李偉娜，蔣云云，劉彥楠，李春英，范代娣

人參皂苷單體定向轉(zhuǎn)化的生物催化及應(yīng)用進(jìn)展

李偉娜1,2，蔣云云1,2，劉彥楠1,2，李春英1,2，范代娣1,2

1 西北大學(xué) 化工學(xué)院 陜西省可降解生物醫(yī)用材料重點(diǎn)實(shí)驗(yàn)室，陜西 西安 710069 2 西北大學(xué) 化工學(xué)院 陜西省生物材料與發(fā)酵工程技術(shù)研究中心，陜西 西安 710069

人參是我國(guó)傳統(tǒng)中藥，藥效顯著、應(yīng)用廣泛。通過(guò)定向修飾與轉(zhuǎn)化人參皂苷糖基可產(chǎn)生高抗癌活性稀有人參皂苷。傳統(tǒng)化學(xué)法由于制備工藝極其復(fù)雜、成本過(guò)高，不能應(yīng)用于臨床，微生物及其酶系轉(zhuǎn)化成為解決該瓶頸問(wèn)題的最可行手段。有關(guān)全細(xì)胞催化、糖苷酶重組表達(dá)、固定化及其催化分子識(shí)別機(jī)制和溶劑工程的生物轉(zhuǎn)化已有大量綜述報(bào)道，但尚無(wú)在人參皂苷轉(zhuǎn)化應(yīng)用中的系統(tǒng)研究。文中通過(guò)對(duì)人參皂苷單體生物轉(zhuǎn)化理論和應(yīng)用研究最新進(jìn)展的回顧，結(jié)合目前廣泛采用的生物催化方法的討論，系統(tǒng)梳理歸納了能夠改善產(chǎn)物專一性、提高催化效率，且具有工業(yè)應(yīng)用前景的人參皂苷單體定向轉(zhuǎn)化方法。基于酶分子設(shè)計(jì)以及離子液體溶劑工程，對(duì)人參皂苷單體抗癌藥物和食品、保健品市場(chǎng)的開(kāi)發(fā)、規(guī)?；苽溥M(jìn)行了展望。

人參皂苷，生物催化，全細(xì)胞轉(zhuǎn)化，酶法轉(zhuǎn)化，離子液體

腫瘤是全球致死率最高的惡性疾病，危害人類的生命與健康[1]。目前臨床上腫瘤治療藥物很多來(lái)源于植物，如紫杉醇、雷公藤甲素等，而現(xiàn)代藥理學(xué)研究表明，來(lái)源于人參、西洋參和三七中的重要次級(jí)代謝產(chǎn)物人參皂苷具顯著抗癌效果，而尤以稀有人參皂苷及苷元的抗腫瘤、保護(hù)神經(jīng)系統(tǒng)、保肝護(hù)肝等藥理活性最為顯著[2]。而人參皂苷、次級(jí)皂苷和皂苷元等成分在人參屬植物中含量較少，體內(nèi)轉(zhuǎn)化量和生物利用度極低，須通過(guò)體外總皂苷降解獲得。

通過(guò)多種技術(shù)手段去除骨架結(jié)構(gòu)達(dá)瑪烷四環(huán)三萜支鏈上所連糖基，定向獲得人參皂苷單體成為研究的熱點(diǎn)[3]。已經(jīng)通過(guò)酸或熱處理方法轉(zhuǎn)化并分離出289種純?nèi)藚⒃碥諉误w[4]。由國(guó)家藥品監(jiān)督管理局(SFDA) 批準(zhǔn)上市的抗癌新藥參一膠囊(人參皂苷Rg3)，成為我國(guó)首個(gè)實(shí)現(xiàn)人參皂苷工業(yè)化生產(chǎn)的一類中藥單體抗癌藥物。國(guó)家SFDA唯一認(rèn)可的人參皂苷Rh2產(chǎn)品今幸膠囊，純度98%的20(S)-Rh2經(jīng)極其復(fù)雜的大孔樹脂吸附、硅膠柱層析分離提取工藝獲得，每斤價(jià)格高達(dá)100多萬(wàn)人民幣[5]。

基于微生物及其酶的生物催化由于反應(yīng)特異性高、條件溫和、副產(chǎn)物少、后處理簡(jiǎn)單成為解決其瓶頸問(wèn)題的最可行手段。原人參萜二醇(Protopanaxadiol，PPD) 型及原人參萜三醇(Protopanaxatriol，PPT) 型皂苷通過(guò)細(xì)胞轉(zhuǎn)化或發(fā)酵以不同的水解途徑轉(zhuǎn)化為去糖基化(Deglycosylation) 的人參皂苷[6]。實(shí)質(zhì)為糖苷水解酶(Glycoside hydrolase，GH) 對(duì)其側(cè)鏈糖基(以1–4分子的D-葡萄糖、L-阿拉伯吡喃糖苷、L-阿拉伯呋喃糖苷、D-木糖和/或L-鼠李糖等組成) 的特異性水解[7]。金鳳燮課題組從微生物培養(yǎng)物、植物提取物得到人參皂苷糖苷酶(纖維素酶和糖苷酶新亞類)，并依據(jù)底物糖基連接位置和內(nèi)、外側(cè)糖殘基水解特異性進(jìn)行了分類[8]。Shin等[7]對(duì)不同來(lái)源人參皂苷GH歸屬、分子量、最適反應(yīng)pH、溫度、比活等生化特性進(jìn)行了總結(jié)。

微生物、酶法轉(zhuǎn)化相比化學(xué)法在持續(xù)性、選擇性和再生等方面的優(yōu)勢(shì)如表1所示。相比化學(xué)法，微生物及其酶的生物轉(zhuǎn)化僅存在溶劑耐受性和轉(zhuǎn)化率方面的不足；相比微生物法，酶法主要存在成本和再生等問(wèn)題[9]。本文綜合人參皂苷生物轉(zhuǎn)化最新進(jìn)展的回顧，及目前廣泛采用的生物催化方法的討論，認(rèn)為基于蛋白質(zhì)工程的酶分子改造和綠色溶劑工程(以離子液體為主) 的催化體系構(gòu)建，在高效、定向轉(zhuǎn)化人參皂苷單體方面有廣闊的工業(yè)應(yīng)用前景。

表1 不同催化方法的特點(diǎn)和選擇

1 人參皂苷糖基代謝及對(duì)藥理活性的影響

通常人參根可直接口服，或以粉末或提取物通過(guò)能量飲料、茶和功能性補(bǔ)充劑食用。然而，口服人參對(duì)主要人參皂苷的吸收來(lái)說(shuō)是無(wú)效的[20]。因?yàn)樘腔藚⒃碥赵谀c道中的生物利用率非常低(比如Rb1為0.1%–4.4%; Rb2為3.7%)，且容易通過(guò)膽道或泌尿系統(tǒng)排出[11-12]，需要通過(guò)腸道微生物群對(duì)其藥物代謝動(dòng)力學(xué)特性的改變來(lái)逆轉(zhuǎn)低生物利用度，最終被位于腸壁的天然微生物群降解為容易被吸收并生物活性稀有人參皂苷(Rg2、Rg3、Compound K等) 和苷元[13]。

稀有人參皂苷單體及其衍生物能夠調(diào)控癌細(xì)胞炎癥、氧化應(yīng)激、血管生成、轉(zhuǎn)移信號(hào)路徑，單獨(dú)或者結(jié)合其他藥材治療癌癥[2]。基于糖基數(shù)量、位置和糖配基類型、雙鍵位置和立體選擇性，Quan等[14]通過(guò)酸轉(zhuǎn)化快速制備了23種稀有人參皂苷，并基于它們對(duì)6種癌細(xì)胞(包括HCT-116、HepG2、MCF-7、Hela、PANC-1和A549) 的細(xì)胞毒性作用分析了其皂苷結(jié)構(gòu)-藥理活性關(guān)系。Wei等[15]研究脂肪酸酯化法修飾Rh2，在體內(nèi)抗腫瘤活性不變的同時(shí)，二辛酰酯(D-Rh2)體外對(duì)人肝細(xì)胞系QSG-7701毒性相比親本Rh2顯著降低。D-Rh2可能通過(guò)刺激淋巴細(xì)胞對(duì)腫瘤細(xì)胞產(chǎn)生細(xì)胞毒性而間接影響腫瘤生長(zhǎng)，較低副作用的D-Rh2可用作抗腫瘤候選藥物。

天然化合物人參皂苷的體外修飾與轉(zhuǎn)化增加了人參皂苷結(jié)構(gòu)多樣性。為深入研發(fā)高抗癌活性藥物的應(yīng)用，本課題組在前期的研究工作中通過(guò)酸轉(zhuǎn)化、柱分離制備了一系列稀有皂苷及其組合物，通過(guò)抗腫瘤活性分析及初步臨床觀察，發(fā)現(xiàn)PPD型人參皂苷雙鍵異構(gòu)體Rk1和Rg5 (即Rg3 C-20處脫羥基產(chǎn)物) 因抗癌活性優(yōu)異而極具開(kāi)發(fā)潛力。皂苷單體殺傷腫瘤細(xì)胞作用的強(qiáng)度與苷元類型、糖鏈長(zhǎng)短及C-20立體異構(gòu)有關(guān)，改造單體結(jié)構(gòu)可增強(qiáng)抗腫瘤活性[6,14,16]。

以酸水解、高溫等改變?cè)碥战Y(jié)構(gòu)的化學(xué)法，均存在價(jià)格高、選擇性差、副產(chǎn)物多等問(wèn)題，工業(yè)化生產(chǎn)不易實(shí)現(xiàn)，多數(shù)單體分子結(jié)構(gòu)和抗腫瘤活性的構(gòu)效關(guān)系仍不能被完全闡明[14]。通過(guò)微生物及其酶系的生物催化，由于反應(yīng)特異性高、條件溫和、副產(chǎn)物少、后處理簡(jiǎn)單，適于稀有人參皂苷Compound K、Rg3及其衍生物等轉(zhuǎn)化制備。生物轉(zhuǎn)化修飾結(jié)構(gòu)已涉及羥基化、環(huán)氧化、甲基化、異構(gòu)化、酯化、水解、醇和酮之間的氧化還原、脫氫等多種反應(yīng)類型[17]。而人參皂苷結(jié)構(gòu)修飾主要在于對(duì)特定位點(diǎn)的糖基進(jìn)行水解，通過(guò)不同位置糖鏈結(jié)構(gòu)的變化來(lái)改善化合物的生物活性。

2 菌體及酶法轉(zhuǎn)化人參皂苷單體

生物催化劑應(yīng)用形式(即細(xì)胞懸浮液、無(wú)細(xì)胞提取物和純化酶) 一般取決于下游加工及菌體固有特性[18](圖1)。細(xì)胞為酶提供了天然環(huán)境并防止蛋白構(gòu)象變化，多種胞內(nèi)、胞外酶的產(chǎn)生取決于生長(zhǎng)條件和細(xì)胞發(fā)育狀況。全細(xì)胞催化有利于節(jié)約人力、生產(chǎn)成本、維護(hù)成本。微生物多樣性是菌體對(duì)各類自然條件(例如溫度、pH、壓力和鹽) 長(zhǎng)期適應(yīng)的結(jié)果，極端微生物在實(shí)驗(yàn)室條件下通常很難生長(zhǎng)。除常規(guī)的富集培養(yǎng)，還可通過(guò)宏基因組(Metagenome) DNA庫(kù)篩選或序列搜索發(fā)現(xiàn)新的功能基因，再結(jié)合適當(dāng)?shù)目寺≥d體和宿主獲得工程菌[19]。酶較全細(xì)胞對(duì)一步反應(yīng)更有優(yōu)勢(shì)，負(fù)責(zé)生命過(guò)程所有必需代謝反應(yīng)的催化，一般反應(yīng)條件溫和，pH、溫度范圍有限，體外僅在最佳pH和溫度條件下發(fā)揮作用。純酶反應(yīng)特異性更高，而酶的分離和純化過(guò)程耗時(shí)費(fèi)錢。

圖1 由分離酶或全細(xì)胞進(jìn)行生物催化的比較(改編自參考文獻(xiàn)[18])

2.1 菌體/全細(xì)胞催化

微生物能夠通過(guò)全細(xì)胞或發(fā)酵以不同代謝途徑對(duì)人參皂苷進(jìn)行去糖基化(Deglycosylation)[6]。例如，以人參皂苷Rb1為底物轉(zhuǎn)化生產(chǎn)Compound K (已證明具諸如抗癌、抗炎、抗過(guò)敏、抗糖尿病、抗血管生成、抗衰老、神經(jīng)保護(hù)和保肝作用等生物學(xué)功效[20])，枝孢霉菌[21]兼具兩條水解途徑：Rb1→G17→F2→Compound K→和Rb1→Rd→F2→Compound K；葉森金桿菌和人體腸道菌群[22]兼具3條水解途徑：Rb1→F2→Compound K、Rb1→G17→G75→Compound K或Rb1→G17→F2 → Compound K。

腸道菌群中的乳酸桿菌和雙歧桿菌屬spp.可合成對(duì)人參皂苷去糖基化所必需的多種糖苷酶(EC 3.2.1)，包括β-葡糖苷酶(EC 3.2.1.2)、纖維素酶(EC 3.2.1.4)、β-半乳糖苷酶(EC 3.2.1.23)、α-L-阿拉伯呋喃糖苷酶(EC 3.2.1.55)、α-L-阿拉伯糖苷酶(無(wú)EC編號(hào))和β-木糖苷酶(EC 3.2.1.37)[10,23]。腸道微生物組成及其糖苷酶活性對(duì)人參皂苷CK、Re等的代謝調(diào)控有重要作用[24]。而以人參皂苷為碳源進(jìn)行的腸道細(xì)菌體外厭氧培養(yǎng)轉(zhuǎn)化，存在培養(yǎng)基昂貴、產(chǎn)率低的問(wèn)題[10]。

人參栽培土壤中可以分離用于人參皂苷轉(zhuǎn)化的真菌[9]。曲霉屬含β-葡萄糖苷酶約250種，在食品和飲料領(lǐng)域應(yīng)用了1 500年，其中黑曲霉和米曲霉列為美國(guó)FDA通常被認(rèn)為是安全的(GRAS) 名單[25-26]。Lin等[27]直接使用雜色曲霉產(chǎn)孢子階段分泌于固體培養(yǎng)物中的胞外β-葡萄糖苷酶進(jìn)行人參皂苷Rb1到Rd的轉(zhuǎn)化。孢子懸浮系統(tǒng)從搖瓶放大至2 L時(shí)的最大生物轉(zhuǎn)化率85% (/)。因培養(yǎng)基便宜且生長(zhǎng)較快，該類土壤真菌的轉(zhuǎn)化較腸道細(xì)菌更經(jīng)濟(jì)可行，被指定為GRAS的土壤微生物能應(yīng)用于食品領(lǐng)域。

預(yù)計(jì)到2021年益生菌市場(chǎng)總量增長(zhǎng)至580億美元[28]。以益生菌修飾人參皂苷糖基結(jié)構(gòu)，有助于其保健品特性的強(qiáng)化和規(guī)范。Ku等[10,29]分別用長(zhǎng)雙歧桿菌RD47和鼠李糖乳桿菌GG將人參皂苷Rb2、Rc和Rb1水解為人參皂苷Rd。作為牛奶、蔬菜等食物發(fā)酵劑的乳酸菌(Lactic acid bacteria，LAB) 在稀有人參皂苷的轉(zhuǎn)化應(yīng)用方面，Park等[30]首次通過(guò)食品級(jí)明串珠菌和乳酸桿菌全細(xì)胞轉(zhuǎn)化生產(chǎn)Compound K。Huq等[31]以LAB在經(jīng)濟(jì)可食用培養(yǎng)基(有20 g/L蘿卜，20 g/L葡萄糖和10 g/L酵母提取物) 上經(jīng)7 d發(fā)酵后，人參皂苷Rb1可轉(zhuǎn)化Rg3，而MRS培養(yǎng)基中只能轉(zhuǎn)化到Rd。作為表達(dá)外源基因高效生產(chǎn)酶蛋白的GRAS宿主，Li等[32]通過(guò)強(qiáng)化密碼子，在乳酸乳球菌中表達(dá)克隆自膠質(zhì)類芽孢桿菌的重組β-葡萄糖苷酶，轉(zhuǎn)化人參皂苷Rb1和Rd為F2。

在三萜合成途徑構(gòu)建方面，以釀酒酵母為代表的微生物工程已被廣泛用于人參皂苷的轉(zhuǎn)化[33-34](表2)。經(jīng)鑒定Cyt P450酶(CYP716A47) 參與達(dá)瑪烷型人參皂苷前體達(dá)瑪烯二醇-II C-12位羥基化，Han等[35]通過(guò)在中 CYP716A47和達(dá)瑪烯二醇合酶基因(PgDDS) 的共表達(dá)，不添加達(dá)瑪烯二醇-II即可產(chǎn)生PPD。Dai等[36]在中引入達(dá)瑪烯二醇-II合成酶、PPD合成酶基因以及擬南芥NADPH-細(xì)胞色素P450還原酶(ATR1) 基因，并通過(guò)過(guò)量表達(dá)截短(Truncation) 的3-羥基-3-甲基戊二酰輔酶A還原酶、法呢基二磷酸合成酶、角鯊烯合成酶和2,3-氧化角鯊烯合成酶基因，以及提高PPD合酶活性的密碼子優(yōu)化，最后通過(guò)雙相萃取發(fā)酵獲得8.40 mg/g DCW PPD (1 189 mg/L)。Zhao等[37]通過(guò)引入人參達(dá)瑪烯二醇-II合成酶、人參細(xì)胞色素P450型PPD合成酶(PPDS) 和擬南芥ATR1基因，在中構(gòu)建了PPD的生物合成途徑，經(jīng)細(xì)胞色素P450氧化系統(tǒng)優(yōu)化，在5 L生物反應(yīng)器中補(bǔ)料分批發(fā)酵，PPD產(chǎn)量達(dá)到1 436.6 mg/L。

表2 微生物基因工程菌全細(xì)胞催化轉(zhuǎn)化人參皂苷

“→” represents one-step reaction; “→→” represents more than one-step reactions; “/” representsdata not mentioned.

通過(guò)在微生物底盤細(xì)胞中重構(gòu)與優(yōu)化天然化合物的生物合成途徑, 實(shí)現(xiàn)目標(biāo)化合物的從頭合成。被首次表征的用于植物四環(huán)三萜底物糖基化的UDP-糖基轉(zhuǎn)移酶(UGT) UGTPg1，轉(zhuǎn)移葡糖基部分到PPD的C-20S-OH得到Compound K，是經(jīng)一鍋反應(yīng)由簡(jiǎn)單糖生物合成Compound K成功的關(guān)鍵[38]。Wang等[39]從人參中克隆并鑒定了兩種基因：45和29，結(jié)合產(chǎn)PPD的酵母底盤細(xì)胞，成功構(gòu)建了由葡萄糖生產(chǎn)Rh2和Rg3的酵母細(xì)胞工廠。Wei等[40]證明從人參分離的新型基因100和101可特異糖基化PPT的C6-OH分別產(chǎn)生人參皂苷Rh1、F1，在酵母底盤細(xì)胞中構(gòu)建了F1和Rh1的合成途徑。Zhuang等[41]將用于PPD到人參皂苷Rh2轉(zhuǎn)化的微生物糖基轉(zhuǎn)移酶UGT51通過(guò)半理性設(shè)計(jì)，將突變糖基轉(zhuǎn)移酶基因引入酵母并經(jīng)代謝工程進(jìn)一步優(yōu)化后，在5 L生物反應(yīng)器中補(bǔ)料分批發(fā)酵，Rh2產(chǎn)量達(dá)到約300 mg/L。

畢赤酵母與遺傳操作相似且外源蛋白表達(dá)量更高，Zhao等[42]通過(guò)在中自組裝達(dá)瑪烯二醇-Ⅱ合酶和角鯊烯環(huán)氧酶促使達(dá)瑪烯二醇-Ⅱ產(chǎn)量增加2.1倍。Li等[43]通過(guò)在大腸桿菌中重建2,3-氧化角鯊烯衍生的三萜類化合物生物合成途徑，成功構(gòu)建產(chǎn)達(dá)瑪烯二醇-Ⅱ的底盤，達(dá)瑪烯二醇-Ⅱ異源生物合成，發(fā)酵48 h產(chǎn)率為8.63 mg/L。但是這些工作目前還僅限于達(dá)瑪烷型人參皂苷前體達(dá)瑪烯二醇-Ⅱ的合成, 尚未真正意義上實(shí)現(xiàn)合成人參皂苷。

通過(guò)外源生物合成途徑重建的工程菌直接從人參皂苷為底物合成稀有人參皂苷。Yu等[44]通過(guò)尿苷二磷酸糖基轉(zhuǎn)移酶(UTG) 基因工程化大腸桿菌分別由20(R)-PPD和20(R)-PPT轉(zhuǎn)化生產(chǎn)20(R)-Compound K和20(R)-F1。Lu等[45]鑒定了參與人參皂苷Rg1和Rb1生物合成的UDP-糖基轉(zhuǎn)移酶，并實(shí)現(xiàn)了中的全細(xì)胞催化。

2.2 酶催化

微生物的代謝易受環(huán)境影響，轉(zhuǎn)化人參皂苷的選擇性差、產(chǎn)率低，參與反應(yīng)的酶未確定。酶促轉(zhuǎn)化由于操作方法簡(jiǎn)單且反應(yīng)特異性強(qiáng)，被認(rèn)為是結(jié)構(gòu)修飾和代謝研究的有用工具。優(yōu)化微生物產(chǎn)酶條件，實(shí)現(xiàn)生物量和產(chǎn)酶之間的平衡，尤以最大限度縮短處理時(shí)間、降低成本的重組酶轉(zhuǎn)化最為顯著[46]。

在中過(guò)表達(dá)各種重組β-葡糖苷酶以酶解糖基化的人參皂苷。Cui等[47]用重組酶在10 L發(fā)酵罐轉(zhuǎn)化20 mg/mL Re得到113 g色譜純(84.0±1.1)%的Rg2 (S)，首次實(shí)現(xiàn)100 g級(jí)Rg2 (S)酶法生產(chǎn)。Kim等[48]補(bǔ)料分批培養(yǎng)強(qiáng)化表達(dá)β-D-糖苷酶和α-L-阿拉伯呋喃糖苷酶的.，通過(guò)立體專一酶的級(jí)聯(lián)轉(zhuǎn)化，由人參葉提取物獲得人參皂苷Compound K。Shin等[49]將能完全轉(zhuǎn)化 所有PPD型皂苷為Compound K的β-糖苷酶在重組表達(dá)，可將丟棄人參葉的PPD型皂苷轉(zhuǎn)化為Compound K。Xie等[50]過(guò)表達(dá)來(lái)自嗜熱酶α-丙氨酸呋喃糖苷酶，85 ℃、pH 5.0、1 h內(nèi)轉(zhuǎn)化25 g/L Rc為21.8 g/LRd，Rd產(chǎn)率為21 800 mg/(L·h)。Quan等[51]首次報(bào)道了高熱穩(wěn)定性重組β-葡萄糖苷酶分別將人參皂苷Re和Rg1轉(zhuǎn)化為人參皂苷Rg2和Rh1，在酶濃度1.36 U/mL、85 ℃、pH 5.5、1 h內(nèi)轉(zhuǎn)化10 g/L人參皂苷Re為8.02 g/L Rg2， 2 g/L人參皂苷Rg1為1.56 g/L Rh1。

重組酶轉(zhuǎn)化人參皂苷可能應(yīng)用于醫(yī)藥或制藥行業(yè)。然而在食品(尤其營(yíng)養(yǎng)保健品) 行業(yè)中，常被認(rèn)為是不可食用細(xì)菌[52]。為提高用于人參皂苷微生物轉(zhuǎn)化的糖苷酶產(chǎn)量，有必要在以下方面進(jìn)一步改善：1) 開(kāi)發(fā)廣泛的食品級(jí)宿主；2) 提高細(xì)胞生物量和酶產(chǎn)量；3) 確定用于酶誘導(dǎo)的最佳培養(yǎng)基組分[53]。

益生菌適合食品級(jí)糖基化酶的生產(chǎn)，其 MRS培養(yǎng)基價(jià)格也僅為的3倍。以含外源基因重組質(zhì)粒的益生菌系統(tǒng)替代，Youn等[54]在雙歧桿菌中表達(dá)克隆自動(dòng)物雙歧桿菌的重組β-葡萄糖苷酶以轉(zhuǎn)化人參皂苷Rb1和Rb2。由于轉(zhuǎn)基因應(yīng)用于食品工業(yè)引發(fā)的爭(zhēng)議，又轉(zhuǎn)向以經(jīng)典發(fā)酵法生產(chǎn)糖苷水解酶，篩選新的益生菌菌株并優(yōu)化細(xì)胞培養(yǎng)條件。來(lái)源于蘑菇真菌的菌絲體酶制劑(菌體培養(yǎng)2周，25 ℃，經(jīng)酶提取、硫酸銨沉淀、透析、凍干) 轉(zhuǎn)化Rb1到Compound K，最適培養(yǎng)條件為72–96 h、pH 4.0–4.5、溫度45–55 ℃[55]。對(duì)于20 mg 花費(fèi)達(dá)163.2美元的98% F1，除了僅停留在確定微生物酶轉(zhuǎn)化能力的小規(guī)模實(shí)驗(yàn)，Wang等[56]首次報(bào)道了以GRAS微生物食品級(jí)的商業(yè)酶Cellulase KN在48 h內(nèi)完成對(duì)Re和Rg1 (源自10 mg/mL PPTGM) 至F1的10 g級(jí)轉(zhuǎn)化。

除了水解人參皂苷的糖基，微生物也能轉(zhuǎn)化產(chǎn)生高生物活性的人參皂苷化學(xué)衍生物。Zhou等[57]以擬青霉sp. 229對(duì)人參皂苷Rb1的6 L發(fā)酵中，通過(guò)反復(fù)硅膠柱層析和高壓液相色譜分離純化出3-酮衍生物和兩種新的脫氫代謝物。不同類別的酶能選擇性修飾天然化合物的復(fù)雜反應(yīng)性官能團(tuán)而產(chǎn)生一系列衍生物，Teng等[58]以乙酸乙烯酯作為乙酰基供體，在有機(jī)溶劑中通過(guò)來(lái)自南極假絲酵母(Novozym 435) 脂肪酶區(qū)域選擇性地酰化人參皂苷，得到單?；藚⒃碥?。Gebhardt等[59]通過(guò)來(lái)自牛初乳的β-1,4-半乳糖基轉(zhuǎn)移酶和Novozym 435脂肪酶的催化，制備獲得了人參皂苷Rb1的一系列特異性衍生物。

2.3 糖苷酶固定化

相對(duì)于游離酶，固定化酶為適于工業(yè)化應(yīng)用的主要形式。基于吸附、包埋、交聯(lián)、共價(jià)結(jié)合的酶的固定化方法的選擇，以考察固定化酶操作穩(wěn)定性為主，綜合考慮工業(yè)放大時(shí)的技術(shù)可行性和固定化過(guò)程中涉及的酶、載體及試劑的費(fèi)用。而提高時(shí)空產(chǎn)率高效酶反應(yīng)器的開(kāi)發(fā)，進(jìn)一步推動(dòng)了固定化酶技術(shù)在生物轉(zhuǎn)化領(lǐng)域中的研究應(yīng)用。而應(yīng)用于人參皂苷轉(zhuǎn)化的酶固定化報(bào)道僅有3篇。Zhang等[60]以交聯(lián)-包埋法(交聯(lián)3 h，戊二醛濃度0.1%，海藻酸鈉濃度1%，CaCl2濃度2%)固定化酶轉(zhuǎn)化人參皂苷Rg1為F1，3.76 U/g固定化酶載體，0.2 mg/mL底物，40 ℃轉(zhuǎn)化2 d，4次平均轉(zhuǎn)化率為80.49%。Yu等[61]以SiO2吸附蝸牛酶然后結(jié)合交聯(lián)-包埋法(海藻酸鈉質(zhì)量濃度2%，CaCl2質(zhì)量濃度2%，SiO2與蝸牛酶質(zhì)量比為1∶1) 制備微球固定化蝸牛酶，轉(zhuǎn)化人參皂苷Rb1為Compound K，55 ℃，1.0 mg/mL底物，轉(zhuǎn)化36 h，5次平均轉(zhuǎn)化率為 36.79%。為解決酶與載體吸附力弱、與底物接觸面積有限的問(wèn)題，Yuan等[62]將纖維素酶固定在用聚乙烯亞胺和戊二醛活化的角叉菜膠珠表面，轉(zhuǎn)化人參皂苷Rb1為 Rd，同時(shí)測(cè)定了反應(yīng)動(dòng)力學(xué)參數(shù)m和max，在連續(xù)使用5次后，固定化酶可以保持初始活性的60%。

Graebin等[63]特別關(guān)注了糖苷酶家族GH1和GH3中β-葡萄糖苷酶的固定化方法。物理吸附、離子交換、疏水作用等增強(qiáng)了固定化體系酶的靈活性，相應(yīng)載體包括土壤膠體顆粒、離子交換樹脂、磁性Fe3O4納米顆粒等。尤其絲瓜蔬菜海綿[64]、含氧化鐵的細(xì)小土壤膠粒[65]等天然可降解、成本低廉載體的使用大大減少了化學(xué)載體丟棄時(shí)涉及的成本和環(huán)境問(wèn)題。包埋固定化可改善酶的熱穩(wěn)定性、最佳使用溫度和儲(chǔ)存穩(wěn)定性，但機(jī)械強(qiáng)度較低且酶滲漏導(dǎo)致固定化成本增加[66]。關(guān)于其相對(duì)活性和包封率，因納米級(jí)聚合物材料(聚氨酯、乳膠和硅膠) 代替?zhèn)鹘y(tǒng)藻酸鹽珠的應(yīng)用得到改善[67]。共價(jià)固定化可提高酶制劑穩(wěn)定性，載體包括最常用殼聚糖及其他海綿、咖啡渣、硅膠、SiO2納米顆粒、環(huán)氧樹脂活化Eupergit C、胺瓊脂糖凝膠等。

吸附或包埋固定β-葡糖苷酶由于酶逐漸釋放導(dǎo)致催化劑半衰期有限；經(jīng)化學(xué)反應(yīng)的共價(jià)固定化引起酶活損失[68]。為克服這些問(wèn)題，Mateo等[69]研究了酶物理聚集再交聯(lián)(交聯(lián)酶聚集體，CLEAs) 制備固體生物催化劑的方法。通過(guò)形成納米尺度CLEAs將β-葡糖苷酶固定于二氧化硅泡沫。高酶載量CLEAs在更廣泛的溫度和pH范圍保持活性，且比游離酶m低，可使用多達(dá)10個(gè)循環(huán)，殘余活性超過(guò)85%[68]。近來(lái)GH1家族β-葡糖苷酶結(jié)構(gòu)的解析有助于定點(diǎn)固定化工作的展開(kāi)。經(jīng)定點(diǎn)誘變后，酶與載體進(jìn)行固定化，穩(wěn)定性提高的同時(shí)獲得產(chǎn)物抑制降低，活性、專一性提高等優(yōu)勢(shì)特性[63]。而基于重組DNA的新酶設(shè)計(jì)經(jīng)過(guò)微生物的遺傳修飾，驗(yàn)證食品安全性的同時(shí)，也應(yīng)充分考慮酶及其載體釋放到加工系統(tǒng)可能帶來(lái)的安全隱患[70]。

3 糖苷酶轉(zhuǎn)化分子機(jī)制的探究

為解決微生物發(fā)酵及自身酶系對(duì)人參皂苷糖苷鍵催化存在的專一性差、效率低等問(wèn)題，基于酶高級(jí)結(jié)構(gòu)、分子識(shí)別機(jī)制改造酶結(jié)構(gòu)，并通過(guò)基因工程菌表達(dá)以高效轉(zhuǎn)化目的人參皂苷單體。Mak等[71]綜合基因組挖掘(Integrative genomic mining approach) 結(jié)合生物信息和分子建模采掘序列數(shù)據(jù)庫(kù)(Mine sequence databases)，使酶對(duì)目標(biāo)反應(yīng)專一性提高100倍，催化效率提高了75倍。因此除了針對(duì)特定產(chǎn)物挖掘?qū)Ｒ恍缘男旅福芯抗ぷ鬟€應(yīng)集中在基于催化分子機(jī)制的酶分子改造。

糖苷酶家族活性結(jié)構(gòu)域拓?fù)鋵W(xué)構(gòu)象有3種：口袋式(Pocket)、裂隙(Cleft 或Groove)、隧道(Tunnel)[72]?；跇?gòu)效關(guān)系水解酶定向轉(zhuǎn)化與分子識(shí)別的報(bào)道較多[73-75]。基于高分辨高葡萄糖耐受β-葡糖苷酶(Bgl6) 和三重突變體M3(隨機(jī)誘變提高熱穩(wěn)定性)的晶體結(jié)構(gòu)，Pang等[75]發(fā)現(xiàn)Bgl6形成的額外通道可作為葡萄糖二級(jí)結(jié)合位點(diǎn)，有助于葡萄糖耐量；三重突變?cè)鰪?qiáng)酶內(nèi)的疏水相互作用，可能是M3熱穩(wěn)定性增強(qiáng)的原因。Zhang等[76]基于活性位點(diǎn)比對(duì)和量子化學(xué)計(jì)算得出化學(xué)必需的相互作用，以驗(yàn)證催化機(jī)理假說(shuō)揭示糖苷水解酶結(jié)構(gòu)與功能的關(guān)系。

關(guān)于糖苷酶轉(zhuǎn)化皂苷的催化機(jī)制的研究，如圖2所示，人糞便GH3β-葡萄糖苷酶基因BIBG3經(jīng)克隆、結(jié)構(gòu)分析，發(fā)現(xiàn)BIBG3絡(luò)合D-葡萄糖的獨(dú)特環(huán)狀結(jié)構(gòu)可參與底物結(jié)合口袋的形成，通過(guò)和底物的分子對(duì)接揭示了口袋的結(jié)合方式，找到關(guān)鍵酶活突變位點(diǎn)R484和H642[77]。而酶分子設(shè)計(jì)改造的報(bào)道迄今僅有3篇。Park等[78]分離出具廣泛底物譜β-GH并測(cè)定其晶體結(jié)構(gòu)，基于產(chǎn)物特性、底物對(duì)接，以在三萜類化合物特定糖基化位點(diǎn)優(yōu)先水解聚糖的方式，重新設(shè)計(jì)了酶底物結(jié)合裂隙。使對(duì)Rb1的催化效率提高4–7倍，促進(jìn)PPD型底物Rb1、Rb2和Rb3 (Rc) 經(jīng)過(guò)F2到C-K的繼續(xù)轉(zhuǎn)化 (圖3)。Choi等[79]通過(guò)同源建模、分子對(duì)接、序列比對(duì)，確定參與α-L-阿拉伯呋喃糖苷酶活性的候選殘基；經(jīng)定點(diǎn)突變得到的L213Aβ-糖苷酶變體具有野生型沒(méi)有的α-L-阿拉伯呋喃糖苷酶活性，促進(jìn)Rc進(jìn)一步水解轉(zhuǎn)化為Rd，轉(zhuǎn)化率比野生型酶高1.5倍 (圖4)。在酵母底盤細(xì)胞重構(gòu)與人參皂苷的生物合成途徑優(yōu)化方面，Zhuang等[41]通過(guò)同源建模、分子動(dòng)力學(xué)和突變研究了UGT的催化分子機(jī)制[40-41,45]。且經(jīng)半理性設(shè)計(jì)的UGT51對(duì)PPD到Rh2的催化效率提高了約1 800倍。

圖2 BlBG整體結(jié)構(gòu)及其催化Rb1反應(yīng)活性位點(diǎn)幾何結(jié)構(gòu)分析(改編自參考文獻(xiàn)[77])

蛋白質(zhì)工程是提高酶對(duì)特定人參皂苷糖苷水解活性的有用工具。多種不同來(lái)源具人參皂苷轉(zhuǎn)化活性的微生物糖苷酶(尤其β-葡萄糖苷酶) 晶體結(jié)構(gòu)已經(jīng)被解析[7]，因此可通過(guò)同源建模、分子對(duì)接設(shè)計(jì)、突變以改造酶分子，改善底物特異性和催化效率，產(chǎn)生高純度的各種特殊三萜類化合物，實(shí)現(xiàn)目的人參皂苷專一、高效的酶法轉(zhuǎn)化。

4 離子液體對(duì)生物催化性能的改善

不同于分子組成的傳統(tǒng)液體溶劑，大多常溫下呈液態(tài)鹽(由特定的有機(jī)陽(yáng)離子與無(wú)機(jī)或有機(jī)陰離子構(gòu)成) 的離子液體(Ionic liquids，ILs)，具飽和蒸氣壓低、不可燃、親疏水、可設(shè)計(jì)等特性。研究最為廣泛的是 ILs 及其水溶液中脂肪酶、蛋白酶和酯酶的催化，而對(duì)于糖苷酶水解作用的研究很少[80]。通常，疏水、低粘度、表面活性、親電陰離子和離液陽(yáng)離子(Chaotropic cation) 的ILs增強(qiáng)酶活性和穩(wěn)定性[80]。然而由于許多結(jié)果矛盾沒(méi)有一般相關(guān)性規(guī)律，因此，以提高酶活性和穩(wěn)定性的方法正在探索，如水中ILs微乳液、設(shè)計(jì)與酶相容的離子溶劑、酶電荷的修飾、酶的固定化等[81]。

圖3 BGL167整體結(jié)構(gòu)及其催化PPD型人參皂苷反應(yīng)活性位點(diǎn)幾何結(jié)構(gòu)分析(改編自參考文獻(xiàn)[78])

圖4 來(lái)自S. solfataricus的β-糖苷酶配體對(duì)接和序列比對(duì)及其突變體催化人參皂苷Rc生物轉(zhuǎn)化途徑(改編自參考文獻(xiàn)[79])

4.1 離子液體參與水相轉(zhuǎn)化應(yīng)用概況

全細(xì)胞催化存在催化劑不穩(wěn)定、產(chǎn)物抑制、有毒副產(chǎn)物形成和質(zhì)量傳遞等問(wèn)題。在水-ILs的兩相催化中，由于全細(xì)胞懸浮在水相而有機(jī)底物溶解在疏水ILs相(或貯存產(chǎn)物)，從而避免了反應(yīng)中低水溶性的底物(或產(chǎn)物) 抑制。Chen等[82]在含ILs體系中全細(xì)胞催化水解甘草甜素制備單葡萄糖醛酸甘草次酸，發(fā)現(xiàn)在咪唑類ILs存在下，ILs提高了細(xì)胞膜通透性，且相比BL21和GS115，青霉Li-3對(duì)ILs (主要為[Bmim] [PF6]) 耐受性最強(qiáng)，60 h收率達(dá)87.63%。ILs在生物催化中的應(yīng)用需綜合對(duì)所需化學(xué)反應(yīng)(產(chǎn)物分離) 的溶劑性質(zhì)和對(duì)全細(xì)胞的溶劑毒性作用。而ILs對(duì)微生物細(xì)胞的 毒性是其工業(yè)應(yīng)用瓶頸之一，目前在含ILs系 統(tǒng)中研究最多的微生物是和釀酒酵母。Egorova等[18]研究了ILs毒理學(xué)特性，從反應(yīng)介質(zhì)(溶劑) 角度分析了全細(xì)胞在苛刻化學(xué)催化中的應(yīng)用前景。

酶能夠在體外不適合細(xì)胞生長(zhǎng)的條件下進(jìn)行催化，專一性強(qiáng)、催化方式簡(jiǎn)單。離子液體中陽(yáng)離子或陰離子類型對(duì)酶的活性、穩(wěn)定性和結(jié)構(gòu)具重要影響。Yanhong等[83]對(duì)含C6MIm·BF4體系黑李種子β-糖苷酶糖基化紅景天苷合成條件進(jìn)行優(yōu)化，發(fā)現(xiàn)陽(yáng)離子咪唑環(huán)上的烷基取代基的最佳鏈長(zhǎng)為C6。Ferdjani等[84]研究了在ILs/水不同比例時(shí)嗜熱棲熱菌β-糖苷酶和兩種分別來(lái)自海棲熱袍菌嗜熱脂肪芽孢桿菌的α-半乳糖苷酶的活性、穩(wěn)定性，發(fā)現(xiàn)在適合水溶性ILs (Water-miscible ILs) 中β-糖苷酶熱穩(wěn)定性最高。Brakowski等[85]發(fā)現(xiàn)含ILs-水緩沖乳液中，[Bmim][Pf6]導(dǎo)致來(lái)自?shī)W氏曲霉的β-半乳糖苷酶轉(zhuǎn)糖基底物專一性的改變。Kudou等[86]基于咪唑ILs磷酸鹽緩沖液，在乙酸1-丁基-3-甲基咪唑鎓[Bmim] [OAc]，pH 7.0，葡糖苷酶水解活性最高，通過(guò)穩(wěn)態(tài)發(fā)射光譜證明糖苷酶活性改善可能與酶構(gòu)象的靈活性有關(guān)。

由于毒性低、生物可降解、易于制備、具100%原子經(jīng)濟(jì)等特點(diǎn)，基于含膽堿鹽和三類氫鍵供體(酰胺、醇和糖) 的低共熔溶劑(DESs)可作為傳統(tǒng)離子液體的廉價(jià)替代品，成為β-葡萄糖苷酶催化的新型綠色溶劑，可拓展其在食品和醫(yī)藥領(lǐng)域方面的應(yīng)用。以對(duì)硝基苯基-β-吡喃葡萄糖苷作為水解反應(yīng)模型，Xu等[87]發(fā)現(xiàn)基于氯化膽堿的DESs (含6% (/) 水的氯化膽堿/丙二醇) 顯著改善β-葡萄糖苷酶的活性和穩(wěn)定性。在ILs中可通過(guò)酶表面電荷修飾和酶固定化來(lái)進(jìn)行酶的穩(wěn)定和激活，以增強(qiáng)在ILs中的耐受性。Zhao等[88]總結(jié)了固定化酶在ILs中被穩(wěn)定和激活的實(shí)例，但大多數(shù)與脂肪酶有關(guān)。最新報(bào)道了Jason等[89]以陽(yáng)離子化的葡萄糖苷酶降解溶于ILs的纖維素，極大提高了纖維素降解效率(100 ℃以上的催化效率是在水溶液時(shí)的30倍) (圖5)。表面陽(yáng)離子化修飾的葡萄糖苷酶(以ILs代替水作溶劑，通過(guò)碳二酰亞胺介導(dǎo)N,N′-二(2-氨乙基)-1,3-丙二胺定向偶聯(lián)酶表面天冬氨酸和谷氨酸殘基)在ILs中溶解度增加，在苛刻實(shí)驗(yàn)條件下依然能以恒定速率工作至少7 d。

ILs中酶以與偶聯(lián)載體(聚合物、納米微?；蛱技{米管)、被水凝膠包裹或以原始狀態(tài)懸浮的方式進(jìn)行固定化。無(wú)固相支撐的CLEAs有望提高ILs中酶穩(wěn)定性，Toral等[90]發(fā)現(xiàn)CLEA和在聚丙烯上吸附交聯(lián)的固定化脂肪酶能夠在使游離酶失活的ILs (如[BMIM][NO3]) 中仍然保持催化活性。溶膠-凝膠基質(zhì)具有防止反應(yīng)過(guò)程中酶從載體泄漏的優(yōu)點(diǎn)，而凝結(jié)和干燥過(guò)程中的凝膠收縮可能導(dǎo)致酶變性。通過(guò)在溶膠-凝膠固定化過(guò)程中添加離子液體，可提高包封酶的固定效率以及機(jī)械抗裂性，表明離子液體在酶性能中起著重要作用[91]。

圖5 離子液體體系纖維素酶非水均相轉(zhuǎn)化多糖催化機(jī)理(改編自參考文獻(xiàn)[89])

4.2 在酶轉(zhuǎn)化人參皂苷中的應(yīng)用潛能

在人參皂苷生物轉(zhuǎn)化方面尚無(wú)ILs的應(yīng)用，首先就人參皂苷溶解性進(jìn)行分析。大多數(shù)人參皂苷在含水正丁醇(常作為皂苷萃取溶劑) 中溶解度較大，而次級(jí)苷由于糖數(shù)目減少、極性減小、在水中溶解度降低，苷元?jiǎng)t難溶于水。例如，Re不易溶解于水，而在DMSO中溶解度達(dá)200 mg/mL。Cui等[40]以20 mg/mL人參皂苷Re在含10% DMSO緩沖液中進(jìn)行轉(zhuǎn)化，實(shí)現(xiàn)了10 L發(fā)酵罐中100 g級(jí)Rg2(S)的酶法生產(chǎn)。來(lái)源于蘑菇真菌菌絲體的酶轉(zhuǎn)化制備Compound K時(shí)，在底物Rb1中加入甲醇助溶[55]。

據(jù)此，有望以非揮發(fā)、環(huán)保的ILs代替DMSO和甲醇有機(jī)溶劑發(fā)揮對(duì)底物的助溶作用。以咪唑類ILs提取和富集三七中藥材及其制劑中的微量人參皂苷20(S)-Rg3和Rk1[92-94]，進(jìn)一步證明ILs有助于增加稀有人參皂苷溶解度，可開(kāi)發(fā)應(yīng)用于人參皂苷的生物催化轉(zhuǎn)化。結(jié)合以上ILs參與其他類型產(chǎn)物的水相反應(yīng)的生物轉(zhuǎn)化時(shí)對(duì)全細(xì)胞及酶催化性能的影響，可能獲得比在有機(jī)溶劑時(shí)更高的催化活性、選擇性和穩(wěn)定性。因而，ILs參與的稀有人參皂苷生物催化體系的構(gòu)建，在轉(zhuǎn)化工藝中除ILs對(duì)底物溶解性作用外，還需要考慮作為反應(yīng)介質(zhì)ILs對(duì)菌體毒性或酶催化性能的影響，以及作為酶修飾劑，ILs的改性方法及酶穩(wěn)定化的應(yīng)用形式。

5 展望

稀有人參皂苷生物催化轉(zhuǎn)化的關(guān)鍵問(wèn)題包括特異性酶活性不高、涉及酶及其催化機(jī)制不明確、結(jié)構(gòu)生物信息學(xué)研究不系統(tǒng)，改變酶水解皂苷糖基專一性的研究尚處于起步階段。益生菌及其酶的使用已在食品工業(yè)中的實(shí)用應(yīng)用中顯示出巨大潛力。而離子液體可以通過(guò)改變反應(yīng)體系的極性增加糖類的溶解度，為合理設(shè)計(jì)糖基轉(zhuǎn)化反應(yīng)體系創(chuàng)造多種機(jī)會(huì)。因此，今后研究工作應(yīng)集中在：1) 基于同源建模、分子對(duì)接催化分子機(jī)制的揭示來(lái)改造酶分子，以改變酶專一性、提高酶活性和穩(wěn)定性；2) 基于ILs的溶劑工程在酶固定化及酶表面修飾的作用，改善酶轉(zhuǎn)化人參皂苷的催化效率和穩(wěn)定性。通過(guò)篩選及構(gòu)建基因工程菌、發(fā)酵或胞外表達(dá)GH，以化學(xué)修飾并制備多種不同尺度、形態(tài)的益生菌酶制劑。同時(shí)，以光譜法、計(jì)算機(jī)模擬等可視化技術(shù)研究ILs-酶-底物相互作用，系統(tǒng)分析ILs反應(yīng)體系中影響糖苷酶結(jié)構(gòu)及其活性、專一性和穩(wěn)定性的催化分子機(jī)制。最終實(shí)現(xiàn)對(duì)人參皂苷定向、高效的生物轉(zhuǎn)化工藝，有助于深入探究并開(kāi)發(fā)新癌癥化學(xué)預(yù)防的藥物化學(xué)和藥理學(xué)方法。

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Biocatalytic strategies in producing ginsenoside by glycosidase-a review

Weina Li1,2, Yunyun Jiang1,2, Yannan Liu1,2, Chunying Li1,2,and Daidi Fan1,2

1 Shaanxi Key Laboratory of Degradable Biomedical Materials, School of Chemical Engineering, Northwest University, Xi’an 710069, Shaanxi, China 2 Shaanxi R&D Center of Biomaterials and Fermentation Engineering, College of Chemical Engineering, Northwest University, Xi’an 710069, Shaanxi, China

is a traditional Chinese medicine with significant pharmaceutical effects and wide application. Through orientational modification and transformation of ginsenoside glycosyl, rare ginsenosides with high antitumor activities can be generated. Traditional chemical methods cannot be applied in clinic. because of extremely complex preparation technologies and very high cost Transformations using microorganisms and their enzymatic systems provide the most feasible methods for solving the main problems. At present, the key problems in enzymatic synthesis of ginsenosides include low specific enzyme activities, identity of enzymes involved in the enzymatic synthesis, and their catalytic mechanisms, as well as nonsystematic studies on structural bioinformatics; specificity of enzymatic hydrolysis for saponin glycosyl has been rarely studied. Many reviews have been reported on glycosidase molecular recognition, immobilization, and biotransformation in ionic liquids (ILs), whereas ginsenoside transformation and application have not been systematically studied. To evaluate theoretical and applied studies on ginsenoside-oriented biotransformation, by reviewing the latest developments in related fields and evaluating the widely applied biocatalytic strategy, this review aims to evaluate the ginsenoside-oriented transformation method with improved product specificity, increased biocatalytic efficiency, and industrial application prospect based on the designed transformations of enzyme and solvent engineering of ILs. Therefore, useful theoretical and experimental evidence can be obtained for the development of ginsenoside anticancer drugs, large-scale preparation, and clinical applications in cancer therapy.

ginsenosides, biocatalysis, whole cell conversion, enzyme conversion, ionic liquids

January 31, 2019;

April 2, 2019

National Natural Science Foundation of China (No. 21576160), National Natural Science Foundation of China Young Scientists Fund (No. 21706211), Postdoctoral Science Foundation of China (No. 2015M582698).

Daidi Fan. Tel/Fax: +86-29-88303360; E-mail: fandaidi66@126.com

國(guó)家自然科學(xué)基金 (No. 21576160)，國(guó)家自然科學(xué)基金青年科學(xué)基金 (No. 21706211)，博士后科學(xué)基金 (No. 2015M582698) 資助。

2019-04-11

http://kns.cnki.net/kcms/detail/11.1998.q.20190411.0856.002.html

李偉娜, 蔣云云, 劉彥楠, 等. 人參皂苷單體定向轉(zhuǎn)化的生物催化及應(yīng)用進(jìn)展. 生物工程學(xué)報(bào), 2019, 35(9): 1590–1606.

Li WN, Jiang YY, Liu YN, et al. Biocatalytic strategies in producing ginsenoside by glycosidase -a review. Chin J Biotech, 2019, 35(9): 1590–1606.

(本文責(zé)編 郝麗芳)