CN109897836A - A kind of monoamine oxidase from aspergillus oryzae is used for the preparation of chiral amine intermediates - Google Patents

Abstract

The invention belongs to field of biotechnology, are related to genetic engineering, bioconversion and biocatalysis, organic synthesis technology, disclose a kind of Monoamine Oxidases Gene from aspergillus oryzae and its application in catalysis asymmetric oxidation reaction preparation chiral amine intermediates.Monoamine oxidase of the present inventionAoMAO8-3 gene is 1419 bp, encodes the monoamine oxidase being made of 470 amino acid.After the recombinant bacterium monoamine oxidase expression of Monoamine Oxidases Gene building of the present invention, full cell or enzyme generate the crucial chiral intermediate of traverte (Telaprevir) for converting cis- -7- azabicyclo [3,3,0] octane.The present invention has reaction condition mild, and stereoselectivity is high, is easy to the advantages that industrializing.

Description

A kind of monoamine oxidase from aspergillus oryzae is used for the preparation of chiral amine intermediates

Technical field

The invention belongs to microbes and enzyme engineering field, and in particular to the one of an Aspergillus oryzae and its genome encoding The new MAO-B B of kind, and prepared in several Chiral Amines using the monoamine oxidase from the bacterial strain as biocatalyst Mesosome.

Background technique

Chiral Amine is important and crucial medicines structure unit, and about 40% drug contains chiral amine structure, Chiral Amine at present High efficiency manufacture technology is key technology necessary to chiral drug process industry progress and development.Currently, the preparation master of Chiral Amine Based on being split and be synthesized with chemical asymmetry, biocatalysis manufacture Chiral Amine, which is then mainly used as using lipase and transaminase, is urged Agent, the research and use to monoamine oxidase (MAO) are less.

Optical activity Δ¹Pyrrolidines are the key that prepare many drug chiral intermediates, are wrapped in these molecules Containing multiple chiral centers, it is high to synthesize difficulty.For example, (1S, 3AR, 6AS)-octahydro cyclopenta [C] pyrroles's -1- carboxylic acid is The crucial chiral intermediate of hepatitis C drug traverte (Telaprevir) is treated, the drug is by Vertex company and Johnson & Johnson Company develops jointly.

It by the method for splitting that non-corresponding isomers crystallize into salt is still current to obtain optical voidness hand using chiral acid reagent Property amine main method, but theoretical yield only have 50% become this technology maximum drawback.Not using transition metal-catalyzed imines Symmetric hydrogenation reduction is another important channel for obtaining Chiral Amine, however there is also many deficiencies for this method: such as needing to activate Imines substrate；Need using transition metal, such as Rh, Ru, Ir and Ti, but expensive price limit they in industrialization Using.

In recent years, with the continuous development of biocatalysis technology, researchers try to explore to find and excavate new enzyme conduct Biocatalyst is used for the synthesis of Chiral Amine.Optical homochiral is obtained using monoamine oxidase asymmetric oxidation racemic amine substrate The research of amine also predominantly stays in laboratory development phase at present, and achievement is concentrated mainly on external several seminars, such as Britain Turner group.

To obtain efficient Chiral Amine Catalytic processes, it should solve the problems, such as that monoamine oxidase source is extremely limited first, so far The only aspergillus niger monoamine oxidase being applied successfully until the present.It is normal for screening novel microbial bacterial strain as catalyst from environment One of effective way.However original strain is directly converted there may be safety, Genetic stability problems, in original strain Other enzymes may to substrate-function generate by-product the problems such as, therefore by the crucial Monoamine Oxidases Gene of original strain clone And heterogenous expression, the above problem then can avoid for bioconversion with recombinant bacterium or enzyme, and this catalyst system is relatively simple, Stablize, product separating-purifying is easy, and is easy to industrialize.

Summary of the invention

The purpose of the present invention is disclose one originate from the aspergillus oryzae newly screened (Aspergillus oryzae15) height is right Reflect selective monoamine oxidaseAoMAO8-3, and be mutually coupled using the enzyme and chemical sulfonating reaction, one pot reaction building contains three A chiral centre and the water-soluble and better optical voidness of stability (1S, 3AR, 6AS)-octahydro cyclopenta [C] pyrroles's -1- sulphur Sour sodium intermediate, then by chemical nitrilation and hydrolysis, the traverte (Telaprevir) for obtaining high-optical-purity is crucial chiral Intermediate (1S, 3AR, 6AS)-octahydro cyclopenta [C] pyrroles's -1- carboxylic acid.

Based on foregoing invention purpose, present invention firstly provides a kind of monoamine oxidaseAoMAO8-3.The enzyme is from one plant of rice Aspergillus (Aspergillus oryzae15) it is taken out in by gene hunt method clone.The bacterium is rich from orchard soil Collection is separated and is obtained after multi-turns screen.

The present invention also provides a kind of above-mentioned monoamine oxidaseAoMAO8-3 is as the cis- -7- nitrogen of biocatalyst conversion of substrate Miscellaneous bicyclic [3,3,0] octane, and be mutually coupled with chemical sulfonating reaction, one pot reaction generates optical voidness (1S, 3AR, 6AS)-eight Hydrogen cyclopenta [C] pyrroles -1- sodium sulfonate intermediate, then by chemical cyaniding and hydrolysis, obtain the Te La of high-optical-purity Rui Wei (Telaprevir) key chiral intermediate (1S, 3AR, 6AS)-octahydro cyclopenta [C] pyrroles's -1- carboxylic acid is answered With.

Wherein, from above-mentioned aspergillus oryzae (Aspergillus oryzae15) crucial Monoamine Oxidases Gene inAoMAO8-3 Transfer and convert the following steps are included:

(1) gene hunt method clonal expansionAoMAO8-3 gene:

Using CTAB method extract aspergillus oryzae (Aspergillus oryzae15) genomic DNA.By to having reported to monoamine Oxidase gene carries out alignment and homogeneous assays, degenerate primer is designed according to N-terminal and C-terminal conserved sequence, with the rice of extraction Aspergillus (Aspergillus oryzae15) genomic DNA is that template carries out PCR, and Monoamine Oxidases Gene therein is cloned It amplifies and.

Wherein, involved in above-mentioned stepsAoMAO8-3 gene PCR expands the primer are as follows:

Forward direction 5 ˊ- GGA TCC ATG GCA CCA CCT TCA AAC GAA -3 ˊ (restriction enzyme siteBAm H I), reversed 5 ˊ- GAA TTC TCA AAG CCT CGA TCG CAA CGT TTC -3 ˊ (restriction enzyme siteECoR I)；

PCR condition is, 95 DEG C initial denaturation 5 minutes, 95 DEG C are denaturalized 30 seconds, and 55 DEG C are annealed 30 seconds, and 72 DEG C extend 90 seconds, 30 A circulation, last 72 DEG C extend 10 minutes.PCR product be connected into pMD19T vector construction TA clone, sequencing result show containing Two sections of intron sequences being made of respectively 60 and 60 nucleotide.

AoMAO8-3 gene (containing introne), base number was 1539 bp, and sequence is (SEQ ID No.1)

ATGGCACCACCTTCAAACGAAGGCTTTTTATGGACTCCACATGGAAGTACGAGTGGCCTCGAAACTGACGCTG CGCAGGAAAGCTCACCAGTTATCCATGACAGCTATGACGTGGTGGTTATAGGGGCTGGGTTCACTGGTCTTATCGCC GCACGAGAATTGAGTCAGAGACATGATCTGAAGGTGTTACTATTAGAGGCAAGAGATCGGATCGGTGGTCGTACATG GACGGCAAGAGCTCTAGGGGAAGAGCTAGAGATGGGAGGAACTTGGGTGGATTGGGCTCAACCTCACCTTTATAGCG AACTTCGCCGGTACGGACTACATATCAATTTAAAGGCTTCCGCAGGCACTGCTGCACCTACCAAGCAGATGTTCAAA CAGGGAAAGGCAACCCCATGTGAGATATCGATCGAGGAGACTGGTGATATTTTGGAGCGCATTGCCCAATCTTTCTT CACGATCGACGGAAGCAGCAGTCGTGAGCTCATGCCATACCCTCACGATCCCTTCAAGCGACCAGCTTTGTGGATGA AATATGATCATTTGTCAGTGCAAGACCGACTGGACAATCTTCGTGGGTTTTCAAGTTGGGAGAAAGATTTGTTCGAA TCAAACGCGAGCACCTTCGGTAGTGCTCCTGGAAAGGATATCGCATTTACCGAAGCACTTCGGTGGTACGCCTTAGG TGGACACAACATGAAGGGAGTGTTCGAACTCGCTGGAGTCTACAAAATTGGGAATGGGGGCATGACTTCGTTTGCGC GAGCAGTCTTGGGTGACTACACGGGGCATATGCTTTTTGGGGCCACTGTGAAGGAAGTCGCGCAGACCAAGTTAGGA GTCAGGGTTACCACAAGGCTTGGGCAAGAGATCAATGCAAAATATGTAGTTTCTACTATTCCTTTGTACGACCACTC TTCCTGAGCCTCTCTCAATTCGAAGTTCTCAAAGCTAATCCTCAGCAGGAACTGTTTAGCGGATGTGAAATTCTACC CCCCAATATCTTCCATTCGACAGTCTGCGATGACGAAAGGACATATCAATAAGGGCGCAAAGATCCACTTCAAGCTC AAGGCAGCGGAGCCGGGCTGGTTTGTAACCGCAAATTCGAGCGACTCGGCCTATGTTTTTGCCTTCTCCGACCACAA CGGCACTCGGGAATCAGAACCATCCGGCACTTGGTGCATTGGATTTGGATACAATGGAAGATTGGATGATAAGAACA ACCACCGTCATATTATCGATCGCTTCCGAAAGGATATTTATCCAACCGGTGACGTTGAAGCTTACGTGACTCACGAC TGGGTGAATGACCCGTACGCGAAAGGAGCTTGGGCTTGTTGGGGGCCTGGATGTGCGACAGCCCATCTCCAAGAACT CCAGAAGCCCCATGGGAGGGTCATTTTTGCCAGTGCCGATTGGGCTGACGGGTGGAGGGGGTTCGTTGATGGAGCCA TCGAACAGGGTCACCAAGCATCTCAATGTGTCGTGGCATCTCTCAAGTCAGAGACTGAAACGTTGCGATCGAGGCTT TGA

It is removed using over-lap PCRAoIntrone in MAO8-3 gene.First segment primer used in over-lap PCR is forward direction 5 ˊ- TGACTTCGTTTGCGCGAGCAACCAAGTTAGGAGTCAGGGT -3 ˊ, reversed 5 ˊ-ACCCTGACTCCTAACTTGGTTGC TCGCGCAAACGAAGTCA -3ˊ；Second segment primer used is 5 ˊ-TAGTTTCTACTATTCCTTTGAACTGTTTAGC of forward direction GGATGTGAA -3 ˊ, reversed -3 ˊ of 5 ˊ-TTCACATCCGCTAAACAGTTCAAAGGAATAGTAGAAACTA；

PCR condition is 50ul reaction system, 95 DEG C of denaturation 30 seconds, 55 DEG C of annealing 30 seconds, extends 72 DEG C 1 point 30 seconds, totally 30 A circulation usesPfuDNA polymerase.PCR product is connected into pMD19T vector construction TA clone, and sequencing result is shown It is correct to remove intron sequences.

AoMAO8-3 gene (removing introne) base number is 1419 bp, and sequence is (SEQ ID No.2)

ATGGCACCACCTTCAAACGAAGGCTTTTTATGGACTCCACATGGAAGTACGAGTGGCCTCGAAACTGACGCTG CGCAGGAAAGCTCACCAGTTATCCATGACAGCTATGACGTGGTGGTTATAGGGGCTGGGTTCACTGGTCTTATCGCC GCACGAGAATTGAGTCAGAGACATGATCTGAAGGTGTTACTATTAGAGGCAAGAGATCGGATCGGTGGTCGTACATG GACGGCAAGAGCTCTAGGGGAAGAGCTAGAGATGGGAGGAACTTGGGTGGATTGGGCTCAACCTCACCTTTATAGCG AACTTCGCCGGTACGGACTACATATCAATTTAAAGGCTTCCGCAGGCACTGCTGCACCTACCAAGCAGATGTTCAAA CAGGGAAAGGCAACCCCATGTGAGATATCGATCGAGGAGACTGGTGATATTTTGGAGCGCATTGCCCAATCTTTCTT CACGATCGACGGAAGCAGCAGTCGTGAGCTCATGCCATACCCTCACGATCCCTTCAAGCGACCAGCTTTGTGGATGA AATATGATCATTTGTCAGTGCAAGACCGACTGGACAATCTTCGTGGGTTTTCAAGTTGGGAGAAAGATTTGTTCGAA TCAAACGCGAGCACCTTCGGTAGTGCTCCTGGAAAGGATATCGCATTTACCGAAGCACTTCGGTGGTACGCCTTAGG TGGACACAACATGAAGGGAGTGTTCGAACTCGCTGGAGTCTACAAAATTGGGAATGGGGGCATGACTTCGTTTGCGC GAGCAACCAAGTTAGGAGTCAGGGTTACCACAAGGCTTGGGCAAGAGATCAATGCAAAATATGTAGTTTCTACTATT CCTTTGAACTGTTTAGCGGATGTGAAATTCTACCCCCCAATATCTTCCATTCGACAGTCTGCGATGACGAAAGGACA TATCAATAAGGGCGCAAAGATCCACTTCAAGCTCAAGGCAGCGGAGCCGGGCTGGTTTGTAACCGCAAATTCGAGCG ACTCGGCCTATGTTTTTGCCTTCTCCGACCACAACGGCACTCGGGAATCAGAACCATCCGGCACTTGGTGCATTGGA TTTGGATACAATGGAAGATTGGATGATAAGAACAACCACCGTCATATTATCGATCGCTTCCGAAAGGATATTTATCC AACCGGTGACGTTGAAGCTTACGTGACTCACGACTGGGTGAATGACCCGTACGCGAAAGGAGCTTGGGCTTGTTGGG GGCCTGGATGTGCGACAGCCCATCTCCAAGAACTCCAGAAGCCCCATGGGAGGGTCATTTTTGCCAGTGCCGATTGG GCTGACGGGTGGAGGGGGTTCGTTGATGGAGCCATCGAACAGGGTCACCAAGCATCTCAATGTGTCGTGGCATCTCT CAAGTCAGAGACTGAAACGTTGCGATCGAGGCTTTGA

AoMAO8-3 gene (removing introne) 470 amino acid of coding, sequence is (SEQ ID No.3):

MAPPSNEGFLWTPHGSTSGLETDAAQESSPVIHDSYDVVVIGAGFTGLIAARELSQRHDLKVLLLEARDRIGG RTWTARALGEELEMGGTWVDWAQPHLYSELRRYGLHINLKASAGTAAPTKQMFKQGKATPCEISIEETGDILERIAQ SFFTIDGSSSRELMPYPHDPFKRPALWMKYDHLSVQDRLDNLRGFSSWEKDLFESNASTFGSAPGKDIAFTEALRWY ALGGHNMKGVFELAGVYKIGNGGMTSFARATKLGVRVTTRLGQEINAKYVVSTIPLNCLADVKFYPPISSIRQSAMT KGHINKGAKIHFKLKAAEPGWFVTANSSDSAYVFAFSDHNGTRESEPSGTWCIGFGYNGRLDDKNNHRHIIDRFRKD IYPTGDVEAYVTHDWVNDPYAKGAWACWGPGCATAHLQELQKPHGRVIFASADWADGWRGFVDGAIEQGHQASQCVV ASLKSETETLRS

To then be sequenced correct plasmid withBAm H I andECoR I digestion is connected into the pET 28a with same enzymic digestion In (+) empty carrier, construction recombination plasmid, and being transferred in e. coli bl21 (DE3) constructs recombinant bacterium, in mistake in conventional manner Amount expression.The gene and enzyme for being related to bioconversion and application hereinafter refer both to removal introne after sequence and protein expressioning product.

(2) recombinant bacterium biocatalysis:

Into LB(50 μ g/ml containing kanamycins) culture medium, 37 DEG C are incubated overnight picking monoclonal, are transferred with 1% inoculum concentration Into LB(50 μ g/ml containing kanamycins) culture medium, 37 DEG C of 3 h of culture, after 0.5 mM IPTG induction is added, 30 DEG C of continuation It cultivates to 20 ~ 36 h.8000 rpm, 4 DEG C thalline were collected by centrifugation, is the buffer solution of potassium phosphate of 8.0,0.1 M concentration with pH value Washing 2 times obtains wet thallus.

It takes the wet thallus of above-mentioned fresh cultured to be resuspended in the kaliumphosphate buffer that pH value is 8.0,1 M concentration, substrate is added Cis- -7- azabicyclo [3,3,0] octane is passed through oxygen, cell concentration in transformation system into reaction system in reaction process For 50 ~ 150 g/L, Final substrate concentrations are 1 ~ 20 g/L, and conversion temperature is 30 DEG C, and revolving speed is 230 rpm, transformation time is 1 ~ 24 h.Response situation is detected using TLC in reaction process, solvent ratio is petroleum ether: ethyl acetate: triethylamine=15:1: 0.1, iodine colour developing.

(3)AoThe protein purification of MAO8-3 enzyme

AoThe purifying of MAO8-3 enzyme uses affinity chromatography (Bio-Rad).After 20 ~ 36 h of above-mentioned steps (2) Fiber differentiation Thallus be collected by centrifugation with 13,000rpm, 4 DEG C, be resuspended in Buffer A(50 mM phosphate buffer, pH 8.0,300 MM NaCl, 10 mM imidazoles), ultrasonication (10 s of ultrasound, 30 s of interval, 30 times) is centrifuged later with 13,000rpm, 4 DEG C 20 minutes, supernatant is added in the column material for use Buffer A to balance, it is slight to mix 30 minutes, with containing 20 mM imidazoles Buffer A rinse foreign protein, then with containing 250 mM imidazoles Buffer A buffer elution destination protein, last electrophoresis It identifies purity, and measures protein concentration with BCA Protein Assay kit.

(4) monoamine oxidaseAoMAO8-3 asymmetric oxidation and nucleophilic addition sulfonation coupling reaction:

ExpressionAoThe thallus culture of the Escherichia coli recombinant strain of MAO8-3 enzyme obtains the step of wet thallus same step (2).

Biotransformation condition: phosphate buffer (pH 8.0,0.1M), bottom are separately added into three mouthfuls of round-bottom reaction flasks Object and wet thallus/enzyme, wherein final concentration of 20 g/L of cis- -7- azabicyclo [3,3,0] octane, wet thallus final concentration of 50 G/L, final concentration of 1000 KU/L of catalase (CAT, 30KU/mg), starts reaction 24 under the conditions of 37 °C, 250 rpm Hour, it keeps being passed through oxygen in reaction process.NaHSO is added dropwise in reaction process₃Solution (NaHSO₃The amount of mole substance is substrate 1.2 times of cis- -7- azabicyclo [3,3,0] octane), asymmetric oxidation generate imine intermediate immediately with NaHSO₃Occur Trans- nucleophilic addition generates (1S, 3AR, 6AS)-octahydro ring penta that water-soluble and stability preferably contains 3 chiral centres Diene simultaneously [C] pyrroles -1- sodium sulfonate product, is added dropwise 3N NaOH in reaction process and adjusts reaction system pH and maintain 8.0 or so.

(5) cyanogenation prepares (1S, 3AR, 6AS)-octahydro cyclopenta [C] pyrroles's -1- nitrile

Cyanogenation condition: cyclopentyl methyl ether (CPME) 50mL is added in the reaction solution of step (4), and solution is cooled to 10 °C. 15mL NaCN aqueous solution is slowly added dropwise, and (amount of NaCN moles of substance is the 1.2 of cis- -7- azabicyclo [3,3,0] octane of substrate Times), it is added dropwise in half an hour.10 °C the reaction was continued 1 hour, is filtered to remove insoluble matter, takes organic phase, water phase with CPME 3 × 20mL extraction merges organic phase, and vacuum distillation removes solvent after anhydrous sodium sulfate is dry, and residue is (1S, 3AR, 6AS)- Octahydro cyclopenta [C] pyrroles's -1- nitrile.

(6) hydrolysis prepares (1S, 3AR, 6AS)-octahydro cyclopenta [C] pyrroles's -1- carboxylic acid

Hydrolysis condition: (1S, 3AR, 6AS)-octahydro cyclopenta [C] pyrroles -1- nitrile and 10 that step (5) is obtained ML concentrated hydrochloric acid (33%) is added separately in 50 mL round-bottomed flasks, heated overnight at reflux.It is cooled to after room temperature into round-bottomed flask Aqueous ammonia conditions system pH to 7.0 is added.It is purified using ion exchange chromatography, the cation exchange resin column filled in is first Washed with 1N HCl, after with pure water until pH is neutral.By reaction solution upper prop, with being used after 3 ~ 5 column volumes of pure water 5% ammonia scrubbing receives eluted product, and the colour developing of elution process ninhydrin is monitored, and vacuum distillation removes class obtained by solvent White solid is the ammonium salt of (1S, 3AR, 6AS)-octahydro cyclopenta [C] pyrroles's -1- carboxylic acid.Step (4) ~ (6), reaction Total recovery about 43%, product (1S, 3AR, 6AS)-octahydro cyclopenta [C] pyrroles -1- carboxylic acid optical purity are 89%ee value.It produces Total recovery 34% after object recrystallizes in ethyl alcohol-methyl tertiary butyl ether(MTBE) mixed liquor, optical purity are increased to 91%ee value.This amino acid The optical purity of product is determined using chiral high performance liquid chromatography (HPLC) analytic approach.Chiral HPLC conditions: chiral column (the D)-Penicillamine(50 mm × 4.6 of Chirex 3126 mm, 5 micron), mobile phase H₂O : CH₃CN = 90 : 10, 2 mM CuSO₄, 0.8 mL of flow velocity/min, 25 °C of column temperature, monitor wavelength 247nm, product retention time Tr =6.5 minutes and 9.7 minutes.

Aspergillus oryzae of the present invention (Aspergillus oryzae15) its crucial Monoamine Oxidases GeneAoMAO8-3 It can cis- -7- azabicyclo [3,3,0] the octane generation imine intermediate (3A of highly-solid selectively conversionR,6AS)-hexahydro ring penta Diene simultaneously [C] pyrrolin, and can be mutually coupled with chemical sulfonating reaction, one pot reaction constructs (1S, 3AR, 6AS)-octahydro ring penta Diene simultaneously [C] pyrroles -1- sodium sulfonate intermediate, then by chemical nitrilation and hydrolysis, obtain the traverte of high-optical-purity (Telaprevir) crucial chiral intermediate (1S, 3AR, 6AS)-octahydro cyclopenta [C] pyrroles's -1- carboxylic acid.It is domestic at present Outside do not have also patent and document report from aspergillus oryzae (Aspergillus oryzae) in excavate and monoamine oxidase and used In crucial chiral intermediate (1S, 3AR, 6AS)-octahydro cyclopenta [C] pyrroles's -1- carboxylic acid of traverte (Telaprevir) Preparation.AoThe highest similarity of MAO8-3 and reported functional protein MAO-N-D5(PDB database ID:2VVM) is 65.1%, it is provided for the synthesis of (1S, 3AR, 6AS)-octahydro cyclopenta [C] pyrroles -1- carboxylic acid biocatalysis alternative Novel enzyme source.

Currently, not having also patent and document report trans- using monoamine oxidase asymmetric oxidation and chemical sulfonation both at home and abroad Nucleophilic addition is mutually coupled, and one pot reaction generates optical voidness (1S, 3AR, 6AS)-octahydro cyclopenta [C] pyrroles -1- Sodium sulfonate intermediate is used to prepare crucial chiral intermediate (1S, 3AR, the 6AS)-octahydro ring penta 2 of traverte (Telaprevir) Alkene simultaneously [C] pyrroles -1- carboxylic acid.

It is all withAoMAO8-3 amino acid similarity, which is higher than 70% and has, is catalyzed cis- -7- azabicyclo [3,3,0] octane Generate (3AR,6ASThe enzyme of)-hexahydro cyclopenta [C] pyrrolin function all belongs to the scope of protection of the present invention.In addition, using Monoamine oxidase asymmetric oxidation is mutually coupled with the chemical trans- nucleophilic addition of sulfonation, one pot reaction generation optical voidness (1S, 3AR, 6AS)-octahydro cyclopenta [C] pyrroles's -1- sodium sulfonate synthetic method also in protection scope of the present invention.

Detailed description of the invention

Fig. 1 isAoSDS-PAGE schemes after MAO8-3 protein purification.

Specific embodiment

Explain the present invention in detail with reference to embodiments.Embodiment to facilitate the understanding of the present invention, but not Limitation of the present invention.

Embodiment 1: extracting genome DNA

It is as follows that genomic DNA operating method is extracted using CTAB method:

Strain culturing: by a small amount of aspergillus oryzae (Aspergillus oryzae15) inclined-plane seed is inoculated in aspergillus culture medium, and 30 DEG C, 230 rpm cultivate 24 ~ 36 h and prepare seed liquor, and it is forwarded in aspergillus culture medium with 0.5% ~ 2% inoculum concentration and expands culture, Condition of culture: 30 DEG C, 230 rpm, 48 ~ 72 h.Aspergillus medium component: 0.3% KH₂PO₄；0.15% MgSO₄•7H₂O； 0.1% yeast extract；0.06% peptone；2% glucose；20% potato.

It takes 0.5 ~ 1.0g mycelium into mortar, quartz sand liquid nitrogen grinding is added to crush.The powder is transferred to 2.0 ml's In EP pipe, the DNA extract (every 50mg thallus adds 500 μ l) of 600 ul is added, vibrates, mixes, 55 DEG C keep the temperature 60 minutes.With It is 1.4 mol/L that 4 mol/L NaCl, which adjust solution NaCl concentration, and 10% CTAB solution of 1/10 volume is then added, and is mixed, 65 DEG C keep the temperature 10 minutes.Isometric chloroform/isoamyl alcohol is added, is mixed by inversion, ice bath 30 minutes.4 DEG C, 15000 r/ minutes from The heart 10 minutes.Supernatant is transferred in another EP pipe, isometric isopropanol is added and mixes, ice bath 30 minutes.Take out EP Pipe, 10000 r/ minutes, 4 DEG C were centrifuged 10 minutes, abandon supernatant, and 75% ethanol washing twice, is air-dried, and 150 ul are added ddH₂O dissolution precipitates up to genome DNA.DNA extract composition: 0.1 mol/L Tris-HCl(pH 8.0), 0.01 The mol/L μ g/ml Proteinase of EDTA, 2% SDS, 100 K, 0.5% β-mercaptoethanol.

Embodiment 2: gene hunt method clonal expansionAoMAO8-3 gene

By being set according to N-terminal and C-terminal conserved sequence to having reported to Monoamine Oxidases Gene progress alignment and homogeneous assays Degenerate primer is counted, PCR is carried out as template using the genomic DNA obtained in embodiment 1, Monoamine Oxidases Gene therein is cloned It amplifies and.

PCR reaction system is 50ul, respectively by 5 ul × 10pfuDNA polymerase buffer (contains Mg2+), and 1 Ul primer F, 1 ul primer R, 4 ul dNTP, 1 ul genomic DNA, 0.5 ulPfu DNA polymerase With 37.5 ul ddH₂O composition.Wherein, PCR amplification the primer are as follows:

Primer F:5 ˊ- GGA TCC ATG GCA CCA CCT TCA AAC GAA -3 ˊ (restriction enzyme siteBAm H I), Primer R:5 ˊ- GAA TTC TCA AAG CCT CGA TCG CAA CGT TTC -3 ˊ (restriction enzyme siteECoR I)；

PCR condition is, 95 DEG C initial denaturation 5 minutes, 95 DEG C are denaturalized 30 seconds, and 55 DEG C are annealed 30 seconds, and 72 DEG C extend 90 seconds, 30 circulations, last 72 DEG C extend 10 minutes.PCR product is connected into pMD19T vector construction TA clone, and sequencing result, which is shown, to be contained The intron sequences being made of by one section 57 nucleotide.AoMAO8-3 gene (containing introne), base number was 1539 bp, Sequence is (SEQ No.1).

It is removed using over-lap PCRAoIntrone in MAO8-3 gene.First segment primers F m1 used in over-lap PCR is forward direction 5 ˊ-TGACTTCGTTTGCGCGAGCAACCAAGTTAGGAGTCAGGGT -3 ˊ, Rm1 are reversed 5 ˊ - ACCCTGACTCCTAACTTGGTTGCTCGCGCAAACGAAGTCA -3ˊ；Second segment primers F m2 used is forward direction 5 ˊ- TAGTTTCTACTATTCCTTTGAACTGTTTAGCGGATGTGAA -3 ˊ, Rm2 are reversed 5 ˊ - TTCACATCCGCTAAACAGTTCAAAGGAATAGTAGAAACTA -3ˊ；

First round PCR: the plasmid pMD19T- that will be extracted beforeAoMAO8-3 carries out PCR, reaction system (50 ul) as template It is as follows:

Leading portion gene (a): 5 ul × 10pfuBuffer (contains Mg²⁺), 1 ul primer F, 1 ul primer Rm1,4 Ul dNTP, 1 ul pMD19T- AoMAO8-3,0.5 ulPfuDNA polymerase, 37.5 ul ddH₂O。

Middle section gene (b): 5 ul × 10 pfuBuffer (contains Mg²⁺), 1 ul primer Fm1,1 ul primer Rm2,4 ul dNTP, 1 ul pMD19T- AoMAO8-3,0.5 ulPfuDNA polymerase, 37.5 ul ddH₂O。

Back segment gene (c): 5 ul × 10 pfuBuffer (contains Mg²⁺), 1 ul primer Fm2,1 ul primer R, 4 ul dNTP, 1 ul pMD19T- AoMAO8-3,0.5 ulPfuDNA polymerase, 37.5 ul ddH₂O。

PCR condition is, 95 DEG C initial denaturation 5 minutes, 95 DEG C are denaturalized 30 seconds, and 55 DEG C are annealed 30 seconds, and 72 DEG C extend 90 Second, 30 circulations, last 72 DEG C extend 10 minutes.

3 sections of PCR products, recovery product number: 8-3-a, 8-3-b and 8-3-c are recycled using gel reclaims kit.

Second wheel PCR: using PCR product 8-3-a, 8-3-b and 8-3-c for recycling before as template, PCR, reaction are carried out System (50 ul) is as follows: 5 ul × 10pfuBuffer (contains Mg²⁺), 1 ul primer F, 1 ul primer R, 4 ul DNTP, 1 ul 8-3-a, 1 ul 8-3-b, 1 ul 8-3-c, 0.5 ulPfuDNA polymerase, 35.5 ul ddH₂O。

PCR product is recycled using gel reclaims kit, and is connect with pMD19-T carrier, competent cell is transformed intoE. In coli DH5 α, LB plate (100 ug/mL containing ampicillin) culture is selected positive colony by blue hickie and is carried out Plasmid and sequencing are extracted in culture.

10 ul linked systems: 1 ul pMD19-T carrier, 5 ul NEB solution I, 4 ul PCR recycling produce Object is connected overnight at 16 DEG C.

Conversion: junction fragment 4ul is transformed intoEIn coli DH5 α, LA plate (X-gal containing 10 ul × 5,7 ul 1M IPTG) 37 DEG C of cultures.

Blue hickie screening: picking white monoclonal colonies (each plate chooses 2 bacterium) arrive the examination of 3ml LA fluid nutrient medium Guan Zhong, is cultivated 17 hours under 180rpm by 37 DEG C.

Plasmid pMD19T- is extracted using the small extraction reagent kit of bacterial plasmidAoMAO8-3.Plasmid order-checking is as the result is shown correctly Remove intron sequences.AoMAO8-3 gene (removing introne) base number is 1419 bp, and sequence is (SEQ No.2)

Embodiment 3: willAoMAO8-3 is gene constructed in engineering strain

To plasmid pMD19T-AoMAO8-3 carries out double digestion.40 ul of digestion system, by 33 ul pMD19T-AoMAO8-3, 1.0 ul BSA, 1.0 ulBAmH I, 4 ul NEB Buffer 3 composition, 37 DEG C are added 1.0 ul after digestion 3 hoursEcoR I continues at 37 DEG C of digestions 3 hours.

Double digestion is carried out to carrier pET28a.40 ul of digestion system, by 33 ul pET28a, 1.0 ul BSA, 1.0 ul BAmH I, 4 ul NEB Buffer 3 composition, 37 DEG C are added 1.0 ul after digestion 3 hoursECoR I continues at 37 DEG C of enzymes It cuts 3 hours.

Digestion products are recycled and are purified using gel reclaims kit.

Connection: 10 ul systems, 6 ulAoMAO8-3(BamH I / ECoR I), 2 ul pET28a (BamH I /ECoR I), 1 ul T4 DNA ligase Buffer, 1 ul T4 DNA ligase.It is connected overnight at 16 DEG C.

Conversion and culture: by 10 ul carrier pET28- of connectionAoMAO8-3 is transformed into competent cellE. coli In DH5 α, it is coated on LB plate (the 50 μ g/ml containing kanamycins), in 37 DEG C of incubator overnight incubations.Contain pET28- from growthAoMAO8-3'sEPicking monoclonal colonies on coli DH5 α plate are cultivated in the LB liquid medium of Yu Han Kan resistance, 37 DEG C, 180rpm.Plasmid pET28- is extracted using the small extraction reagent kit of bacterial plasmidAoMAO8-3。

It is transformed into genetic engineering bacterium and culture: by 2ul plasmid pET28-AoMAO8-3 is transformed into competent cellE. In coli BL21, it is coated on LB plate (the 50 μ g/ml containing kanamycins), in 37 DEG C of incubator overnight incubations.Contain from growth pET28-AoMAO8-3'sEPicking monoclonal colonies on coli BL21 plate, in the LB liquid medium of Yu Han Kan resistance Culture, 180rpm, 37 DEG C, overnight incubation.LB(50 μ g/ml containing kanamycins is forwarded to 1% inoculum concentration) in culture medium, 37 DEG C culture 3 hours, be added 0.5 mM IPTG induction after, 30 DEG C continue culture to 16 ~ 20 hours.8000 rpm, 4 DEG C of centrifugations Thallus is collected, is washed 2 times with the buffer solution of potassium phosphate that pH value is 8.0,0.1 M concentration, obtaining wet thallus can be used for subsequent life Object conversion reaction.

Embodiment 4: monoamine oxidaseAoMAO8-3 recombinant bacterium Whole Cell Biocatalysis

Monoamine oxidaseAoThe cultural method and microorganism collection method of MAO8-3 recombinant bacterium are shown in specification Summary.

(1) take 1.0 g of thallus, be resuspended in 10 ml phosphate buffers (pH 8.0,0.1M), be added 10 mg of substrate, 30 DEG C, 230 rpm convert 3h.With the extraction of 10 ml ethyl acetate, anhydrous sodium sulfate is dry, is concentrated under reduced pressure to give product (3AR, 6AS)-hexahydro cyclopenta [C] pyrrolin, through TLC thin-layer chromatography chromatography, the conversion ratio of substrate is 25%.

(2) thallus 1.0g is taken, is resuspended in 10 ml phosphate buffers (pH 8.0,0.1M), 10 mg of substrate, reaction is added Pure oxygen is passed through in bottle, 30 DEG C, 230 rpm convert 3 h.With the extraction of 10 ml ethyl acetate, anhydrous sodium sulfate is dry, is concentrated under reduced pressure Obtain product (3AR,6AS)-hexahydro cyclopenta [C] pyrrolin, through TLC thin-layer chromatography chromatography, the conversion ratio of substrate It is 30%.

(3) 1.0 g of thallus is taken, is resuspended in 10 ml phosphate buffers (pH 8.0,0.1M), 10 mg of substrate, mistake is added 1 ul of hydrogen oxide enzyme (CAT, 30KU/mg) is passed through pure oxygen in reaction flask, and 30 DEG C, 230 rpm convert 3 h.With 10 ml acetic acid second Ester extraction, anhydrous sodium sulfate is dry, is concentrated under reduced pressure to give product (3AR,6AS)-hexahydro cyclopenta [C] pyrrolin, through TLC Thin-layer chromatography chromatography, the conversion ratio of substrate are 45%.

(4) 1.0 g of thallus is taken, is resuspended in 10 ml phosphate buffers (pH 8.0,0.1M), 10 mg of substrate, mistake is added 1 ul of hydrogen oxide enzyme (CAT, 30KU/mg) is passed through pure oxygen in reaction flask, and 37 DEG C, 230 rpm convert 24 h.With 10 ml acetic acid second Ester extraction, anhydrous sodium sulfate is dry, is concentrated under reduced pressure to give product (3AR,6AS)-hexahydro cyclopenta [C] pyrrolin, through TLC Thin-layer chromatography chromatography, the conversion ratio of substrate are 79%.

(5) 1.5 g of thallus is taken, is resuspended in 10 ml phosphate buffers (pH 8.0,0.1M), 500 mg of substrate, mistake is added 1 ul of hydrogen oxide enzyme (CAT, 30KU/mg) is passed through pure oxygen in reaction flask, and 37 DEG C, 230 rpm convert 24 h.With 10 ml acetic acid second Ester extraction, anhydrous sodium sulfate is dry, is concentrated under reduced pressure to give product (3AR,6AS)-hexahydro cyclopenta [C] pyrrolin, through TLC Thin-layer chromatography chromatography, the conversion ratio of substrate are 47%.

Embodiment 5: monoamine oxidaseAoThe pure enzymes biocatalysis of MAO8-3

Transformation system: kaliumphosphate buffer (0.1M, pH 8.0), pure 2 g/L of enzyme concentration, the cis- -7- azabicyclo of substrate [3,3, 0] octane, 1 ul of catalase (CAT, 30KU/mg) are passed through pure oxygen, 30 DEG C of bioconversion 3h in reaction flask.With 10 ml second Acetoacetic ester extraction, anhydrous sodium sulfate is dry, is concentrated under reduced pressure to give product (3AR,6AS)-hexahydro cyclopenta [C] pyrrolin, Through TLC thin-layer chromatography chromatography, the conversion ratio of substrate is 21%.

Embodiment 6: monoamine oxidaseAoMAO8-3 asymmetric oxidation and sulfonating reaction are coupled

Monoamine oxidaseAoMAO8-3The cultural method and microorganism collection method of recombinant bacterium are shown in specification Summary.

10 mL resuspended bacterium solutions are separately added into 100 mL round bottom, three mouthfuls of reaction flasks, and (with 0.1M, 8.0 potassium phosphate of pH delays 1.5g wet thallus is resuspended in fliud flushing), 2 grams of cis- -7- azabicyclo [3,3,0] octanes, catalase (CAT, 30KU/mg) 5 Ul, oxygen is continually fed into reaction flask, and 5 mL NaHSO are slowly added dropwise after starting in reaction₃Solution (NaHSO₃2.2g), in 1h It finishes, continues thereafter with and reacted 24 hours under the conditions of 37 °C, 230 rpm, 3N NaOH is added dropwise in reaction process and adjusts reactant It is that pH maintains 8.0 or so.It is extracted after reaction with 10 ml ethyl acetate, removes organic phase, water phase is added 3N HCl and adjusts PH < 2 is extracted with 3 × 10 ml methyl tertiary butyl ether(MTBE)s (MTBE), and anhydrous sodium sulfate is dry, and off-white powder is obtained after vacuum distillation As (1S, 3AR, 6AS)-octahydro cyclopenta [C] pyrroles's -1- sulfonic acid, yield 81%.

Sequence table

<110>Chengdu origin Biotechnology Co., Ltd

<120>a kind of monoamine oxidase from aspergillus oryzae is used for the preparation of chiral amine intermediates

<130>specification

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1539

<212> DNA

<213> Aspergillus oryzae

<400> 1

atggcaccac cttcaaacga aggcttttta tggactccac atggaagtac gagtggcctc 60

gaaactgacg ctgcgcagga aagctcacca gttatccatg acagctatga cgtggtggtt 120

ataggggctg ggttcactgg tcttatcgcc gcacgagaat tgagtcagag acatgatctg 180

aaggtgttac tattagaggc aagagatcgg atcggtggtc gtacatggac ggcaagagct 240

ctaggggaag agctagagat gggaggaact tgggtggatt gggctcaacc tcacctttat 300

agcgaacttc gccggtacgg actacatatc aatttaaagg cttccgcagg cactgctgca 360

cctaccaagc agatgttcaa acagggaaag gcaaccccat gtgagatatc gatcgaggag 420

actggtgata ttttggagcg cattgcccaa tctttcttca cgatcgacgg aagcagcagt 480

cgtgagctca tgccataccc tcacgatccc ttcaagcgac cagctttgtg gatgaaatat 540

gatcatttgt cagtgcaaga ccgactggac aatcttcgtg ggttttcaag ttgggagaaa 600

gatttgttcg aatcaaacgc gagcaccttc ggtagtgctc ctggaaagga tatcgcattt 660

accgaagcac ttcggtggta cgccttaggt ggacacaaca tgaagggagt gttcgaactc 720

gctggagtct acaaaattgg gaatgggggc atgacttcgt ttgcgcgagc agtcttgggt 780

gactacacgg ggcatatgct ttttggggcc actgtgaagg aagtcgcgca gaccaagtta 840

ggagtcaggg ttaccacaag gcttgggcaa gagatcaatg caaaatatgt agtttctact 900

attcctttgt acgaccactc ttcctgagcc tctctcaatt cgaagttctc aaagctaatc 960

ctcagcagga actgtttagc ggatgtgaaa ttctaccccc caatatcttc cattcgacag 1020

tctgcgatga cgaaaggaca tatcaataag ggcgcaaaga tccacttcaa gctcaaggca 1080

gcggagccgg gctggtttgt aaccgcaaat tcgagcgact cggcctatgt ttttgccttc 1140

tccgaccaca acggcactcg ggaatcagaa ccatccggca cttggtgcat tggatttgga 1200

tacaatggaa gattggatga taagaacaac caccgtcata ttatcgatcg cttccgaaag 1260

gatatttatc caaccggtga cgttgaagct tacgtgactc acgactgggt gaatgacccg 1320

tacgcgaaag gagcttgggc ttgttggggg cctggatgtg cgacagccca tctccaagaa 1380

ctccagaagc cccatgggag ggtcattttt gccagtgccg attgggctga cgggtggagg 1440

gggttcgttg atggagccat cgaacagggt caccaagcat ctcaatgtgt cgtggcatct 1500

ctcaagtcag agactgaaac gttgcgatcg aggctttga 1539

<210> 2

<211> 1419

<212> DNA

<213> Aspergillus oryzae

<400> 2

atggcaccac cttcaaacga aggcttttta tggactccac atggaagtac gagtggcctc 60

gaaactgacg ctgcgcagga aagctcacca gttatccatg acagctatga cgtggtggtt 120

ataggggctg ggttcactgg tcttatcgcc gcacgagaat tgagtcagag acatgatctg 180

aaggtgttac tattagaggc aagagatcgg atcggtggtc gtacatggac ggcaagagct 240

ctaggggaag agctagagat gggaggaact tgggtggatt gggctcaacc tcacctttat 300

agcgaacttc gccggtacgg actacatatc aatttaaagg cttccgcagg cactgctgca 360

cctaccaagc agatgttcaa acagggaaag gcaaccccat gtgagatatc gatcgaggag 420

actggtgata ttttggagcg cattgcccaa tctttcttca cgatcgacgg aagcagcagt 480

cgtgagctca tgccataccc tcacgatccc ttcaagcgac cagctttgtg gatgaaatat 540

gatcatttgt cagtgcaaga ccgactggac aatcttcgtg ggttttcaag ttgggagaaa 600

gatttgttcg aatcaaacgc gagcaccttc ggtagtgctc ctggaaagga tatcgcattt 660

accgaagcac ttcggtggta cgccttaggt ggacacaaca tgaagggagt gttcgaactc 720

gctggagtct acaaaattgg gaatgggggc atgacttcgt ttgcgcgagc aaccaagtta 780

ggagtcaggg ttaccacaag gcttgggcaa gagatcaatg caaaatatgt agtttctact 840

attcctttga actgtttagc ggatgtgaaa ttctaccccc caatatcttc cattcgacag 900

tctgcgatga cgaaaggaca tatcaataag ggcgcaaaga tccacttcaa gctcaaggca 960

gcggagccgg gctggtttgt aaccgcaaat tcgagcgact cggcctatgt ttttgccttc 1020

tccgaccaca acggcactcg ggaatcagaa ccatccggca cttggtgcat tggatttgga 1080

tacaatggaa gattggatga taagaacaac caccgtcata ttatcgatcg cttccgaaag 1140

gatatttatc caaccggtga cgttgaagct tacgtgactc acgactgggt gaatgacccg 1200

tacgcgaaag gagcttgggc ttgttggggg cctggatgtg cgacagccca tctccaagaa 1260

ctccagaagc cccatgggag ggtcattttt gccagtgccg attgggctga cgggtggagg 1320

gggttcgttg atggagccat cgaacagggt caccaagcat ctcaatgtgt cgtggcatct 1380

ctcaagtcag agactgaaac gttgcgatcg aggctttga 1419

<210> 3

<211> 470

<212> PRT

<213> Aspergillus oryzae

<400> 3

Met Ala Pro Pro Ser Asn Glu Gly Phe Leu Trp Thr Pro His Gly Ser

1 5 10 15

Thr Ser Gly Leu Glu Thr Asp Ala Ala Gln Glu Ser Ser Pro Val Ile

20 25 30

His Asp Ser Tyr Asp Val Val Val Ile Gly Ala Gly Phe Thr Gly Leu

35 40 45

Ile Ala Ala Arg Glu Leu Ser Gln Arg His Asp Leu Lys Val Leu Leu

50 55 60

Leu Glu Ala Arg Asp Arg Ile Gly Gly Arg Thr Trp Thr Ala Arg Ala

65 70 75 80

Leu Gly Glu Glu Leu Glu Met Gly Gly Thr Trp Val Asp Trp Ala Gln

85 90 95

Pro His Leu Tyr Ser Glu Leu Arg Arg Tyr Gly Leu His Ile Asn Leu

100 105 110

Lys Ala Ser Ala Gly Thr Ala Ala Pro Thr Lys Gln Met Phe Lys Gln

115 120 125

Gly Lys Ala Thr Pro Cys Glu Ile Ser Ile Glu Glu Thr Gly Asp Ile

130 135 140

Leu Glu Arg Ile Ala Gln Ser Phe Phe Thr Ile Asp Gly Ser Ser Ser

145 150 155 160

Arg Glu Leu Met Pro Tyr Pro His Asp Pro Phe Lys Arg Pro Ala Leu

165 170 175

Trp Met Lys Tyr Asp His Leu Ser Val Gln Asp Arg Leu Asp Asn Leu

180 185 190

Arg Gly Phe Ser Ser Trp Glu Lys Asp Leu Phe Glu Ser Asn Ala Ser

195 200 205

Thr Phe Gly Ser Ala Pro Gly Lys Asp Ile Ala Phe Thr Glu Ala Leu

210 215 220

Arg Trp Tyr Ala Leu Gly Gly His Asn Met Lys Gly Val Phe Glu Leu

225 230 235 240

Ala Gly Val Tyr Lys Ile Gly Asn Gly Gly Met Thr Ser Phe Ala Arg

245 250 255

Ala Thr Lys Leu Gly Val Arg Val Thr Thr Arg Leu Gly Gln Glu Ile

260 265 270

Asn Ala Lys Tyr Val Val Ser Thr Ile Pro Leu Asn Cys Leu Ala Asp

275 280 285

Val Lys Phe Tyr Pro Pro Ile Ser Ser Ile Arg Gln Ser Ala Met Thr

290 295 300

Lys Gly His Ile Asn Lys Gly Ala Lys Ile His Phe Lys Leu Lys Ala

305 310 315 320

Ala Glu Pro Gly Trp Phe Val Thr Ala Asn Ser Ser Asp Ser Ala Tyr

325 330 335

Val Phe Ala Phe Ser Asp His Asn Gly Thr Arg Glu Ser Glu Pro Ser

340 345 350

Gly Thr Trp Cys Ile Gly Phe Gly Tyr Asn Gly Arg Leu Asp Asp Lys

355 360 365

Asn Asn His Arg His Ile Ile Asp Arg Phe Arg Lys Asp Ile Tyr Pro

370 375 380

Thr Gly Asp Val Glu Ala Tyr Val Thr His Asp Trp Val Asn Asp Pro

385 390 395 400

Tyr Ala Lys Gly Ala Trp Ala Cys Trp Gly Pro Gly Cys Ala Thr Ala

405 410 415

His Leu Gln Glu Leu Gln Lys Pro His Gly Arg Val Ile Phe Ala Ser

420 425 430

Ala Asp Trp Ala Asp Gly Trp Arg Gly Phe Val Asp Gly Ala Ile Glu

435 440 445

Gln Gly His Gln Ala Ser Gln Cys Val Val Ala Ser Leu Lys Ser Glu

450 455 460

Thr Glu Thr Leu Arg Ser

465 470

Claims (3)

1. a kind of monoamine oxidaseAoMAO8-3, it is characterised in that its nucleotides sequence is classified as shown in SEQ No.2, amino acid sequence It is classified as shown in SEQ No.3.

2. monoamine oxidase described in claim 1AoMAO8-3 is being converted as biocatalyst

Cis- -7- azabicyclo [3,3,0] octane of substrate generates (3AR,6ASAnswering in)-hexahydro cyclopenta [C] pyrrolin With.

3. monoamine oxidase described in claim 1AoMAO8-3 and chemical nucleopilic reagent pass through asymmetric oxidation and sulfonating reaction It is mutually coupled, cis- -7- azabicyclo [3,3, the 0] octane of one pot reaction conversion of substrate generates (1S, 3AR, 6AS)-octahydro ring penta The application of diene simultaneously [C] pyrroles -1- sodium sulfonate intermediate.