徐铮1
, 张倩2, 李克文2, 徐虹1 1. 南京工业大学食品与轻工学院, 材料化学工程国家重点实验室, 江苏 南京 211816;
2. 保龄宝生物股份有限公司, 山东 禹城 251200
收稿日期：2020-03-26；修回日期：2020-05-20；网络出版日期：2020-07-24
基金项目：江苏省六大人才高峰（NY-056）；2016年泰山产业领军人才（传统产业创新类）
*通信作者：徐铮, Tel/Fax:+86-25-58139433, E-mail:xuzheng@njtech.edu.cn.
摘要：乳果糖是由D-半乳糖和D-果糖两个基团通过β-1，4糖苷键连接而成的还原型二糖；乳果糖口服液具有治疗慢性便秘和肝性脑病的功效，在100多个国家作为常见非处方药（OTC）使用，需求量十分巨大；乳果糖还可以作为益生元改善人体肠道菌群关系。乳果糖的生产依赖化学法，其催化剂对人体有害，下游分离难度大。近年来，纤维二糖差向异构酶被发现能够高效催化乳糖制备乳果糖，该技术绿色环保、步骤简单，具有很强的产业化前景。本文结合自身研究经历对纤维二糖差向异构酶的研发情况进行总结，并综述了乳果糖酶法制备技术的现状。
关键词：乳果糖纤维二糖差向异构酶生物催化益生元便秘肝性脑病
Advances in bio-production of lactulose using cellobiose 2-epimerases
Zheng Xu1
, Qian Zhang2, Kewen Li2, Hong Xu1 1. State Key Laboratory of Materials-Oriented Chemical Engineering, College of Food Science and Light Industry, Nanjing Tech University, Nanjing 211816, Jiangsu Province, China;
2. Baolingbao Biology Co., Ltd., Yucheng 251200, Shandong Province, China
Received: 26 March 2020; Revised: 20 May 2020; Published online: 24 July 2020
*Corresponding author: Zheng Xu, Tel/Fax: +86-25-58139433; E-mail: xuzheng@njtech.edu.cn.
Foundation item: Supported by the Six Talent Peaks Project in Jiangsu Province (NY-056) and by the Taishan Industry Leader Talent Project
Abstract: Lactulose, a reducing disaccharide, is composed of D-galactosyl and D-fructosyl moieties via the ligation of a β-1, 4 glycosidic bond. The concentrated lactulose solution can be used to treat chronic constipation and hepatic encephalopathy. It is an over-the-counter drug (OTC) worldwide, resulting in a massive requirement for its production. Lactulose is also a prebiotic that benefits human intestinal flora. Current industrial production of lactulose relies on chemical catalysis, with harmful catalyst and difficult in the down-stream processing. Recently, cellobiose 2-epimerase (CE) is recognized as an efficient biocatalyst for lactulose production using lactose as the substrate. This technique is environmentally friendly and composed of simpler procedures, showing promising future for an industrial application. This paper reviews recent developments in CE enzyme research, and the biotechnological route of lactulose synthesis.
Keywords: lactulosecellobiose 2-epimerasebiocatalysisprebioticsconstipationhepatic encephalopathy
乳果糖(lactulose)，分子式C12H22O11、分子量342.3、CAS号4618-18-2，别名乳酮糖、异构化乳糖、4-O-β-D-吡喃半乳糖基-D-果糖等，是D-半乳糖和D-果糖以β-1, 4-糖苷键连接而成的二糖(图 1)，易溶于水(20 ℃时溶解度达到2060 g/L)，熔点168.5–170.0 ℃[1-5]。1930年Montgomery和Hudson就在JACS杂志发表了乳果糖的化学合成方法[6]，从此乳果糖的应用研究进入了科学家的视野。药效分析研究表明，乳果糖具有治疗慢性便秘和肝性脑病的功效，例如每天摄入10–40 g就可以缓解便秘，每天摄入90 g则可以治疗肝性脑病[7]。服用乳果糖不会产生任何代谢毒物，对人体非常安全(LD50大于10 g/kg体重)，全球已有100多个国家注册了乳果糖相关的药物。此外，在荷兰、日本、意大利等国，乳果糖还可以作为食品添加剂销售。它属于渗透性泻药也是优异的肠道益生元，不会被小肠吸收从而到达结肠，促使乳酸菌、双歧杆菌等肠道益生菌大量繁殖并发酵产生低分子有机酸，降低肠道pH值；保留肠道水分软化大便，同时抵制梭菌、沙门氏菌等有害微生物的生长[8-9]。复旦大学附属华东医院报道乳果糖口服液对老年便秘患者具有显著的疗效，有效率达79.2%，与对照组相比有统计学差异；乳果糖治疗的不良反应率低，安全性好，而且可以调节老年人失调的肠道菌群[10]。由于便秘在老年人中易导致排便用力过度引起的急性心肌梗死、脑血管意外等危重疾病，而长期使用酚酞、番泻叶、大黄等刺激型泻药又可能导致结肠黑病变甚至不可逆的肠神经损害，因此乳果糖对老年人便秘病患较为友好。上海交通大学医学院也报道了乳果糖对妊娠期妇女便秘的治疗效果，结果表明效果显著、副作用小、患者治疗满意度达90.8%[11]；雷衍蔚等报道乳果糖对120例小儿功能性便秘的治疗有效率达85%，远高于对照组的41.6%[12]；MacGillivray等报道奶粉中如果添加乳果糖，婴儿长期饮用后的肠道菌群会和母乳喂养组非常接近，而没有添加乳果糖则无此效果，说明乳果糖也有很好的益生元功效[13]。事实上乳果糖除作为便秘药以外，还是人类商业化使用的第一款益生元；但由于其在早期开发阶段被归类为药品，因此大多数国家仍然习惯作为药物来宣传、销售和使用，2009年全球对乳果糖的需求量已达到5万t并且持续增长，预计2020年后达到10万t。
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| 图 1 乳果糖(lactulose)和乳糖的化学结构式 Figure 1 The chemical structures of lactulose and lactose. |
| 图选项 |
商品化的乳果糖口服液是乳果糖占主要比例的糖浓溶液，颜色微黄、无异味、口感较甜(甜度为蔗糖的0.6–0.8倍)。其中乳果糖浓度一般为667 g/L (或为634–700 g/L)，另外还含有20%左右的杂糖，包括乳糖(≤90 g/L)、D-半乳糖(≤150 g/L)、依匹乳糖(≤70 g/L)、D-塔格糖(≤30 g/L)、D-果糖(≤10 g/L)，是治疗便秘、肝性脑病的有效药物(非处方药OTC类)[14]。具有国内生产许可资质的主要口服液制剂销售厂家包括雅培(荷兰)公司(商品名：杜密克)、北京韩美药品有限公司(商品名：利动)、奥地利费森尤斯卡比公司、山德士(中国)制药有限公司、四川健能制药有限公司、湖南科伦制药有限公司、丹东康复制药有限公司等。截止目前，乳果糖的工业化生产完全依靠化学催化法，使用诸如硼酸、偏铝酸钠等有毒或重金属型催化剂，需要专用树脂完全吸附掉多余硼或铝，没有任何残留后才是合格产品[15]。由于化学催化剂的用量较大，因此彻底去除非常困难，且色素和副产物产生较多，对下游技术有很高的要求，产业化具有一定难度。化学法也可以使用氢氧化钙、氢氧化钠、氢氧化钾、碳酸钾、氧化镁等进行催化，但遇到转化率低、副产物多等类似的问题[16]。跨国企业利用技术优势占据了我国乳果糖口服液产品的几乎全部市场，我国企业则边缘化严重，开发生物技术生产乳果糖是打破这一垄断的最可行路线。生物技术制备乳果糖的工艺一般依靠β-半乳糖苷酶或纤维二糖差向异构酶(cellobiose 2-epimerase，简称CE酶)两种酶，均以乳糖作为催化底物。前者的转化率很低，而且需要在乳糖解离后外源加入D-果糖作为第二底物才能合成出乳果糖[17-19]，因此反应体系中存在单糖和二糖等多种糖类，产品分离难度极大，而且转化率很低(小于20%)，普遍认为不具备工业化前景；采用纤维二糖差向异构酶的技术催化转化率高，根据报道，对于700 g/L高浓度的乳糖底物，催化转化率可以达到58%左右，与硼或铝为催化剂的化学法(转化率一般75%以上)的差距大为缩小[20]。生物法中使用最多的是来源于嗜热微生物Caldicellulosiruptor saccharolyticus的纤维二糖差向异构酶(简称CSCE酶)，该酶的研究报道近来很多，使用最为广泛；此外还有来源于Dictyoglomus turgidum、Caldicellulosiruptor obsidiansis、Dictyoglomus thermophilum来源的纤维二糖差向异构酶也适合生产乳果糖[21-24]。值得注意的是，绝大多数已发现的纤维二糖差向异构酶不产乳果糖而是产依匹乳糖(epilactose，又名表乳糖，由D-半乳糖和D-甘露糖以β-1, 4-糖苷键结合而成的二糖)，例如包括Cellulosilyticum lentocellum、Dysgonomonas gadei、Flavobacterium johnsoniae、Pedobacter heparinus、Thermoanaerobacterium saccharolyticum、Ruminococcus albus、Rhodothermus marinus、Spirochaeta thermophila、Eubacterium cellulosolvens、Bacteroides fragilis等来源的纤维二糖差向异构酶催化乳糖若干小时，均只产生依匹乳糖[25-32]，这说明以上纤维二糖差向异构酶具有很强的差向异构反应能力，但醛酮糖异构反应能力不强。
1 已报道的纤维二糖差向异构酶 CE酶曾被认为是AGE酶(N-乙酰-D-氨基葡萄糖2-差向异构酶、EC 5.1.3.8)家族的成员，但由于AGE酶一般需要ATP参与反应，而CE酶并不需要任何辅酶，且两种酶的序列相似度很低，因此近年来将CE酶单独划为纤维二糖差向异构酶。早在1967年Tyler和Leatherwood就研究了革兰氏阳性菌Ruminococcus albus (白色瘤胃球菌)来源的CE酶，发现它可以催化纤维二糖产生葡萄糖基甘露糖[33]，然而CE酶成为研究热点却是在40多年后。2011年韩国的Park等发现Caldicellulosiruptor saccharolyticus来源CE酶(CSCE)可以催化单糖D-葡萄糖产生D-甘露糖(同时产生副产物D-果糖)[34]，由于D-甘露糖的应用价值，很快CE酶得到了重视。仅一年后通过继续研究，Kim和Oh又发现CSCE酶可以催化乳糖产生乳果糖[20]，自此拉开了CE酶应用于乳果糖合成的序幕。乳糖的溶解度随温度变化很大，其中0 ℃下溶解度仅为11.9% (W/W)，而74 ℃却达到86.2% (W/W)[35]，这表明高的反应温度可以溶解更多的乳糖，从而提高产物浓度和生产强度。已报道可以产乳果糖的CE酶均具有较高的反应温度，在70–80 ℃；温度还可以影响CE酶催化醛酮糖异构(乳糖变为乳果糖)和差向异构(乳糖变为依匹乳糖)的产物比例，Park等发现CSCE酶在65 ℃下的醛酮糖异构活性是37 ℃下的12倍，而差向异构活性仅为37 ℃下的27%[36]。因此提高反应温度对高产乳果糖和降低依匹乳糖含量有很大帮助。由表 1可知，目前已知的利用纤维二糖差向异构酶催化乳糖得到乳果糖的报道中，都会得到依匹乳糖这一反应副产物，包括Caldicellulosiruptor saccharolyticus、Dictyoglomus turgidum、Dictyoglomus thermophilum、Caldicellulosiruptor obsidiansis等来源(这里均指野生型酶)，所产依匹乳糖的含量大约为11%–16% (W/W)[20-24]。依匹乳糖的结构与乳糖和乳果糖都非常相似，属于同分异构体，因此分离极为困难[37]，目前还没有产业化规模下分离成功的报道。为了克服这一困难，Kim等提出在生物催化的过程中添加化学催化剂硼酸，可以使得依匹乳糖的含量降低到2%的水平[38]。其原理是硼酸可以和酮糖(乳果糖)形成络合物，使酮糖脱离反应体系，导致反应平衡向更多酮糖产物生成方向移动。由于依匹乳糖不含酮基，因此在硼酸体系中含量会大幅下降。硼酸的使用也见于D-塔格糖等稀少糖的催化研究中[39]，然而硼酸的使用量较大，而硼是禁止出现在食品中的，因此必须完全去除才能符合法律标准。但作为一种弱酸，想要完全去除非常困难，即使价格昂贵的专用树脂也比较困难，因此这一方法的产业化价值有限。尽管许多糖基异构酶都被证明是金属酶，金属离子对CE酶的活性却没有促进作用，而且经EDTA处理活性也未发生变化，这表明CE酶并不是金属酶[34]。已报道的CE酶热稳定性大多较好，例如Dictyoglomus turgidum CE酶在70 ℃的活性半衰期达到了55 h[21]。
表 1. 已报道催化乳糖能够产生乳果糖的纤维二糖差向异构酶 Table 1. Reported cellobiose 2-epimerase that produced lactulose from lactose
| Original microorganisms | Optimal temperature/℃ | Optimal pH | Specific activity/ (U/mg) | Catalytic efficiency [1/min 1/(mmol/L)] | Lactose:epilactose: lactulose | References |
| Caldicellulosiruptor saccharolyticus | 80 | 7.5 | 10.8 | 0.55 7.05 (1/s) | 27:15:58 | [20, 23, 40] |
| CSCE mutant R5M/I52V/ A12S/K328I/F231L | 80 | 7.5 | 30.1 | 21.0 (1/s) | 24:0:76 | [40] |
| Dictyoglomus turgidum | 70 | 7.0 | 14.2 | 1.12 0.51 | 32.9:12.8:54.3 | [21, 23] |
| Dictyoglomus thermophilum | 85 | 7.0 | 160.1 (epimerization); 3.52 (isomerization) | 0.84 (1/s) | NR | [24] |
| Caldicellulosiruptor obsidiansis | 70 | 7.5 | 93.6 | 0.77 (1/s) | 35:11:54 | [22] |
| NR: not reported. | ||||||
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2 酶催化制备乳果糖的工艺研究 已报道的生物法制备乳果糖工艺见表 2，目前产物乳果糖浓度最高达到614 g/L，转化率高达88%[38]，但反应体系添加了较高浓度的硼酸。在不添加硼酸的情况下，产物浓度最高为408 g/L，此时转化率为58%[20]。生产强度最高的工艺中，添加硼酸组为205 g/L，不添加为204 g/L，水平相当。所有催化工艺都可以在2–4 h完成催化，显示了较快的反应速度。反应体系中酶的用量差异则较大，在12.5–150.0 U/mL的范围内均完成了催化。由以上数据可知，CE酶催化乳糖制备乳果糖的速度是比较快的，产物浓度和生产强度也接近了产业化的水平，因此使用该酶作为工业酶理论上可行。由于CE酶生产乳果糖的催化温度很高，降低反应温度又会显著降低底物转化率，因此固定化CE酶体系必须能够长时间耐受高温，这对固定化材料和酶本身都提出了很高的要求。目前有一些固定化体系获得了成功，江南大学的Gu等通过吸附法将CE酶固定化在枯草芽孢杆菌的芽孢上，固定量最高达到1.47 mg每1011个芽孢，酶回收率为79.4%。固定化芽孢在4 h内获得56.4%转化率，乳果糖浓度395 g/L，重复使用8批次后芽孢残留有70%的酶活[41]。Wang等也尝试将CSCE酶固定化在商业化的Doulite A568树脂上，固定化使用的是纯酶。纯化方法是在70 ℃热处理破碎细胞液2 h，失活杂蛋白后直接得到较纯的CSCE酶，纯酶回收率达到86.6%。固定化时利用酶与树脂的静电吸附作用，同时加入戊二醛交联，该固定化酶重复使用15批次后仍有90%的酶活[42]。
表 2. 已报道的生物法制备乳果糖生产工艺 Table 2. Reported bio-production progresses of lactulose
| Bio-catalyst | Substrate concentration/(g/L) | Lactulose concentration/yield | Productivity/ [g/(L·h)] | Enzyme loading/ (U/mL) | Reaction time/h | References |
| CSCE enzyme | 700 | 408 g/L (58%) | 204 | 150 | 2 | [20] |
| CSCE enzyme | 700 (addition of 120 g/L boric acid) | 614 g/L (88%) | 205 | 150 | 3 | [38] |
| CSCE mutant enzyme R5M/I52V/A12S/K328I/F231L | 500 | 380 g/L (76%) | 95 | 3 g/L | 4 | [40] |
| Immobilized CSCE enzyme | 600 | 350 g/L (58.3%) | 87.5 | 12.5 | 4 | [42] |
| Permeabilized E. coli cells (expressing CSCE) | 600 | 390.6 g/L (65.1%) | 195.3 | 12.5 | 2 | [43] |
| Immobilized CSCE enzyme | 700 | 395 g/L (56.4%) | 98.8 | 100 | 4 | [41] |
| COCE enzyme | 200 | 108 g/L (54%) | 27 | 120 | 4 | [22] |
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3 纤维二糖差向异构酶的催化机理 一般认为只有高温CE酶才可以催化乳糖产生乳果糖，而常温型CE酶则只能产生依匹乳糖。但近来研究表明，通过延长反应时间，常温型酶也可以大量产生乳果糖。Kuschel等选取了7种常温型CE酶，在10 ℃的较低温度下催化乳糖长达21 d后，最高获得了56.8%的乳果糖，依匹乳糖含量13.3%，这与高温型酶的催化结果类似[44]。而与之对比，反应仅5 min，就可以获得最高30%的依匹乳糖。这一结果表明，CE酶催化乳糖首先产生依匹乳糖；继续延长反应时间，高温酶能够在短时间内产生乳果糖并达到反应平衡；而常温型酶则需要更长的时间才能产生乳果糖，但在较长的反应时间后也可以达到反应平衡，因此CE酶催化的差向异构反应速率要远高于醛酮糖异构反应。而且，乳果糖的形成似乎有两条途径，即直接从乳糖异构化得到，以及从依匹乳糖间接转化而来[45]。糖基醛酮糖异构酶或差向异构酶的催化机制包括烯二醇中间体机制(cis-enediol intermediate mechanism)和氢负离子转移机制(hydride-shift mechanism)，前者主要代表有L-阿拉伯糖异构酶、D-塔格糖3-差向异构酶等[46-47]，后者包括D-木糖异构酶和L-鼠李糖异构酶等[48-49]。两种反应机理的研究均较为透彻，CE酶通过晶体结构分析表明属于烯二醇中间体机制[50]。以Rhodothermus marinus来源CE酶催化纤维二糖为例，在反应开始时，通过H390作为广义酸或碱来打开葡萄糖基糖环(底物为乳糖时同样打开葡萄糖基糖环)；开环后再由H390残基夺取糖链中C-2位的质子形成烯二醇中间体(C1-C2间形成双键)，然后再由H259提供质子使得中间体发生转化形成开环的产物，最终再经过闭环反应成为终产物；H200也起到了稳定反应中间体的作用[51]。而在Ruminococcus albus来源的CE酶中组氨酸同样是催化残基，H243和H374扮演了广义酸碱催化剂的角色，H184也被发现对催化起到关键作用[52]。因此CE酶的底物催化过程主要是由3个组氨酸残基配合完成的，组氨酸的不同质子化状态使得反应能够以可逆的方式进行。由于组氨酸的等电点接近中性，因此在中心环境下才适合释放与接收质子；这从机理上解释了为什么CE酶只能在中性条件下才具有比较高的酶活力，过酸和过碱都能够大幅降低酶的活性。
4 针对纤维二糖差向异构酶的改造研究 尽管纤维二糖差向异构酶催化乳糖的效率较高，但仍然存在诸多问题；首先是副产物依匹乳糖的比例过高，尽管依匹乳糖本身也是一种人体益生元[53-56]，但各国药典对其含量均有严格限制。蛋白质工程改造是解决这一难题的途径，其中定向进化显示了这方面的优势。Shen等通过定向进化筛选到了五位点突变体R5M/I52V/A12S/K328I/ F231L，该突变体并不产生副产物依匹乳糖[40]，说明定向进化是非常有效的纤维二糖差向异构酶改造方法。此外，根据PDB数据库中已发布的CE酶晶体结构数据，通过分子同源建模和理性设计的方法，也可以对CE酶进行有效改造。截止目前，PDB数据库共有5种CE酶的晶体结构(表 3)，已报道的CE酶多为单体酶，分子量43–47 kDa。分析多种来源的CE酶表明氨基酸序列相似度在35%–50%，氨基酸序列保守区包括No. 50–60、No. 180–200、No. 240–260、No. 300–330、No. 370–390等区域的残基。笔者通过分析CSCE酶结晶结构发现，不同来源CE酶的活性口袋相似度很高，其中的氨基酸残基相当保守且有一定的疏水性，针对这些残基的定点突变实验都导致了酶活力大幅下降或彻底失活(结果尚未发表)，这些残基的编号和推测功能如表 4所述。Ito等分析了Ruminococcus albus来源CE酶(以依匹乳糖为产物)的重要氨基酸残基，结果表明无论底物是纤维二糖还是乳糖，R52、H243、E246、W249、W304、E308和H374残基都是催化必需残基，F114和W303也对催化有重要贡献[57]。Park等分析CSCE酶活性中心与甘露糖基团C-2位的作用情况，选择Y114和N184两个残基位点进行饱和突变研究。结果表明在所得突变体中，Y114E催化200 g/L乳糖生产乳果糖的转化率为43.5% (产物浓度86.9 g/L)，尽管转化率不高但依匹乳糖的浓度仅为4.6 g/L (转化率2.3%)，这可能是因为突变体丧失了大部分的差向异构化能力[36]。针对热稳定性的突变研究也有报道，Shen等发现双位点突变E161D/N365P在高温下的活性半衰期提高了4倍，最适反应温度也从80 ℃提高到87.5 ℃，对底物乳糖的催化效率kcat/Km上升了29%[58]。
表 3. PDB数据库中已发布的CE酶晶体结构 Table 3. Published crystal structures of cellobiose 2-epimerase in the PDB database
| Originals | Molecular weight determined by SDS-PAGE/kDa | Oligomeric state | PDB ID | Published data | References |
| Rhodothermus marinus | NR | NR | 3WKF, apo structure 3WKG, holo structure with glucosylmannose 3WKH, holo structure with epilactose 3WKI, holo structure with cellobiitol | 12/5/2013 | [51] |
| Ruminococcus albus | 43.1 | Monomer | 3VW5, apo structure | 6/26/2013 | [52] |
| Caldicellulosiruptor saccharolyticus DSM 8903 | 47 | Monomer | 4Z4J, apo structure 4Z4L, apo structure | 4/13/2016 | Not published |
| Bacillus thermoamylovorans B4167 | NR | NR | 5ZHB, apo structure | 6/19/2019 | Not published |
| Spirochaeta thermophila DSM 6192 | 47 | Monomer | 5ZIG, apo structure | 4/10/2019 | Not published |
| NR: not reported. | |||||
表选项
表 4. CE酶活性中心关键氨基酸残基一览* Table 4. Essential residues in the active site of cellobiose 2-epimerase*
| Residues | Predicted functions | Residues interacted with the side chain |
| R66 (R56) | Interacted with No. 5 oxygen atom and No. 6 hydroxyl group of substrate | Y124, W385 |
| Y124 (Y113) | Interacted with No. 2 hydroxyl group of substrate | R66 |
| N196 (N184) | Interacted with No. 2 and 3 hydroxyl group of substrate | H259 |
| H200 (H188) | A catalytic residue, interacted with No. 1 and 2 hydroxyl group of substrate | None |
| H259 (H247) | A catalytic residue, interacted with No. 3 hydroxyl group of substrate | N196, S256 |
| E262 (E250) | Interacted with No. 1 hydroxyl group of substrate | Y389, R393 |
| W322 (W308) | Stabilizing substrate via π-π interaction | None |
| W385 (W373) | Stabilizing substrate via π-π interaction | R66 |
| H390 (H377) | A catalytic residue, interacted with No. 5 oxygen atom and No. 1 hydroxyl group of substrate | None |
| *According to the crystal structure of Rhodothermus marinus CE enzyme (PDB ID: 3WKG), and residue numbers in brackets are corresponded to the CSCE enzyme. | ||
表选项
5 CE酶在食品级宿主中的表达应用 大肠杆菌因其清晰的分子遗传背景、成熟的基因操作方法、顶级的生长繁殖速度而被生物工程领域视为首选的微生物宿主，已报道的CE酶研究大多在大肠杆菌中实现了克隆表达和发酵过程研究。然而大肠杆菌易产内毒素，难以在食品领域应用，因此开发食品级的微生物宿主来表达CE酶具有重要的现实意义。包括枯草芽孢杆菌、毕赤酵母等食品级宿主已被尝试用于CE酶表达研究，韩亮等将CSCE基因经过密码子优化后克隆到pPIC9K分泌型表达载体，再引入到毕赤酵母GS115中表达成功。甲醇诱导144 h后摇瓶发酵上清酶活为0.42 U/mL，纯化后的重组酶与野生型酶性质接近，说明酵母宿主潜在的糖基化作用并没有对酶活性造成负面影响[59]。由于毕赤酵母是适合于高密度发酵的微生物宿主，因此经过高密度发酵后酶活力可能有大幅度的提升。此外，外源基因的拷贝数、甲醇含量的在线控制都会对CE酶基因表达产生重要影响，然而相关研究还较为缺乏。江南大学的王鑫淼等将嗜热网球菌(Dictyoglomus thermophilum)来源CE酶基因连接pBSuL3载体，导入枯草芽孢杆菌CCTCC M2016536中，表达后胞内酶活达到7.5 U/mL。在乳糖浓度400 g/L、反应温度85 ℃、加酶量20 U/mL条件下，乳果糖转化率达51%[60]。笔者将CSCE基因克隆到pMA09载体并转化枯草芽孢杆菌WB800进行表达，16 h后在7.5 L发酵罐获得了5.3 U/mL的发酵上清酶活力。将酶液(浓度7.5 U/mL)用酶膜固定化制备成EMR反应器，利用乳清中的乳糖进行催化反应，乳果糖产率达到58.5%。该反应器反应10个批次后还残留有42.4%的酶活力[61]，该工艺能够降低乳果糖的生产成本，并且排除了食品安全隐患，具有一定的应用价值。
6 相关知识产权情况 部分已公开的生物法制备乳果糖技术发明专利如表 5所示，可见专利申请人主要来自日本、韩国、中国，表明东亚国家对乳果糖的生物法制备技术有着强烈的兴趣。这可能与东亚国家老龄化严重，老年人肠胃蠕动功能差、肠道菌群失调、便秘比例较高有关。而且肝病在我国一直属于高发病，对肝性脑病的治疗也需要使用乳果糖，因此近年来乳果糖口服液产品在我国保持了很大的需求量。已公开的专利主要保护了用于生产乳果糖的纤维二糖异构酶、固定化酶或细胞生产乳果糖的方法、乳果糖的纯化与结晶方法、纤维二糖异构酶在食品级宿主中的表达方法等。
表 5. 部分已公开的生物法制备乳果糖发明专利 Table 5. Part of published patents for bio-production of lactulose
| Patent title | Patent applicant | Patent No. | Application time |
| Cellobiose 2-epimerase, its preparation and uses | Watanabe et al. | US2012329098A1 | 02-05-2009 (In Japan) |
| Method for preparing lactulose from lactose using cellobiose 2-epimerase or N-acetyl glucosamine 2-epimerase | Univ Konkuk Ind Coop Corp Oh and Kim | WO2012115390 | 02-23-2011 (In Korea) |
| Production process for concentrated solution of lactulose | Jiangsu Hi-Stone Pharmaceutical Co., Ltd. Wang et al. | WO2015188566 | 06-11-2014 |
| High-purity epilactose and method for producing the same | Japan Maize Prod Saburi and Yamamoto | JP2011217701A | 04-14-2010 |
| Cellobiose 2-epimerase, and application of the same | Japan Maize Prod Univ Hokkaido Saburi et al. | JP2012130332A | 11-30-2010 |
| Method for production of lactulose from lactose using by cellobiose 2-epimerase | Univ Konkuk Ind Coop Corp Oh and Kim | KR20120096769A | 02-23-2011 |
| Method for production of lactulose from lactose using N-acetyl glucosamine 2-epimerase | Univ Konkuk Ind Coop Corp Oh and Kim | KR20130019309A | 08-16-2011 |
| Method of synthesizing lactulose and system for synthesizing lactulose | Univ Korea Res & Bus Found Kim et al. | KR20130101690A | 03-06-2012 |
| Microreactor for synthesizing lactulose and method of synthesizing lactulose | Univ Korea Res & Bus Found Kim et al. | KR20130101689A | 03-06-2012 |
| Catalyst for synthesizing lactulose and method of synthesizing lactulose | Univ Korea Res & Bus Found Kim et al. | KR101291906B1 | 03-06-2012 |
| System for synthesizing lactulose using sodium carbonate | Korea Advanced Inst Sci & Tech Han and Seo | KR20150053047A | 11-07-2013 |
| System for synthesizing lactulose using ammonium carbonate | Korea Advanced Inst Sci & Tech Han et al. | KR20150048567A | 10-28-2013 |
| Lactulose production using immobilized cells | Forbiokorea Co., Ltd Lee et al. | KR101762222B1 | 10-18-2016 |
| A genetic engineering strain of Bacillus subtilis and its construction and application in lactulose production (authorized patent) | Nanjing Tech University | CN105255805A (Chinese patent) | 11-16-2015 |
| A whole cell immobilization method of cellobiose 2-epimerase (authorized patent) | Jiangnan University | CN104313009A (Chinese patent) | 10-21-2014 |
| A method of heterologous expression and preparation of cellobiose 2-epimerase in yeast cells | Feed Research Institute, Chinese Academy of Agricultural Sciences; National Animal Husbandry Station | CN106434736A (Chinese patent) | 12-21-2016 |
| Recovery and reuse of boric acid in lactulose preparation | Baolingbao Biology Co., Ltd. | CN106589006A (Chinese patent) | 12-08-2016 |
| Enzymatic production of lactulose | Shandong Bailong Group Co., Ltd. | CN104805152A (Chinese patent) | 12-04-2014 |
| A process for simultaneous production of lactulose and tagatose | Yucheng Lvjian Biotechnology Co., Ltd. | CN102296129A (Chinese patent) | 06-16-2011 |
| A production process of crystalline lactulose (authorized patent) | Yucheng Lvjian Biotechnology Co., Ltd. | CN102153598A (Chinese patent) | 02-25-2011 |
| A preparation method of high purity lactulose (authorized patent) | Baolingbao Biology Co., Ltd. | CN102020680A (Chinese patent) | 01-07-2011 |
表选项
7 展望 生物法制备乳果糖具有过程绿色环保、无毒性和重金属催化剂使用、副产物种类少、色素少等优势，具有较好的产业化前景。然而，生物法还存在许多问题，例如纤维二糖差向异构酶的酶活还不够高，故想要获得高转化率只能降低底物浓度，造成了生产强度下降；或者增加了酶的用量，造成了成本的上升以及过多杂质的引入。这也是许多工业酶面临的问题，若开发出更高通量的筛选方法，依靠定向进化或半理性设计较有可能获得酶活力显著提升的突变体。目前乳果糖的检测方法主要通过强酸反应显色，操作繁琐且误差较大，不能实现高通量筛选。针对乳果糖的生物传感器(biosensor)或特异性荧光探针的研发可给予这一方向突破性的进展，例如中国科学院微生物研究所针对乳糖操纵子阻遏蛋白LacI基因筛选获得了突变体LacI-L5，它只能与乳果糖结合并解除乳糖操纵子表达阻遏，触发所连接的荧光蛋白基因表达。因此可以利用该突变体作为胞内乳果糖含量的定量筛选工具，以获得高产乳果糖的突变菌株[62]。高密度发酵(high cell density cultivation，HCDC)可以大幅提升微生物宿主的菌体密度，获得更高的重组酶量从而降低成本，是工业酶产业化的关键技术。这方面需要对发酵过程调控开展深入研究，目前CE酶在这块的研究披露较少，未来需要更多的相关成果来保障产业化技术的实施。由于乳果糖产品主要涉及药品和食品领域，因此法律法规建设十分重要，生物法工艺是否可以获得各国许可？是否要纳入转基因法规管理？这些问题都有待于澄清和解决。总的来说，利用CE酶来生产乳果糖在技术上是完全可行的，在产品质量和工艺成本上都优于化学法。工艺路线可以与大多数酶催化路线兼容，无需生产线的改造，相信在不远的未来生物法制备乳果糖会获得广泛的认可。
References
| [1] | Aider M, de Halleux D. Isomerization of lactose and lactulose production:review. Trends in Food Science & Technology, 2007, 18(7): 356-364. |
| [2] | A?t-Aissa A, A?der M. Lactulose:production and use in functional food, medical and pharmaceutical applications. Practical and critical review. International Journal of Food Science and Technology, 2014, 49(5): 1245-1253. DOI:10.1111/ijfs.12465 |
| [3] | Olano A, Corzo N. Lactulose as a food ingredient. Journal of the Science of Food and Agriculture, 2009, 89(12): 1987-1990. DOI:10.1002/jsfa.3694 |
| [4] | Wang H, Yang RJ, Hua X, Zhao W, Zhang WB. Enzymatic production of lactulose and 1-lactulose:current state and perspectives. Applied Microbiology and Biotechnology, 2013, 97(14): 6167-6180. DOI:10.1007/s00253-013-4998-3 |
| [5] | Pranami D, Sharma R, Pathak H. Lactulose:a prebiotic, laxative and detoxifying agent. Drugs & Therapy Perspectives, 2017, 33(5): 228-233. |
| [6] | Montgomery EM, Hudson CS. Relations between rotatory power and structure in the sugar group. XXVII. Synthesis of a new disaccharide ketose (lactulose) from lactose1. Journal of the American Chemical Society, 1930, 52(5): 2101-2106. DOI:10.1021/ja01368a060 |
| [7] | Schumann C. Medical, nutritional and technological properties of lactulose. European Journal of Nutrition, 2002, 41(1): i17-i25. |
| [8] | Dutta A, Berry SH, El-Omar EM, Hold GL, Mukhopadhya A. The effect of lactulose on the faecal microbiota of patients with minimal hepatic encephalopathy. Gastroenterology, 2017, 152(5): S1049. |
| [9] | Agarwalla A, Weber A, Davey S, Hamilton K, Goldberg D, Rhim AD, Yang YX. Lactulose is associated with decreased risk of Clostridium difficile infection in decompensated cirrhosis. Clinical Gastroenterology and Hepatology, 2017, 15(6): 953-954. DOI:10.1016/j.cgh.2017.01.012 |
| [10] | Zhang Y, Bao ZJ, Zhang GS, Guo ZY, Zhang ZY, Yu XF. Systematic evaluation of the therapeutic effect of lactulose oral solution on functional constipation. Chinese Journal of Gerontology, 2015, 35(22): 6470-6473. (in Chinese) 张颖, 保志军, 张赣生, 郭正扬, 张自妍, 于晓峰. 乳果糖口服液治疗功能性便秘疗效的系统评价. 中国老年学杂志, 2015, 35(22): 6470-6473. DOI:10.3969/j.issn.1005-9202.2015.22.074 |
| [11] | Lactulose Clinical Research Group. Lactulose for the treatment of constipation in pregnant women:a randomized, double-blind, placebo-controlled, multicenter study. Chinese Journal of Digestion, 2006, 26(10): 690-693. (in Chinese) 乳果糖临床协作组. 乳果糖治疗妊娠期妇女便秘的随机、双盲、安慰剂对照多中心临床研究. 中华消化杂志, 2006, 26(10): 690-693. DOI:10.3760/j.issn:0254-1432.2006.10.011 |
| [12] | Lei YW, Cheng L, Zhu YB. The effect of lactulose on functional constipation in children. Clinical Rational Drug Use, 2012, 5(12): 50-51. (in Chinese) 雷衍蔚, 成玲, 朱彦波. 乳果糖治疗小儿功能性便秘的疗效观察. 临床合理用药杂志, 2012, 5(12): 50-51. DOI:10.3969/j.issn.1674-3296.2012.12.036 |
| [13] | MacGillivray PC, Finlay HVL, Binns TB. Use of lactulose to create a preponderance of lactobacilli in the intestine of bottle-fed infants. Scottish Medical Journal, 1959, 4(4): 182-189. DOI:10.1177/003693305900400405 |
| [14] | Seki N, Saito H. Lactose as a source for lactulose and other functional lactose derivatives. International Dairy Journal, 2012, 22(2): 110-115. DOI:10.1016/j.idairyj.2011.09.016 |
| [15] | Wang MM, Gasmalla MAA, Tessema HA, Hua X, Yang RJ. Lactulose production from efficient isomerization of lactose catalyzed by recyclable sodium aluminate. Food Chemistry, 2017, 233: 151-158. DOI:10.1016/j.foodchem.2017.04.080 |
| [16] | De la Fuente MA, Juárez M, de Rafael D, Villamiel M, Olano A. Isomerization of lactose catalyzed by alkaline-substituted sepiolites. Food Chemistry, 1999, 66(3): 301-306. DOI:10.1016/S0308-8146(99)00060-6 |
| [17] | Hua X, Yang RJ, Shen QY, Ye FY, Zhang WB, Zhao W. Production of 1-lactulose and lactulose using commercial β-galactosidase from Kluyveromyces lactis in the presence of fructose. Food Chemistry, 2013, 137(1/4): 1-7. |
| [18] | Lee YJ, Kim CS, Oh DK. Lactulose production by β-galactosidase in permeabilized cells of Kluyveromyces lactis. Applied Microbiology and Biotechnology, 2004, 64(6): 787-793. DOI:10.1007/s00253-003-1506-1 |
| [19] | Mayer J, Kranz B, Fischer L. Continuous production of lactulose by immobilized thermostable β-glycosidase from Pyrococcus furiosus. Journal of Biotechnology, 2010, 145(4): 387-393. DOI:10.1016/j.jbiotec.2009.12.017 |
| [20] | Kim YS, Oh DK. Lactulose production from lactose as a single substrate by a thermostable cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus. Bioresource Technology, 2012, 104: 668-672. DOI:10.1016/j.biortech.2011.11.016 |
| [21] | Kim JE, Kim YS, Kang LW, Oh DK. Characterization of a recombinant cellobiose 2-epimerase from Dictyoglomus turgidum that epimerizes and isomerizes β-1, 4-and α-1, 4-gluco-oligosaccharides. Biotechnology Letters, 2012, 34(11): 2061-2068. DOI:10.1007/s10529-012-0999-z |
| [22] | Chen QM, Levin R, Zhang WL, Zhang T, Jiang B, Stressler T, Fischer L, Mu WM. Characterisation of a novel cellobiose 2-epimerase from thermophilic Caldicellulosiruptor obsidiansis for lactulose production. Journal of the Science of Food and Agriculture, 2017, 97(10): 3095-3105. DOI:10.1002/jsfa.8148 |
| [23] | Kuschel B, Claa?en W, Mu WM, Jiang B, Stressler T, Fischer L. Reaction investigation of lactulose-producing cellobiose 2-epimerases under operational relevant conditions. Journal of Molecular Catalysis B:Enzymatic, 2016, 133(S1): S80-S87. |
| [24] | Xiao YQ, Chen QM, Shakhnovich EI, Zhang WL, Mu WM. Simulation-guided enzyme discovery:a new microbial source of cellobiose 2-epimerase. International Journal of Biological Macromolecules, 2019, 139: 1002-1008. DOI:10.1016/j.ijbiomac.2019.08.075 |
| [25] | Chen QM, Zhang WL, Zhang T, Jiang B, Mu WM. Characterization of an epilactose-producing cellobiose 2-epimerase from Thermoanaerobacterium saccharolyticum. Journal of Molecular Catalysis B:Enzymatic, 2015, 116: 39-44. DOI:10.1016/j.molcatb.2015.03.005 |
| [26] | Ito S, Hamada S, Yamaguchi K, Umene S, Ito H, Matsui H, Ozawa T, Taguchi H, Watanabe J, Wasaki J, Ito S. Cloning and sequencing of the cellobiose 2-epimerase gene from an obligatory anaerobe, Ruminococcus albus. Biochemical and Biophysical Research Communications, 2007, 360(3): 640-645. DOI:10.1016/j.bbrc.2007.06.091 |
| [27] | Ito S, Taguchi H, Hamada S, Kawauchi S, Ito H, Senoura T, Watanabe J, Nishimukai M, Ito S, Matsui H. Enzymatic properties of cellobiose 2-epimerase from Ruminococcus albus and the synthesis of rare oligosaccharides by the enzyme. Applied Microbiology and Biotechnology, 2008, 79(3): 433-441. DOI:10.1007/s00253-008-1449-7 |
| [28] | Taguchi H, Senoura T, Hamada S, Matsui H, Kobayashi Y, Watanabe J, Wasaki J, Ito S. Cloning and sequencing of the gene for cellobiose 2-epimerase from a ruminal strain of Eubacterium cellulosolvens. FEMS Microbiology Letters, 2008, 287(1): 34-40. DOI:10.1111/j.1574-6968.2008.01281.x |
| [29] | Senoura T, Taguchi H, Ito S, Hamada S, Matsui H, Fukiya S, Yokota A, Watanabe J, Wasaki J, Ito S. Identification of the cellobiose 2-epimerase gene in the genome of Bacteroides fragilis NCTC 9343. Bioscience, Biotechnology, and Biochemistry, 2009, 73(2): 400-406. DOI:10.1271/bbb.80691 |
| [30] | Ojima T, Saburi W, Sato H, Yamamoto T, Mori H, Matsui H. Biochemical characterization of a thermophilic cellobiose 2-epimerase from a thermohalophilic bacterium, Rhodothermus marinus JCM9785. Bioscience, Biotechnology, and Biochemistry, 2011, 75(11): 2162-2168. DOI:10.1271/bbb.110456 |
| [31] | Park CS, Kim JE, Lee SH, Kim YS, Kang LW, Oh DK. Characterization of a recombinant mannobiose 2-epimerase from Spirochaeta thermophila that is suggested to be a cellobiose 2-epimerase. Biotechnology Letters, 2013, 35(11): 1873-1880. DOI:10.1007/s10529-013-1267-6 |
| [32] | Ojima T, Saburi W, Yamamoto T, Mori H, Matsui H. Identification and characterization of cellobiose 2-epimerases from various aerobes. Bioscience, Biotechnology, and Biochemistry, 2013, 77(1): 189-193. DOI:10.1271/bbb.120742 |
| [33] | Tyler TR, Leatherwood JM. Epimerization of disaccharides by enzyme preparations from Ruminococcus albus. Archives of Biochemistry and Biophysics, 1967, 119: 363-367. DOI:10.1016/0003-9861(67)90466-3 |
| [34] | Park CS, Kim JE, Choi JG, Oh DK. Characterization of a recombinant cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus and its application in the production of mannose from glucose. Applied Microbiology and Biotechnology, 2011, 92(6): 1187-1196. DOI:10.1007/s00253-011-3403-3 |
| [35] | Hunziker OF, Nissen BH. Lactose solubility and lactose crystal formation:I. Lactose solubility. Journal of Dairy Science, 1926, 9(6): 517-537. DOI:10.3168/jds.S0022-0302(26)93924-6 |
| [36] | Park AR, Kim JS, Jang SW, Park YG, Koo BS, Lee HC. Rational modification of substrate binding site by structure-based engineering of a cellobiose 2-epimerase in Caldicellulosiruptor saccharolyticus. Microbial Cell Factories, 2017, 16(1): 224. DOI:10.1186/s12934-017-0841-3 |
| [37] | Dendene K, Guihard L, Balannec B, Bariou B. Study of the separation of lactose, lactulose and galactose by liquid chromatography using cationic ion-exchange resin columns. Chromatographia, 1995, 41(5/6): 561-567. |
| [38] | Kim YS, Kim JE, Oh DK. Borate enhances the production of lactulose from lactose by cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus. Bioresource Technology, 2013, 128: 809-812. DOI:10.1016/j.biortech.2012.10.060 |
| [39] | Lim BC, Kim HJ, Oh DK. High production of D-tagatose by the addition of boric acid. Biotechnology Progress, 2007, 23(4): 824-828. DOI:10.1002/bp070056y |
| [40] | Shen QY, Zhang YZ, Yang RJ, Pan SY, Dong J, Fan YT, Han L. Enhancement of isomerization activity and lactulose production of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus. Food Chemistry, 2016, 207: 60-67. DOI:10.1016/j.foodchem.2016.02.067 |
| [41] | Gu JY, Yang RJ, Hua X, Zhang WB, Zhao W. Adsorption-based immobilization of Caldicellulosiruptor saccharolyticus cellobiose 2-epimerase on Bacillus subtilis spores. Biotechnology and Applied Biochemistry, 2015, 62(2): 237-244. DOI:10.1002/bab.1262 |
| [42] | Wang MM, Hua X, Yang RJ, Shen QY. Immobilization of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus on commercial resin Duolite A568. Food Bioscience, 2016, 14: 47-53. DOI:10.1016/j.fbio.2016.03.001 |
| [43] | Wang MM, Yang RJ, Hua X, Shen QY, Zhang WB, Zhao W. Lactulose production from lactose by recombinant cellobiose 2-epimerase in permeabilised Escherichia coli cells. International Journal of Food Science and Technology, 2015, 50(7): 1625-1631. DOI:10.1111/ijfs.12776 |
| [44] | Kuschel B, Seitl I, Glu?ck C, Mu WM, Jiang B, Stressler T, Fischer L. Hidden reaction:mesophilic cellobiose 2-epimerases produce lactulose. Journal of Agricultural and Food Chemistry, 2017, 65(12): 2530-2539. DOI:10.1021/acs.jafc.6b05599 |
| [45] | Rentschler E, Schuh K, Krewinkel M, Baur C, Claa?en W, Meyer S, Kuschel B, Stressler T, Fischer L. Enzymatic production of lactulose and epilactose in milk. Journal of Dairy Science, 2015, 98(10): 6767-6775. DOI:10.3168/jds.2015-9900 |
| [46] | Manjasetty BA, Chance MR. Crystal structure of Escherichia coli L-arabinose isomerase (ECAI), the putative target of biological tagatose production. Journal of Molecular Biology, 2006, 360(2): 297-309. DOI:10.1016/j.jmb.2006.04.040 |
| [47] | Yoshida H, Yamada M, Nishitani T, Takada G, Izumori K, Kamitori S. Crystal structures of d-tagatose 3-epimerase from Pseudomonas cichorii and its complexes with d-tagatose and d-fructose. Journal of Molecular Biology, 2007, 374(2): 443-453. DOI:10.1016/j.jmb.2007.09.033 |
| [48] | Yoshida H, Yamada M, Ohyama Y, Takada G, Izumori K, Kamitori S. The structures of L-rhamnose isomerase from Pseudomonas stutzeri in complexes with L-rhamnose and D-allose provide insights into broad substrate specificity. Journal of Molecular Biology, 2007, 365(5): 1505-1516. DOI:10.1016/j.jmb.2006.11.004 |
| [49] | Whitlow M, Howard AJ, Finzel BC, Poulos TL, Winborne E, Gilliland GL. A metal-mediated hydride shift mechanism for xylose isomerase based on the 1.6 ? Streptomycs rubiginosus structure with xylitol and D-xylose. Proteins:Structure, Function, and Bioinformatics, 1991, 9(3): 153-173. DOI:10.1002/prot.340090302 |
| [50] | Saburi W. Functions, structures, and applications of cellobiose 2-epimerase and glycoside hydrolase family 130 mannoside phosphorylases. Bioscience, Biotechnology, and Biochemistry, 2016, 80(7): 1294-1305. DOI:10.1080/09168451.2016.1166934 |
| [51] | Fujiwara T, Saburi W, Matsui H, Mori H, Yao M. Structural insights into the epimerization of β-1, 4-linked oligosaccharides catalyzed by cellobiose 2-epimerase, the sole enzyme epimerizing non-anomeric hydroxyl groups of unmodified sugars. The Journal of Biological Chemistry, 2014, 289(6): 3405-3415. DOI:10.1074/jbc.M113.531251 |
| [52] | Fujiwara T, Saburi W, Inoue S, Mori H, Matsui H, Tanaka I, Yao M. Crystal structure of Ruminococcus albus cellobiose 2-epimerase:structural insights into epimerization of unmodified sugar. FEBS Letters, 2013, 587(7): 840-846. DOI:10.1016/j.febslet.2013.02.007 |
| [53] | Mu WM, Li QX, Fan C, Zhou C, Jiang B. Recent advances on physiological functions and biotechnological production of epilactose. Applied Microbiology and Biotechnology, 2013, 97(5): 1821-1827. DOI:10.1007/s00253-013-4687-2 |
| [54] | Nishimukai M, Watanabe J, Taguchi H, Senoura T, Hamada S, Matsui H, Yamamoto T, Wasaki J, Hara H, Ito S. Effects of epilactose on calcium absorption and serum lipid metabolism in rats. Journal of Agricultural and Food Chemistry, 2008, 56(21): 10340-10345. DOI:10.1021/jf801556m |
| [55] | Watanabe J, Nishimukai M, Taguchi H, Senoura T, Hamada S, Matsui H, Yamamoto T, Wasaki J, Hara H, Ito S. Prebiotic properties of epilactose. Journal of Dairy Science, 2008, 91(12): 4518-4526. DOI:10.3168/jds.2008-1367 |
| [56] | Suzuki T, Nishimukai M, Shinoki A, Taguchi H, Fukiya S, Yokota A, Saburi W, Yamamoto T, Hara H, Matsui H. Ingestion of epilactose, a non-digestible disaccharide, improves postgastrectomy osteopenia and anemia in rats through the promotion of intestinal calcium and iron absorption. Journal of Agricultural and Food Chemistry, 2010, 58(19): 10787-10792. DOI:10.1021/jf102563y |
| [57] | Ito S, Hamada S, Ito H, Matsui H, Ozawa T, Taguchi H, Ito S. Site-directed mutagenesis of possible catalytic residues of cellobiose 2-epimerase from Ruminococcus albus. Biotechnology Letters, 2009, 31(7): 1065-1071. DOI:10.1007/s10529-009-9979-3 |
| [58] | Shen QY, Zhang YZ, Yang RJ, Hua X, Zhang WB, Zhao W. Thermostability enhancement of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus by site-directed mutagenesis. Journal of Molecular Catalysis B:Enzymatic, 2015, 120: 158-164. DOI:10.1016/j.molcatb.2015.07.007 |
| [59] | Han L, Yang RJ, Zhao W, Shen QY, Zhang WB, Hua X. Expression of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus in Pichia pastoris and its enzymatic characterization. Industrial Microbiology, 2016, 46(1): 16-21. (in Chinese) 韩亮, 杨瑞金, 赵伟, 沈秋云, 张文斌, 华霄. 纤维二糖差向异构酶在毕赤酵母中的表达及酶学性质研究. 工业微生物, 2016, 46(1): 16-21. DOI:10.3969/j.issn.1001-6678.2016.01.003 |
| [60] | Wang XM, Zhang K, Chen S, Wu J. Recombinant expression and fermentation optimization of Dictyoglomus thermophilum cellobiose 2-epimerase in Bacillus subtilis. China Biotechnology, 2019, 39(7): 24-31. (in Chinese) 王鑫淼, 张康, 陈晟, 吴敬. 嗜热网球菌纤维二糖差向异构酶在枯草芽孢杆菌中的表达及发酵优化. 中国生物工程杂志, 2019, 39(7): 24-31. |
| [61] | Wu L T, Xu C, Li S, Liang JF, Xu H, Xu Z. Efficient production of lactulose from whey powder by cellobiose 2-epimerase in an enzymatic membrane reactor. Bioresource Technology, 2017, 233: 305-312. DOI:10.1016/j.biortech.2017.02.089 |
| [62] | Wu JY, Jiang PX, Chen W, Xiong DD, Huang LL, Jia JY, Chen YY, Jin JM, Tang SY. Design and application of a lactulose biosensor. Scientific Reports, 2017, 7: 45994. DOI:10.1038/srep45994 |
