CN116574199B - 一种木聚糖酶底物及其制备方法 - Google Patents
一种木聚糖酶底物及其制备方法 Download PDFInfo
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- CN116574199B CN116574199B CN202310632155.6A CN202310632155A CN116574199B CN 116574199 B CN116574199 B CN 116574199B CN 202310632155 A CN202310632155 A CN 202310632155A CN 116574199 B CN116574199 B CN 116574199B
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- 229920001221 xylan Polymers 0.000 claims abstract description 58
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Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- Health & Medical Sciences (AREA)
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- General Health & Medical Sciences (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Sustainable Development (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明采用乙醇分级沉淀法从制浆废液中获得一种木聚糖,该木聚糖命名为“XYL”,硫酸苯酚法测得其总糖含量为95.3%,单糖组成分析显示,XYL含有97.1%的木糖,微量的葡萄糖(1.2%)、半乳糖(0.7%)和半乳糖醛酸(1.0%),甲基化分析发现糖残基以单一的1‑4糖苷键连接,结合一维和二维核磁共振图谱分析,鉴定该木聚糖为纯度高、结构单一、线性的β‑D‑1→4吡喃木聚糖。通过米氏方程评价了其木聚糖酶的底物亲和力,结果显示,以XYL为底物测得米氏方程的Km最低(数值为1.49mg/ml),说明该木聚糖与木聚糖酶具有较强的亲和力,是木聚糖酶比较适合的底物,该木聚糖具备成为一种商用的木聚糖酶标准底物的潜力。
Description
技术领域
本发明涉及木聚糖研究技术领域,尤其涉及一种木聚糖酶底物及其制备方法。
背景技术
“绿色化学”最有趣的研究目标之一是将垃圾转化为有价值的材料。由于环保解决方案的快速发展,纸浆和造纸工业废水中的废植物成分最近引起了越来越多的关注。半纤维素是地球上仅次于纤维素的第二丰富的多糖,它们共同构成了纸浆和造纸厂原料中最关键的成分(50~60%的纤维素和20~30%的半纤维素)。在全球年制浆能力接近2亿吨的巨大产能下,除纤维素外的大部分植物纤维原料溶解到废液中没有得到有效利用,使处理成为一个重大问题。其中,半纤维素就是一个没能有效利用的资源,目前,人们正在作出更多努力来利用这些组分,这对回收废物生物质资源和减少污染排放至关重要。
木聚糖在半纤维素中最为丰富,是综纤维素结构的第二大主要成分,具有容易获得、可生物降解和可能的生物活性等特点,在食品、制药、化学和化妆品工业中被广泛应用。根据分子结构分类,木聚糖可分为同聚糖和杂聚糖,植物中的木聚糖大多是具有多种结构的杂聚糖,主要由β-1,4木糖主链组成,以阿拉伯糖和4-o-甲基葡萄糖醛酸等残基作为侧链附着在木糖上,也含有d-半乳糖、d-木糖、l-鼠李糖、d-葡萄糖、乙酰基以及不同的二聚体和三聚体的侧链。木聚糖的存在抑制了纤维之间氢键的产生,因此,在造纸过程中去除纸浆中的木聚糖对于生成具有强度特性的高品质纸张至关重要。
生物酶催化剂具有反应条件温和、速度快、高选择性等优点,是20世纪最鼓舞人心的发现。木聚糖酶是作用于木聚糖聚合物,催化发生β-1,4-木聚糖水解,形成不同长度的低聚木糖和d-木糖的生物催化剂,在生物炼制、纺织、造纸、纸浆和食品工业中发挥着关键作用。木聚糖的分子结构决定其木聚糖酶底物亲和力,线性分子的木聚糖是木聚糖内切酶的理想底物,如来自gh11家族的木聚糖酶至少需要三个连续的未取代的木糖残基单位作为结合位点才能切割木聚糖主链。因此,探索木聚糖酶的最适底物意义非凡,FelipeA.S等人从桉树硫酸盐浆中提取木聚糖作为木聚糖酶底物,表现出相当高的酶活性。然而,从高价木浆原料中分离木聚糖在商业上不具有成本效益。桉树含有大约35%的木聚糖成分,在硫酸盐法制浆过程中,这些原材料中的木聚糖大多溶解在制浆废水中。
在过去的几十年里,大多数关于木聚糖酶的研究都使用桦木(Birch wood)4-O-甲基葡萄糖醛酸木聚糖作为催化能力评估的标准底物。然而,最近主要的桦木木聚糖停产,大多数供应商出现无货状态。尽管出现一些替代的木聚糖,要么价格昂贵,或纯度不够,要么结构不明确,为使用者所诟病,这引起了科研工作者的密切关注,因此迫切需要研制出一种新的木聚糖酶标准底物,替补目前市场上的空缺。
发明内容
本发明所要解决的技术问题在于,针对现有木聚糖要么价格昂贵,要么纯度不够、或者结构不清的问题,提出了一种木聚糖酶底物及其制备方法。
本发明提供了一种木聚糖酶底物,其总糖含量为95.3wt%,其中单糖组分包括:97.1wt%的木糖、1.2wt%的葡萄糖、0.7wt%的半乳糖和1.0wt%的半乳糖醛酸,木聚糖酶底物命名为XYL。
木聚糖酶底物的主要分子结构为以β-D-Xylp通过1→4糖苷键连接形成的高线性的线性木聚糖,其重复单元结构为:
本发明还提供了该木聚糖酶底物的制备方法,包括以下步骤:
步骤一、取制浆废液,过滤,加盐酸,过滤除去沉淀,上清液加氢氧化钠,减压浓缩,加入无水乙醇,4℃沉淀放置12h,离心收集沉淀,再依次透析、浓缩、冻干得到粗多糖。
进一步地,加入盐酸的量为至废液pH为3,加入氢氧化钠的量为至上清液pH为6;减压浓缩后的浓缩液为原上清液的1/10体积,加入无水乙醇的量为浓缩液的4倍。
步骤二、取粗多糖4g,配置成浓度为20mg/ml的溶液,进行乙醇梯度沉淀,沉淀溶于纯水,透析48h除盐,冷冻干燥获得多糖粉末XYL。
进一步地,乙醇梯度沉淀的具体方法为:加入无水乙醇至无水乙醇体积浓度达10%,4℃沉淀12h及以上;离心除去沉淀,继续往上清液加入无水乙醇至无水乙醇浓度达30%,4℃沉淀12h及以上;离心,用无水乙醇洗涤沉淀、离心,重复洗涤2次。
步骤一和步骤二中透析均采用的是截留分子量1000的透析袋进行透析。
实施本发明,具有如下有益效果:
本发明公开了一种木聚糖酶底物的制备方法,采用乙醇分级沉淀法从制浆废液中获得一种木聚糖,命名为XYL,该木聚糖为纯度高、结构清晰、线性度高的β-D-1→4吡喃木聚糖,与木聚糖酶具有较强的亲和力,是木聚糖酶比较适合的底物,具备成为一种商用的木聚糖酶标准底物的潜力。
附图说明
为了更清楚地说明本发明或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为XYL的GPC分析图谱;
图2为XYL的单糖组份分析图谱;
图3为XYL的甲基化GC-MS色谱图;
图4为XYL的一维和二维核磁共振图;
图5为底物浓度和反应速度的双倒数曲线图;
具体实施方式
下面将结合本发明中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明的木聚糖酶底物,其总糖含量为95.3wt%,其中单糖组分包括:97.1wt%的木糖、1.2wt%的葡萄糖、0.7wt%的半乳糖和1.0wt%的半乳糖醛酸,木聚糖酶底物命名为XYL。
木聚糖酶底物的主要分子结构为以β-D-Xylp通过1→4糖苷键连接形成的高线性的木聚糖,其重复单元结构为:
本发明还提供了该木聚糖酶底物的制备方法,包括以下步骤:
步骤一、取制浆废液,过滤,加盐酸至废液pH为3,过滤除去沉淀,上清液加氢氧化钠至上清液pH为6,减压浓缩,浓缩液为原上清液的1/10体积,加入为浓缩液4倍体积的无水乙醇,4℃沉淀放置12h,离心收集沉淀,再依次透析、浓缩、冻干得到粗多糖。
步骤二、取粗多糖4g,配置成浓度为20mg/ml的溶液,进行乙醇梯度沉淀,加入无水乙醇至无水乙醇体积浓度达10%(即1体积份无水乙醇,9体积份粗多糖溶液),4℃沉淀12h及以上;离心除去沉淀,继续往上清液加入无水乙醇至无水乙醇浓度达30%,4℃沉淀12h及以上;离心,用无水乙醇洗涤沉淀、离心,重复洗涤2次,得到沉淀溶于纯水,透析48h除盐,冷冻干燥获得多糖粉末XYL。
步骤一和步骤二中透析均采用的是截留分子量1000的透析袋进行透析。
上述制备方法中,制浆废液取自海南金海浆纸业有限公司(APP)制浆车间碱抽提工段废水,pH值14,总糖浓度2.5g/L。木聚糖酶从S.griseorubens LH-3纯化而得,由广西大学食品与发酵工程研究所提供。透析袋(截留分子量1000)购买于Solarbio,单糖标准品购于上海叶源生物,氘代丙酮(D,99.9%)、重水(D,99.9%)购于上海阿拉丁,其它试剂为分析纯。
对制备得到的多糖粉末XYL进行研究分析:
分子量测定及单糖组份分析:
多糖的分子量用高效凝胶渗透色谱法(HPGPC)测定:样品用0.05MNaCl溶液溶解,经12000rpm离心10min,上清液过0.22μm PVDF滤膜进GPC分析。分析条件:Shodex-ks-805-804-802色谱柱,Shodex示差检测器(RI-102),进样浓度为5mg/mL,流动相是0.05M NaCl,流速为0.6mL/min。以分子量为1152,11600,23800,48600,80900,148000,273000,409800的standard pullulans做标准曲线,计算XYL的分子量。
单糖组份采用high-pressure anion exchange chromatography(HPAEC)法测定。称量5mg样品置于安瓿瓶中,加入3M TFA2ml,120℃水解2h。水解液转移至干燥管中氮吹干燥,加入10ml水涡旋混匀,吸取80uL加入920uL去离子水,12000rpm离心5min。取上清进IC分析,条件如下:DionexCarbopacTMPA20(3*150)色谱柱配置电化学检测器,配制A:H2O;B:15mM NaOH;C:15mM NaOH&100mM NaOAC做流动相,流速0.3ml/min,进样量5μL,柱温30℃。取单糖标准品配成混合标注溶液,根据绝对定量方法,测定不同单糖质量,根据单糖摩尔质量计算出摩尔比。
碱抽提废液经乙醇梯度沉淀,获得木聚糖XYL,得率2.8%,经硫酸苯酚法测得总糖含量95.3%,经GPC分析获得分子量为60KDa的单一对称峰,请参见图1,图1为XYL的GPC分析图谱。
单糖主要组份是D木糖,含量高达97.1%,同时测出微量的葡萄糖1.2%、半乳糖0.7%和半乳糖醛酸1.0%,请参见图2和表1,图2为XYL的单糖组份分析图谱。
表1XYL的单糖组成成份表
图2和表1表明XYL是一个具有相当纯度的同聚糖。从天然植物分离的木聚糖以杂多糖为主,都含有相当比例的阿拉伯糖、葡萄糖、葡萄糖醛酸等组分,如Corn stalks木聚糖,木糖含量在60~80%,sct(sctx)木聚糖则存在74%的d-木糖残基,16%的d-葡萄糖醛酸残基和10%的l-阿拉伯糖。APMP制浆废液木聚糖,含葡萄糖(28.84%),木糖(68.52%)和阿拉伯糖(3.64%)。至今,从植物中获得木糖含量高于95%的木聚糖少有报道,从碱抽提废液制备出高纯度的同木聚糖,可能的原因是植物纤维中杂木聚糖的支链部分在制浆过程前段的蒸煮和氧脱木素工序中被降解,剩下的木聚糖则以主链线性形式在后段的碱抽提废液中溶出,以至于单糖组分中仍然检到微量的葡萄糖、半乳糖、半乳糖醛酸残留。
甲基化分析:
首先,称量多糖样品10mg溶解于1mL无水DMSO中,加入1ml的二甲基钠溶液,充分震荡12h,逐滴加入1ml的碘化钾,置于暗处室温反应5h。接着,反应液经流动水透析24h,冻干即得甲基化多糖。用fourier-transform infrared spectroscopic(FT-IR)测定甲基化多糖的红外吸收光谱,羟基处的吸收光谱消失证明多糖甲基化完全。甲基化多糖经2M三氟乙酸(2ml)水解,sodiumborohydride(50mg)还原,然后乙酰化获得methylated alditolacetates(PMAAs)。最后,采用GC-MS对PMAAs进行分析,测定条件如下:RXI-5SIL毛细管色谱柱(30m*0.25mm*0.25um)配套离子阱质谱检测器。程序升温条件设定为始温度140℃,以3℃/min升温至250℃/min,保持5min。进样口温度为250℃,检测器温度为250℃/min,载气为氦气,流速为1mL/min。通过数据比对进行甲基化结构分析。
甲基化是多糖精细结构推断的一种不可缺少的方法,XYL最初被甲基化、水解和还原得到PMAAS,然后进行GC-MS分析。如图3所示,图3为XYL的甲基化GC-MS色谱图。图中出现只有一种主要的甲基化糖衍生物,保留时间为15.1min。根据比对离子碎片和保留时间,推断为2,3-二甲基木糖苷,请参见表2。
表2基于甲基化分析的主链数据表
表明XYL的主链可能是→4)-xylp-(1→,即是一个具有高度重复线性结构的1-4连接的木聚糖。没有发现端基木糖,说明该木聚糖不存在支链,而且分子量大(GPC测分子量高达60KDa)。也没有发现其它糖基的键合方式,可能是因为其它糖基的含量太低的原因,和单糖组分分析的一致。这一发现也被NMR光谱学证实。结合单糖组分推断该聚糖为高度线性木聚糖,精细结构用核磁进一步解析。
核磁检测:
称取多糖样品50mg,将其溶于0.5ml的重水中并冷冻干燥(重复3次),以充分交换活泼氢。然后将样品溶于0.5ml的重水(99.9%D)中,加入30μl氘代丙酮(δH2.225,δC 31.45)做内标,25℃,置于400MHz的核磁共振仪(Bruker)测定1H、13C、COSY、HSQC、HMBC和NOESY谱。用MestReNova软件进行数据处理。
利用核磁共振1H、13C、H-H-COSY、HSQC、HMBC和NOESY等波谱进一步确定了XYL的精细化学结构。1H谱中出现6个强的信号峰δH4.28、3.92、3.61、3.36、3.22和3.13,由积分面积可推断每个信号峰含1个质子,可初步确定为糖残基上的6个质子。δH3.36、3.22和3.13存在交叉重叠,通过HMQC谱可以把它们分开指认。
13C谱中出现5条尖锐、分离的谱线δC103.39、77.39、75.74、74.34和64.66,推断为糖残基上5个碳原子信号。根据HMQC谱可判断δC103.39为端基碳,对应的δH4.28为端基质子,端基质子化学位移小于4.5,说明是β构型。结合HMQC谱可指认δH3.22、3.92分别是亚甲基碳上的两个H,δC64.66为亚甲基碳。对COSY谱进行邻位氢3J交叉峰分析,归属2,3,4位H的化学位移,再结合HMQC谱可得出对应的碳化学位移,从而获得该糖残基的碳氢化学位移全归属。
糖苷键连接方式通过3JCH长程耦合HMBC谱分析。该HMBC谱分辨率高,耦合信息一目了然,存在明显清晰的H1-C4的交叉峰,可判断糖苷键为1-4连接,而没有显示其它糖残基片段信号,表明该结构为木糖苷键为主链,侧链的其它糖残基占比例很小,与单糖组分和甲基化分析结果一致。同时存在C1-X5的远程耦合信号,且在82~84之间无信号,说明不存在呋喃糖,证实存在吡喃糖。请参见图4,图4为XYL的一维和二维核磁共振图。
NOESY谱是空间距离谱,该谱显示了清晰明显的6个H之间的耦合信号,说明这些H空间距离靠近,归属为糖环上H的信号,与以上的H谱归属结果一致。“β-D→4)-xylp-(1→”木糖残基以“X”符号表示,其碳氢化学位移及耦合信息统计请参见表3。
表3XYL的1H、13C、COSY、HSQC、HMBC、TOCSY和NOESY的波谱数据数据分析表
综合分析,XYL的分子结构鉴定为以β-D-Xylp通过1→4糖苷键连接形成的线性木聚糖,核磁推导的结构与甲基化分析的结构完全符合,说明推导的结构是可靠的,其重复单元结构如下:
底物亲和力实验
动力学参数:计算了木聚糖的平均常数(Km)和最大速度(Vmax)。底物设置为6种不同浓度,在醋酸缓冲液(50mM,pH5.0)中制备,并与纯化的木聚糖酶在50℃下孵育15min。酶活性以国际单位(IU)表达。木聚糖酶活性的测定方法是将0.2mL适当稀释的酶与1.8mL含有1%的醋酸酯缓冲液(pH 6.0)的1.8mL溶液反应。在50℃反应15min后,用二硝基水杨酸测定释放的还原物质。在煮沸后加入酶作为对照。对底物的1IU活性的定义是在规定的测定条件下释放1μmol的木糖当量,使用木糖标准曲线。
XYL是木糖含量高达97%的线性1→4糖苷键连接的木聚糖,具备成为木聚糖酶最适底物的潜力。米氏常数是衡量底物亲和力的重要参数,Km越小,底物与酶的亲和力越强。请参见图5,图5为底物浓度和反应速度的双倒数曲线图(XYL“◆”、蔗渣木聚糖“■”、榉木木聚糖“▲”),由图5可知,当分别以XYL、蔗渣木聚糖和榉木木聚糖为底物时,根据Lineweaver-Burk双倒数方程,求得米氏常数Km值分别为1.49mg/mL、1.59mg/mL、1.74mg/mL。结果表明,XYL的Km值最小,亲和力最大,是该木聚糖酶的最适底物。wu等人以现今停产的桦木木聚糖为底物测得该酶的米氏常数Km值为1.44mg/mL,说明XYL具备替代桦木木聚糖作为标准底物的潜力。
制浆工业废水排放含有大量的木聚糖资源,但因组成复杂,直接从制浆综合废液或者黑液中提取纯化木聚糖不经济。本发明采用乙醇分级沉淀法从制浆分段废液中提取,获得木糖含量高达97%,纯度95.5%的木聚糖,通过甲基化和核磁技术鉴定是线性(1→4)-β-d-吡喃木聚糖分子结构。在木聚糖酶底物亲和力实验中,平行测试了XYL和商品木聚糖的酶促反应米氏方程,实验得出,与蔗渣木聚糖、榉木木聚糖和桦木木聚糖相比,XYL与酶催化的亲和力很强。结果表明,XYL是较佳的木聚糖酶酶促反应底物,具备成为标准底物的潜力,有望成为商品木聚糖填补市场空缺。本发明的结果有助于提高制浆废液回收利用的可行性,并进一步促进木聚糖酶领域的应用和基础研究。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (4)
1.一种木聚糖酶底物,其特征在于,所述木聚糖酶底物的总糖含量为95.3wt%,其中单糖组分包括:97.1wt%的木糖、1.2wt%的葡萄糖、0.7wt%的半乳糖和1.0wt%的半乳糖醛酸,所述木聚糖酶底物命名为XYL;所述木聚糖酶底物是主链为→4)-xylp-(1→的高线性的木聚糖。
2.根据权利要求1所述的木聚糖酶底物,其特征在于,制备方法包括以下步骤:
步骤一、取制浆废液,过滤,加盐酸,过滤除去沉淀,上清液加氢氧化钠,减压浓缩,加入无水乙醇,4℃沉淀放置12h,离心收集沉淀,再依次采用截留分子量1000的透析袋进行透析、浓缩、冻干得到粗多糖;
步骤二、取粗多糖4g,配置成浓度为20mg/ml的溶液,进行乙醇梯度沉淀,乙醇梯度沉淀的具体方法为:加入无水乙醇至无水乙醇体积浓度达10%,4℃沉淀12h及以上;离心除去沉淀,继续往上清液中加入无水乙醇至无水乙醇浓度达30%,4℃沉淀12h及以上;离心,用无水乙醇洗涤沉淀、离心,重复洗涤2次;得到的沉淀溶于纯水,冷冻干燥获得多糖粉末XYL。
3.根据权利要求2所述的木聚糖酶底物,其特征在于,所述步骤一中,加入盐酸的量为至废液pH为3,加入氢氧化钠的量为至上清液pH为6。
4.根据权利要求3所述的木聚糖酶底物,其特征在于,所述步骤一中,减压浓缩后的浓缩液为原上清液的1/10体积,加入无水乙醇的量为浓缩液的4倍。
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