CN115287291B - 一种α-L-***呋喃糖苷酶及其应用 - Google Patents
一种α-L-***呋喃糖苷酶及其应用 Download PDFInfo
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- CN115287291B CN115287291B CN202210739282.1A CN202210739282A CN115287291B CN 115287291 B CN115287291 B CN 115287291B CN 202210739282 A CN202210739282 A CN 202210739282A CN 115287291 B CN115287291 B CN 115287291B
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- arabinofuranosidase
- alpha
- gly
- thr
- leu
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
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- A—HUMAN NECESSITIES
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- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
- C12C11/00—Fermentation processes for beer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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Abstract
本发明涉及基因工程技术领域,具体涉及一种α‑L‑***呋喃糖苷酶及其应用。本发明所提供的α‑L‑***呋喃糖苷酶(AF)以SEQ ID NO.1所示的泡盛曲霉α‑L‑***呋喃糖苷酶基因序列为基础,进行基因序列优化设计。本发明所提供的AF的最适催化温度为50℃,在pH 2.0~7.0之间保持了较高的相对活性;本发明所述AF对较高浓度的***糖和木糖具有较好的耐受性;在半纤维素水解应用中具有强大的潜力。并且,本发明所述AF对胃蛋白酶和胰蛋白酶具有较好的耐受性,可以动物体内仍可以具备一定的活性,在饲料应用中具有一定的发展前景。
Description
技术领域
本发明涉及基因工程技术领域,具体涉及一种***呋喃糖苷酶的基因优化及其在毕赤酵母中的高效表达方法。
背景技术
在农作物加工过程中产生大量的副产物,比如米糠、麦麸和玉米皮等,这些植物纤维结构复杂,使用糖苷水解酶来降解其中的多糖效率不高。同时在畜牧养殖的过程中,由于许多谷物副产物中多糖的复杂侧链结构,很少有酶能有效的将其降解,因此阻碍了饲料中营养成分的释放。
木聚糖是一种重要的半纤维素,其主链由D-吡喃木糖木糖以β-1,4-糖苷键连接而成,大量***糖、葡萄糖醛酸和阿魏酸酯基等取代基构成了侧链。这些侧链的存在严重影响了木聚糖酶等主链酶发挥作用。由于结构的复杂性,木聚糖的彻底降解需要多种酶的参与,其中木聚糖酶作为主酶以内切的方式作用于木聚糖的主链,***呋喃糖昔酶是侧链降解酶之一,并对主链的降解起到重要的促进作用。
α-L-***呋喃糖苷酶根据其氨基酸序列不同,可以分成GH3、10、43、51、54和62等几个家族。细菌、真菌和植物均能产生α-L-***呋喃糖苷酶,不同来源的α-L-***呋喃糖苷酶在酶学性质上存在差别。细菌来源的α-L-***呋喃糖苷酶在偏中性条件下具有最佳催化活性,而真菌来源的酶在酸性环境下催化活性高,最适催化pH为4.5~5.5。不同来源酶的最适催化反应温度差异也很大,一般介于40~70℃之间。
α-L-***呋喃糖苷酶具有多种重要的工业用途。在饲料工业中,麦类饲料中含有较多的***木聚糖,这种木聚糖具有抗营养作用,增加食糜的粘度,不利于动物对营养物质的消化和吸收。目前单一酶的使用具有一定的局限性,因而主链酶和侧链酶协同配合已经是目前研究的热点。例如只使用木聚糖酶不能对玉米糖类的支链结构进行有效降解。而当***呋喃糖苷酶和木聚糖酶同时作用于***木聚糖时,首先侧链的***糖快速被降解释放,然后木聚糖酶才与主链木聚糖作用释放木糖;***糖的移除给木聚糖酶提供了作用位点,同时也增加了底物的溶解性。
***呋喃糖苷酶还被广泛应用于食品行业,可作为一种低摄入量的甜味剂,能够替代传统的蔗糖,适用于老年人和高血糖患者食用。***呋喃糖苷酶能阻断人体对蔗糖的吸收,在减肥和控制糖尿病方面应用前景广阔。***呋喃糖苷酶在酿酒工业也发挥着一定的作用,啤酒酿造时加入***呋喃糖苷酶可以提高木聚糖的降解效率和麦芽汁的过滤速度。
***呋喃糖苷酶还能增加酿酒过程中萜烯醇的浓度,进而提高酒的香味。同时***呋喃糖苷酶能去除***糖侧链,将***聚糖水解成短链的可溶性物质,增加果汁的澄清度,因此被广泛应用于果汁生产工业。
发明内容
本发明的目的是提供一种新的α-L-***呋喃糖苷酶。具体地,编码所述α-L-***呋喃糖苷酶的核苷酸序列如SEQ ID NO.3所示。
本发明所提供的α-L-***呋喃糖苷酶的氨基酸序列如SEQ ID NO.2所示。
本发明所提供的新型α-L-***呋喃糖苷酶的最适pH为2.0-7.0。
本发明还请求保护含有上述α-L-***呋喃糖苷酶的生物材料,所述生物材料为表达盒、载体、工程菌。
根据本领域技术人员的理解,本发明还请求保护上述α-L-***呋喃糖苷酶或上述的生物材料在酿酒或饲料加工中的应用。
以及上述α-L-***呋喃糖苷酶或上述的生物材料在提高植物纤维降解效率中的应用。
在本发明所提供的应用中,采用α-L-***呋喃糖苷酶对植物纤维进行降解。
在本发明所提供的应用中,采用金属离子Ca2+、Zn2+、Mn2+、K+和所述α-L-***呋喃糖苷酶对植物纤维进行降解。
在本发明所提供的应用中,所述植物纤维为酒糟、玉米皮和/或麸皮。
本发明还请求保护一种饲料或饲料添加剂,所述饲料或饲料添加剂中含有上述α-L-***呋喃糖苷酶或上述的生物材料。
本发明的有益效果在于:
本发明提供的α-L-***呋喃糖苷酶是从泡盛曲霉中得到的,其最适pH为5.0,最适温度为50℃,具有良好的金属离子抗性,并在胃蛋白酶和胰蛋白酶处理后仍有较高的活性,同时该酶对高浓度的L-***糖和D-木糖具有较好的耐受性。这些特性利于其在食品和饲料工业上的应用,具有一定的应用前景。
(1)本发明所提供的重组α-L-***呋喃糖苷酶,最适催化温度为50℃,最适pH为5.0且pH的稳定性较好,在pH 2.0-7.0之间保持了较高的相对活性;并且本发明所提供的Ca2+、Zn2+、Mn2+、K+等金属离子可以促进酶活性,其中硫酸铵处理后酶活力约142.26%。
(2)本发明所提供的重组α-L-***呋喃糖苷酶,对较高浓度的***糖和木糖具有较好的耐受性,表明该酶不易受到酶促反应产物的反馈抑制,在半纤维素水解应用中具有强大的潜力。
(3)本发明所提供的重组α-L-***呋喃糖苷酶,在胃蛋白酶处理80min后仍有61.32%活性,胰蛋白酶处理80min后仍有52.95%酶活力,说明该酶对胃蛋白酶和胰蛋白酶具有较好的耐受性,可以动物体内仍可以具备一定的活性,在饲料应用中具有一定的发展前景。
(4)本发明所提供的重组α-L-***呋喃糖苷酶,降解DDGS、玉米皮和麸皮时,可以打破底物木聚糖侧链的一部分糖苷键并释放一定量的还原糖,具有较好的酶解效果,应用前景广阔。
附图说明
图1为本发明实施例3中摇瓶发酵上清液SDS-PAGE分析电泳图。
图2为本发明实施例3中发酵罐发酵菌体湿重增长曲线及酶活增长曲线;其中,a为甘油培养阶段;b为甘油补料阶段;c为甲醇诱导阶段。
图3为本发明实施例3中发酵上清液SDS-PAGE分析图。
图4为本发明实施例4中温度对重组α-L-***呋喃糖苷酶AF活性的影响。
图5为本发明实施例4中pH对重组α-L-***呋喃糖苷酶AF活性的影响。
图6为本发明实施例4中重组α-L-***呋喃糖苷酶AF的pH稳定性。
图7为本发明实施例5中重组α-L-***呋喃糖苷酶AF的单糖耐受性。其中A为重组α-L-***呋喃糖苷酶AF的D-木糖耐受性;B为重组α-L-***呋喃糖苷酶AF的L-***糖耐受性。
图8为本发明实施例5中重组α-L-***呋喃糖苷酶AF的胃蛋白酶和胰蛋白酶耐受性。
图9为本发明实施例6中重组α-L-***呋喃糖苷酶降解不同底物的还原糖释放量。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
本发明中巴斯德毕赤酵母表达载体pPICZαA及菌株X33购自Invitrogen公司;pPICZαAn载体(α信号肽序列进行了优化设计)由本实验室保存;大肠杆菌Top10感受态细胞购自北京擎科新业生物技术有限公司。
本发明中内切酶EcoRI和Xba I购自New England Biolabs公司,线性化酶Sac I购自TaKaRa公司,连接酶T4及连接缓冲液购自New England Biolabs公司。
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例均按照常规实验条件,如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:A Laboratory Manual,2001),或按照制造厂商说明书建议的条件。
本发明中涉及到的百分号“%”,若未特别说明,是指质量百分比;但溶液的百分比,除另有规定外,是指100mL溶液中含有溶质的克数。
实施例1泡盛曲霉α-L-***呋喃糖苷酶基因优化合成
以SEQ ID NO.1所示的泡盛曲霉α-L-***呋喃糖苷酶基因序列(NCBI登录号:AB046702)为基础(去除信号肽序列),进行基因序列优化设计。SEQ ID NO.2为SEQ ID NO.1所示的核苷酸序列所编码的氨基酸序列。
在不改变氨基酸序列的前提下,用毕赤酵母高使用频率的密码子替换低使用频率的密码子,同时消除连续嘧啶碱基序列和降低mRNA 5’的二级结构的稳定性。密码子优化后基因的G+C含量由55%降低到45%,更接近于巴斯德毕赤酵母基因组的GC含量。设计了两条不同的优化序列(SEQ ID NO.3和SEQ ID NO.4)。优化序列由北京擎科新业有限公司合成,并在5’和3’末端分别添加EcoRI和Xba I限制性内切酶酶切位点。对优化序列进行了筛选,以提高其表达效率。
实施例2重组α-L-***呋喃糖苷酶表达工程菌株构建
分别将含pPICZα-AF质粒(SEQ ID NO.3和SEQ ID NO.4)和pPICZαAn质粒的大肠杆菌(Escherichia coil,E.coli)接种于10mL含Zeocin(100μg/mL)LB液体培养基的50mL摇瓶中,37℃培养12小时,用质粒提取试剂盒(Omega,美国)提取重组克隆质粒。重组质粒用内切酶EcoRI和Xba I进行双酶切,合成的α-L-***呋喃糖苷酶基因也同样酶切,然后用T4连接酶进行连接,而后转入TOP 10感受态中提取质粒进行测序。重组质粒用Sac I线性化,用DNA纯化试剂盒(Omega,美国)进行纯化,电击转化至毕赤酵母X-33感受态细胞中,涂布于含Zeocin(100μg/mL)的YPDS(10g/L酵母提取物,20g/L蛋白胨,20g/L葡萄糖,1mol/L山梨醇,20g/L琼脂,115℃高压灭菌)固体平板培养基上,28℃倒置培养。挑选单菌落划线至含有Zeocin(100μg/mL)的YPD(10g/L酵母提取物,20g/L蛋白胨,20g/L葡萄糖,115℃高压灭菌)固体培养基,28℃培养,所得菌株即为重组α-L-***呋喃糖苷酶AF毕赤酵母工程菌株。
实施例3重组α-L-***呋喃糖苷酶工程菌株发酵
将实施例2所得到的重组毕赤酵母菌株接种于50mL BMGY(1%酵母提取物,2%蛋白胨,100mmol/L磷酸钾溶液(pH 6.0),1.34%YNB,1%甘油,4×10-5%生物素),于28℃、220rpm摇床培养至OD600值为2~6。5,000rpm离心收集菌体。用50mL BMMY(1%酵母提取物,2%蛋白胨,100mmol/L磷酸钾溶液(pH 6.0),1.34%YNB,0.5%甲醇,4×10-5%生物素)培养基重悬菌体,保持甲醇终浓度为0.5%(v/v)进行摇瓶诱导96h,每12h添加一次甲醇。重组α-L-***呋喃糖苷酶(SEQ ID NO.3)的摇瓶表达量为29.0U/mL;重组α-L-***呋喃糖苷酶(SEQ ID NO.4)的摇瓶表达量为15.4U/mL(以对硝基苯基-α-L-***呋喃糖苷为底物测定),且在SDS-PAGE上大小约为100kDa(图1)。因重组重组α-L-***呋喃糖苷酶(SEQ IDNO.4)的摇瓶表达量相对较低,用SEQ ID NO.3展开后续试验。
α-L-***呋喃糖苷酶酶活测定:将浓度为10mM对硝基苯基-α-L-***呋喃糖苷(Sigma公司,美国)溶液于37℃水浴预热5min,然后再分别吸取pH 5.5的柠檬酸-磷酸二氢钠缓冲液稀释100,000倍的酶液和水浴后α-L-***呋喃糖苷酶底物溶液各100μL充分混合,于37℃恒温水浴10min,加入100μL 1M碳酸钠溶液终止反应,振荡混匀,利用酶标仪测定吸光度OD405。根据对硝基苯酚标准曲线得到OD405与底物对硝基苯酚浓度的关系,计算得出α-L-***呋喃糖苷酶降解后对硝基苯酚的浓度,进而计算出α-L-***呋喃糖苷酶的酶活。
挑取α-L-***呋喃糖苷酶表达菌株接种于含有Zeocin(100μg/mL)YPD培养基中28℃220rpm培养过夜后,将100μL菌液转入含有100mL YPD液体培养基的500mL三角瓶中,28℃220rpm培养。待菌液OD600达到2~6时接入装有12L发酵基础培养基的30L发酵罐进行培养。待湿菌重达到90~100g/L时(此时基础碳源耗尽),进入50%甘油(w/v)补料阶段,补料后湿菌种达到225g/L以上,停止甘油补料,饥饿到溶氧达90%以上开始用甲醇诱导。通过调节转速、空气流量和甲醇补料速度控制相对溶氧在20%以上,每隔12h取样测定湿菌种和酶活(图2),并对上清液进行12%SDS-PAGE电泳分析(图3),表达产物的大小约为100kDa与之前摇瓶发酵结果相同。待湿菌种及酶活达到峰值保持稳定或呈下降趋势时,停止发酵,离心取上清液进行冻干,冻干后取样测酶活为6,683.9U/g。
实施例4重组α-L-***呋喃糖苷酶酶学性质分析
将粗酶液适当稀释后,分别在40℃、45℃、50℃、55℃、60℃、65℃、70℃、75℃温度下测定并计算酶活,确定其最适温度。
用pH为2.0、2.5、3.0、3.5、4.0、4.5、5.0、5.5、6.0、6.5、7.0的柠檬酸-磷酸氢二钠缓冲液稀释酶液,底物也用相应pH的缓冲液配制底物溶液,在50℃条件下测定酶活。试验结果表明,AF的最适催化温度为50℃(图4),最适pH为5.0且pH的稳定性较好,在pH2.0~7.0之间保持了较高的相对活性(图5和图6)。
用pH 5.0的柠檬酸-磷酸氢二钠缓冲液配制成20mM的不同金属离子的化合物溶液,将酶液与金属离子等体积混合,使金属离子终浓度为10mM,室温处理1h后,最适条件下测定酶活,以不添加金属离子的酶液作为空白对照。结果表明,AF酶对终浓度为10mM的不同金属离子及EDTA常温作用1h的耐受性不一致,Fe3+、Co2+、Na+等金属离子和EDTA会抑制酶活性,除了Fe3+的抑制性较强以外,其他离子处理后酶的相对活性都在70%以上,而Ca2+、Zn2+、Mn2+、K+等金属离子可以促进酶活性,其中硫酸铵处理后酶活力约142.26%(表1)。
表1 金属离子对α-L-***呋喃糖苷酶AF活性的影响
实施例5重组α-L-***呋喃糖苷酶对单糖耐受性及胃胰蛋白酶耐受性的影响
α-L-***呋喃糖苷酶作用底物时会产生较多单糖,产物有L-***糖D-木糖等单糖,因此探究了重组α-L-***呋喃糖苷酶在不同浓度(0~2,000mmol/L)L-***糖、D-木糖存在下的酶活力情况。
用pH 5.0的柠檬酸-磷酸氢二钠缓冲液配制成不同浓度的(0~2,000mmol/L)单糖的溶液,将酶液与单糖溶液等体积混合,室温处理1h后,最适条件下测定酶活,以不添加单糖溶液的酶液作为空白对照。结果表明,低浓度(<200mmol/L)时,L-***糖的存在对重组α-L-***呋喃糖苷酶活力有轻微促进作用。随着L-***糖浓度升高,L-***糖对α-L-***呋喃糖苷酶活力产生抑制作用。当D-木糖存在时,重组α-L-***呋喃糖苷酶活力受到轻微抑制,甚至在一些浓度中酶活力反而有所提高(图7和图8)。该酶对较高浓度的***糖和木糖具有较好的耐受性,表明该酶不易受到酶促反应产物的反馈抑制,在半纤维素水解应用中具有强大的潜力。
α-L-***呋喃糖苷酶在动物体内发挥作用时会受到体内酶(胃、胰蛋白酶)的影响,本发明探究了重组α-L-***呋喃糖苷酶在胃/胰蛋白酶处理后的酶活力。用浓度为5mg/mL的胃(胰)蛋白酶处理稀释100,000倍的酶液,将酶液与金属离子等体积混合,室温处理20min、40min、60min和80min,最适条件下测定酶活,以未处理过的酶液作为空白对照。结果表明,随着时间的增加,重组α-L-***呋喃糖苷酶酶活力在下降,但胃蛋白酶处理80min后仍有61.32%活性,胰蛋白酶处理80min后仍有52.95%酶活力。(图8)结果说明该酶对胃蛋白酶和胰蛋白酶具有较好的耐受性,可以动物体内仍可以具备一定的活性,在饲料应用中具有一定的发展前景。
实施例6α-L-***呋喃糖苷酶AF降解DDGS、玉米皮和麦麸皮的研究
将冻干后的α-L-***呋喃糖苷酶作用于DDGS、玉米皮和麸皮三种底物,底物事先经粉碎,过40目筛且高温高压(121℃20min)处理。以只含底物不加酶的相同体系相同处理的作为空白对照。反应体系(10ml)含有0.1g底物(预处理过的DDGS、玉米皮和麦麸皮),50mM柠檬酸-磷酸盐缓冲液(pH为5.0),重组α-L-***呋喃糖苷酶添加量为6U/mL,50℃孵育24h,12,000rpm离心10分钟,利用DNS法测定三种底物产生的还原糖的量(图9)。
从图9中可以看出当α-L-***呋喃糖苷酶AF降解三种底物时,可以打破底物木聚糖侧链的一部分糖苷键并释放一定量的还原糖,具有较好的酶解效果,应用前景广阔。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
<110> 中国农业大学
<120> 一种α-L-***呋喃糖苷酶及其应用
<130> KHP221116991.2
<160> 4
<170> SIPOSequenceListing 1.0
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gtttccttga aggtctccac ccagggtggc aactcatcca gccccatcct gtatgggttc 60
atgtttgagg atatcaatca ctcaggagac ggaggaattt acgggcaatt gctgcagaac 120
cctggccttc agggaacgac acccaacctg actgcttggg cggctgtcgg tgatgctacc 180
atcgcgattg atggtgacag tccattgact tctgccattc ccagcactat aaagctggat 240
gttgcggatg atgctaccgg tgcggtgggt ctcaccaatg agggatattg gggcatccca 300
gtcgacggca gcgaattcca gagttccttc tggataaagg gagattactc gggcgacatc 360
accgtccgac tggttggaaa ctataccggc acggagtacg gctctgccac tatcacccat 420
acgtccacag cagacaactt cacccaagcc tccgtcaagt tccccaccac caaggctcca 480
gatggcaacg tcttgtacga gctcacagtg gatggaagcg tggctgctgg ttcgtctttg 540
aacttcggat acctgacgct ttttggcgag acttataaat caagggaaaa tggcctgaag 600
ccccagctgg ccaatgtgtt ggctgatatg aaaggatctt tcctgaggtt tcccggcggc 660
aacaaccttg agggaaacag cgcagaaaac cgctggaagt ggaacgagac aatcggcgat 720
ctctgggatc gtcccggccg tgaaggcact tggacttact ataacaccga tggacttggc 780
ctccacgaat acttttactg gtgtgaggat ttgggactcg tgccggtgct cggagtctgg 840
gatgggttcg ctctggagtc aggtggcaac accccgatta cgggcgatgc actgacccct 900
tatattgatg acgtcttgaa cgaactcgag tacatcttgg gcgatacgag cacgacctac 960
ggagcatggc gcgctgccaa tggacaagag gagccgtgga accttaccat ggtcgagatt 1020
ggcaatgaag atatgctggg aggcggatgc gagtcctacg ccgagcgttt tactgccttc 1080
tatgatgcaa tccatgcggc ttacccggat ctcatcctta ttgctagcac cagcgaggcg 1140
gattgcttgc ccgagtcgat gcccgagggt agctgggtcg actaccacga ctacagcacg 1200
cccgatggac tggtgggcca gttcaactat ttcgacaacc tgtaccgctc tgtgccatac 1260
ttcatcggcg agtattcgcg ctgggagatc gactggccca acatgaaggg gtcggtttcc 1320
gaggctgttt tcatgatcgg gttcgagagg aacagcgatg tggtcaagat ggcggcgtat 1380
gcgccattgc tccagctggt gaactcgact cagtggacgc cggacctgat cggatacacc 1440
cagtcacccg atgacatttt cctctcgacc agctactacg tgcaggaaat gttctcgcgc 1500
aaccgtggtg ataccatcaa ggaggtgacc tctgacagcg actttggacc gttgtactgg 1560
gttgcgtcga gcgccggcga ctcgtactac gtgaagctgg ccaactacgg ctcggagacg 1620
caagacctta ccgtgagcat cccaggaacg agcacaggca agttgacggt gctggcggac 1680
aatgaccccg acgcgtacaa ctctgacacc cagacgctgg tcacgccgag cgaatcgacg 1740
gtgcaggcga gcaatggtac ctttaccttt agtttgccgg catgggcggt ggctgtcctg 1800
gctgcgaact ag 1812
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Asn Leu Thr Ala Trp Ala Ala Val Gly Asp Ala Thr Ile Ala Ile Asp
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Gly Asp Ser Pro Leu Thr Ser Ala Ile Pro Ser Thr Ile Lys Leu Asp
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Val Ala Asp Asp Ala Thr Gly Ala Val Gly Leu Thr Asn Glu Gly Tyr
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Lys Gly Asp Tyr Ser Gly Asp Ile Thr Val Arg Leu Val Gly Asn Tyr
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Thr Gly Thr Glu Tyr Gly Ser Ala Thr Ile Thr His Thr Ser Thr Ala
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Asp Asn Phe Thr Gln Ala Ser Val Lys Phe Pro Thr Thr Lys Ala Pro
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Asp Gly Asn Val Leu Tyr Glu Leu Thr Val Asp Gly Ser Val Ala Ala
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Lys Ser Arg Glu Asn Gly Leu Lys Pro Gln Leu Ala Asn Val Leu Ala
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Gly Asn Ser Ala Glu Asn Arg Trp Lys Trp Asn Glu Thr Ile Gly Asp
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Asp Gly Leu Gly Leu His Glu Tyr Phe Tyr Trp Cys Glu Asp Leu Gly
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Gly Asn Thr Pro Ile Thr Gly Asp Ala Leu Thr Pro Tyr Ile Asp Asp
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Val Leu Asn Glu Leu Glu Tyr Ile Leu Gly Asp Thr Ser Thr Thr Tyr
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Gly Ala Trp Arg Ala Ala Asn Gly Gln Glu Glu Pro Trp Asn Leu Thr
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Met Val Glu Ile Gly Asn Glu Asp Met Leu Gly Gly Gly Cys Glu Ser
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Tyr Ala Glu Arg Phe Thr Ala Phe Tyr Asp Ala Ile His Ala Ala Tyr
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Pro Asp Leu Ile Leu Ile Ala Ser Thr Ser Glu Ala Asp Cys Leu Pro
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Glu Ser Met Pro Glu Gly Ser Trp Val Asp Tyr His Asp Tyr Ser Thr
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Pro Asp Gly Leu Val Gly Gln Phe Asn Tyr Phe Asp Asn Leu Tyr Arg
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Ser Val Pro Tyr Phe Ile Gly Glu Tyr Ser Arg Trp Glu Ile Asp Trp
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Pro Asn Met Lys Gly Ser Val Ser Glu Ala Val Phe Met Ile Gly Phe
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Glu Arg Asn Ser Asp Val Val Lys Met Ala Ala Tyr Ala Pro Leu Leu
450 455 460
Gln Leu Val Asn Ser Thr Gln Trp Thr Pro Asp Leu Ile Gly Tyr Thr
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Gln Ser Pro Asp Asp Ile Phe Leu Ser Thr Ser Tyr Tyr Val Gln Glu
485 490 495
Met Phe Ser Arg Asn Arg Gly Asp Thr Ile Lys Glu Val Thr Ser Asp
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Asn Asp Pro Asp Ala Tyr Asn Ser Asp Thr Gln Thr Leu Val Thr Pro
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<210> 3
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<212> DNA
<213> Artificial Sequence
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gaattcgtga gtttgaaagt ttctacccaa ggaggaaact ccagtagtcc aatcttatat 60
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caaaatcctg gattacaagg aactacccca aacttgactg cttgggctgc tgtcggtgat 180
gccactattg ctattgatgg agactcacca cttacttcag ctataccatc tactatcaag 240
ttggacgtgg cagatgacgc caccggtgct gtcggactta ccaatgaagg atactggggt 300
atcccagtcg atggttctga atttcagtct tctttttgga ttaaaggaga ttactctggt 360
gatatcacgg taaggctggt tggaaattac acgggcaccg agtatggatc tgctaccatt 420
actcatacta gtaccgctga caattttacg caggcttcag ttaagtttcc aacaactaaa 480
gctccagatg gcaatgtgtt gtatgaactg accgttgacg gttcagtcgc cgctggtagt 540
tcactgaact tcggatatct tacattattt ggagagactt ataagtctcg tgaaaatggt 600
ctaaaacctc aattagccaa tgtcttggca gacatgaagg gttctttctt gagatttcca 660
ggtggcaata atttagaggg aaacagtgct gaaaaccgtt ggaaatggaa cgagacgatc 720
ggagatttgt gggacagacc tggtagagag ggcacttgga catattacaa cacagacggc 780
ctaggtttgc acgaatattt ctactggtgc gaggatctgg gcttagtgcc agttttgggt 840
gtttgggacg gcttcgctct ggagtccggt ggaaatacac caatcactgg agatgctttg 900
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acttatggtg cttggcgtgc cgcaaatggt caggaagaac cttggaactt gactatggtc 1020
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gcttatgctc cattattgca attagttaat tcaacccagt ggactccaga cttgatcggt 1440
tacacccaga gtccagatga tatttttttg tcaacctcct actacgtcca agaaatgttt 1500
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gctgataacg atcctgacgc ctataactct gatactcaaa ccttagttac tccttccgaa 1740
tccacagttc aggccagtaa cggaacattc accttctctt tgcccgcttg ggcagttgct 1800
gttctggcag ccaactaatc taga 1824
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<211> 1824
<212> DNA
<213> Artificial Sequence
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gaattcgtct ccctaaaggt gtcaactcaa ggtggtaatt cttcatcccc aattttgtac 60
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caaaacccag gacttcaagg tacaactcca aacttgaccg cctgggctgc tgttggcgac 180
gccactattg ctatcgatgg agactcacct ttaacttctg caattccaag tactattaaa 240
ctggatgtcg ctgatgatgc tactggtgct gtaggactaa caaatgaagg ttactgggga 300
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gacatcacag tgcgtctggt cggaaattac actggaactg aatacggtag tgcaacgatt 420
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ttgggattgc atgaatactt ttactggtgc gaggaccttg gactggttcc tgtactgggt 840
gtatgggatg gtttcgcctt ggagagtggt ggtaatactc caatcaccgg agatgctctg 900
actccttaca ttgacgatgt tttaaatgaa ttggagtaca ttttaggtga cacaagtacc 960
acctatggtg cctggagagc cgcaaacggt caggaagagc catggaacct gactatggtc 1020
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gctttctatg acgctattca tgctgcttac ccagatctta ttcttattgc ttcaacatct 1140
gaggccgact gtttgcctga atccatgcct gaaggctcat gggttgacta tcacgactac 1200
tctacgccag acggtttggt gggacagttt aattatttcg ataatttata cagaagtgtg 1260
ccatatttta tcggtgaata ttccagatgg gagattgatt ggcctaatat gaaaggatct 1320
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gcctacgctc cactgttaca actggtcaat tctactcaat ggactccaga cttgataggt 1440
tacacacaat cccctgacga tattttcctt tcaacgagtt actacgttca agaaatgttc 1500
tcacgtaacc gtggtgatac aatcaaagag gtgacatccg attctgattt tggtccactg 1560
tactgggtcg cttcaagtgc tggagactcc tactatgtga agttggccaa ttacggatct 1620
gagactcaag acttgacagt ttctattccc ggtacgagta ccggtaagct gacagtatta 1680
gctgataatg atcctgatgc ttacaacagt gatactcaga ccttggtcac tccatctgaa 1740
tcaacagttc aggctagtaa tggaactttt acattttctc tacctgcctg ggccgttgcc 1800
gtccttgctg ccaactaatc taga 1824
Claims (10)
1.一种α-L-***呋喃糖苷酶,其特征在于,编码所述α-L-***呋喃糖苷酶的核苷酸序列如SEQ ID NO.3所示。
2.根据权利要求1所述的α-L-***呋喃糖苷酶,其特征在于,所述α-L-***呋喃糖苷酶的氨基酸序列如SEQ ID NO.2所示。
3.根据权利要求2所述的α-L-***呋喃糖苷酶,其特征在于,所述α-L-***呋喃糖苷酶的最适pH为2.0-7.0。
4.含有权利要求1所述的α-L-***呋喃糖苷酶的生物材料,其特征在于,所述生物材料为表达盒、载体、工程菌。
5.权利要求1所述α-L-***呋喃糖苷酶或权利要求4所述的生物材料在酿酒或饲料加工中的应用。
6.权利要求1所述α-L-***呋喃糖苷酶或权利要求4所述的生物材料在提高植物纤维降解效率中的应用。
7.根据权利要求5-6任一项所述的应用,其特征在于,采用α-L-***呋喃糖苷酶对植物纤维进行降解。
8.根据权利要求7所述的应用,其特征在于,采用金属离子Ca2+、Zn2+、Mn2+、K+和所述α-L-***呋喃糖苷酶对植物纤维进行降解。
9.根据权利要求8所述的应用,所述植物纤维为酒糟、玉米皮和/或麸皮。
10.一种饲料或饲料添加剂,其特征在于,含有权利要求1所述α-L-***呋喃糖苷酶或权利要求4所述的生物材料。
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Role of Two a-L-Arabinofuranosidases in Arabinoxylan Degradation and Characteristics of the Encoding Genes from Shochu Koji Molds, Aspergillus kawachii andAspergillus awamori;TAKUYA KOSEKI等;JOURNAL OF BIOSCIENCE AND BIOENFINEERING;第93卷(第3期);摘要,第234页第3段 * |
***呋喃糖苷酶的重组表达及其发酵工艺优化;李鸿雁等;食品与发掘工业;第46卷(第15期);摘要,第15页第2段 * |
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