解槐豆胶的方法
技术领域
本发明属于生物技术领域，涉及两种甘露聚糖酶及半乳糖苷酶协同水解槐豆胶的方法及在生产半乳糖、甘露糖及甘露二糖中的应用。
背景技术
槐豆胶是从豆科多年生植物——国槐种子中提取出来的一种多糖类聚合体。甘露聚糖是自然界中含量仅次于木聚糖的一类半纤维素。根据构成主链糖的种类不同以及有无侧链，天然的植物来源甘露聚糖可分为：甘露聚糖、葡甘露聚糖、半乳甘露聚糖和半乳葡甘露聚糖四类。槐豆胶为半乳甘露聚糖化合物，其主链是由甘露糖残基通过β-1,4糖苷键连接组成，侧链为通过α-1,6-糖苷键连接的半乳糖；其中甘露糖与半乳糖的比例大约为4:1。半乳甘露聚糖完全降解需要甘露聚糖酶、甘露糖苷酶、葡萄糖苷酶和半乳糖苷酶等的协同作用。利用不同甘露聚糖酶的底物偏好性及作用方式的不同，与半乳糖苷酶协同作用于槐豆胶，能够将底物降解的更为彻底。
甘露糖是一种具有重要功能的六碳糖，存在于植物的细胞中，在医药工业、食品工业、饲料工业和生命科学等的研究中具有重要的作用。研究表明甘露糖对脂多糖诱导的大鼠急性肺损伤具有保护作用、通过甘露糖受体抑制脂多糖诱导的小鼠巨噬细胞炎症；此外还可以作为原料合成L-核酸(抗艾滋病、抗病毒、抗癌药物的原料)。半乳糖常以半乳糖苷的形式存在于大脑和神经组织中，也是某些糖蛋白的重要组成成分。
甘露二糖属于甘露寡糖，研究表明甘露二糖能够抑制大肠杆菌对细胞的粘附。由于目前分离纯化技术尚存在欠缺，从多种聚合度寡糖混合物中分离纯化出高纯度的甘露二糖较为困难。本发明通过三种酶协同水解槐豆胶制备的寡糖成分仅含有甘露二糖，产物后期处理工艺经济简便。
本发明旨对酶水解槐豆胶制备甘露糖、半乳糖、甘露二糖提供新思路及方法。
发明内容
本发明目的在于克服现有技术中的不足之处，提供一种协同水解槐豆胶的方法。
本发明解决其技术问题所采用的技术方案是：
一种协同水解槐豆胶的方法，步骤如下：
(1)制备槐豆胶溶液；
(2)一次酶解：向槐豆胶溶液中加入α-半乳糖苷酶Gal27A，添加量为10-100U/g底物，搅拌均匀后，置30-40℃下反应1-10h；
(3)二次酶解：将(2)所得反应液加入β-甘露聚糖酶ManA，添加量为1×103-1×104U/g底物，搅拌均匀后，置30-40℃下反应1-10h；
(4)三次酶解：将(3)所得反应液加入β-甘露聚糖酶BaMan113A，添加量为10-100U/g底物，搅拌均匀后，置30-40℃下反应1-10h；
(5)将(4)所得反应液沸水浴加热10-20min，10,000-12,000rpm离心5-10min，收集上清液，即得半乳糖、甘露糖及甘露二糖混合物。
步骤(2)中α-半乳糖苷酶Gal27A氨基酸序列为SEQ ID NO:1所示。
步骤(3)中β-甘露聚糖酶ManA氨基酸序列为SEQ ID NO:2所示。
步骤(4)中β-甘露聚糖酶BaMan113A氨基酸序列为SEQ ID NO:3所示。
本发明取得的优点和积极效果为：
本发明方法首次利用三种酶协同水解半乳甘露聚糖底物槐豆胶制备甘露糖，半乳糖及甘露二糖，丰富了甘露糖，半乳糖及甘露二糖的来源，条件温和，酶解过程无污染物产生，且无毒、耗能低、产物安全性高，水解槐豆胶最终产物中甘露糖的生成率为19.4％、半乳糖的生成率为22.3％、甘露二糖的生成率为58.2％；且未检测到其他聚合度甘露寡糖。
附图说明
图1为本发明中半乳糖苷酶作用于槐豆胶的产物图；其中，G：半乳糖标品；M：甘露糖(M1)及甘露寡糖(M2-M6)混标；1：α-半乳糖苷酶Gal27A作用于槐豆胶。
图2为本发明中半乳糖苷酶及甘露聚糖酶A协同作用于槐豆胶的产物图；其中，G：半乳糖标品；M：甘露糖(M1)及甘露寡糖(M2-M6)混标；1：α-半乳糖苷酶Gal27A、β-甘露聚糖酶ManA依次作用于槐豆胶。
图3为本发明中半乳糖苷酶及甘露聚糖酶A、甘露聚糖酶B协同作用于槐豆胶的产物图；其中，G：半乳糖标品；M：甘露糖(M1)及甘露寡糖(M2-M6)混标；1：α-半乳糖苷酶Gal27A、β-甘露聚糖酶ManA、β-甘露聚糖酶BaMan113A依次作用于槐豆胶。
图4为本发明中离子色谱法对半乳糖进行定量的标曲图；
图5为本发明中离子色谱法对甘露糖进行定量的标曲图；
图6为本发明中离子色谱法对甘露二糖进行定量的标曲图；
图7为本发明中离子色谱法对甘露三糖进行定量的标曲图；
图8为本发明中甘露聚糖酶及半乳糖苷酶协同作用于槐豆胶底物最终产物的离子色谱图；图中M1：甘露糖；M2：甘露二糖；M3：甘露三糖；G：半乳糖；1:半乳糖标品；2：甘露糖及甘露寡糖标品；3：样品(α-半乳糖苷酶Gal27A、β-甘露聚糖酶ManA、β-甘露聚糖酶BaMan113A依次作用于槐豆胶)。
具体实施方式
下面详细叙述本发明的实施例，需要说明的是，本实施例是叙述性的，不是限定性的，不能以此限定本发明的保护范围。
本发明中所使用的原料，如无特殊说明，均为常规的市售产品；本发明中所使用的方法，如无特殊说明，均为本领域的常规方法。
具体操作及验证试验如下：
一、底物的配制：
称取0.5g槐豆胶少量多次加入100mL pH为7.0的柠檬酸缓冲液中，并不停搅拌，形成均匀的乳浊液，115℃灭菌15-20min，待冷却后于4℃保存备用。
二、半乳糖、甘露糖及甘露二糖的制备：
1、取pH为7.0的缓冲液配置的0.5％的槐豆胶底物，将800μL底物在37℃预热3-4min，然后加入0.12Uα-半乳糖苷酶Gal27A在37℃反应2h。
2、取900μL如1所述α-半乳糖苷酶Gal27A反应后溶液，在37℃预热3-4min，然后加入25.4Uβ-甘露聚糖酶ManA在37℃反应1h。
3、取800μL如2所述α-半乳糖苷酶Gal27A及β-甘露聚糖酶ManA反应后溶液，在37℃预热3-4min，然后加入0.2Uβ-甘露聚糖酶BaMan113A在37℃反应2h。
4、反应液沸水浴加热10min，10,000rpm离心10min，收集上清液，即得半乳糖、甘露糖及甘露二糖混合物。
三、甘露聚糖酶及半乳糖苷酶协同作用于槐豆胶底物最终产物的薄层色谱：
将100mm×100mm硅胶板于110℃恒温烘箱中活化30min，取适量反应后的半乳糖、甘露糖及甘露二糖混合物，用毛细管点样于硅胶板并吹干。提前30min配制好展开剂并倒入层析缸，将点样并吹干的硅胶板小心放入层析缸进行展板。展板结束后将硅胶板取出并吹干，在新鲜配制的显色剂中浸泡30s，在90℃恒温烘箱中加热10min左右至斑点清晰，用红外激光成像仪捕获图像。
四、甘露聚糖酶及半乳糖苷酶协同作用于槐豆胶底物最终产物的离子色谱：
1、离子色谱的样品的处理
样品用超滤管(3kDa)超滤离心，以去除样品中的蛋白质，上样前过0.22μm滤膜。离子色谱的操作条件如下：流动相组分：A：500mmol NaOH，B：去离子水；流动相条件：60％A+40％B等度洗脱；色谱柱：Dionex Cabropac PA 1(150mm×3mm，6μm)，Diono×PA1Guard(预柱，30mm×3mm，6μm)；检测器：GoldAg-AgCl，脉冲安培检测器；流速：1mL/min；柱温：30℃；进样量：25μL。
2、离子色谱标曲的绘制
用去离子水配置5mg/L，7.5mg/L，10mg/L，15mg/L，20mg/L，30mg/L浓度梯度的半乳糖水溶液，离子色谱进样，计算半乳糖峰积分面积，以积分面积为横坐标，半乳糖浓度为纵坐标，绘制标准曲线如图4。同上步骤绘制甘露糖、甘露二糖、甘露三糖标准曲线，如图分别为图5、6、7。
3、水解产物分析
根据对α-半乳糖苷酶Gal27A与β-甘露聚糖酶ManA、β-甘露聚糖酶BaMan113A的添加量及反应条件，在反应温度37℃、反应pH 7.0、条件下制备水解产物。取800μL的已灭菌底物中加入0.12Uα-半乳糖苷酶Gal27A酶液反应2h，反应结束后加25.38Uβ-甘露聚糖酶ManA酶液反应1h，反应结束后再加0.21Uβ-甘露聚糖酶BaMan113A酶液反应2h，将水解产物用去离子水稀释50倍后用离子色谱对水解产物进行分析，对产物定量分析结果如表1所示。
甘露糖的生成率是指水解释放出的甘露糖与半乳糖，甘露糖及甘露二糖的比值，计算公式如下:
Figure BDA0002737845330000041
半乳糖的生成率是指水解释放出的半乳糖与甘露糖，半乳糖及甘露二糖的比值，计算公式如下:
Figure BDA0002737845330000042
甘露二糖的生成率是指水解释放出的甘露二糖与半乳糖，甘露糖及甘露二糖的比值，计算公式如下:
Figure BDA0002737845330000051
因此水解槐豆胶最终产物中甘露糖的生成率为19.4％、半乳糖的生成率为22.3％、甘露二糖的生成率为58.2％；且未检测到其他聚合度甘露寡糖。
表1槐豆胶水解产物组分分析
Figure BDA0002737845330000052
注：nd表示未检测到该成分
SEQ ID NO:1
mtlkpkryaeglaktppmgwnsfntfgcepteelikqsadvmvksglleagyryiniddgwmaderdsagnlvpdpqkfpngmkpvtdyihekgllagtylgcgqktygekpgslgyeerdaqliadqgfdllkydyrelpgdpigrgvkedyvtmrnalmktgrdmvfsicehgkshpetwaqeighmwrttpdikdsfdedinwgwsinhiidethalhhyagpggwndpdmlvvginglndwlgpgctyneykshfslwcllaaplligcdirkmseetktillnkemiainqdplgiqghllkkehgidywvkplanndiaigllnrfnepkeavlslsdlledgnylmkdvwtnerkelqsewisktlkshecavfrlmsknke
SEQ ID NO:2
Mkkklsqiyhliictliisvgimgittspseassgfyvdgntlydangqpfvmkginhghawykdtastaipaiaeqgantirivlsdggqwekddidtvrevielaeqnkmvavvevhdatgrdsrsdldravdywiemkdaligkedtviinianewygswdgaawadgyidvipklrdaglthtlmvdaagwgqypqsihdygqdvfnadplkntifsihmyeyaggdantvrsnidrvidqdlalvigefghrhtdgdvdedtilsyseetgtgwlawswkgnsaewdyldlsedwagnhltdwgnrivhganglqetskpstvftddnggapeppttttlydfegstqgwhgsnvmggpwsvtewgasgnyslkgdvnlssnsshelyseqsrnlhgysqlnatvrhanwgnpgngmnarlyvktgsdytwysgpftrinssnsgttlsfdlnnienshhvreigvqfsaadnssgqtalyvdnvtlr
SEQ ID NO:3
mdyikgmtwgwignsedwrsneaersmeemtnlainwtaiafqglqetahspditfaeppmvtdenvrwaiakakslglsvilkpivnvrdgtwrahinffdkdvpceptwsqwfksyesfmlhyaklaedtgcemlcigcemvqterrekewrdliqkvrqvysgiityncdkyqedevtwwdavdvmsssgyypigswehhesrikkiveswqkpfffmeagcpsrlesgsvpndwnknrgqidmdeqrvfyeemfkffhgqkwfygfmlwdwpaklyrledasenddycvygkpaaeviksfftsnkiakr
尽管为说明目的公开了本发明的实施例，但是本领域的技术人员可以理解：在不脱离本发明及所附权利要求的精神和范围内，各种替换、变化和修改都是可能的，因此，本发明的范围不局限于实施例和附图所公开的内容。
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Gly Ser Trp Asp Gly Ala Ala Trp Ala Asp Gly Tyr Ile Asp Val Ile
165 170 175
Pro Lys Leu Arg Asp Ala Gly Leu Thr His Thr Leu Met Val Asp Ala
180 185 190
Ala Gly Trp Gly Gln Tyr Pro Gln Ser Ile His Asp Tyr Gly Gln Asp
195 200 205
Val Phe Asn Ala Asp Pro Leu Lys Asn Thr Ile Phe Ser Ile His Met
210 215 220
Tyr Glu Tyr Ala Gly Gly Asp Ala Asn Thr Val Arg Ser Asn Ile Asp
225 230 235 240
Arg Val Ile Asp Gln Asp Leu Ala Leu Val Ile Gly Glu Phe Gly His
245 250 255
Arg His Thr Asp Gly Asp Val Asp Glu Asp Thr Ile Leu Ser Tyr Ser
260 265 270
Glu Glu Thr Gly Thr Gly Trp Leu Ala Trp Ser Trp Lys Gly Asn Ser
275 280 285
Ala Glu Trp Asp Tyr Leu Asp Leu Ser Glu Asp Trp Ala Gly Asn His
290 295 300
Leu Thr Asp Trp Gly Asn Arg Ile Val His Gly Ala Asn Gly Leu Gln
305 310 315 320
Glu Thr Ser Lys Pro Ser Thr Val Phe Thr Asp Asp Asn Gly Gly Ala
325 330 335
Pro Glu Pro Pro Thr Thr Thr Thr Leu Tyr Asp Phe Glu Gly Ser Thr
340 345 350
Gln Gly Trp His Gly Ser Asn Val Met Gly Gly Pro Trp Ser Val Thr
355 360 365
Glu Trp Gly Ala Ser Gly Asn Tyr Ser Leu Lys Gly Asp Val Asn Leu
370 375 380
Ser Ser Asn Ser Ser His Glu Leu Tyr Ser Glu Gln Ser Arg Asn Leu
385 390 395 400
His Gly Tyr Ser Gln Leu Asn Ala Thr Val Arg His Ala Asn Trp Gly
405 410 415
Asn Pro Gly Asn Gly Met Asn Ala Arg Leu Tyr Val Lys Thr Gly Ser
420 425 430
Asp Tyr Thr Trp Tyr Ser Gly Pro Phe Thr Arg Ile Asn Ser Ser Asn
435 440 445
Ser Gly Thr Thr Leu Ser Phe Asp Leu Asn Asn Ile Glu Asn Ser His
450 455 460
His Val Arg Glu Ile Gly Val Gln Phe Ser Ala Ala Asp Asn Ser Ser
465 470 475 480
Gly Gln Thr Ala Leu Tyr Val Asp Asn Val Thr Leu Arg
485 490
<210> 3
<211> 314
<212> PRT
<213> Bacillus alcalophilus
<400> 3
Met Asp Tyr Ile Lys Gly Met Thr Trp Gly Trp Ile Gly Asn Ser Glu
1 5 10 15
Asp Trp Arg Ser Asn Glu Ala Glu Arg Ser Met Glu Glu Met Thr Asn
20 25 30
Leu Ala Ile Asn Trp Thr Ala Ile Ala Phe Gln Gly Leu Gln Glu Thr
35 40 45
Ala His Ser Pro Asp Ile Thr Phe Ala Glu Pro Pro Met Val Thr Asp
50 55 60
Glu Asn Val Arg Trp Ala Ile Ala Lys Ala Lys Ser Leu Gly Leu Ser
65 70 75 80
Val Ile Leu Lys Pro Ile Val Asn Val Arg Asp Gly Thr Trp Arg Ala
85 90 95
His Ile Asn Phe Phe Asp Lys Asp Val Pro Cys Glu Pro Thr Trp Ser
100 105 110
Gln Trp Phe Lys Ser Tyr Glu Ser Phe Met Leu His Tyr Ala Lys Leu
115 120 125
Ala Glu Asp Thr Gly Cys Glu Met Leu Cys Ile Gly Cys Glu Met Val
130 135 140
Gln Thr Glu Arg Arg Glu Lys Glu Trp Arg Asp Leu Ile Gln Lys Val
145 150 155 160
Arg Gln Val Tyr Ser Gly Ile Ile Thr Tyr Asn Cys Asp Lys Tyr Gln
165 170 175
Glu Asp Glu Val Thr Trp Trp Asp Ala Val Asp Val Met Ser Ser Ser
180 185 190
Gly Tyr Tyr Pro Ile Gly Ser Trp Glu His His Glu Ser Arg Ile Lys
195 200 205
Lys Ile Val Glu Ser Trp Gln Lys Pro Phe Phe Phe Met Glu Ala Gly
210 215 220
Cys Pro Ser Arg Leu Glu Ser Gly Ser Val Pro Asn Asp Trp Asn Lys
225 230 235 240
Asn Arg Gly Gln Ile Asp Met Asp Glu Gln Arg Val Phe Tyr Glu Glu
245 250 255
Met Phe Lys Phe Phe His Gly Gln Lys Trp Phe Tyr Gly Phe Met Leu
260 265 270
Trp Asp Trp Pro Ala Lys Leu Tyr Arg Leu Glu Asp Ala Ser Glu Asn
275 280 285
Asp Asp Tyr Cys Val Tyr Gly Lys Pro Ala Ala Glu Val Ile Lys Ser
290 295 300
Phe Phe Thr Ser Asn Lys Ile Ala Lys Arg
305 310

Claims (6)

1.一种协同水解槐豆胶的方法，其特征在于：向槐豆胶溶液中依次添加α-半乳糖苷酶Gal27A、β-甘露聚糖酶ManA和β-甘露聚糖酶BaMan113A进行水解反应。
2.如权利要求1所述的一种协同水解槐豆胶的方法，其特征在于步骤如下：
(1)制备槐豆胶溶液；
(2)一次酶解：向槐豆胶溶液中加入α-半乳糖苷酶Gal27A，添加量为10-100U/g底物，搅拌均匀后，置30-40℃下反应1-10h；
(3)二次酶解：将(2)所得反应液加入β-甘露聚糖酶ManA，添加量为1×103-1×104U/g底物，搅拌均匀后，置30-40℃下反应1-10h；
(4)三次酶解：将(3)所得反应液加入β-甘露聚糖酶BaMan113A，添加量为10-100U/g底物，搅拌均匀后，置30-40℃下反应1-10h；
(5)将(4)所得反应液沸水浴加热，离心，收集上清液，即得半乳糖、甘露糖及甘露二糖混合物。
3.根据权利要求2所述的一种协同水解槐豆胶的方法，其特征在于：步骤(2)中α-半乳糖苷酶Gal27A氨基酸序列为SEQ ID NO:1所示。
4.根据权利要求2所述的一种协同水解槐豆胶的方法，其特征在于：步骤(3)中β-甘露聚糖酶ManA氨基酸序列为SEQ ID NO:2所示。
5.根据权利要求2所述的一种协同水解槐豆胶的方法，其特征在于：步骤(4)中β-甘露聚糖酶BaMan113A氨基酸序列为SEQ ID NO:3所示。
6.权利要求1所述的方法在生产半乳糖、甘露糖、甘露二糖中的应用。
CN202011139582.3A 2020-10-22 2020-10-22 一种协同水解槐豆胶的方法 Pending CN112391425A (zh)

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CN109055333A (zh) * 2018-07-26 2018-12-21 天津科技大学 一种糖苷水解酶及其复合酶在半乳甘露聚糖降解中的应用
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CN109097348A (zh) * 2018-07-26 2018-12-28 天津科技大学 α-半乳糖苷酶及其复合酶在半乳甘露聚糖降解中的应用
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113151025A (zh) * 2021-03-12 2021-07-23 上海国龙生物科技有限公司 一种融合表达β-甘露聚糖酶和α-半乳糖苷酶的毕赤酵母、制备方法及其应用

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