CN106148369A - 高温碱性果胶酸裂解酶Pel-863及其编码基因和应用 - Google Patents
高温碱性果胶酸裂解酶Pel-863及其编码基因和应用 Download PDFInfo
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Abstract
本发明公开了一种高温碱性果胶酸裂解酶Pel-863及其编码基因和应用,所涉及的果胶酸裂解酶基因编码序列如SEQ ID NO.1或SEQ ID NO.2所示,氨基酸序列如SEQ ID NO.3或SEQ ID NO.4所示。该果胶酸裂解酶基因来源于热解纤维素菌Caldicellulosiruptor kronotskyensis,该酶可在70-75℃、pH9.0-9.5以及Ca2+存在条件下高效降解多聚半乳糖醛酸(PGA)和果胶,并且具有较好的温度和pH稳定性。另外,Pel-863对麻纤维、苹果渣及天然秸秆中的果胶质成分均有较好的降解效果,可应用于纺织、造纸及生物能源等领域。
Description
技术领域
本发明属于基因工程和生物质利用领域,具体涉及一种高温碱性果胶酸裂解酶Pel-863及其编码基因和应用。
背景技术
果胶是一类主要存在于植物初生细胞壁和胞间层的多糖,它与纤维素、半纤维素、木质素的微纤丝以及某些伸展蛋白相互交联,使植物组织保持结构完整性和刚性,进而对生物质的抗降解性有重要作用。果胶的分子量一般较大并且结构较为复杂,其基本结构由两大不同结构域组成:线性的同聚半乳糖醛酸区域(homogalacturonan,HG)和分支的鼠李糖-半乳糖醛酸聚糖区域(rhamnogalacturonan,RG)。HG是由100-200个D-半乳糖醛酸通过α(1,4)糖苷键连接而成的线性同聚物,其中70-80%的半乳糖醛酸残基发生了甲基酯化,也有部分C2和C3位置的羟基发生了乙酰化(Chiliveri&Linga,2014)。RG区域又分为RG-和RG-II两种类型,RG-I的主链是由含半乳糖醛酸和鼠李糖的重复二糖单位构成的,其结构可大致表示为GalAp-α-(1,2)-Rhap-α-(1,4)-GalAp-α-(1,2)-Rhap;RG-II是由HG的主链和11种不同的糖侧链组成的分支型结构,主要有L-阿拉伯糖,D-半乳糖和D-木糖共价连接到主链的C1或C2位置上(Itoh et al.,2006)。
果胶的降解涉及多种酶的作用,主要有果胶甲酯酶(pectin methyl esterase,[PME][EC3.1.1.11]),多聚半乳糖醛酸酶(polygalacturonase,[PG][EC 3.2.1.15])和果胶/果胶酸裂解酶(pectin lyase,[PNL][EC 4.2.2.10]/pectate lyase,[PEL][EC 4.2.2.2])。果胶酸裂解酶是果胶酶中作用方式较为特殊的一类,它通过反式消除方式切割半乳糖醛酸单位间的α(1,4)糖苷键,生成(4,5)不饱和寡聚半乳糖醛酸(Sukhumsiirchart et al.,2009;Wang et al.,2011)。果胶酸裂解酶因其在纺织、造纸、茶或咖啡发酵、果胶废水处理及植物油提取等领域的广泛应用而备受关注(Chiliveri&Linga,2014)。值得一提的是,棉麻等植物纤维应用于纺织之前尤为重要的前处理便是脱胶过程,即去除果胶质及一些杂质。传统的煮碱脱胶法不仅带来严重的环境污染,而且会对纤维本身造成一定的伤害,应用果胶酸裂解酶的生物酶法脱胶在去除非纤维类物质的同时,保持了原纤维的完整性,也大大减少了化学品的消耗,是一种能够替代碱法脱胶的环境友好的方法。同时,由于果胶物质在碱性高温条件下更易溶解,所以嗜热菌或嗜碱菌来源的果胶酸裂解酶具有非常大的工业应用潜质及竞争力。目前发现及研究的果胶酸裂解酶主要来自于芽孢杆菌属、假单胞菌属及曲霉真菌属(Li et al.,2014;Sasaki et al.,2015)。
热解纤维素菌Caldicellulosiruptor kronotskyensis是分离自堪察加半岛热泉中的严格厌氧的革兰氏阳性菌,其可在较高温度(45-82℃)下利用纤维素、滤纸以及果胶等物质(Miroshnichenko et al.,2008)。对其基因组分析发现,该菌是迄今发现的具有最高糖苷水解酶家族(Glycoside hydrolases family)多样性的厌氧嗜热微生物,包含84个GH结构域,分属于38个糖苷水解家族(Blumer-Schuette et al.,2012)。已报道的Caldicellulosiruptor菌属来源的降解酶类如纤维素酶、木聚糖酶、α-葡萄糖醛酸苷酶等均具有较高的作用温度和良好的热稳定性,同时表现出对天然底物的降解优势。本发明实现了C.kronotskyensis中果胶酸裂解酶的高效异源表达,该酶的最适温度为70℃,最适pH为9.0,可替代强碱溶液进行麻纤维脱胶,并且可明显促进天然秸秆的降解。
发明内容
本发明的目的是提供一种高温碱性果胶酸裂解酶Pel-863及其基因工程制备方法和应用,具体包括:
1.本发明提供一种来源于热解纤维素菌Caldicellulosiruptorkronotskyensis的果胶酸裂解酶Pel-863,该酶属于多糖裂解酶家族3,其编码基因序列如SEQ ID NO.1或SEQ ID NO.2所示,其氨基酸序列如SEQ ID NO.3或SEQ ID NO.4所示。其中,该酶包含447个氨基酸,N端32个氨基酸为其预测的信号肽序列,即“MSNRKILAIVVSLIMVVSLFTGIGLRNEVAKA”(SEQID NO.5)。成熟的果胶酸裂解酶Pel-863的氨基酸序列即为去掉信号肽(SEQ ID NO.5)之后的序列,如SEQ ID NO.4所示,该成熟酶对应的基因序列如SEQ ID NO.2所示。
2.本发明提供包含上述果胶酸裂解酶基因的重组载体,包括大肠杆菌表达载体、乳酸菌表达载体、酵母菌表达载体、枯草杆菌表达载体及丝状真菌表达载体等。将本发明的高温碱性果胶酸裂解酶基因与线性质粒片段连接获得重组表达载体。重组表达质粒pET-28b-Pel-863作为本发明的一个最优选的重组表达载体。
3.本发明提供包含上述果胶酸裂解酶基因的重组菌株,所述菌株为大肠杆菌(如Escherichia coli BL 21(DE3)、E.coli Top10、E.coli Rosetta(DE3)等)、乳酸菌(如Lactococcuslactis等)、酵母菌(如Pichiapastoris、Saccharomyces cerevisiae等)、枯草杆菌(如Bacillus subtilis BS168等)和丝状真菌(如Trichodermareesei、Aspergillusniger等),优选为大肠杆菌E.coli Rosetta(DE3)。
4.本发明还提供上述果胶酸裂解酶Pel-863的基因工程制备方法,包括以下步骤:
1)用上述的重组载体pET-28b-Pel-863转化宿主细胞,得重组菌株;
2)培养重组菌株,诱导果胶酸裂解酶Pel-863的表达;
3)回收并纯化所表达的果胶酸裂解酶Pel-863。
5.本发明还提供上述果胶酸裂解酶Pel-863在酶解多聚半乳糖醛酸、果胶以及苹果渣、麻纤维、玉米秸秆及水稻秸秆中果胶质成分的应用,尤其是在麻纤维脱胶和天然秸秆降解过程中的应用。
本发明的技术方案:
为实现上述发明目的,本发明采用以下技术方案:
(1)包含上述高温碱性果胶酸裂解酶Pel-863编码基因的工程菌的构建
提取热解纤维素菌Caldicellulosiruptor kronotskyensis的基因组,设计引物扩增Pel-863。得到的目的基因片段和载体经处理后,连接转化宿主细胞。筛选阳性克隆并测序,然后对测序正确的工程菌提取重组质粒,获得重组表达载体。所述表达载体,是指大肠杆菌表达载体、乳酸菌表达载体、酵母菌表达载体、枯草杆菌表达载体及丝状真菌表达载体等,优选为将本发明的高温碱性果胶酸裂解酶基因与线性质粒pET-28b相连接,得到的重组表达质粒pET-28b-Pel-863。将重组表达质粒转化宿主细胞,获得含高温碱性果胶酸裂解酶Pel-863基因的重组菌。所述菌株为大肠杆菌(如Escherichia coli BL 21(DE3)、E.coli Top10、E.coli Rosetta(DE3)等)、乳酸菌(如Lactococcuslactis等)、酵母菌(如Pichiapastoris、Saccharomycescerevisiae等)、枯草杆菌(如Bacillus subtilis BS168等)和丝状真菌(如Trichodermareesei、Aspergillusniger等),优选为大肠杆菌E.coli Rosetta(DE3)。
(2)制备高温碱性果胶酸裂解酶Pel-863
培养上述重组菌株,进行诱导表达。收集菌体,超声破碎,60℃热失活,收集上清,利用亲和层析以及凝胶过滤层析纯化重组蛋白。最后利用SDS-PAGE电泳检测目的蛋白的表达和纯化情况。
(3)高温碱性果胶酸裂解酶Pel-863的酶学性质测定
测定Pel-863的最适pH、pH稳定性、最适温度、热稳定性、不同金属离子和化学试剂对Pel-863活性的影响以及其动力学参数。
(4)高温碱性果胶酸裂解酶在降解苹果渣、麻纤维、玉米秸秆及水稻秸秆中果胶质成分的应用。
称取一定量粉碎、水洗并干燥的苹果渣、麻纤维、玉米秸秆及水稻秸秆原料,加入一定量的重组果胶酸裂解酶Pel-863酶液,于70℃、pH9.0条件下振荡反应过夜。离心取上清液通过紫外分光光度法测定生成的不饱和寡聚半乳糖醛酸含量并用用薄层层析法定性分析苹果渣、麻纤维、玉米秸秆及水稻秸秆原料酶解液的成分。
附图说明
图1重组高温碱性果胶酸裂解酶Pel-863的凝胶过滤层析(A)及各收集组分的SDS-PAGE电泳分析(B)。
图2重组高温碱性果胶酸裂解酶Pel-863的最适pH(A),pH稳定性(B),最适温度(C),热稳定性(D)以及新提取酶的米氏方程动力学(E)和贮存酶的变构动力学(F)。
图3不同金属离子和化学试剂(A)及不同钙离子浓度(B)对重组高温碱性果胶酸裂解酶Pel-863活性的影响。
图4重组高温碱性果胶酸裂解酶Pel-863对不同底物的降解及TLC分析。
图5不同处理下的麻纤维的红外光谱图(A)和扫描电镜图(B:对照组1即未处理纤维,C:对照组2即仅使用缓冲液处理,D:实验组1即Pel-863处理,E:实验组2即1%NaOH处理)。
图6重组高温碱性果胶酸裂解酶Pel-863对玉米秸秆(A)和水稻秸秆(B)糖化的促进作用。
具体实施方式
实施例1:含高温碱性果胶酸裂解酶Pel-863基因的工程菌构建
1.1C.kronotskyensis基因组DNA的提取
使用细菌基因组DNA提取试剂盒(Tiangen Biotech)提取热解纤维素菌C.kronotskyensis的基因组DNA,-20℃冻存备用。
1.2Pel-863基因及载体pET-28b的PCR扩增
根据已发表的C.kronotskyensis基因组信息及其注释,设计扩增果胶酸裂解酶基因的引物如下:
Calkro_0863-F 5'-GCCGCGCGGCAGCATGGCGACACTTTTAACA-3'
Calkro_0863-R 5'-GCGGCCGCAAGCGTTTAGTATTGATGTATCTGTG-3'
以提取的基因组DNA为模板,进行目的基因的PCR扩增。
载体pET-28b经Hind III和Nhe I双酶切过夜后进行琼脂糖凝胶电泳,使用普通琼脂糖凝胶DNA回收试剂盒回收大小约为5.3kb的片段。以所回收片段为模板,进行质粒PCR扩增。
PCR反应产物均进行琼脂糖凝胶电泳检测,目的片段使用普通琼脂糖凝胶DNA回收试剂盒回收。
1.3果胶酸裂解酶基因与载体的处理和连接
回收得到的Pel-863基因片段和质粒pET-28b片段用T4DNA Polymerase处理后,22℃连接20min,获得重组表达载体pET-28b-Pel-863。
1.4重组质粒pET-28b-Pel-863转化大肠杆菌Top10
取重组质粒1-3μl加入到100μl Top10感受态细胞中,冰浴30min。然后42℃热激60s后立即冰浴2min。加入500μl LB液体培养基,于37℃下200rpm培养1h。取菌液低速离心弃部分上清,将剩余菌液全部涂布于含有50μg/mL卡那霉素的LB平板上,于37℃培养过夜。
1.5重组质粒的鉴定和提取
挑选转化成功的单菌落过夜培养进行菌液PCR鉴定,经琼脂糖凝胶电泳检测确定为阳性克隆的菌液由生工测序公司测序。测序显示正确后使用质粒小提试剂盒提取质粒。
1.6重组质粒转化大肠杆菌Rosetta(DE3)
按1.4所述方法将重组质粒转化大肠杆菌Rosetta(DE3)感受态细胞中,于含有50μg/mL卡那霉素的LB平板上37℃培养12-16h获得单菌落。挑取3-5个单克隆于5ml含有50μg/mL卡那霉素LB液体培养基中培养过夜,加入终浓度为15-20%的甘油,于-80℃保存菌种。至此获得以pET-28b为载体,以大肠杆菌Rosetta(DE3)为宿主菌构建的含高温碱性果胶酸裂解酶Pel-863基因的工程菌。
实施例2:重组高温碱性果胶酸裂解酶Pel-863的诱导表达和纯化
将实施例1中得到的工程菌培养于含50μg/mL卡那霉素的LB液体培养基中,37℃200rpm振荡培养至OD600达到0.6左右时,加入IPTG至终浓度0.1mM,37℃200rpm振荡培养4-6h。4000g离心15min收集菌体,重悬于Binding Buffer(50mM Tris-HCl,pH7.5,300mMNaCl)中,于冰浴中超声破碎。而后4℃10000g离心15min去除细胞碎片,所得上清即为粗酶液。
将粗酶液于60℃加热处理30min去除热不稳定蛋白,4℃10000g离心15min留上清。600μL Ni-NAT亲和层析柱用6mL Binding Buffer平衡后,将上清过柱3-5次。过柱后先用6mlBinding Buffer洗涤,再用400μL Elution Buffer(50mM Tris-HCl,pH 7.5,300mMNaCl,150mM咪唑)洗脱,收集洗脱的样品液。将所得样品液使用透析袋去除咪唑并换成酶液贮存缓冲液(50mM Tris-HCl pH 7.0,300mM NaCl)。30mL Superdex 200葡聚糖凝胶柱(GE Healthcare)预先用酶液贮存缓冲液平衡后,将样品液过柱,在出现波峰时收集蛋白。取各个操作时期的样品进行SDS-PAGE电泳检测目的蛋白的表达和纯化情况。凝胶过滤层析谱图和SDS-PAGE电泳图显示,天然Pel-863以单体和同源三聚体两种状态存在(图1)。
实施例3:重组高温碱性果胶酸裂解酶Pel-863的酶学性质测定
3.1高温碱性果胶酸裂解酶Pel-863活性测定
采用紫外分光光度法对果胶酸裂解酶Pel-863活性进行分析:1.5mL反应管中含80μLpH7.0,2%(W/V)PGA-NaOH溶液,320μLPel-863缓冲液(50mM甘氨酸-氢氧化钠,pH9.0,150mM NaCl,1.5mM CaCl2),加入10μL适当稀释的纯化后酶液,70℃反应10min,加入400μL50mM HCl终止反应,离心取上清于235nm处测定紫外吸收值。以未加入酶液的一组为对照。1个酶活单位(U)定义为在给定条件下每分钟产生1μmol不饱和寡聚半乳糖醛酸的酶量,其中不饱和寡聚半乳糖醛酸的摩尔吸光系数ε235为4075M-1cm-1。
3.2高温碱性果胶酸裂解酶Pel-863最适pH和pH稳定性的测定
Pel-863的最适pH的测定为在不同pH缓冲条件下(pH7.8-8.5为Tris-HCl缓冲液;pH9.0-10.5为甘氨酸-氢氧化钠缓冲液)进行酶促反应。Pel-863于上述各种不同pH的缓冲液中室温放置36h,然后于最适pH下测定酶活以研究其pH稳定性。结果显示,Pel-863的最适pH为9.0,在pH9.5时保持93%以上活性(图2A)。Pel-863的最适储存pH为7.0,在pH7.0-8.5范围内室温放置36h后剩余酶活在85%以上(图2B)。
3.3高温碱性果胶酸裂解酶Pel-863最适温度和热稳定性的测定
Pel-863的最适温度的测定为在甘氨酸-氢氧化钠缓冲液(pH9.0)及不同温度下进行酶促反应。热稳定性为将Pel-863在60℃,65℃,70℃及75℃下保温不同时间,再于最适温度下进行酶活测定。结果显示,Pel-863的最适温度为70℃,75℃时保持90%以上的活性(图2C),在60℃温育8h,酶活力保留70%以上(图2D)。
3.4高温碱性果胶酸裂解酶Pel-863动力学参数的测定
Pel-863动力学参数Km和Vmax是通过在不同PGA浓度(0.1-2g/L)下收集酶活数据然后应用Graph Pad Prism 6软件中的米氏方程拟合得到的。拟合结果显示,新提取的高温碱性果胶酸裂解酶Pel-863以多聚半乳糖醛酸为底物时的Km值为0.5942g/L,Vmax为172.8U/mg(图2E)。于4℃放置约1个月的Pel-863呈现变构酶的动力学性质,Khalf值变为0.3594g/L,Vmax值下降为80.88U/mg(图2F)
3.5不同金属离子及化学试剂对高温碱性果胶酸裂解酶Pel-863活性影响的测定
在酶促反应体系中加入不同金属离子及化学试剂来研究其对Pel-863酶活的影响。其中各金属离子终浓度为1mM,化学试剂终浓度为0.1%(W/V)并加入终浓度为1mM的钙离子。此外,不同钙离子浓度(0.5-5mM)对Pel-863活性的影响也被研究。结果显示,Ca2+是该酶必不可少的激活剂,Fe3+对其有部分激活作用,K+,Cu2+,Zn2+,Mg2+,Ni2+,Mn2+对其没有激活作用;EDTA可完全抑制其活性,0.1%的SDS便可使其丧失50%左右的活性(图3A)。在以多聚半乳糖醛酸为底物时,兼顾酶活性和反应液流动性,较为适宜的Ca2+浓度为3-5mM(图3B)。
3.6高温碱性果胶酸裂解酶Pel-863对天然底物中果胶质的降解作用
Pel-863分别以2%PGA、果胶、麻纤维、苹果渣、玉米秸秆和水稻秸秆为底物,在70℃,pH9.0甘氨酸-氢氧化钠缓冲条件(含5mM Ca2+)下酶解过夜,生成的不饱和寡聚半乳糖醛酸通过测定OD235nm及薄层层析(TLC)来确定,结果如图4所示。Pel-863作用PGA生成不饱和半乳糖醛酸单体和二聚体;Pel-863对苹果渣、麻纤维、玉米秸秆及水稻秸秆中果胶质成分的降解均优于对果胶的降解,尤其是苹果渣,产生的不饱和寡聚半乳糖醛酸浓度达到2mM。
实施例4:重组高温碱性果胶酸裂解酶Pel-863应用于麻纤维脱胶
Pel-863对麻纤维的脱胶效果是通过与1%NaOH处理对比研究实现的。称取0.015g粉碎、水洗并干燥的麻纤维,实验组1加入750μL pH9.0甘氨酸-氢氧化钠缓冲液(含5mM Ca2+)及12μg/mg纤维的Pel-863,实验组2加入750μL 1%NaOH溶液,对照组1不加任何试剂,对照组2仅加入750μL pH9.0甘氨酸-氢氧化钠缓冲液(含5mM Ca2+),所有样品于70℃振荡反应过夜。处理后的纤维经水洗干燥后进行红外光谱和扫描电镜研究,结果如图5所示。Pel-863处理的纤维与1%NaOH处理的纤维基本达到一致(图5A),而且,Pel-863脱胶过程明显降低了对麻纤维的损害(图5D;图5E)
实施例5:重组高温碱性果胶酸裂解酶Pel-863应用于玉米和水稻秸秆降解
按照实施例4中实验组1的加入比例对玉米秸秆和水稻秸秆进行预降解,反应液测定OD235nm后于85℃烘箱中蒸干水分,而后加入750μL pH5.0的醋酸-醋酸钠缓冲液,实验组再加入比例为7.5μg/mg秸秆的诺维信酶Cellic CTec2,于50℃下振荡反应过夜,使用高效液相色谱(HPLC)分析其还原糖产生量,结果如图6所示。对玉米秸秆来说,Pel-863预降解使葡萄糖产生量增加了14.2%,木糖产生量增加了311.6%(图6A);对水稻秸秆来说,Pel-863预降解使葡萄糖产生量增加了6.5%,木糖产生量增加了55%(图6B)。总之,高温碱性果胶酸裂解酶Pel-863可明显提高玉米和水稻秸秆的降解糖化率,尤其是木糖产生量。
Claims (7)
1.一种高温碱性果胶酸裂解酶Pel-863,其编码基因的核苷酸序列如SEQ ID NO.1或SEQ IDNO.2所示。
2.权利要求1所述高温碱性果胶酸裂解酶Pel-863,其氨基酸序列如SEQ ID NO.3或SEQ IDNO.4所示。
3.权利要求2所述的高温碱性果胶酸裂解酶Pel-863,其氨基酸序列SEQ ID NO.3或SEQ IDNO.4中的氨基酸经过一个或多个氨基酸残基的取代、缺失及添加形成的具有果胶酸裂解酶活性的衍生蛋白质。
4.含有权利要求1所述的高温碱性果胶酸裂解酶Pel-863的编码基因的重组表达体系,其特征在于:
1)扩增权利要求1所述的高温碱性果胶酸裂解酶Pel-863的编码基因所使用的引物为:
Calkro_0863-F 5'-GCCGCGCGGCAGCATGGCGACACT TTTAACA-3'
Calkro_0863-R 5'-GCGGCCGCAAGCGTTTAGTATTGATGTATCTGTG-3'
2)异源表达权利要求2所述的高温碱性果胶酸裂解酶Pel-863所用的重组载体包括大肠杆菌表达载体、乳酸菌表达载体、酵母菌表达载体、枯草杆菌表达载体及丝状真菌表达载体等,所对应使用的重组表达菌株为大肠杆菌(如Escherichia coli BL 21(DE3)、E.coli Top10、E.coli Rosetta(DE3)等)、乳酸菌(如Lactococcuslactis等)、酵母菌(如Pichiapastoris、Saccharomyces cerevisiae等)、枯草杆菌(如Bacillus subtilis BS168等)和丝状真菌(如Trichodermareesei、Aspergillusniger等)。
5.一种制备高温碱性果胶酸裂解Pel-863的基因工程方法,其特征在于:
1)使用权利要求4中2)所述的重组载体转化宿主细胞,得到重组菌株;
2)培养所得到的重组菌株,诱导高温碱性果胶酸裂解酶的表达;
3)回收和纯化所表达的高温碱性果胶酸裂解酶Pel-863。
6.权利要求2所述的高温碱性果胶酸裂解酶Pel-863在降解PGA、果胶以及麻纤维、苹果渣、天然秸秆等生物质中胶质成分中的应用,其酶解的最佳温度和pH分别为70-75℃和pH9.0-9.5,所需的Ca2+适宜浓度为3-5mM。
7.权利要求2所述的高温碱性果胶酸裂解酶Pel-863在促进秸秆等生物质降解糖化中的应用,Pel-863预处理天然玉米和水稻秸秆,使其后期在商业纤维素酶作用中的葡萄糖产生量增加10%左右,木糖产生量增加55-300%左右。
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CN115948929B (zh) * | 2022-09-15 | 2024-01-19 | 华南理工大学 | 一种果胶裂解酶处理再生浆胶黏物的方法及应用 |
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