论文总字数：27592字

摘 要

(S)-N-Boc-3-羟基哌啶，又称(S)-1-叔丁氧羰基-3-哌啶醇，是手性醇类家族成员之一，可以作为关键中间体，用于手性药物的制备过程。制备(S)-1-叔丁氧羰基-3-哌啶醇，过去常常采用化学合成法，化学法缺点显著：能源耗费多、生产成本高、环境污染严重。现在经常采用生物法制备(S)-1-叔丁氧羰基-3-哌啶醇，是以N-Boc-3-哌啶酮作为潜手性底物，利用生物酶的催化作用进行合成。生物催化法优势显著：能源耗费少、生产成本较低、环境污染较少。截止目前，具有以N-Boc-3-哌啶酮为底物，合成(S)-1-叔丁氧羰基-3-哌啶醇能力的酶很少见。因此，继续研究具有催化还原N-Boc-3-哌啶酮，合成(S)-1-叔丁氧羰基-3-哌啶醇能力的酶，有重要的现实意义。

本研究通过挖掘基因组数据，筛选出符合实验条件的酶，该酶是羰基还原酶CprCR，来源于近平滑假丝酵母。经重组得到的质粒为pET-28a-CprCR，利用大肠杆菌表达系统，对该质粒进行转化表达。测定酶活，结果表明，重组质粒pET-28a-CprCR的粗酶液，对底物N-Boc-3-哌啶酮的酶活为5.04U/ml发酵液。因此，可利用重组质粒pET-28a-CprCR的粗酶液进行催化，将N-Boc-3-哌啶转化成(S)-1-叔丁氧羰基-3-哌啶醇。并且，重组质粒pET-28a-CprCR的光学活性和选择性很高。

将羰基还原酶CprCR纯化后，再进行研究。本实验以探讨酶学性质为主。研究结果显示：CprCR是一种NADH辅酶依赖型的羰基还原酶。测定CprCR的动力学参数，其催化底物N-Boc-3-哌啶酮的米氏常数Km值为2.3 mmol/L，最大反应速率Vmax为19.08 mmol/L/min，kcat数为23.1s-1。CprCR酶的最适反应温度在50°C至55°C之间，最适pH在6-7之间。当温度在40°C以下及中性偏碱条件下（pH 6-8），酶性质更稳定。体系中存在Li 、Mg2 、Mn2 、Na 和K 时，对酶有激活作用；存在Fe2 和Fe3 ，则会使酶活完全消失。

关键词：酶学性质 (S)-1-叔丁氧羰基-3-哌啶醇 羰基还原酶CprCR

Abstract

(S) -N-Boc-3- hydroxy piperidine ((S) -NBHP) as a chiral alcohol, is a key intermediate in the synthesis of chiral drugs, such as non natural medicine card triptorelin, FDA approved anticancer drugs ibrutinib (a Bruton's tyrosine kinase (BTK) inhibitor of the first drug). The traditional preparation of (S) - N-Boc-3- piperidine chemical method, its high energy consumption, high cost, high pollution shortcomings, limiting its industrial applications. N-Boc-3- (S) -NBHP, which is prepared by enzyme catalysis as chiral substrate, has attracted more and more attention because of its mild reaction conditions, low cost and low pollution. However, few enzymes have been reported to catalyze the synthesis of N-Boc-3- piperidine (S) -NBHP at present, so it is the focus of the present study to continue to dig out enzymes with catalytic synthesis (S) -NBHP capability.

In this study, a genomic data mining method was used to screen carbonyl reductase CprCR from Candida rugosa. The recombinant plasmid pET-28a-CprCR was transformed into Escherichia coli. The enzyme activity assay showed that the enzyme activity of the crude enzyme solution to substrate N-Boc-3- was 5.04U/ml fermentation broth. The CprCR crude enzyme solution was used to catalyze N-Boc-3- piperidine synthesis (S) -NBHP, and glucose dehydrogenase and coenzyme glucose were added into the catalytic system for coenzyme regeneration. After 24 h reaction, the final conversion rate of 20-80g/l can reach 94%, and the optical purity of product (S) -NBHP is E.E., which indicates that gt;99% has high activity and stereoselectivity.

The enzyme CprCR after purification, characterization of CprCR NADH as a coenzyme dependent carbonyl reductase, determination of kinetic parameters of Michaelis constant Km shows its catalytic substrate N-Boc-3- piperidones value was 2.3 mmol/L, the maximum reaction rate of Vmax was 19.08 mmol/L/min, the kcat number is 23.1s-1. The optimum reaction temperature of CprCR enzyme ranged from 50 C to 55 DEG C, and the optimum pH was between 6-7 and 10. When the temperature is below 40 degrees C and neutral alkaline condition (pH 6-8), the enzyme properties are more stable. Li 、Mg2 、Mn2 、Na and K have an active

effect on enzymes, and in the presence of Fe2 and Fe3 , enzyme activity is completely lost.

Key words: (S) -N-Boc-3- carbonyl reductase, CprCR, enzymatic properties.

目 录

摘 要 Ⅰ

ABSTRACT Ⅱ

第一章 文献综述 1

1.1 催化羰基不对称还原合成手性药物的还原酶 1

1.1.1 手性药物 1

1.1.2 羰基不对称还原合成手性药物 1

1.1.3 催化羰基不对称还原的生物催化剂 2

1.2 制备(S)-1-叔丁氧羰基-3-哌啶醇 3

1.2.1 (S)-1-叔丁氧羰基-3-哌啶醇的简介及其应用 3

1.2.2 化学法制备(S)-1-叔丁氧羰基-3-哌啶醇 3

1.2.3 生物法制备(S)-1-叔丁氧羰基-3-哌啶醇 4

1.3 羰基还原酶在不对称还原反应的应用 4

1.3.1 羰基还原酶及其催化机理 4

1.3.2 羰基还原酶催化的不对称还原反应 4

1.3.3 羰基还原酶催化在含氮杂环上引入手性羟基 4

1.3.4 羰基还原酶的基因克隆及表达 4

1.3.5 酶偶联法 5

1.3.6 酶学性质研究 5

第二章 羰基还原酶CprCR的克隆表达及功能鉴定 7

2.1 前言 7

2.2 材料和方法 7

2.2.1 实验材料 8

2.2.2 实验试剂 8

2.2.3 工具酶及试剂盒 10

2.2.4 菌株与质粒 10

2.2.5 培养基及培养方法 11

2.2.5.1 培养基-----------------------------------------11

2.2.5.2 菌株培养方法-----------------------------------11

2.3 实验方法 12

2.3.1 近平滑假丝酵基因组的提取 12

2.3.2 目的基因片段CprCR的克隆 12

2.3.3 重组质粒pET-28a-CprCR的构建及鉴定 12

2.3.4 目的蛋白CprCR的表达 13

2.3.5 目的蛋白CprCR的酶活测定 13

2.3.6 底物及产物的检测方法 14

2.3.6.1 N-Boc-3-哌啶酮与NBPH的测定方法----------------14

2.3.6.2 N-Boc-3-哌啶酮与(S)-NBPH、(R)-NBPH手性鉴定方法-------------------------------------------------------------------14

2.3.7 双酶耦联催化不对称还原N-Boc-3-哌啶酮 14

2.4 结果与讨论 15

2.4.1 基因组数据挖掘获得羰基还原酶CprCR 15

2.4.2 近平滑假丝酵母基因组的提取 16

2.4.3 目的基因的PCR扩增 17

2.4.4 重组质粒的构建和PCR鉴定 17

2.4.5 CprCR的蛋白表达 18

2.4.6 CprCR的酶活力测定 18

2.4.7 N-Boc-3-哌啶酮与NBPH的测定及标准曲线的绘制 18

2.4.8 N-Boc-3-哌啶酮与(S)-NBPH、(R)-NBPH手性鉴定方法 19

2.4.9 双酶偶联催化不对称合成(S)-NBHP初探 20

第三章 羰基还原酶CprCR的纯化及酶学性质研究 21

3.1 前言 21

请支付后下载全文，论文总字数：27592字