生物工程学报  2019, Vol. 35 Issue (7): 1234-1246
http://dx.doi.org/10.13345/j.cjb.180464
中国科学院微生物研究所、中国微生物学会主办
0

文章信息

钮成拓, 李昕玥, 许鑫, 包敏, 李永仙, 刘春凤, 郑飞云, 王金晶, 李崎
Niu Chengtuo, Li Xinyue, Xu Xin, Bao Min, Li Yongxian, Liu Chunfeng, Zheng Feiyun, Wang Jinjing, Li Qi
微生物1, 3-1, 4-β-葡聚糖酶蛋白质改造及工业应用研究进展
Research progresses in microbial 1, 3-1, 4-β-glucanase: protein engineering and industrial applications
生物工程学报, 2019, 35(7): 1234-1246
Chinese Journal of Biotechnology, 2019, 35(7): 1234-1246
10.13345/j.cjb.180464

文章历史

Received: November 9, 2018
Accepted: January 24, 2019
微生物1, 3-1, 4-β-葡聚糖酶蛋白质改造及工业应用研究进展
钮成拓1,2 , 李昕玥2 , 许鑫1,2 , 包敏1,2 , 李永仙1,2 , 刘春凤1,2 , 郑飞云1,2 , 王金晶1,2 , 李崎1,2,3     
1. 江南大学 生物工程学院 工业生物技术教育部重点实验室,江苏 无锡 214122;
2. 江南大学 生物工程学院,江苏 无锡 214122;
3. 江南大学 食品安全与营养协同创新中心,江苏 无锡 214122
摘要:1, 3-1, 4-β-葡聚糖酶(E.C.3.2.1.73)是一种重要的工业用酶,其可以通过特异性切割毗邻β-1, 3-糖苷键的β-1, 4-糖苷键将β-葡聚糖或地衣多糖降解为纤维三糖和纤维四糖。微生物β-葡聚糖酶属于糖苷水解酶家族16,其三维结构为卷心蛋糕状的逆向β-片层结构。文中综述了近些年来β-葡聚糖酶在工业上的应用情况及酶蛋白质工程改造的研究进展,并对其研究前景进行了展望。
关键词1, 3-1, 4-β-葡聚糖酶    工业应用    蛋白质改造    稳定性    催化性质    
Research progresses in microbial 1, 3-1, 4-β-glucanase: protein engineering and industrial applications
Chengtuo Niu1,2 , Xinyue Li2 , Xin Xu1,2 , Min Bao1,2 , Yongxian Li1,2 , Chunfeng Liu1,2 , Feiyun Zheng1,2 , Jinjing Wang1,2 , Qi Li1,2,3     
1. Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China;
2. School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China;
3. Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, Jiangsu, China
Abstract: 1, 3-1, 4-β-glucanase (E.C.3.2.1.73) is an important industrial enzyme which cleave β-glucans into oligosaccharides through strictly cutting the β-1, 4 glycosidic bonds in 3-O-substituted glucopyranose units. Microbial 1, 3-1, 4-β-glucanase belongs to retaining glycosyl hydrolases of family 16 with a jellyroll β-sandwich fold structure. The present paper reviews the industrial application and protein engineering of microbial β-glucanases in the last decades and forecasts the research prospects of microbial β-glucanases.
Keywords: 1, 3-1, 4-β-glucanase    industrial application    protein engineering    stability    catalytic property    

生物质资源是地球上最丰富的资源之一,其主要以纤维素和半纤维素等多糖形式存在[1]。目前认为针对多糖的有效降解主要有如下两个手段,一为加速纤维素和半纤维素的释放,二为将多糖快速水解为寡糖[2-4]。糖苷水解酶是降解多糖的主要酶系,决定了其在工业上具有广泛的应用价值,例如在食品行业(果汁行业、大豆产品开发、啤酒行业和酒类产品)、饲料行业、农业工业副产品加工(生产生物燃料等)和农副产品废品降解等[5]

β-葡聚糖是自然界多糖的重要组成部分,其是禾本科植物(特别是高价值经济作物,如大麦、黑麦、高粱、大米和小麦)细胞壁的重要多糖组分[6]。β-葡聚糖是多达1 200个β-D-葡萄糖残基通过1, 3-β-糖苷键和1, 4-β-糖苷键连接形成的线性多糖(其中β-1, 3糖苷键的比例占25%–30%),其结构与冰岛地衣Cetraria islandica地衣多糖相似[7]。β-葡聚糖可分为水溶性β-葡聚糖和水不溶性β-葡聚糖,其中水溶性β-葡聚糖主要存在于谷物完整胚乳细胞壁中,而水不溶性β-葡聚糖则与蛋白质结合存在于谷物胚乳细胞间[6, 8]。水溶性β-葡聚糖溶于水后具有分子量大和黏度大等特点,给工业生产带来很多困难[6]。目前根据切割糖苷键类型的不同将降解β-葡聚糖的糖苷水解酶分为以下4种:1, 4-β-葡聚糖酶(EC 3.2.1.4)、1, 3-β-葡聚糖酶(EC 3.2.1.39)、1, 3(4)-β-葡聚糖酶(EC 3.2.1.6)和1, 3-1, 4-β-葡聚糖酶(EC 3.2.1.73)。在4种β-葡聚糖降解酶中,1, 3-1, 4-β-葡聚糖酶(下称β-葡聚糖酶)的催化活性最高,因此具有最广泛的应用价值。β-葡聚糖酶通过特异性切割1, 3-β-糖苷键毗邻的1, 4-β-糖苷键将β-葡聚糖或地衣多糖降解为以纤维三糖和纤维四糖为主的低聚葡萄糖[9],因此可以消除β-葡聚糖黏度高的特点,在以谷物为主要原料的行业中有重要应用价值[6, 10]。基于此作用,β-葡聚糖酶作为半纤维素酶系的重要组成部分在生物质能源的生产过程中同样可以与其他酶系协同作用将木质纤维素降解为可发酵性糖[11-12]或生物燃料[13]。另一方面,β-葡聚糖酶降解β-葡聚糖产生的低聚葡萄糖作为益生元对人体具有有益的性质[14-16]

β-葡聚糖酶主要来源于高等植物和微生物,然而植物酶和微生物酶的结构特点完全不同。植物β-葡聚糖酶属于糖苷水解酶家族17,为(β/α)8桶状结构[6, 17];而微生物β-葡聚糖酶属于糖苷水解酶家族16,为三明治夹心结构[18]。微生物β-葡聚糖酶的热稳定性和催化活性均优于植物来源酶[10],然而目前筛选得到微生物β-葡聚糖酶的热稳定性、pH耐受性和催化活性依旧不能满足工业上的应用要求。近年来通过定点突变等蛋白质工程改造手段对β-葡聚糖酶进行改造已经取得一定成效,部分酶在工业上取得了良好的应用效果。在之前研究中,Planas[10]和孙军涛等[19]分别对β-葡聚糖酶的结构、催化机制、理化性质和部分酶学性质改造的工作进行了综述,而钮成拓等[20]对β-葡聚糖酶催化活性提高的方法和成果进行了总结。文中总结了本实验室和其他研究团队对β-葡聚糖酶的研究工作,主要对微生物β-葡聚糖酶的工业应用情况和蛋白质工程改造进行了综述,旨在为高效β-葡聚糖酶制剂的构建提供参考。

1 β-葡聚糖酶的工业应用

β-葡聚糖在工业应用上会造成不利影响,同时也是一种重要的非淀粉多糖。基于β-葡聚糖酶的作用方式和产物特点,β-葡聚糖酶主要在3类工业中具有应用价值。在第一类应用领域中,β-葡聚糖的存在不利于工业操作,而β-葡聚糖酶通过降解β-葡聚糖消除其不利作用从而促进工业生产,例如啤酒和饲料行业。在第二类应用领域中,以通过β-葡聚糖酶降解作用得到的低聚葡萄糖作为主要目标产物,例如益生元行业。在第三类应用领域中,以β-葡聚糖酶降解得到的低聚葡萄糖作为底物供进一步生物技术加工,如生物乙醇和生物柴油行业。

1.1 酒类行业

在啤酒酿造过程中,麦芽内源β-葡聚糖酶在麦芽烘焙和麦汁糖化过程失去活力,大量β-葡聚糖残留在麦汁中,导致麦汁黏度过高,且其会与麦汁中高分子量蛋白质结合压紧麦糟,使麦汁过滤困难[6, 21-22]。在啤酒发酵过程中,β-葡聚糖无法被酵母利用且会诱使酵母过早沉降,影响发酵效果。在成品啤酒中,β-葡聚糖会与残留蛋白质结合形成雾状物质,影响成品啤酒的非生物稳定性[6, 23],β-葡聚糖的残留同时是造成成品啤酒泡沫挂杯性和泡沫稳定性差的主要原因[24]。研究者将筛选得到的微生物β-葡聚糖酶在啤酒酿造过程中进行了应用,取得了良好的效果。Bai等[25]和Chaari等[26]分别将脂环酸芽孢杆菌Alicyclobacillus sp. A4和青霉菌Penicillium occitanis Pol6来源β-葡聚糖酶在麦汁协定糖化过程中进行了应用,发现相同活力微生物β-葡聚糖酶和商业酶(Ultraflo或Finizym)的添加对于麦汁过滤速度的提高和麦汁黏度的降低效果几乎相同。钮成拓等[6, 27]将特基拉芽孢杆菌B. tequilensis CGX5-1来源β-葡聚糖酶添加在麦汁协定糖化过程开始阶段,最后将过滤时间和麦汁黏度分别降低了29.7%和12.3%,其应用效果优于两种商业酶。研究者将小孢根霉Rhizopus microsporus var. microsporus[28]和埃默森篮状菌Talaromyces emersonii[29-31]来源β-葡聚糖酶在麦汁糖化过程中进行了应用,同样取得了良好的效果。

在葡萄酒酿造过程中,葡萄皮/果肉细胞壁多糖和污染灰葡萄孢属微生物的葡萄产生的多糖会与蛋白质结合形成难溶性颗粒状物质,导致葡萄酒过滤困难,影响葡萄酒的澄清度,影响成品葡萄酒的非生物稳定性[6, 32]。Liauberes等[33]将含β-葡聚糖酶的复合酶制剂在葡萄酒生产过程中进行应用,发现酶制剂的添加不仅使葡萄酒的澄清、过滤更为容易,同时减少了过滤助剂的使用量。在皮渣浸泡之后的葡萄汁中添加β-葡聚糖酶使发酵后的葡萄酒具有更理想的香气[34]

1.2 饲料行业

在饲料行业中,大麦在饲料中的应用逐渐规模化。然而,谷物中的β-葡聚糖在动物肠道中无法被有效吸收和利用(动物肠道缺乏降解β-葡聚糖相关酶类)[35-36],与此同时β-葡聚糖较高的持水性会导致哺乳动物及禽畜类肠道内液体黏稠,作为“抗营养因子”阻碍饲料营养成分被动物肠道吸收,影响饲料利用效率和动物营养吸收效率[37]。β-葡聚糖存在于动物肠道中同时为肠道有害细菌的繁殖提供营养,而有害细菌的繁殖也会竞争性消耗饲料营养,造成动物肠道菌群失调并影响动物营养吸收,最终导致动物胃肠道疾病[38]。对于饲料中β-葡聚糖的降解关键酶为1, 3-1, 4-β-葡聚糖酶还是1, 4-β-葡聚糖酶在前期研究中颇有争议。针对此问题,Fernandes等[39]在大肠杆菌Escherichia coli中重组表达了热纤梭菌Clostridium thermocellum来源1, 3-1, 4-β-葡聚糖酶(CtGlc16A)和1, 4-β-葡聚糖酶(CtCel8A)并将两种酶添加在肉鸡饲料中,结果发现CtGlc16A对于以大麦为主的饲料黏度降低能力远优于CeCel8A,这说明1, 3-1, 4-β-葡聚糖酶是提高大麦饲料营养价值的关键酶。经过研究发现,β-葡聚糖酶添加于肉鸡饲料中不仅可以提高饲料利用效率,同时肉鸡的日增重速度、肠道黏度和饲养成本均优于未添加酶对照组[40-42]。近年来发现β-葡聚糖酶在饲料中的添加同时具有以下功效:1)新型低价值向日葵或油菜籽在饲料中有效利用[43];2)缓解蛋鸡使用小麦饲料时的骨质矿化现象[44];3)表达高耐热β-葡聚糖酶的转基因大麦在饲料中添加与传统玉米饲料效果相似[45];4) β-葡聚糖酶可以提升动物氧化还原稳态,促进动物肠道营养吸收,从而提升动物免疫力[46]。Clarke等[47]将β-葡聚糖酶添加于肥育猪饲料中,其明显改善了猪的营养吸收率和生长情况,进一步研究发现,和对照组肥育猪相比添加β-葡聚糖酶大麦饲料组肥育猪肠道营养吸收相关基因均明显上调。Guan等进一步将多粘类芽孢杆菌Paenibacillus polymyxa CP7来源β-葡聚糖酶在仔猪腮腺中进行了异源表达,发现转基因猪对β-葡聚糖和其他营养物质的摄取和吸收能力明显提高,降低了β-葡聚糖的抗营养因子作用[37, 48]。Zhang等[49]将耐酸β-葡聚糖酶基因在玉米中进行了异源表达,转基因玉米β-葡聚糖酶酶活是非转基因玉米对应β-葡聚糖酶酶活的236倍,且其最适pH值为4.0,符合动物消化系统酸性环境,这为玉米在饲料中的高效应用奠定了基础。

1.3 益生元行业

通过β-葡聚糖酶将β-葡聚糖降解为低聚葡萄糖,对人体健康产生益处。研究发现,摄入低聚葡萄糖可以降低血清胆固醇[50]、提高机体免疫力[51]、提高人体抗菌消炎和抗氧化能力[52]。与此同时,低聚葡萄糖可以促进人体肠道内有益微生物(主要为双歧杆菌和乳杆菌等)的选择性生长,缓解或治愈人类相关疾病[53-54]。Cho等[55]和Chaari等[56]分别将芽孢杆菌属Bacillus sp.和青霉菌P. occitanis Pol6来源β-葡聚糖酶进行固定化并用于低聚葡萄糖的生产,发现固定酶具有和游离酶相同的酶学性质且酶活稳定性较好,得到的低聚葡萄糖具有较高的抗氧化能力,且对大肠杆菌E. coli、枯草芽孢杆菌B. subtilis、肺炎克雷伯氏菌Klebsiella pneumonia和鼠伤寒沙门氏菌Salmonella thyphimirium具有较高的抑菌活性。Kim等[14]进一步将聚合度为3和4的低聚葡萄糖添加在糖尿病大鼠的饲料中,发现其可以显著降低大鼠的胆固醇含量,这说明低聚葡萄糖具备抗高胆固醇血症、抗高甘油三酯血症和降血糖等功效,可为新型生物健康产品开发奠定基础。

1.4 生物燃料行业

随着近年来对能源需求的提升,通过酶法将生物质能源降解生产生物燃料成为研究热点[57]。降解生物质的酶系主要包括纤维素酶系和半纤维素酶系[4]。在半纤维素酶系中,β-葡聚糖酶(地衣多糖酶)可与木聚糖酶、昆布多糖酶等协同作用降解半纤维素,为生物乙醇或生物柴油的生产奠定基础[3, 58-59]。尽管目前研究主要集中在纤维素降解酶系中,但是从经济效益的角度来看同时利用半纤维素(木质纤维素中含量第二高的多糖组分)可以取得更好的经济价值。与此同时,半纤维素通常包裹着纤维素,只有将半纤维素降解后才能启动纤维素的降解过程[60]。由此可见,作为一种重要的半纤维素降解酶,β-葡聚糖酶是一种降解生物质资源生产生物燃料的重要酶类。

近年来研究者已经筛选得到多株具有发达的降解木质纤维素酶系的菌株。通过色谱分析发现其中具有较高木质纤维素降解能力的一株烟曲霉Aspergillus fumigatus Z5中β-葡聚糖酶具有最高的酶活[3]。Li等[13]将β-葡聚糖酶与木聚糖酶进行协同作用,将玉米秸秆降解为木糖、低聚葡萄糖和葡萄糖,从而供酵母菌株转化生成生物乙醇,结果发现碳水化合物向生物乙醇的转化率达到84%。Menon等[61]采用碱性嗜热单胞菌属Thermomonospora sp.来源β-葡聚糖酶与β-葡萄糖苷酶协同作用,以地衣多糖为底物生产生物乙醇产量达到0.450 2–0.480 2 g/g,达到理论转化率的93%–96%。

1.5 其他行业

β-葡聚糖酶在生物防治、制糖行业等领域同样具有一定效果。在生物防治领域,β-葡聚糖酶可以通过降解或抑制真菌细胞壁1, 3-β-葡聚糖的合成抑制真菌细胞的生长周期,从而抑制真菌生长[6]。Enrique等[62]分析了市售β-葡聚糖酶对于常见葡萄酒污染酵母(如白色隐球菌Cryptococcus albidus、布鲁塞尔德克酵母菌Dekkera bruxellensis、膜醭毕赤酵母Pichia membranifaciens、酿酒酵母Saccharomyces cerevisiae、拜尔接合酵母Zygosaccharomyces bailii和双孢酵母Z. bisporus)的影响,发现β-葡聚糖酶对布鲁塞尔德克酵母D. bruxellensis和拜耳接合酵母Z. bailii具有明显抑制作用,与此同时酶在葡萄酒中的添加对葡萄酒品质并没有影响。在制糖工业中,被肠膜明串珠菌Leuconostoc mesenteroides污染的蔗糖液体会产生大量高黏度β-葡聚糖,β-葡聚糖酶添加在甘蔗制糖过程中可以迅速除去榨糖物料中的β-葡聚糖,大幅减少蔗糖的损失,提高了经济效益[63]

2 β-葡聚糖酶的酶学性质改造

β-葡聚糖酶在工业上的高效应用取决于其能否在工业应用环境中维持较高的催化活性。工业应用环境通常存在一些不利于酶应用的特点,例如高温、pH环境等。在啤酒行业和饲料行业中,β-葡聚糖酶通常应用于麦汁糖化过程中或添加于饲料中。麦汁糖化温度通常从48 ℃逐渐提升至78 ℃,而饲料造粒温度在60–90 ℃。与此同时,麦汁是一种弱酸性液体(pH 5.0–5.5),而动物消化系统通常也为酸性环境。对β-葡聚糖酶酶学性质进行改造使其更适应工业应用环境将更有利于其在工业上的应用效果。近年来,研究者通过新酶筛选、非理性/半理性/理性改造、计算机模拟等手段对β-葡聚糖酶的酶学性质进行了分析和改造,取得了一定成果。

2.1 新酶筛选

芽孢杆菌属Bacillus sp.是β-葡聚糖酶的主要来源,其分子量通常在25–30 kDa[64]。绝大部分芽孢杆菌属β-葡聚糖酶的最适pH值集中在pH 6–7.5,最适温度集中在45–65 ℃,而比活力值通常在1 200–4 500 μmol/ (min·mg)[10]。以β-葡聚糖为底物时β-葡聚糖酶的Km值为1.2–1.5 mg/mL,而以地衣多糖为底物时酶的Km值为0.8–2 mg/mL[65-67]。近年来,研究者逐步从非芽孢杆菌属(Non-Bacillus sp.)中分离得到大量β-葡聚糖酶。和芽孢杆菌属来源β-葡聚糖酶相比,非芽孢杆菌微生物来源酶通常具有一些具备其他功能的蛋白质区域[68]。然而,目前发现绝大部分非芽孢杆菌属微生物来源β-葡聚糖酶的酶学性质与芽孢杆菌属微生物来源酶性质相似,其最适pH值和最适温度集中在pH 6.0–10和45–60 ℃[68-70]。热纤梭菌C. thermocellum[36]、埃默森篮状菌T. emersonii[31]和嗜热拟青霉Paecilomyces thermophilum[71]来源的β-葡聚糖酶的最适温度分别达到80 ℃、80 ℃和70 ℃,其最适pH值分别为6.6–10、4.8和7.0,然而其催化活性较低。

2.2 β-葡聚糖酶的热稳定性改造

Planas已经对β-葡聚糖酶热稳定性的改造进行了一定的描述。研究发现蛋白质N端和钙离子对β-葡聚糖酶热稳定性有重要影响。Borriss等[65-66, 72]发现将淀粉液化芽孢杆菌B. amyloliquefaciens来源β-葡聚糖酶N端16个氨基酸替换地衣芽孢杆菌B. licheniformis来源酶对应区域可以大幅提升酶的热稳定性,进一步分析发现酶N端Gln1、Thr2、Ser5和Phe7位点通过构建N端-核心区域氢键网络和N端-C端氢键网络维持β-葡聚糖酶的稳定性[73]。Pons等[74]将β-葡聚糖酶分子内部二硫键Cys61-Cys90打断,发现二硫键对酶的催化性质和稳定性均没有太大影响。钙离子的丧失会大幅降低β-葡聚糖酶的热稳定性,而其对酶的催化性质几乎没有影响。Keitel等[75]采用Na+替代杂交酶H (A16-M)中的Ca2+,结果酶在高温下的半衰期显著下降。通过差示量热法,Gargallo等[76]发现钙离子的存在明显改变了β-葡聚糖酶在高温下的解折叠过程。β-葡聚糖酶在钙离子存在时高温解折叠过程中出现了一个中间体状态,为两步解折叠,而当钙离子不存在时其为一步解折叠。对于Asp51-Arg64无规卷曲的精氨酸突变实验发现Asn57为高温下不稳定性氨基酸,将其突变为精氨酸后酶的热稳定性有所提升[73]

近年来研究者进一步通过非理性、半理性和理性手段对β-葡聚糖酶的热稳定性进行了改造。张秀艳等[77]采用随机突变结合DNA改组技术对枯草芽孢杆菌B. subtilis ZLF-1A5来源β-葡聚糖酶进行体外进化改造并建立一种高通量筛选耐高温β-葡聚糖酶的方法,获得的EGs1和EGs2突变体的Tm值和野生酶相比分别提高了3 ℃和5 ℃[78]。Chen等[79]对产琥珀酸丝状杆菌Fibrobacter succinogenes来源β-葡聚糖酶氨基酸保守序列进行了分析和突变研究,发现Gly63对酶热稳定性有较大作用。Gargallo等[76]对芽孢杆菌属来源β-葡聚糖酶进行了分子动力学模拟研究,发现β-葡聚糖酶N端和C端区域的根平均偏差值(Root mean square deviation,RMSD)偏高,说明其酶N/C两端有较高的结构柔性,可能对于酶的热稳定性有影响。在此基础上,Wen等[80]和李卫芬等[81]分别将产琥珀酸丝状杆菌F. succinogenes和热纤梭菌C. thermocellum来源β-葡聚糖酶的N端和C端区域逐步截去,发现β-葡聚糖酶N端和C端区域对酶热稳定性有一定贡献且具有叠加效应。

近年来本研究室对芽孢杆菌来源β-葡聚糖酶进行了一系列改造,成功获得了热稳定性提高的酶突变体。秦久福等[82]采用易错PCR技术对淀粉液化芽孢杆菌B. amyloliquefaciens BS5582来源β-葡聚糖酶进行定向进化改造,得到的3株酶突变体的Tm值和野生酶相比分别提高了2.2 ℃、5.5 ℃和3.5 ℃。钮成拓等[83]采用亚硝酸作为修饰剂对淀粉液化芽孢杆菌B. amyloliquefaciens BS5582来源β-葡聚糖酶进行了化学修饰研究,推测酶蛋白质表面赖氨酸对酶的热稳定性有重要影响。在此基础上,采用定点突变方法将特基拉芽孢杆菌B. tequilensis CGX5-1来源β-葡聚糖酶表面赖氨,发现20位、117位和165位赖氨酸突变后其蛋白质总能量值和氢键数量均优于野生酶,K20S/K117S/K165S组合突变体的最适温度和T50值与野生酶相比分别提高了15 ℃和14 ℃[6, 84]。基于蛋白质结构和柔性分析,钮成拓等在β-葡聚糖酶三维结构中引入了二硫键N31C-T187C和P102C-N125C,其Tm值和野生酶相比分别提高了1.4 ℃和2.3 ℃[85]。β-葡聚糖酶自身二硫键对于热稳定性没有影响,然而该两对二硫键的引入成功提高了β-葡聚糖酶的Tm值。对β-葡聚糖酶结构中的二硫键进行分析,发现二硫键Cys61-Cys90主要处于蛋白质凹侧β-片层区域,结构相对稳定,打断其对于酶结构稳定性影响较小。二硫键N31C-T187C和P102C-N125C主要连接β-葡聚糖酶高柔性区域,通过分子动力学模拟发现将其引入酶结构后可以一定程度降低酶的整体柔性。因此在合适的位置引入二硫键可以提高β-葡聚糖酶的热稳定性。为了解析β-葡聚糖酶热稳定性关键区域和氨基酸位点,钮成拓等[6, 86]建立了一种基于同源蛋白质氨基酸序列比对、空间结构区域化和分子动力学模拟的突变热点区域分析法(Spatial compartmentalization of mutational hotspots)对微生物β-葡聚糖酶热稳定性关键区域和氨基酸位点进行了分析,发现β-葡聚糖酶结构凸侧钙离子结合区域与酶热稳定性具有较高的相关性,这说明该区域可能是微生物β-葡聚糖酶热稳定性关键区域。通过定点突变方法将该区域内6个氨基酸位点突变为高耐热β-葡聚糖酶对应氨基酸位点,发现40位、43位、46位和205位氨基酸位点对酶热稳定性具有重要的影响,说明这4个位点可能为酶热稳定性关键氨基酸位点。采用迭代饱和突变手段对这4个氨基酸位点进行改造,得到的组合突变酶E46P/S43E/H205P/S40E的最适温度、Tm值和T50值与野生酶相比分别提高了20 ℃、13.8 ℃和14.5 ℃,其在60 ℃和70 ℃的半衰期是野生酶的3.86倍和7.13倍[6]。最后钮成拓[6, 27]将上述酶热稳定性有利突变位点进行了组合突变,得到的组合突变体的最适温度、T50值和Tm值分别达到70 ℃、81.7 ℃和56.2 ℃,和野生酶相比分别提高了25 ℃、19.7 ℃和15.9 ℃。其在60 ℃和70 ℃的半衰期达到153.2 min和99.6 min,是野生酶对应值的4.71倍和9.05倍。对野生酶和组合突变酶蛋白质结构进行比较和分析,发现钙离子结合区域带有更多负电,和钙离子的结合更加紧密,同时组合突变酶蛋白质中有序二级结构所占比例也有大幅提高,从而提升酶的热稳定性。根据结合突变热点区域分析法所得结果和钙离子替换实验,推测β-葡聚糖酶与钙离子结合的紧密度与酶热稳定性有重要关联。

2.3 β-葡聚糖酶的催化性质改造

β-葡聚糖酶特异性切割β-葡聚糖的催化机制为包含一个亲核氨基酸和一个广义酸/碱氨基酸的双位移机制(Double displacement mechanism)[6],其催化机制和底物特异性识别位点在Planas发表的综述[10]中已有明确描述。尽管较多微生物能够分泌β-葡聚糖酶,但是其催化性质依旧不能满足工业应用水平。目前对于β-葡聚糖酶催化性质的改造方法主要采用蛋白质工程改造。对近年来研究者对β-葡聚糖酶催化性质改造工作进行分析,发现酶N/C端改造和酶催化活性中心内关键分子内作用力的形成对β-葡聚糖酶的催化性质有重要影响。毛淑蕊等[87-88]采用易错PCR手段对高地芽孢杆菌Bacillus altitudinis YC-9来源β-葡聚糖酶进行了定向进化,得到的K142N/Q203L/N214D三突变体的催化活性达到474.6 U/mL,和野生酶相比提高了48.6%,同时其pH性质和热稳定性均没有影响。Wen等[80]将产琥珀酸丝状杆菌F. succinogenes β-葡聚糖酶C端截断,并在毕赤酵母P. pastoris中进行了表达,结果发现截断酶的kcat值是全长酶的3–4倍。在毕赤酵母P. pastoris中表达的截断酶是一种糖基化酶,其比活力值达到(10 800±200) U/mg,而对于底物地衣多糖的亲和力(Km值)和全长酶没有差别。

本研究室在对β-葡聚糖酶进行热稳定性改造的同时,发现部分氨基酸位点的变化对酶的催化性质造成影响。针对β-葡聚糖酶赖氨酸改造过程中,发现20位、117位和165位赖氨酸突变为丝氨酸后其比活力值和kcat值与野生酶相比分别提高了58.1%和34.6%,而其Km值有一定程度的降低[84]。通过突变热点区域分析法改造得到的E46P/S43E/H205P/S40E突变位点的引入将β-葡聚糖酶的比活力值和kcat值从2 490.1 U/mg和137.4 /s分别提高至4 093.8 U/mg和187.4 /s,Km值从297 μmol/L降低至271 μmol/L。最后得到的组合突变酶的比活力值则进一步提高至4294.1 U/mg[86]。对于野生酶和酶突变体的结构(特别是催化活性中心)进行分析和比较,尽管突变位点位于蛋白质表面,但是改造后突变酶的二级结构,特别是催化活性中心部分发生了改变。酶突变体催化活性中心有序二级结构比例稍有提高,柔性降低,部分与底物结合氨基酸位点所处微环境发生变化,并形成了部分关键氢键(如E109-Q119和W103-E105等),这可能是突变提高β-葡聚糖酶催化性质的主要原因[27]

2.4 β-葡聚糖酶的pH性质改造

β-葡聚糖酶在酒类[89]、饲料[90]和生物燃料[91]生产行业中主要应用在酸性环境,而在洗涤剂等行业中通常需要适应碱性环境[92],因此这需要β-葡聚糖酶具有不同的pH性质。尽管在BRENDA等酶数据库中能够找到一些最适pH值为pH 4–5的β-葡聚糖酶,然而其热稳定性或者催化活性均存在缺陷。目前针对β-葡聚糖酶的耐酸性或耐碱性的蛋白质改造的研究较少。江正强等[93]采用易错PCR手段对嗜热拟青霉P. thermophilum来源β-葡聚糖酶的耐酸性进行了分析,发现56位和263位氨基酸对于酶的最适pH值改变有重要影响,发现突变体PtLic16AM2的最适pH值(pH 5.0)和野生酶(pH 7.0)相比有所降低。在本研究室对特基拉芽孢杆菌B. tequilensis CGX5-1来源β-葡聚糖酶进行改造过程中,组合突变酶的最适pH值从6.5降低至6.0。对蛋白质结构进行分析,发现酶突变体的蛋白质表面带有更多的负电荷,这可能是酶最适pH值向酸性环境移动的主要原因[27, 85]

3 总结与展望

β-葡聚糖酶是一种重要的工业用酶,在酒类、饲料、益生元等行业中具有重要应用价值。现代工业生产中的高温、酸性/碱性pH环境等对β-葡聚糖酶的热稳定性、pH性质和催化活性提出了更高的要求。近年来通过蛋白质工程改造技术在提高β-葡聚糖酶热稳定性和催化性质等领域已经取得了一系列成果,但是目前得到的β-葡聚糖酶酶学性质依然存在缺陷,尤其是β-葡聚糖酶的耐酸性不足,严重阻碍了其在工业上的应用。因此,通过蛋白质工程改造技术进一步提高β-葡聚糖酶的酶学性质,使其更贴合于工业生产,将更有利于其在工业中发挥作用。

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