动物营养学报  2013, Vol. 25 Issue (10): 2202-2211   PDF (1606 KB)    
木聚糖酶基因的体外定向进化
杜文, 王谦, 王佳堃 , 刘建新    
浙江大学奶业科学研究所, 动物分子营养学教育部重点实验室, 杭州 310058
摘要:木聚糖酶在天然材料中基因表达水平低、活性差、生产成本高,严重限制了它的推广应用。宏基因组学通过免培养技术,研究生境中全部微小生物遗传物质的总和,在开发微生物酶资源上具有强劲的优势。体外定向进化技术模拟达尔文的自然进化论,利用基因的突变和重组,从体外改造酶基因,产生基因多样性,并结合定向的筛选最终获得预期性状的进化酶。宏基因组技术与体外定向进化技术结合必将加速木聚糖酶的开发和产业化应用,推动半纤维类生物能源的利用。为此,本文就木聚糖酶基因的来源、宏基因组学在木聚糖酶基因开发中的应用、体外定向进化进行了系统的综述。
关键词木聚糖酶基因     宏基因组学     体外定向进化    
Directed Evolution of Xylanase Genes in Vitro
DU Wen, WANG Qian, WANG Jiakun , LIU Jianxin    
Key Laboratory of Molecular Animal Nutrition of Ministry of Education, Institute of Dairy Science, Zhejiang University, HangZhou 310058, China
Abstract: The application of xylanases is limited by certain factors, such as low gene expression level, low enzyme activity and high cost. Metagenomics study the genetic materials of communities of microbial organisms in their natural environments by culture-independent technology, which showd great potential in exploring the resource of enzymes. Directed evolution, which simulates natural evolution of Darwin, reforms enzyme genes and creats gene diversity by gene mutation and recombination in vitro, and gets superior enzymes with expected properties by directed screening. The combination of metagenomics and directed evolution will accelerate the exploitation and industrial application of xylanases, promote the utilization of hemicellulose energy. Therefore, the sources of xylanase genes, the application of metagenomics in exploiting xylanases and directed evolution in vitro were reviewed in this article. [Chinese Journal of Animal Nutrition, 2013, 25(10):2202-2211]
Key words: xylanase genes     metagenomics     directed evolution in vitro    
木聚糖是D-木糖通过β-1,4-木糖苷键连接而成的一种多聚五碳糖,是植物细胞壁中常见的半纤维素多糖,占植物碳水化合物总量的1/3,含量仅次于纤维素,是自然界中第2丰富的可利用资源[1]。在饲料工业中,由于单胃动物缺少降解半纤维素的酶,因此木聚糖对单胃动物几乎没有营养作用,而且没有消化的纤维物质会增加食物的黏性,干扰消化酶的作用及营养的吸收[2];反刍动物由于瘤胃微生物的存在,对木聚糖具有一定的降解能力[3],但是,实际生产中仍需要补充外源酶制剂,进一步提高其对粗饲料的消化率[4, 5]。木聚糖酶是能专一水解木聚糖为主体的低聚木糖和D-木糖的一类糖苷水解酶的总称[6],主要包括:β-D-1,4-内切木聚糖酶(endo-1,4-β-D-xylanxylanohydrolase,E.C.3.2.1.8)、β-木糖苷酶(β-xylosidase,E.C.3.2.1.37)和β-1,4-外切木聚糖酶(E.C.3.2.1.7)。木聚糖酶在天然材料中基因表达水平低、活性差、生产成本高,严重限制了它的推广应用[5]。为了满足饲用酶制剂的生产需要、开发半纤维类生物能源,利用体外定向进化技术改良木聚糖酶基因,提高木聚糖酶的催化活性和热稳定性,以获得高活性高产量的木聚糖酶就成了目前研究的热点。

1 木聚糖酶基因的来源

自1983年Bernier等[7]从枯草芽孢杆菌(Bacillus subtilis)中分离得到木聚糖酶基因,到目前为止,国内外已报道了300余种不同菌株来源的木聚糖酶基因,其中近百种基因被克隆和表达在合适的宿主中[2, 8]。不同来源的木聚糖酶,酶学性质差别较大(表1)[9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25]。细菌木聚糖酶的分子质量在22.5~60.0 ku,最适温度范围为40~75 ℃,最适pH范围为6.0~8.0,其中以7.0居多;热稳定性在40 ℃附近较好,pH稳定性范围为4.5~11.0,主要集中于6.0~9.0。一些放线菌也能产生木聚糖酶,如橄榄绿链霉菌A1(Streptomyces olivaceoviridis A1)[15]和耐盐高温双歧菌YIM 90462T(Thermobifida halotolerans YIM 90462T)[16],其酶分子质量分别为20.8和34.0 ku,最适温度分别为60和90 ℃,最适pH分别为5.2和9.0;热稳定性在60 ℃附近较好,pH稳定性范围分别为4.0~8.8和7.0~8.0。真菌木聚糖酶的酶分子质量在20.0~31.6 ku,最适温度主要集中于50 ℃(50~65 ℃),最适pH范围为3.0~6.0;热稳定性范围为40~60 ℃,平均在50 ℃左右,pH稳定性范围为2.5~10.0,主要集中于3.5~7.5。在木聚糖酶产量方面,真菌与细菌相比具有明显的优势[26];各类产木聚糖酶微生物里都有木聚糖酶活性较高的代表,如细菌里的中度嗜盐菌AX2000(Bacillus alcalophilus AX2000)[12], 它产生的木聚糖酶活性高达25 000 U/mg;放线菌里的橄榄绿链霉菌A1[15],它产生的木聚糖酶活性高达15 000 U/mL;真菌里的青霉菌Pol6(Penicillium occitanis Pol6)[23]和黑曲霉BCC14405(Aspergillus niger BCC14405)[21],它们产生的木聚糖酶活性分别为8 549.85和8 007 U/mg。

表1 不同微生物木聚糖酶基因的克隆表达及酶学特性 Table 1 Cloning and expression of different microbial xylanase genes and their enzymology characterization

2 宏基因组学在木聚糖酶基因开发上的应用

随着分子生物学技术的发展,越来越多的产木聚糖酶微生物被发现,然而环境中还有很多微生物资源不能通过纯培养技术分离得到,近年来发展起来的宏基因组技术解决了这一难题,它通过免培养技术,研究生境中全部微生物遗传物质的总和,挖掘未培养微生物的基因资源[27],在木聚糖酶基因的开发上显示出了巨大的潜力。表2[28, 29, 30, 31, 32, 33, 34, 35, 36, 37]列举了2008—2013年间以环境样本构建宏基因组文库筛选到的木聚糖酶,样品来源于反刍动物瘤胃[29, 31, 32, 34, 35, 36]、土壤[28, 33, 37]和堆肥[30, 37],其中以反刍动物瘤胃居多。常用的文库载体是质粒BAC[29, 32]和FOS[30, 33, 34, 35, 36],宿主是大肠杆菌[28, 29, 30, 31, 32, 33, 34, 35, 36, 37]。文库平均插入片段在5.5~54.5 kb之间,筛选到的克隆数大都在10 000以上,木聚糖酶阳性率最高可达0.14%。

表2 利用宏基因组文库筛选到的木聚糖酶 Table 2 Xylanases screening from the metagenomic library

3 木聚糖酶基因的体外定向进化

蛋白质工程技术,包含蛋白质的理性设计和非理性设计2种方法。传统的理性设计需要预先知道蛋白质的结构、活性位点、催化机制等信息,在清楚结构与功能的前提下,定点突变改变蛋白质分子中个别氨基酸残基,产生新性状的蛋白质[38]。然而,在实际应用中,蛋白质的结构信息很难获取,结构与功能的关系异常复杂,因此理性设计具有很强的限制性[39]。蛋白质的非理性设计,即体外定向进化,它模拟达尔文的自然进化论,利用基因的突变和重组,从体外改造酶的基因,产生基因多样性,并结合定向的筛选最终获得预期性质的进化酶,因它不需要预先知道蛋白质的三维结构信息,弥补了理性设计的不足[40]。它主要包括突变文库的构建、功能表达和文库筛选(选择)3个步骤[41],其核心是突变文库的构建和文库筛选(图1)。

图1 酶定向进化的突变文库构建(A)和高通量文库筛选(B)策略 Fig.1 Strategies for the directed evolution of enzymes involving generation of variant gene libraries (A) and high-throughput screening of libraries (B)[42]

3.1 木聚糖酶基因突变文库的构建

突变文库的库容及多样性是酶分子体外定向进化的基础。构建突变文库的方法有很多,如易错PCR(error-prone PCR)、DNA重排(DNA shuffling),以及基于DNA重排的原理,围绕基因片段重组这一思想产生的交错延伸法(staggered extension process,StEP)、随机引物体外重组(random-priming in vitro recombination,RPR)、退火低核苷酸基因重排(degenerate oligonucleotide gene shuffling,DOGS)、外显子重组(exon shuffling)、酵母增强组合文库(combinatorial libraries enhanced by recombination in yeast,CLERY)、随机片段交换法(random insertional-deletional strand exchange mutagenesis,RAISE)等[38, 40],常用的是易错PCR和DNA重排(表3)[43, 44, 45, 46, 47, 48, 49, 50]

表3 定向进化技术成功改良的微生物木聚糖酶 Table 3 Successfully optimized microbial xylanases by directed evolution

易错PCR是定向进化最早采用的一种建库方法,由Leung等[51]提出,后经Cadwell等[52]改良,其原理是在体外扩增目的基因时改变PCR的条件,使碱基产生错配,导致目的基因随机突变。Stephens等[43]用易错PCR产生基因突变,改良1个来自疏棉状嗜热丝孢菌(Thermomyces lanuginosus)的耐热木聚糖酶基因XynA,以提高木聚糖酶热稳定性,其最优突变体酶2B7-10发生1处点突变(Y58F),该突变使其在80 ℃孵育60 min还能保持71%的活性,远高于亲本酶XynA。McHunu等[44]利用易错PCR提高另一个来自疏棉状嗜热丝孢菌(Thermomyces lanuginosus)的木聚糖酶的耐碱性,其突变体酶在60 ℃,pH 10.0的碱性条件下孵育60 min,还能保持84%的活性,而亲本酶同样处理后仅剩22%的活性。Wang等[45]利用易错PCR提高1株杂合木聚糖酶ATx的催化活性,1处氨基酸替换(L49P)使突变体酶的催化活性提高。易错PCR的原理简单,操作简便,对亲本基因的限制条件不多,而且可以和其他突变方法结合使用,因此应用十分广泛,但该方法属于无性突变,遗传只发生在单一分子内部,一般适用于较小的基因片段(<800 bp)[40]

随着人们对酶的进化期望越来越大,定向进化技术也在不断的成熟和发展。1994年,Stemmer[53, 54]提出DNA重排(DNA shuffling)并成功运用,为体外定向进化技术的飞跃做出了巨大贡献。其原理是用脱氧核糖核酸酶Ⅰ(DNaseⅠ)切割一组含有不同点突变的基因片段,产生不同大小的随机片段,这些片段再经重新组合、扩增形成全长的基因,实现不同基因片段的重组。其优势是可以有效积累有益突变,排除有害和中性突变,同时也能实现蛋白质多种特性的共进化[55]。在木聚糖酶基因定向进化研究上,也有不少应用该技术的例子。Xia等[46]利用DNA重排改良1个来自变铅青链霉菌(Streptomyces lividans)的木聚糖酶B的热稳定性和耐碱性,得到的最优突变体酶在70 ℃可以维持活性时长达6 h,而亲本酶处理3 min后就丧失了50%的活性,此外,突变体酶在pH 9.0的条件下稳定性增加,这些都显示了突变体酶在热稳定性和耐碱性上的极大优势。

DNA重排的前提是存在1组含有不同点突变的基因片段,因而常将DNA重排和易错PCR等进化方法结合使用。Miyazaki等[47]将易错PCR、饱和诱变、DNA重排3种方法结合改良1个来自枯草芽孢杆菌的木聚糖酶的热稳定性,最终得到1个含有3个氨基酸替换的优势突变体Xylst,该突变使其热稳定性显著增加,在60 ℃条件下,突变体酶活性可维持2 h,然而亲本型5 min内就失去了活性。Zhang等[48]利用易错PCR和基于DNA重排的家族重排(family shuffling)技术,与蛋白质半理性设计相结合改良1个来源于嗜热脂肪芽孢杆菌(Geobacillus stearothermophilus)的木聚糖酶XT6的热稳定性,稳定性最佳的突变体酶含有13个氨基酸的替换,该替换导致其半衰期是亲本型的52倍,最适温度从77 ℃上升至87 ℃,催化效率提高90%。

3.2 突变文库筛选

木聚糖酶基因突变文库构建之后,确定一个高通量、高选择性、高灵敏度的筛选方法是快速成功地从庞大的突变库中筛选到目的产物的重要保证[56]。目前常用的筛选方法有平板筛选和基于荧光或显色反应的筛选2种。其中平板筛选最为简便,针对木聚糖酶的平板筛选有RBB-xylan法和刚果红染色法,它是基于木聚糖酶的催化活性和底物分解前后性质的改变,在固体平板中加入木聚糖底物,将克隆点种在相应的选择平板上,宿主菌表达木聚糖酶水解木聚糖形成透明圈,根据透明圈的有无、大小,初步确定是否阳性克隆或优势突变体。但这种筛选局限于底物特异性、酶活性等突变方向的筛选,并且在筛选酶活高的突变体时,也只能作为初步的筛选。对于酶的最适pH、最适温度、稳定性等突变方向,仍需要以可测定的酶促反应结果来筛选,即基于荧光或显色反应的方法,该方法也用于复筛酶活提高的突变体,常用的如3,5-二硝基水杨酸(3,5-dinitrosalicylic acid,DNS)法,它的原理是DNS与木聚糖酶水解木聚糖后产生的还原糖发生氧化还原反应,产物在煮沸条件下显棕红色,且在一定范围内颜色深浅与还原糖含量成比例关系,利用比色法测定还原糖含量,以达到筛选突变体的目的。该方法需要结合96孔板、酶标仪等设备以提高筛选效率[57]

4 小 结

不同生物来源的木聚糖酶,其酶学性质差别较大,各类产木聚糖酶微生物里都有酶活较高的代表。宏基因组技术在开发环境木聚糖酶基因上显示了巨大的潜力。随着体外定向进化技术的进一步发展和完善,酶的突变、重组、筛选等过程进一步改进,木聚糖酶基因的改良将得到更快的发展。宏基因组技术和体外定向进化技术相结合必将加速木聚糖酶基因的开发和产业化应用,推动半纤维类生物能源的利用。

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