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酰胺酶的挖掘及在有機(jī)合成中的應(yīng)用進(jìn)展

2018-01-23 00:45:15吳哲明金建強(qiáng)鄭仁朝
生物加工過程 2018年1期
關(guān)鍵詞:水解酶酰基中間體

吳哲明,金建強(qiáng),鄭仁朝

(1. 浙江工業(yè)大學(xué) 生物工程學(xué)院,浙江 杭州 310014;2. 浙江省生物有機(jī)合成技術(shù)研究重點(diǎn)實(shí)驗(yàn)室, 浙江 杭州 310014)

酰胺酶(amidase,EC 3.5.1.X),又稱酰胺水解酶(amidohydrolase),是一類催化酰胺化合物水解生成相應(yīng)羧酸和氨的重要水解酶。該酶促反應(yīng)的本質(zhì)是催化酰基從供體(酰胺)轉(zhuǎn)移至受體(如水),因此,當(dāng)體系中存在比水親核性更高的羥胺時(shí),則生成相應(yīng)的氧肟酸(圖1)[1-3]。作為腈轉(zhuǎn)化酶家族的重要成員,酰胺酶的底物譜很廣,能夠水解各種天然及人工合成的脂肪族、芳香族及雜環(huán)類酰胺。酰胺酶所具有的高立體選擇性、廣底物譜等特性使其在動(dòng)力學(xué)拆分外消旋酰胺制備手性羧酸、手性酰胺衍生物及光學(xué)純氨基酸等方面具有獨(dú)特優(yōu)勢,正日益受到研究者的重視(表1)[4-7]。

酰胺酶最早由Kelly等[20]和Jakoby等[21]分別在Pseudomonasaeruginosa和P.fluorescens中發(fā)現(xiàn),其來源十分廣泛,存在于各類原核、真核微生物和動(dòng)植物中[22-23]。其中,細(xì)菌是酰胺酶的主要來源,如紅球菌屬Rhodococcus[24-25]、假單孢菌屬Pseudomonas[26]、嗜硫菌屬Sulfolobus[27-28]、芽孢桿菌屬Bacillus[29]、代爾夫特菌屬Delftia[30-31]、克雷伯氏菌屬Klebsiella[32]和蒼白桿菌屬Ochrobactrum[33]等。

圖1 酰胺酶催化的水解和酰基轉(zhuǎn)移反應(yīng)Fig.1 The hydrolysis and acyl transfer reaction catalyzed by amidase

表1 酰胺酶催化的外消旋酰胺的立體選擇性水解

1 酰胺酶的分類及結(jié)構(gòu)

酰胺酶的種類很多,不同來源的酰胺酶結(jié)構(gòu)和性質(zhì)差異顯著,目前仍未有一個(gè)系統(tǒng)的分類方法。根據(jù)不同的標(biāo)準(zhǔn),酰胺酶有多種分類方式。根據(jù)底物特異性差異,可分為廣譜類酰胺酶、α-氨基酰胺酶、脂肪族酰胺酶和芳香族酰胺酶等;根據(jù)酰胺酶基因上下游是否存在腈水合酶基因編碼,可以分為與腈水合酶耦聯(lián)的酰胺酶和非耦聯(lián)的酰胺酶。近年來,基于氨基酸序列的酰胺酶分類方法受到普遍認(rèn)可。Chebrou等[34]通過氨基酸序列比對分析,提出將酰胺酶分為腈水解酶超家族(nitrilase superfamily)和酰胺酶標(biāo)簽(amidase signature,AS)家族兩大類。

腈水解酶家族酰胺酶是一類含有保守親核半胱氨酸的巰基酶。該家族酰胺酶之間序列同源性高,與腈水解酶具有序列相似性,并含有保守的Glu、Cys和Lys催化三聯(lián)體負(fù)責(zé)共價(jià)鍵催化(圖2)。目前,已知的腈水解酶家族酰胺酶底物譜較窄,催化短鏈脂肪族酰胺的水解。酶蛋白通常以同源四聚體、六聚體的形式存在,單體結(jié)構(gòu)一般表現(xiàn)為α-β-β-α式的夾心折疊。Novo等[35]以蠕蟲腈水解酶融合蛋白晶體結(jié)構(gòu)為模版,假定催化三聯(lián)體Glu-Lys-Cys在所有腈水解酶家族成員中均為保守,通過對比模擬,建立了P.aeruginosa酰胺酶3D結(jié)構(gòu)模型,證明了催化三聯(lián)體Glu-Lys-Cys負(fù)責(zé)共價(jià)鍵的催化,其中Cys為親核體。Kimani等[36]對GeobacilluspallidusRAPc8酰胺酶晶體結(jié)構(gòu)研究表明,其單體具有典型的腈水解酶超家族α-β-β-α折疊,同時(shí)也證實(shí)了在腈水解酶超家族成員中Glu-Lys-Cys催化三聯(lián)體的保守性。

Dt-Ami7—Delftia tsuruhatensis ZJB-05174酰胺酶(KP943495);BS AMI—B. stearothermophilus酰胺酶(Q9RQ17);Bs AMI—Bacillus sp. BR449酰胺酶 (AF257487);Pa AMI—P. aeruginosa酰胺酶(AAA25697)圖2 腈水解酶家族酰胺酶序列比對Fig.2 Sequence alignment of nitrilase superfamily amidases

有別于腈水解酶家族酰胺酶,酰胺酶標(biāo)簽家族酰胺的一級結(jié)構(gòu)中含有一段高度保守的GGSS區(qū)域,且具有含約130個(gè)富含甘氨酸、絲氨酸和丙氨酸等氨基酸組成的高度保守序列(AS序列),其催化三聯(lián)體為Ser、Ser和Lys(圖3),一般呈同源二聚體或同源八聚體。酰胺酶標(biāo)簽家族酰胺酶的底物譜通常較廣,能夠水解脂肪族、芳香族和雜環(huán)酰胺[24,37]。目前已報(bào)道結(jié)構(gòu)的標(biāo)簽家族酰胺酶較多,如來源于Stenotrophomonasmaltophilia的PAM是酰胺酶標(biāo)簽家族中第一個(gè)已解析三級結(jié)構(gòu)的酰胺酶,具有清晰的α-β夾心折疊結(jié)構(gòu),中心的β-折疊片核心被α-螺旋包圍,該折疊在標(biāo)簽酰胺酶家族成員中保守[38]。Ohtaki等[39]發(fā)現(xiàn)Rhodococcussp. N-771酰胺酶晶體結(jié)構(gòu)含有3個(gè)結(jié)構(gòu)域:N端α螺旋結(jié)構(gòu)域,小結(jié)構(gòu)域和大結(jié)構(gòu)域。大結(jié)構(gòu)域含有一個(gè)α/β結(jié)構(gòu),由一個(gè)疏水核心(18個(gè)β折疊片)和8個(gè)位于β折疊片兩側(cè)的α螺旋構(gòu)成,其包含了一段酰胺酶標(biāo)簽序列。小結(jié)構(gòu)域有5個(gè)α螺旋,位于大結(jié)構(gòu)域頂端。Ser-cisSer-Lys催化三聯(lián)體位于大結(jié)構(gòu)域,而小結(jié)構(gòu)域的一些疏水性氨基酸殘基則參與底物的識(shí)別。Lee等[40]報(bào)道了芳基?;0访?AAA)的晶體結(jié)構(gòu),發(fā)現(xiàn)其屬于α/β水解酶家族,催化三聯(lián)體為Ser187、Ser163與Lys84。該酶外部結(jié)構(gòu)為α螺旋,內(nèi)部則由β折疊結(jié)構(gòu)組成,并且擁有一個(gè)獨(dú)特的由兩個(gè)loop環(huán)和一個(gè)α螺旋構(gòu)成的底物結(jié)合口袋。

Dt-Ami 2、Dt-Ami 6—D. tsuruhatensis ZJB-05174酰胺酶(KP943493、KP943494);Ca. AMI—R. rhodochrous Jl 酰胺酶(BAA03744);PAM—S. maltophilia酰胺酶 (CAC93616);Re. AMI—R. erythropolis MP50酰胺酶(AY026386)圖3 腈水解酶家族酰胺酶序列比對Fig.3 Sequence alignment of amidase signature family amidases

2 酰胺酶的催化機(jī)制

目前,酰胺酶的催化反應(yīng)機(jī)制尚不十分明晰。Maestracci等[41]以Rhodococcussp. R312酰胺酶為研究對象,以乙酰胺和羥胺為雙底物,發(fā)現(xiàn)并證明其介導(dǎo)的?;D(zhuǎn)移反應(yīng)符合雙底物乒乓反應(yīng)機(jī)制,即底物先與酰胺酶結(jié)合形成?;?酶復(fù)合物,進(jìn)而將底物的?;D(zhuǎn)移至其受體羥胺,生成相應(yīng)的氧肟酸。Kobayashi等[42]認(rèn)為底物酰胺的羰基受到親核進(jìn)攻時(shí),與酶形成一個(gè)四面體中間體,中間體因氨的形成并解離而快速轉(zhuǎn)變?yōu)轷;?酶復(fù)合物。在水分子加入后,復(fù)合物發(fā)生水解生成相應(yīng)的酸(圖4)。

圖4 酰胺酶水解反應(yīng)和?;D(zhuǎn)移反應(yīng)作用機(jī)制Fig.4 Mechanism of amide hydrolysis reactionand acyl transfer reaction from amide to hydrazine and hydroxylamine

隨著酰胺酶結(jié)構(gòu)的不斷解析,基于催化三聯(lián)體的酰胺酶催化機(jī)制被研究者廣泛報(bào)道。脂肪酸酰胺水解酶(FAAH)是一個(gè)典型的標(biāo)簽家族酰胺酶,其催化機(jī)制被學(xué)者廣泛研究[43-45]。Mileni等[46]推測FAAH的反應(yīng)機(jī)制主要為以下四步:1)親核體Ser241被極化激活并進(jìn)攻底物酰胺鍵,形成酶-底物復(fù)合體;2)酰胺底物C—N鍵斷裂,氨基部分脫離,形成酰基-酶中間體;3)活化的水分子進(jìn)攻?;?酶中間體并形成新的四面體中間體;4)復(fù)合體分解產(chǎn)生羧酸,而酰胺酶則獲得再生(圖5)。

圖5 酰胺酶FAAH假設(shè)的催化機(jī)制Fig.5 Proposed reaction mechanism of FAAH

Labahn等[38]研究標(biāo)簽家族酰胺酶PAM催化機(jī)制主要分為4步(圖6):1)cisSer202側(cè)鏈羥基的質(zhì)子轉(zhuǎn)移至堿催化劑Lys的氨基上,增強(qiáng)了其對底物酰胺羰基氧的質(zhì)子化能力;Ser226親核進(jìn)攻酰胺羰基碳原子,同時(shí)cisSer202質(zhì)子化Ser226的羰基氧;隨后,失去質(zhì)子的cisSer202奪取Ser226的質(zhì)子,形成酶-底物復(fù)合體;2)復(fù)合體上的氨基奪取cisSer202的質(zhì)子,而失去質(zhì)子的cisSer202轉(zhuǎn)而從帶正電的Lys123捕獲質(zhì)子,恢復(fù)穩(wěn)定;3)質(zhì)子化的氨基形成氨脫離復(fù)合體,而Lys123重新奪回cisSer202上的質(zhì)子;失去質(zhì)子后的cisSer202轉(zhuǎn)而捕獲底物上的羥基質(zhì)子,兩者恢復(fù)最初狀態(tài);同時(shí),底物的羰基也重新產(chǎn)生,形成酶-酰基中間體;4)水分子進(jìn)攻酶-?;虚g體使其分解,形成產(chǎn)物羧酸。Ser226獲得水分子的質(zhì)子,重新恢復(fù)穩(wěn)定。

圖6 酰胺酶PAM假設(shè)的催化機(jī)制Fig.6 Proposed reaction mechanism of PAM

Lee等[40]報(bào)道了標(biāo)簽家族酰胺酶AAA的晶體結(jié)構(gòu),并解釋了其催化機(jī)制。該酶以Ser163、Ser187與Lys84作為催化三聯(lián)體,其中Lys84作為廣義堿催化劑接受來自于Ser163通過氫鍵傳遞的質(zhì)子,激活Ser163(圖7)。具體催化步驟如下:1)在堿性環(huán)境下的Lys84處于去質(zhì)子化狀態(tài),其通過氫鍵網(wǎng)絡(luò)介導(dǎo)cisSer163的Oγ與親核體Ser187的OγH通過氫鍵發(fā)生極化并使之激活;2)Ser187的OγH與底物(對乙酰氨基酚)的羰基碳通過共價(jià)鍵形成四面體中間體,同時(shí)Ser187的Oγ脫去質(zhì)子;3)形成酰基-酶復(fù)合體,隨后在1分子水的作用下發(fā)生脫酰反應(yīng);4)酶與產(chǎn)物分離,酰胺酶獲得再生。

圖7 酰胺酶AAA假設(shè)的催化機(jī)制Fig.7 Proposed reaction mechanism of AAA

3 酰胺酶的篩選與挖掘

傳統(tǒng)的酰胺酶篩選是從土壤或菌種庫中篩選具有酰胺酶活力的微生物,但效率低,耗時(shí)長,而且無法區(qū)分酰胺酶是否具有立體選擇性。筆者所在團(tuán)隊(duì)的Zheng等[47]在首次證明酰胺酶催化的?;D(zhuǎn)移和水解反應(yīng)立體選擇性一致的基礎(chǔ)上,利用?;D(zhuǎn)移反應(yīng)產(chǎn)物氧肟酸與鐵離子在酸性條件下螯合顯色的特性,建立了高通量立體選擇性酰胺酶篩選模型(圖8)。該方法與已有的基于底物官能團(tuán)的酰胺酶篩選模型[48]相比,其更顯著的優(yōu)勢在于對底物的普適性,是目前報(bào)道的普適性最強(qiáng)的酰胺酶篩選模型。

圖8 基于?;D(zhuǎn)移反應(yīng)的比色法篩選立體選擇性酰胺酶Fig.8 Selective colorimetric screen for enantioselective amidase-producing microorganisms

近20年來,雖然已從自然環(huán)境中篩選獲得大量產(chǎn)酰胺酶的微生物(表2),但由于野生菌中酰胺酶的表達(dá)水平通常較低,且可能同一微生物中存在幾種不同立體選擇性的酰胺酶,酶活、對映選擇性低等問題成為其大規(guī)模應(yīng)用的瓶頸。因此,研究人員把目光投向了如何快速、高效獲得高立體選擇性、高催化活力的酰胺酶。近年來,得益于DNA測序技術(shù)的進(jìn)步,急劇增加的生物信息為新酶的開發(fā)帶來了前所未有的機(jī)遇,基因挖掘已成為快速開發(fā)新酶的有力手段。截至2017年10月,根據(jù)基因組測序計(jì)劃統(tǒng)計(jì)網(wǎng)站GOLD(www.genomesonline.org)的統(tǒng)計(jì)數(shù)據(jù),目前已完成全基因組測序的生物多達(dá)282 640種,其中細(xì)菌完成測序250 327種,真核生物20 956種,這為酰胺酶的挖掘提供了寶庫(圖9)。筆者根據(jù)酰胺酶保守序列,通過基因挖掘從DelftiatsuruhatensisZJB-05174、ParvibaculumlavamentivoransZJB14001[63]以及BurkholderiaphytofirmansZJB-15079[64]全基因組中分別獲得了若干重組酰胺酶。同時(shí),Ruan等[65]利用基因組同源比對和HiTAIL-PCR對未知區(qū)域的高效擴(kuò)增,從B.epidermidisZJB-07021基因組中克隆獲得2個(gè)酰胺酶基因,并實(shí)現(xiàn)了異源表達(dá)。

表2 不同來源酰胺酶的性質(zhì)

圖9 完成全基因組測序生物組成Fig.9 Sequenced genome projects in GOLD (www.genomesonline.org)

4 酰胺酶的應(yīng)用

手性羧酸和酰胺衍生物是重要的旋光性模塊化合物,可用于大量手性生物活性分子的合成,包括羥基酸、氨基酸、甲基氨基酸、伯胺和偽核苷化合物等,在精細(xì)化工、醫(yī)藥、農(nóng)用化學(xué)品及功能材料等方面有廣泛應(yīng)用。酰胺酶底物譜廣、催化活性高以及對映選擇性嚴(yán)格等特點(diǎn)使其在制備復(fù)雜結(jié)構(gòu)手性羧酸及酰胺衍生物具有無可比擬的優(yōu)勢。筆者所在課題組多年來致力于酰胺酶生物催化工業(yè)應(yīng)用開發(fā),建立了一系列以酰胺酶為催化劑的(手性)羧酸及酰胺的生物合成新工藝。

4.1 (S)-2,2-二甲基環(huán)丙甲酰胺的合成

(S)-2,2-二甲基環(huán)丙甲酰胺是抗重癥感染首選藥物亞胺培南/西司他丁鈉的關(guān)鍵手性中間體,開發(fā)其高效合成技術(shù)對控制西司他丁鈉成本具有重要意義。目前工業(yè)上應(yīng)用的化學(xué)法合成工藝步驟冗長,反應(yīng)條件苛刻,工藝總收率低,且需大量有毒有害試劑,環(huán)境負(fù)擔(dān)大。

筆者所在團(tuán)隊(duì)的Zheng等[31]通過建立普適性高通量立體選擇性酰胺酶篩選模型,獲得了能夠R型立體選擇性水解2,2-二甲基環(huán)丙烷甲酰胺的酰胺酶產(chǎn)生菌D.tsuruhatensisZJB-05174,對映體選擇率E>100(圖10)。在添加5%的乙腈作為共溶劑后,(S)-2,2-二甲基環(huán)丙甲酰胺的收率為43.6%,光學(xué)純度達(dá)到99%以上。

4.2 1-氰基環(huán)己基乙酸的合成

加巴噴丁(Gabapentin)是一種新型抗癲癇藥物,具有療效好、安全性高和耐受性好等特點(diǎn)[66-67]。1-氰基環(huán)己基乙酸(1-CCHAA)是合成加巴噴丁的關(guān)鍵中間體。筆者等[68]通過基因挖掘,篩選獲得了可高效水解1-氰基環(huán)己基乙酰胺(1-CCHAM)合成1-CCHAA 的酰胺酶Pa-Ami,比酶活達(dá)297.6 U/mg。以含Pa-Ami的重組菌為催化劑水解1-CCHAM(圖11),底物質(zhì)量濃度為80 g/L,濕菌體質(zhì)量濃度為1 g/L時(shí),反應(yīng)20 min,轉(zhuǎn)化率可達(dá)100%。

圖10 R,S-2,2-二甲基環(huán)丙烷甲酰胺的酶法拆分Fig.10 Enzymatic resolution of (R,S)-2,2-dimethylcyclopropane carboxamide

圖11 酰胺酶法制備加巴噴丁路線Fig.11 Enzymatic route for synthesis of gabapentin by amidase

4.3 R -3,3,3-三氟-2-羥基-2-甲基丙酸的合成

3,3,3-三氟-2-羥基-2-甲基丙酸(TFHMA)是一種手性醫(yī)藥中間體,其S型或R型異構(gòu)體均可用于系列藥物的合成[69-70],如S型異構(gòu)體可用于制備新型ATP敏感性鉀(KATP)通道開放劑[71];R型異構(gòu)體可合成丙酮酸脫氫酶激酶(PDK)抑制劑等[72]。瑞士Lonza公司的Shaw等[73]利用產(chǎn)酸克雷伯氏菌KlebsiellaoxytocaR-酰胺酶拆分外消旋3,3,3-三氟-2-羥基-2-甲基丙酰胺(TFHMM)(圖12),制備R-3,3,3-三氟-2-羥基-2-甲基丙酸和S-3,3,3-三氟-2-羥基-2-甲基丙酰胺,底物質(zhì)量濃度100 g/L,產(chǎn)率接近50%,產(chǎn)物e.e.>98%。筆者等[64]利用來源于B.phytofirmansZJB-15079的重組酰胺酶工程菌拆分3,3,3-三氟-2-羥基-2-甲基丙酰胺,在底物質(zhì)量濃度200 g/L、濕菌體質(zhì)量濃度5 g/L、80 ℃下反應(yīng)10 min,R-3,3,3-三氟-2-羥基-2-甲基丙酸質(zhì)量濃度可達(dá)86.2 g/L,e.e.>95%,E值達(dá)86。

圖12 R,S-3,3,3-三氟-2-羥基-2-甲基丙酰胺的酰胺酶法拆分Fig.12 Enzymatic resolution of (R,S)-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide

4.4 2-氯煙酸的合成

2-氯煙酸是一種重要精細(xì)化工中間體,廣泛用于殺菌劑、殺蟲劑、抗生素和心血管疾病藥物的合成[74-76]。金建強(qiáng)[77]通過大量篩選,獲得了可高活性水解2-氯煙酰胺合成2-氯煙酸的重組酰胺酶工程菌(圖13),并考察了其對系列氯代煙酰胺的反應(yīng)動(dòng)力學(xué)。全細(xì)胞(濕菌體質(zhì)量濃度5 g/L)催化2-氯煙酰胺水解底物濃度可達(dá)400 mmol/L,轉(zhuǎn)化率達(dá)94%。

圖13 酰胺酶水解合成2-氯煙酸Fig.13 Enzymatic route for synthesis of 2-chloronicotinic acid by amidase

5 結(jié)論

不對稱生物合成方興未艾,酰胺酶正崛起為不對稱生物催化的重要工具酶。作為腈轉(zhuǎn)化酶家族中的重要一員,酰胺酶所具有的高立體選擇性、廣底物譜等特性使其在動(dòng)力學(xué)拆分外消旋酰胺制備復(fù)雜結(jié)構(gòu)手性羧酸及酰胺衍生物中占據(jù)無可比擬的優(yōu)勢。隨著酰胺酶空間結(jié)構(gòu)的解析,其反應(yīng)催化機(jī)制也正逐漸被揭示。然而,由于對酰胺酶底物特異性和立體選擇性規(guī)律研究的缺乏,酰胺酶的開發(fā)仍只能局限于以“底物”為探針篩選“催化劑”的傳統(tǒng)研究范疇,無法主動(dòng)進(jìn)行生物催化劑的選擇和理性改造。通過探究酰胺酶的“結(jié)構(gòu)和功能”的關(guān)系,在掌握酶的結(jié)構(gòu)信息的情況下對酰胺酶進(jìn)行分子改造,可進(jìn)一步促進(jìn)酰胺酶的工業(yè)化應(yīng)用。

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N-脂肪?;被猁}的合成、性能及應(yīng)用
α-甲氧甲?;?γ-丁內(nèi)酯和α-乙氧甲?;?γ-丁內(nèi)酯的合成及表
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