Variant nitrile hydratase, microorganism expressing same and use thereof in amide synthesis

Abstract

The present invention relates to a variant nitrile hydratase, a nucleic acid encoding said nitrile hydratase and a microorganism engineered to express said novel nitrile hydratase, said variant nitrile hydratase being engineered to comprise higher activity and / or stability. Furthermore, the present invention relates to the use of such a nitrile hydratase and a microorganism expressing said nitrile hydratase as a biocatalyst, in particular in a method for producing amide compounds from nitrile compounds, preferably for the conversion of acrylonitrile to acrylamide.

Description

[0001] Related Applications

[0002] The present invention claims priority to U.S. Provisional Application No. 63 / 322,203 filed on March 21, 2022, the entire contents of which are incorporated herein by reference in their entirety.

[0003] About Electronic Sequence Listings

[0004] The contents of the electronic sequence listing (1149704.018013.xml; size: 20,675 bytes; and creation date: March 14, 2023) are incorporated herein by reference in its entirety. Technical Field

[0005] The present invention relates to a novel improved nitrile hydratase and / or an operon containing the improved nitrile hydratase, which is engineered to contain higher activity and / or stability, and also to nucleic acids encoding the improved nitrile hydratase and / or operon, and microorganisms engineered to contain and express the improved nitrile hydratase or the operon containing the improved nitrile hydratase. In addition, the present invention relates to the use of the improved nitrile hydratase and / or the microorganism engineered to express the improved nitrile hydratase as a biocatalyst, in particular in a method for producing an amide compound from a nitrile compound, preferably for converting acrylonitrile into acrylamide. Background Art

[0006] Acrylamide is used as a monomer for forming polymers and copolymers of acrylamide. For these polymerization and copolymerization reactions, aqueous acrylamide solutions prepared by bioconversion can be used. Since the discovery of nitrile hydratase, a microbial enzyme that hydrolyzes nitriles to amides, microorganisms having nitrile hydratase activity have been widely used for the industrial production of amide compounds. Microorganisms that produce nitrile hydratase are increasingly used as biocatalysts due to milder reaction conditions compared to the chemical synthesis of amides.

[0007] In fact, one of the most famous commercial examples of nitrile bioconversion by microorganisms producing nitrile hydratase is the production of acrylamide (AMD) from acrylonitrile (AN). However, a challenging problem with using microorganisms producing nitrile hydratase as biocatalysts is that both AN and AMD can inactivate the biocatalyst. Thus, providing a nitrile hydratase biocatalyst that remains stable for a long time is problematic. In addition, nitrile hydratase biocatalysts that have been modified to have enhanced stability are generally not as active as unmodified biocatalysts, i.e., they do not initiate the conversion of AN to AMD as quickly as unmodified biocatalysts.

[0008] Therefore, the technical problem of the present invention is to provide improved nitrile hydratase and microorganisms producing nitrile hydratase and their use as biocatalysts, i.e., biocatalysts having enhanced stability (resistance to inactivation by AN and AMD) and desirable activity (i.e., very rapid initiation of the conversion of AN to AMD, i.e., wherein the biocatalyst activity is the initial reaction rate) when used for converting nitriles to amides.

[0009] This technical problem is solved by providing the embodiments reflected in the claims, described in the specification and shown in the following examples and drawings. Summary of the invention

[0010] The present invention relates to a novel nitrile hydratase, a nucleic acid encoding the nitrile hydratase, and a microorganism engineered to express the novel nitrile hydratase, wherein the novel nitrile hydratase is engineered to contain higher activity and / or stability. In addition, the present invention relates to the use of the nitrile hydratase and the microorganism engineered to express the nitrile hydratase as a biocatalyst, in particular in a method for producing an amide compound from a nitrile compound, preferably for converting acrylonitrile to acrylamide.

[0011] More specifically, the present invention relates to a novel nitrile hydratase derived from Pseudonocardia thermophila that has been engineered to contain mutations that provide enhanced stability and activity. In short, we have created a DNA sequence encoding a novel nitrile hydratase that can be transferred into another microorganism, such as an industrial production microorganism, and used to industrially produce acrylamide (AMD) from acrylonitrile (AN).

[0012] Even more specifically, the present invention provides a variant nitrile hydratase (an enzyme comprising an α subunit (nhhA) and a β subunit (nhhB), the α subunit having an amino acid sequence at least 98%, 99% or 100% identical to SEQ ID NO: 2, and the β subunit comprising an amino acid sequence at least 98%, 99% or 100% identical to SEQ ID NO: 1, provided that the α subunit comprises one, two or three of the following mutations: L6T, A19V and F126Y, and the β subunit comprises one or two of the following mutations: E108D and A200E or comprises all three of the following mutations: E108R, A200E and S212Y; wherein the variant nitrile hydratase has enhanced stability and / or activity compared to the nitrile hydratase produced by the wild-type strain (Pseudonocardia thermophila DSM 43832 strain).

[0013] In an exemplary embodiment, the invention provides a variant nitrile hydratase wherein the alpha subunit comprises the following mutations: L6T, A19V, and F126Y, and the beta subunit comprises the following mutations: E108D and A200E; or E108R, A200E, and S212Y.

[0014] In an exemplary embodiment, the invention provides a variant nitrile hydratase wherein the α and β enzyme subunits are expressed in conjunction with an nhhG activator protein, optionally comprising an amino acid sequence at least 98%, 99% or 100% identical to SEQ ID NO:3.

[0015] In some exemplary embodiments, the invention provides a variant nitrile hydratase according to any of the foregoing, comprising a soluble enzyme.

[0016] In some exemplary embodiments, a variant nitrile hydratase according to any of the foregoing may be immobilized to a solid support.

[0017] In some exemplary embodiments, the invention provides a variant nitrile hydratase according to any of the foregoing, which is encapsulated in, for example, a vesicle, a sol-gel matrix, or other material that provides improved thermal stability compared to the enzyme in solution.

[0018] In other exemplary embodiments, the invention provides a nucleic acid comprising a sequence encoding a nitrile hydratase comprising an α subunit and a β subunit according to any of the foregoing and optionally an activator protein, wherein the nucleic acid encoding one or more of the α subunit, the β subunit and the optional activator protein is codon-optimized for increased expression in a desired microorganism, which is optionally a yeast, a fungus or a bacterium.

[0019] In other exemplary embodiments, the invention provides a nucleic acid as described above, which is codon-optimized for expression in a desired microorganism, which is optionally selected from the following bacteria: Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Brevibacterium, Burkholderia Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Arthrobacter, Brevibacterium, Corynebacterium, Microbacterium, Micrococcus, Nocardia, Pseudonocardia, Trichoderma, Myrothecium, Aureobasidium, Candida, Cryptococcus, Debaryomyces, Geotrichum, Hanseniaspora, Kluyveromyces, Pichia, Rhodotorula, Comomonas, and Pyrococcus. In an exemplary embodiment of the present invention, the microorganism can be selected from bacteria of the genera Rhodococcus, Pseudomonas, Escherichia and Geobacillus, or optionally selected from the following species: Rhodococcus rhodochrous, Rhodococcus pyridinovorans, Rhodococcus erythropolis, Rhodococcus equiequi), Rhodococcus ruber, Rhodococcus opacus, Aspergillus niger, Acidovorax avenae, Acidovorax facilis, Agrobacterium tumefaciens, Agrobacterium radiobacter, Bacillus subtilis, Bacillus pallidus, Bacillus smithii, Bacillus sp BR449, Bradyrhizobium oligotrophicum, Bradyrhizobium diazoefficiens), Bradyrhizobium japonicum, Burkholderia cenocepacia, Burkholderia gladioli, Escherichia coli, Geobacillus sp. RAPc8, Klebsiella oxytoca, Klebsiella pneumonia, Klebsiella variicola, Mesorhizobium ciceri, Mesorhizobium opportunistum, Mesorhizobium sp. F28 F28), Moraxella, Pantoeaendophytica, Pantoea agglomerans, Pseudomonaschlororaphis, Pseudomonas putid, Rhizobium, Rhodopseudomonas palustris, Serratia liquefaciens, Serratiamarcescens), Amycolatopsis, Arthrobacter, Brevibacterium sp CH1, Brevibacterium sp CH2, Brevibacterium sp R312, Brevibacterium imperiale, Corynebacterium nitrilophilus, Corynebacterium pseudodiphteriticum, Corynebacterium glutamicum, Corynebacterium hoffmanii, Microbacterium imperiale, Microbacterium smegmatis, Micrococcus luteus, Nocardia globerula, Nocardia rosea rhodochrous, Pseudonocardia thermophila, Trichoderma, Myrothecium verrucaria, Aureobasidium pullulans, Candida famata, Candida guilliermondii, Candida tropicalis, Cryptococcus flavus, Cryptococcus sp UFMG-Y28, Debaryomyces hanseii, Geotrichum candidum, Geotrichum sp JR1, Hanseniaspora, Kluyveromyces thermotolerans, Pichia kluyveri, kluyveri), Rhodotorula glutinis, Comomonas testosterone, Pyrococcusabyssi), Pyrococcus furiosus, Pyrococcus horikoshii, Brevibacterium casei or Nocardia sp. 163; and in a specific exemplary embodiment, the bacteria of the species Corynebacterium glutamicum may be included, further optionally including strain Corynebacterium glutamicum ATCC13032 or its derivative strain MB001 (DE3).

[0020] In other exemplary embodiments, the invention provides a nucleic acid as described above, wherein:

[0021] i) the β subunit is encoded by the nucleic acid of SEQ ID NO: 4;

[0022] ii) the α subunit is encoded by the nucleic acid of SEQ ID NO: 5;

[0023] iii) the activator protein is encoded by the nucleic acid of SEQ ID NO: 6; or

[0024] iv) Any combination of (i) to (iii).

[0025] In other exemplary embodiments, the present invention provides a nitrile hydratase operon comprising nucleic acids encoding the α subunit (nhhA) and β subunit (nhhB) of a variant nitrile hydratase according to any of the foregoing, and optionally an activator protein (nhhG), optionally wherein the nucleic acid is any of those described above, optionally the operon is derived from a yeast, fungus or bacterium expressing a nitrile hydratase, optionally a bacterium of the genus Pseudonocardia, further optionally Pseudonocardia thermophila.

[0026] In other exemplary embodiments, the present invention provides a nitrile hydratase operon comprising a nucleic acid encoding an alpha subunit (nhhA) and a beta subunit (nhhB) of a variant nitrile hydratase according to any of the foregoing, and optionally an activator protein (nhhG), comprising SEQ ID NO:7.

[0027] In other exemplary embodiments, the invention provides one or more extrachromosomal sequences, optionally a plasmid, comprising at least one nucleic acid or an operon according to any of the nucleic acids previously described above.

[0028] In other exemplary embodiments, the present invention provides a microorganism, optionally a yeast, fungus or bacterium, further optionally an industrial microorganism, which optionally does not endogenously express nitrile hydratase or endogenously expresses nitrile hydratase, the microorganism being engineered to comprise a nucleic acid encoding a nitrile hydratase or an operon comprising said nucleic acid, the nitrile hydratase comprising the α subunit (nhhA) and the β subunit (nhhB) of a variant nitrile hydratase according to any of the above, and optionally an activator protein (nhhG), the operon optionally being as described above, optionally wherein one or more of said nucleic acids are contained in one or more extrachromosomal sequences (plasmids) or integrated (one or more copies) into the chromosomal DNA of the microorganism.

[0029] In some exemplary embodiments, the microorganism is an industrial microorganism that endogenously expresses a nitrile hydratase, and the nucleic acid or operon replaces the endogenous nitrile hydratase gene or an operon comprising the endogenous nitrile hydratase gene. Microorganisms encoding nitrile hydratases include, for example, the following microbial species: Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Arthrobacter, Brevibacterium, Corynebacterium, Microbacterium, Micrococcus, Nocardia, Pseudonocardia, Trichoderma, Myroculitis, Aureobasidium, Candida, Cryptococcus, Debaryomyces, Geotrichum, Hansenula, Kluyveromyces, Pichia, Rhodotorula, Comamonas, and Pyrococcus.

[0030] In some exemplary embodiments, the microorganism used to express the variant nitrile hydratase or nitrile hydratase operon is a microorganism selected from the genera Rhodococcus, Pseudomonas, Escherichia, and Geobacillus. Typically, the biocatalyst is selected from the group consisting of Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Arthrobacter, Brevibacterium, Corynebacterium, Microbacterium, Micrococcus, Nocardia, Pseudonocardia, Trichoderma, Myroculin, Candida, Cryptococcus, Debaryomyces, Geotrichum, Hansenula, Kluyveromyces, Pichia, Rhodotorula, Comamonas, and Pyrococcus, or any part of said microorganisms having nitrile hydratase activity. The microorganism used to express the variant nitrile hydratase or nitrile hydratase operon is a bacterium selected from the group consisting of Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Brevibacterium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Arthrobacter, Brevibacterium, Corynebacterium, Microbacterium, Micrococcus, Nocardia, Pseudonocardia, Trichoderma, Myroculin, Candida, Cryptococcus, Debaryomyces, Geotrichum, Hansenula, Kluyveromyces, Pichia, Rhodotorula, Comamonas, and Pyrococcus.

[0031] In more specific exemplary embodiments of the present invention, the microorganism can be a bacterium selected from the genera Rhodococcus, Pseudomonas, Escherichia and Geobacillus, or selected from the following species: Rhodococcus rhodochrous, Rhodococcus pyridinophilus, Rhodococcus erythropolis, Rhodococcus equi, Rhodococcus erythropolis, Rhodococcus opacus, Aspergillus niger, Acidovorax avenae, Acidovorax agile, Agrobacterium tumefaciens, Agrobacterium radiobacterium, Bacillus subtilis, Bacillus pallidum, Bacillus smithii, Bacillus spore Bacillus species BR449, oligotrophic Bradyrhizobium, efficient nitrogen-fixing Bradyrhizobium, soybean Bradyrhizobium, Burkholderia cenocepacia, Burkholderia gladiolus, Escherichia coli, Geobacillus species RAPc8, Klebsiella oxytoca, Klebsiella pneumoniae, Klebsiella variegata, Mesorhizobium chickpea, Mesorhizobium opportunum, Mesorhizobium species F28, Moraxella, Pantoea endophytes, Pantoea agglomerans, Pseudomonas chlororaphis Pseudomonas putida, Rhizobium, Rhodopseudomonas palustris, Serratia liquefaciens, Serratia marcescens, Amycolatopsis, Arthrobacter, Brevibacterium species CH1, Brevibacterium species CH2, Brevibacterium species R312, Brevibacterium moth, Corynebacterium azotobacterium, Corynebacterium pseudodiphtheriae, Corynebacterium glutamicum, Corynebacterium hofmannii, Microbacterium moth, Microbacterium smegmatis, Micrococcus luteus, Nocardia sphaeroides, Nocardia rosea, Pseudonocardia thermophila, Trichoderma, Myrocera verrucosus, Budding Morphology, Candida anonymosa, Candida guillimon, Candida tropicalis, Cryptococcus flavus, Cryptococcus species UFMG-Y28, Debaryomyces hansenii, Geotrichum candidum, Geotrichum species JR1, Hansenula sporangiophora, Kluyveromyces thermotolerans, Pichia kluyveri, Rhodotorula colloidospermum, Comamonas testosteroni, Pyrococcus abyss, Pyrococcus furiosus, Pyrococcus horikoshii, Brevibacterium casei, or Nocardia species 163.

[0032] In an even more specific exemplary embodiment, the biocatalyst is a bacterium of the species Corynebacterium glutamicum, optionally the strain Corynebacterium glutamicum ATCC13032 or its derivative MB001 (DE3), which has been deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under strain number 102071.

[0033] In some exemplary embodiments, the present invention provides a method for producing an amide compound from a nitrile compound, the method comprising: contacting the nitrile compound with a nitrile hydratase or a bacterium expressing the nitrile hydratase according to any of the above descriptions. The bacteria may optionally be in a dried form. The enzyme may be in a soluble form or fixed to a substrate such as a solid support. Further optionally, the nitrile hydratase may be encapsulated using a material such as a gel-sol matrix that enhances thermal stability during amide synthesis.

[0034] In some exemplary embodiments, the present invention provides a method for producing an amide compound from a nitrile compound, comprising: contacting the nitrile compound with a nitrile hydratase or a bacterium expressing the nitrile hydratase according to any one of the above descriptions, wherein the amide compound is selected from the group consisting of acrylamide, methacrylamide, acetamide and nicotinamide, preferably acrylamide, and the nitrile compound is selected from the group consisting of acrylonitrile, methacrylonitrile, acetonitrile and 3-cyanopyridine, preferably acrylonitrile.

[0035] In some exemplary embodiments, these amide synthesis methods use a soluble nitrile hydratase, an encapsulated nitrile hydratase, an immobilized nitrile hydratase, or use a whole microbial cell biocatalyst or a lysed microbial cell biocatalyst.

[0036] In some exemplary embodiments, these amide synthesis methods use an intact microbial biocatalyst, which optionally can be freshly produced (i.e., directly from fermentation); stored, for example, in a frozen form (frozen in wet form); or dried, such as a freeze-dried product.

[0037] An exemplary method for producing an amide compound from a nitrile compound using a biocatalyst according to the present invention is further described and exemplified below. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The present invention will be described in more detail with reference to the accompanying drawings which are described in detail below.

[0039] Figure 1 Contains an alignment of the wild-type (DSM 43832 strain) and variant nitrile hydratase beta subunit (nhhB) polypeptide sequences.

[0040] Figure 2 Contains an alignment of wild-type (DSM 43832 strain) and variant nitrile hydratase alpha subunit (nhhA) polypeptide sequences.

[0041] Figure 3 Contains the comparison of wild-type (DSM 43832 strain) nhhBAG operon and variant nhhBAG operon nucleic acid sequence. Variant nhhBAG operon includes NdeI / XhoI restriction sites for cloning DSM 43832 strain nhhBAG operon, and lacks the amidase gene present in nhhB upstream in endogenous operon. In the wild-type operon sequence, the TGA stop codon of nhhB overlaps with the ATG start codon of nhhA, and similarly, the TGA stop codon of nhhA overlaps with the GTG start codon of nhhG. In contrast, in the variant operon, this overlap is resolved by introducing an intergenic ribosome binding site (considered as a gap in the comparison). DETAILED DESCRIPTION

[0042] Before describing the present invention, the following definitions are provided. Unless otherwise indicated, all terms should be interpreted according to those skilled in the art.

[0043] definition

[0044] As used herein, the singular forms "a", "an", "and", and "the" include plural referents unless the context clearly dictates otherwise. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs, unless the context clearly dictates otherwise.

[0045] As used herein, the term "biocatalyst" refers to any biocatalyst having nitrile hydratase activity. The biocatalyst capable of converting acrylonitrile to acrylamide may be a microorganism encoding an enzyme having nitrile hydratase activity or any part of the microorganism having nitrile hydratase activity. The biocatalyst may be selected from the microorganism, a lysed cell of the microorganism, a cell lysate of the microorganism, or any combination of these. In a very specific embodiment, the biocatalyst is a variant nitrile hydratase as disclosed in the examples or a bacterial strain (Corynebacterium glutamicum) expressing the variant nitrile hydratase.

[0046] As used herein, the term "biomass" generally refers to collected cells, typically microbial cells, and most typically bacterial cells obtained after fermentation, typically after removal of excess fermentation broth, wherein the removal is optionally achieved by filtration or centrifugation, typically resulting in a biomass composition having a dry content ranging from about 10% to 35%, more typically around 25% to 30%.

[0047] As used herein, the term "microorganism" when used herein encompasses a "microorganism producing nitrile hydratase", wherein the microorganism endogenously expresses and / or is engineered to express a variant nitrile hydratase according to the present invention. In the context of the present invention, such a microorganism is preferably a bacterium, a fungus or a yeast. In the present invention, a "microorganism producing nitrile hydratase" is used as or for converting a nitrile compound into a corresponding amide compound. Biocatalyst.

[0048] As used herein, the term "nitrile compound" is a compound converted into an amide compound by the nitrile hydratase according to the present invention or a microorganism expressing the nitrile hydratase according to the present invention through the action of the nitrile hydratase. The nitrile compound is any organic compound having a -C=N functional group, such as methacrylonitrile, acetonitrile or 3-cyanopyridine, preferably acrylonitrile.

[0049] As used herein, the term "amide compound" is a compound produced from a nitrile compound by nitrile hydratase. The amide compound has a functional group Rn C(O) x NR'2, wherein R and R' refer to H or an organic group or an organic amide, and n = 1, x = 1. Examples of such amide compounds include methacrylamide, acetamide or nicotinamide, preferably acrylamide.

[0050] As used herein, the term "microorganism producing nitrile hydratase" may be any microorganism capable of producing the variant nitrile hydratase of the present invention. In the context of the present invention, "microorganism producing nitrile hydratase" includes those microorganisms that do not naturally encode nitrile hydratase, which are genetically engineered to contain genes or polynucleotides encoding nitrile hydratase (e.g., by transformation, transduction, transfection, conjugation or other methods known in the art suitable for transferring or inserting polynucleotides into cells; see Sambrook and Russell 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (CSH Press), Cold Spring Harbor, NY, USA), so that the microorganism can produce and stably retain nitrile hydratase. For this purpose, it may be further necessary to insert additional polynucleotides, which may be necessary to allow transcription and translation of the nitrile hydratase gene or mRNA, respectively. Such additional polynucleotides may particularly include promoter sequences or replication origins or other plasmid control sequences. In this context, such a genetically engineered microorganism that does not naturally contain a gene encoding a nitrile hydratase, but has been manipulated to contain a polynucleotide encoding a nitrile hydratase, can be a prokaryotic or eukaryotic microorganism. Examples of such prokaryotic microorganisms include, for example, Escherichia coli and Corynebacterium species. Examples of such eukaryotic microorganisms include, for example, yeast (e.g., Saccharomyces cerevisiae or Pichia pastoris).

[0051] A "nitrile hydratase-producing microorganism" encoding a nitrile hydratase (naturally or non-naturally) is capable of producing and stably retaining the nitrile hydratase in some embodiments. However, according to the present invention, such a microorganism may also produce the nitrile hydratase only during the cultivation (or fermentation) of the microorganism. Such microorganisms include, in particular, bacteria of the genera Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Brevibacterium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Arthrobacter, Brevibacterium, Corynebacterium, Microbacterium, Micrococcus, Nocardia, Pseudonocardia, Trichoderma, Myroculitis, Candida, Cryptococcus, Debaryomyces, Geotrichum, Hansenula, Kluyveromyces, Pichia, Rhodotorula, Comamonas and Pyrococcus. In an exemplary embodiment of the present invention, the microorganism can be selected from bacteria of the genera Rhodococcus, Pseudomonas, Escherichia and Geobacillus. In addition, the "microorganism producing nitrile hydratase" especially includes the following species: Rhodococcus rhodochrous, Rhodococcus pyridinophilus, Rhodococcus erythropolis, Rhodococcus equi, Rhodococcus erythropolis, Rhodococcus opacus, Aspergillus niger, Acidovorax avena, Acidovorax agile, Agrobacterium tumefaciens, Agrobacterium radiobacterium, Bacillus subtilis, Bacillus pallidum, Bacillus smithii, Bacillus species BR449, oligotrophic bradyrhizobium, efficient nitrogen-fixing bradyrhizobium, Bradyrhizobium sojae, Burkholderia cenocepacia, Burkholderia gladiolus, Escherichia coli, Geobacillus species RAPc8, Klebsiella oxytoca, Klebsiella pneumoniae, Klebsiella variegata, Mesorhizobium chickpea, Mesorhizobium opportunum, Mesorhizobium species F28, Moraxella, Pantoea endophytes, Pantoea agglomerans, Pseudomonas chlororaphis, Pseudomonas putida, Rhizobium spp., Rhodopseudomonas palustris Spores, Serratia liquefaciens, Serratia marcescens, Amycolatopsis, Arthrobacter, Brevibacterium species CH1, Brevibacterium species CH2, Brevibacterium species R312, Brevibacterium moth, Corynebacterium azotophilum, Corynebacterium pseudodiphtheriae, Corynebacterium glutamicum, Corynebacterium hofmannii, Microbacterium moth, Microbacterium smegmatis, Micrococcus luteus, Nocardia sphaeroides, Nocardia rosea, Pseudonocardia thermophila, Trichoderma, Myrocera verrucosum, Budding Aureobasidium pullulans, Candida anonymus, Candida guillimon, Candida tropicalis, Cryptococcus flavus, Cryptococcus species UFMG-Y28, Debaryomyces hansenii, Geotrichum candidum, Geotrichum species JR1, Hansenula sporangiophora, Kluyveromyces thermotolerans, Pichia kluyveri, Rhodotorula glutelinum, Comamonas testosteroni, Pyrococcus abyssinus, Pyrococcus furiosus, Pyrococcus horikoshii, Brevibacterium casei, or Nocardia species 163.

[0052] In a specific exemplary embodiment, the "nitrile hydratase producing microorganism" is a bacterium of the species Corynebacterium glutamicum, preferably strain Corynebacterium glutamicum ATCC13032 or its derivative MB001 (DE3), which has been deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ) under strain number 102071.

[0053] In the context of the present invention, "nitrile hydratase" refers to a microbial enzyme (IUBMB enzyme nomenclature EC 4.2.1.84) that catalyzes the hydration of nitrile to its corresponding amide. The term "nitrile hydratase" as used herein also encompasses improved or enhanced enzymes that, for example, are capable of converting nitrile compounds (e.g., acrylonitrile) to amide compounds (e.g., acrylamide) faster, or that can be produced at a higher yield / time ratio, or are more stable, as long as they are capable of catalyzing the conversion (i.e., hydration) of nitrile compounds (e.g., acrylonitrile) to amide compounds (e.g., acrylamide). This enzyme generally comprises an alpha subunit (nhhA) and a beta subunit (nhhB), which are optionally expressed in conjunction with an activation protein (nhhG). In an exemplary embodiment, it refers to a variant nitrile hydratase (an enzyme comprising an α subunit (nhhA) and a β subunit (nhhB), the α subunit having an amino acid sequence having at least 98%, 99% or 100% sequence identity with SEQ ID NO: 2, the β subunit comprising an amino acid sequence having at least 98%, 99% or 100% sequence identity with SEQ ID NO: 1, provided that the α subunit comprises one, two or three of the following mutations: L6T, A19V and F126Y, and the β subunit comprises one or two of the following mutations: E108D and A200E or comprises all three of the following mutations: E108R, A200E and S212Y; wherein the variant nitrile hydratase has enhanced stability and / or activity compared to the nitrile hydratase produced by the wild-type strain (Pseudonocardia thermophila DSM 43832 strain).

[0054] In the context of the present invention, "nitrile hydratase stability" or "enzyme stability" or "stability" refers to the extent to which the biocatalyst tolerates AN and AMD under specific reaction conditions (e.g., temperature, solvent, etc.), i.e., good stability under specific reaction conditions means that the nitrile hydratase has a lower inactivation rate compared to another nitrile hydratase under the same specific reaction conditions (because both AN and AMD are known to inactivate endogenous nitrile hydratase biocatalysts).

[0055] In the context of the present invention, "nitrile hydratase activity" or "enzyme activity" or "activity" refers to the time it takes for the enzyme biocatalyst to start converting AN to AMD. That is, if the conversion starts very quickly, the enzyme is considered to have high activity, i.e., the biocatalyst activity refers to the initial reaction rate.

[0056] Description of the Invention

[0057] Development of new biocatalysts

[0058] We created a novel nitrile hydratase and the DNA sequence encoding the enzyme and transferred it into industrial production microorganisms. This biocatalyst can be used in the industrial production of acrylamide (AMD) from acrylonitrile (AN).

[0059] Technically, the present inventors attempted to obtain a novel nitrile hydratase biocatalyst that has high activity and can be used to produce high concentrations of 54% AMD. As previously mentioned, although many natural nitrile hydratases have been sequenced and used for AMD synthesis, endogenous nitrile hydratases generally lack one or two of the desired characteristics (activity and stability).

[0060] The development of the biocatalyst of the present invention includes the screening of multiple nitrile hydratases and host cells. The host cell or industrial production microorganism is a microbial species into which the nitrile hydratase gene is desired to be inserted and used to produce the biocatalyst by fermentation. The development of novel variant nitrile hydratases and operons includes the introduction of genetic improvements into many nitrile hydratase genes and the evaluation of the performance (stability / activity) of the variants. Nitrile hydratases derived from several different microbial species have been studied. The methods used by the inventors to improve the biocatalyst include in vivo recombination, site-specific and random mutagenesis, chaperone co-expression, operon optimization, expression plasmid optimization, codon selection optimization and fermentation optimization.

[0061] As disclosed in the Examples, the amino acid sequences of the α and β nitrile hydratase subunits of the nitrile hydratase endogenously produced by the Pseudonocardia thermophila strain were mutated in order to obtain variant nitrile hydratases having enhanced stability and / or activity compared to the parent nitrile hydratase endogenously produced by the Pseudonocardia thermophila strain.

[0062] Sequences of variant nitrile hydratase genes and operons, both of which are derived from a specific strain of Pseudonocardia thermophila and are extensively improved relative to the wild-type nitrile hydratase gene and the operon containing the wild-type nitrile hydratase gene contained endogenously in Pseudonocardia thermophila. It was found that the α and β nitrile hydratase subunits containing specific mutations contained in SEQ ID NO:2 and SEQ ID NO:1 (the combination of which was selected after screening a large number of different mutation combinations) produced the best combination of stability and / or increased activity compared to the parent nitrile hydratase produced endogenously in the Pseudonocardia thermophila strain. (See Figure 1 and Figure 2 Sequence alignment of the wild-type and variant α and β nitrile hydratase subunits contained in ).

[0063] As shown in the Examples, when compared in the amide synthesis experiments disclosed in the Examples, a variant nitrile hydratase comprising specific mutations in the α subunit contained in SEQ ID NO: 2 (L6T, A19V, F126Y) and specific mutations in the β subunit contained in SEQ ID NO: 1 (E108D, A200E) exhibited the best stability / activity, while a variant nitrile hydratase comprising the same mutations in the α subunit contained in SEQ ID NO: 2 (L6T, A19V, F126Y) and E108R / A200E / S212Y mutations in the β subunit exhibited the second best stability / activity.

[0064] As further described below in the Examples, the nitrile hydratase operon from the thermophilic Pseudonocardia strain is further engineered to enhance the stability and / or activity of the nitrile hydratase and promote expression in selected exemplary industrial strains, such as Corynebacterium glutamicum, optionally Corynebacterium glutamicum ATCC13032 or its derivative strain MB001 (DE3). The parent Corynebacterium glutamicum MB001 (DE3) strain has been deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) with strain number 102071. A detailed description of this strain is published in: Kortmann M. et al. (2015). "A chromosomally encoded T7 RNA polymerase-dependent gene expression system for Corynebacterium glutamicum: construction and comparative evaluation at the single-cell level." Microb Biotechnol. 8: 253-65.

[0065] These operon improvements included: codon optimization of the nhhA, nhhB, and nhhG coding sequences of the parent Pseudonocardia thermophila strain, elimination of the overlap of the TGA stop codon of nhhB with the ATG start codon of nhhA and the overlap of the TGA stop codon of nhhA with the GTG start codon of nhhG in the variant operon by introducing an intergenic ribosome binding site, introduction of NdeI / XhoI restriction sites for cloning, deletion of the amidase gene upstream of nhhB as part of the operon, and other changes. Figure 3 These changes can be further seen in FIG, which contains an alignment of the improved operon with the parental operon of the Pseudonocardia thermophila strain used to derive the improved operon.

[0066] With respect to the foregoing, in the exemplary embodiments described in the Examples below, plasmids comprising these variant nitrile hydratase sequences were transferred to the host strain Corynebacterium glutamicum. As further illustrated in the Examples, when used to produce acrylamide from acrylonitrile, the exemplary Corynebacterium glutamicum strains expressing the variant nitrile hydratase sequences of the present invention demonstrated enhanced expression and stability relative to the parental strain and other contrast strains (e.g., other microbial strains engineered to include nitrile hydratases comprising different mutations).

[0067] While the exemplary C. glutamicum strain expressing the exemplary variant nitrile hydratase provides the best activity / stability combination, it is contemplated that the variant nitrile hydratase sequences of the invention may also be expressed in other microorganisms, preferably other industrial microorganisms that are advantageously used in industrial processes.Examples thereof are bacteria selected from the group consisting of Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Brevibacterium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Arthrobacter, Brevibacterium, Corynebacterium, Microbacterium, Micrococcus, Nocardia, Pseudonocardia, Trichoderma, Myroculin, Candida, Cryptococcus, Debaryomyces, Geotrichum, Hansenula, Kluyveromyces, Pichia, Rhodotorula, Comamonas, and Pyrocaulosa. The genus Coccus, or more specifically the microorganism is a bacterium selected from the genera Rhodococcus, Pseudomonas, Escherichia and Geobacillus, or a bacterium selected from the following species: Rhodococcus rhodochrous, Rhodococcus pyridinophilus, Rhodococcus erythropolis, Rhodococcus equi, Rhodococcus erythropolis, Rhodococcus opacus, Aspergillus niger, Acidovorax avenacum, Acidovorax agile, Agrobacterium tumefaciens, Agrobacterium radiobacterium, Bacillus subtilis, Bacillus pallidum, Bacillus smithii, Bacillus species BR449, oligotrophic bradyrhizobium, efficient nitrogen-fixing bradyrhizobium, soybean bradyrhizobium, Burkholderia cenocepacia, Burkholderia gladiolus, Escherichia coli, Geobacillus species RAPc8, Klebsiella oxytoca, Pneumonia Klebsiella, Klebsiella mutans, Mesorhizobium chickpeaensis, Mesorhizobium opportunum, Mesorhizobium species F28, Moraxella, Pantoea endophytes, Pantoea agglomerans, Pseudomonas chlororaphis, Pseudomonas putida, Rhizobium, Rhodopseudomonas palustris, Serratia liquefaciens, Serratia marcescens, Amycolatopsis, Arthrobacter, Brevibacterium species CH1, Brevibacterium species CH2, Brevibacterium species R312, Brevibacterium mothii, Corynebacterium azotophilum, Corynebacterium pseudodiphtheriae, Corynebacterium glutamicum, Corynebacterium hofmannii, Microbacterium mothii, Microbacterium smegmatis, Micrococcus luteus, Nocardia sphaeroides, Nocardia rosea, Pseudonocardia thermophila, Trichoderma, Myrocera verrucosum, Budding Mycorrhizal , Candida anonymum, Candida guillimon, Candida tropicalis, Cryptococcus flavus, Cryptococcus species UFMG-Y28, Debaryomyces hansenii, Geotrichum candidum, Geotrichum species JR1, Hansenula sporangial yeast, Kluyveromyces thermotolerans, Pichia kluyveri, Rhodozyma, Comamonas testosteroni, Pyrococcus abyss, Pyrococcus furiosus, Pyrococcus horikoshii, Brevibacterium casei or Nocardia species 163; and optionally comprising a bacterium of the species Corynebacterium glutamicum, further optionally strain Corynebacterium glutamicum ATCC13032 or its derivative strain MB001 (DE3), which has been deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ) with strain number 102071.

[0068] New biocatalyst form that can be used in amide synthesis

[0069] The biocatalyst of the invention, e.g., an exemplary Corynebacterium glutamicum strain expressing an exemplary variant nitrile hydratase, can be freshly produced (i.e., directly from fermentation); stored, such as stored in a frozen form (frozen in a wet form); or dried, e.g., in a spray-dried form. In an exemplary embodiment, during use of the microbial biocatalyst of the invention as a biocatalyst, we prepare a slurry by mixing the microbial biocatalyst with water.

[0070] After fermentation, the harvested biomass or harvested cells may optionally be washed, or otherwise processed, such as by freezing or drying.

[0071] In addition, the biocatalyst capable of converting acrylonitrile to acrylamide may be a microorganism encoding a variant nitrile hydratase or any part of the microorganism having nitrile hydratase activity. The biocatalyst may be selected from the microorganism, a lysed cell of the microorganism, a cell lysate of the microorganism, or any combination of these. In a very specific embodiment, the biocatalyst is a nitrile hydratase.

[0072] In one embodiment, the amount of biocatalyst is from 0.1 kg dry cells / m3 to 5 kg dry cells / m3 of reaction mixture.

[0073] In another embodiment, the amount of the biocatalyst is 0.1 g dry cells / kg 100% AMD to 3 g dry cells / kg 100% AMD, more specifically 0.2 g dry cells / kg 100% AMD to 3 g dry cells / kg 100% AMD, more specifically 0.2 g dry cells / kg 100% AMD to 2.5 g dry cells / kg 100% AMD, based on the final AMD amount. In another embodiment, the amount of the biocatalyst is 0.5 g dry cells / kg 100% AMD to 2 g dry cells / kg 100% AMD, or more specifically 1.1 g dry cells / kg 100% AMD to 1.5 g dry cells / kg 100% AMD.

[0074] In yet another embodiment, the amount of biocatalyst is 0.5 g dry cells / kg 50% AMD to 1 g dry cells / kg 50% AMD, more specifically 1.6 g dry cells / kg 50% AMD to 1.8 g dry cells / kg 50% AMD based on the final AMD amount.

[0075] In another embodiment, at the end of the maturation of the reaction mixture, the amount of the biocatalyst is 0.1 kg dry cells / m3 to 1.5 kg dry cells / m3 reaction mixture. In one embodiment, the amount of the biocatalyst is 0.1 kg dry cells / m3 to 1.0 kg dry cells / m3 reaction mixture.

[0076] During the process, a plurality of the biocatalysts may be added, for example, when acrylonitrile begins to accumulate in the reactor. The biocatalyst may be added, for example, in the form of a homogenous slurry in water.

[0077] Exemplary Amide Reaction Conditions

[0078] The reaction is generally carried out at ambient pressure, more particularly at 1 bar.

[0079] The slurry, ie the aqueous mixture containing the biocatalyst, may be produced by any method known in the art, such as mixing water and the biocatalyst in a container or reactor. Optionally, the slurry is homogenous and free of strong agglomerates.

[0080] In the presence of a biocatalyst with nitrile hydratase activity, once acrylonitrile is fed into a reactor containing the slurry, a reaction of converting acrylonitrile into acrylamide in an aqueous solution is started. In an exemplary embodiment, acrylonitrile can be fed into a reactor containing the slurry to provide a reaction mixture comprising water, acrylamide, acrylonitrile and the biocatalyst. In an exemplary embodiment, the biocatalyst slurry can be added to the reactor together with additional water, and the AN feed is started after such addition. The amount of additional water and biocatalyst slurry can be about 5000kg relative to 100kg slurry in an exemplary embodiment, and can be in the range of about 25000kg relative to 100kg slurry to about 1000kg relative to 100kg slurry.

[0081] Aqueous solutions of acrylamide at high concentrations (e.g., at least 35 wt%, or at least 40 wt%, or at least 45 wt%, or at least 50 wt%), at least 55 wt%, at least 56%, or at least about 57%, or about 55%-60% AMD, may alternatively be produced using a controlled acrylonitrile feed and process temperature profile. With respect to the foregoing, the maximum AMD concentration may be limited by precipitation issues, i.e., at high AMD concentrations, it may begin to precipitate.

[0082] Usually need cooling reactor to keep reaction mixture at desired reaction temperature.When reaction starts, temperature and acrylonitrile feed rate are all relatively high, to realize fast reaction rate and shorter building-up time.Reactor begins to cool after for example reaction starts 60min, because compared with the reaction mixture of higher temperature, the deactivation of biocatalyst caused by accumulation of acrylamide at lower temperature is obviously less.During last several hours, acrylonitrile feed rate is relatively low, to avoid acrylonitrile to accumulate in reactor.

[0083] Because the reaction is exothermic, cooling capacity and also safety issues may be limiting factors in determining the AN feed rate.In some exemplary embodiments, the amount of AN in the reactor is maintained at a maximum of about 3%.

[0084] In some exemplary embodiments, the reactor containing the biocatalyst is at about 15°C prior to adding the AN feed, starting the AN feed and maintaining the temperature at about 23°C until the end of the reaction in order to minimize deactivation of the biocatalyst.

[0085] The feeding of acrylonitrile can be continued in the whole process, more specifically continued until the aging stage in the whole process. The feeding rate of acrylonitrile can change during this process. The feeding of acrylonitrile can be continuous or intermittent. The feeding rate of acrylonitrile depends on the reaction rate that acrylonitrile is converted into acrylamide and the speed of biocatalyst deactivation. In one embodiment, the feeding of acrylonitrile is continued in the whole process until the aging stage.

[0086] In one embodiment, the acrylonitrile feed rate is adjusted during the process to avoid acrylonitrile accumulation in the reaction mixture. During the process, acrylonitrile is fed at such a rate that acrylonitrile is converted into acrylamide. More specifically, the amount of acrylonitrile in the reaction mixture is maintained to be less than 3wt%, or less than 2wt%, more specifically less than 1wt%, even more specifically less than 0.5wt%, relative to the total amount of the reaction mixture. However, in some embodiments, the reaction can be carried out at a much higher AN concentration, for example, 5%, 10% or even higher if all the AN is added at once.

[0087] In another embodiment of the process, 38% to 48% of the total amount of acrylonitrile fed to the reactor is fed during 0 min to 60 min from the start of the process; 22% to 30% of the total amount of acrylonitrile is fed during 60 min to 120 min of the process; 12% to 18% of the total amount of acrylonitrile is fed during 120 min to 180 min of the process; 8% to 12% of the total amount of acrylonitrile is fed during 180 min to 240 min of the process. In order to achieve a balance of 100% of the fed acrylonitrile, the remaining acrylonitrile is fed during the process before the maturation stage.

[0088] During the maturation stage, substantially no acrylonitrile, and more specifically no acrylonitrile, is fed into the reactor. During maturation, acrylonitrile monomer still present in the reaction mixture reacts with acrylamide. The reaction mixture is matured until the desired characteristics are achieved.

[0089] The temperature of the reaction mixture is generally monitored. Monitoring and measuring can be performed using any suitable means and methods in the art.

[0090] In an exemplary embodiment, the temperature of the reaction mixture is maintained at 15°C to 25°C. In one embodiment, the temperature is maintained at 19°C to 25°C, more specifically maintained at 20°C to 22°C, and even more specifically maintained at 22°C. In one embodiment, the temperature is maintained within the desired range by measuring the temperature of the reaction mixture and either cooling the mixture or heating the mixture to maintain the temperature within the desired range. The cooling and / or heating of the reaction mixture can be performed using methods known in the art.

[0091] When cooling of the reaction mixture is initiated, the temperature of the reaction mixture may be equal to, higher than or lower than the temperature of the reaction mixture at the start of the process.

[0092] In some embodiments, the cooling of the reaction mixture is continued so that when the acrylamide concentration reaches 37 wt% to 55 wt%, the temperature of the reaction mixture is in the range of 10°C to 18°C ​​or 10°C to 21°C. In other words, the time period for cooling the reaction mixture to a temperature of 10°C to 18°C ​​or 10°C to 21°C is the time period when the acrylamide concentration of at least 27 wt% (more specifically 27 wt% to 38 wt%) increases to an acrylamide concentration of 37 wt% to 55 wt% (more specifically 40 wt% to 50 wt%).

[0093] In one embodiment, the cooling of the reaction mixture is continued so that when the acrylamide concentration reaches 37 wt% to 55 wt%, the temperature is in the range of 10°C to 16°C, more specifically 13°C to 16°C, and even more specifically the temperature is 15°C. In one embodiment, the cooling is started, for example, after the reaction mixture is maintained at 15°C to 25°C. In one embodiment, the reaction mixture is cooled by at least 10°C, more specifically at least 5°C, even more specifically at least 4°C. The cooling can be performed linearly or stepwise, usually linearly.

[0094] In one embodiment, when the acrylamide concentration reaches 37 wt % to 55 wt %, the reaction mixture is aged at a temperature ranging from 10° C. to 18° C. or from 10° C. to 21° C.

[0095] During the aging period, substantially no acrylonitrile, and more specifically no acrylonitrile is fed into the reactor. During the aging period, unreacted acrylonitrile in the reactor reacts to form acrylamide. After the reaction mixture has cooled and the temperature of the reaction mixture is in the range of 10°C to 18°C ​​or 10°C to 21°C, and / or after the acrylonitrile is fed into the reactor, aging begins. More specifically, aging continues until the final concentration of acrylonitrile in the reaction mixture is at most 1000ppm, at most 500ppm, at most 250ppm, at most 100ppm, at most 50ppm, at most 10ppm or at most 0ppm.

[0096] In one embodiment of the process, the temperature of the reaction mixture is maintained at 15°C to 25°C for 30 min to 90 min, such as for 45 min to 60 min, and the cooling of the reaction mixture to a temperature of 10°C to 18°C ​​or 10°C to 21°C is carried out over a period of 45 min to 120 min, such as 60 min to 120 min.

[0097] After the reaction is complete, the biocatalyst can be separated from the aqueous AMD solution by any known separation method, such as centrifugation, flotation, filter pressing or filtration. The aqueous acrylamide solution produced by this process is then typically used to make polyacrylamide.

[0098] In some exemplary embodiments, the separation or harvesting of biomass cells after fermentation can be promoted by adding a flocculant to help collect cells, and can be performed prior to the separation method, such as before centrifugation / filtration. In an exemplary embodiment, salt or other stabilizers will be added before freezing the biomass. For example, after the cells are harvested, suitable preservative salts can be added to the biomass / cell material, such as ammonium salts, calcium salts, iron salts, magnesium salts, potassium salts or sodium salts.

[0099] In some exemplary embodiments, the separation or harvesting of the biomass cells after fermentation can be carried out after first adding a cationic solution and then adding an anionic solution. Relative to the material to be flocculated, the amount of the cationic solution added can be in the range of, for example, 2% to 10%, and the amount of the anionic solution can be in the range of, for example, 0.5% to 3.5%. In some exemplary embodiments, the cationic solution is in the range of 4% to 7%, and the anionic solution is in the range of 1% to 2%. In addition, the suspended matter can be stirred during the process. [In this process, a low molecular weight cationic flocculant acts as a coagulant, resulting in the formation of a microflocculant, followed by the addition of an anionic solution, which acts as a matrix to provide a high molecular weight anionic flocculant for coagulation].

[0100] After a certain time, such as about 5-30, 5-15 or 10 minutes, after adding the anionic solution, a flocculant is formed that can be separated by desired means, such as by centrifugation, flotation, filter press or filtration. For example, separation can be achieved by using a filter with pores having a diameter of <0.45 μm. If required, the filtered biomass can be resuspended, optionally suspended in desalted water, and filtered repeatedly to further remove fermentation broth culture residues from the biomass.

[0101] As mentioned above, if the resulting recycled biomass is stored in the form of a paste, the biomass is typically mixed with a stabilizer or preservative, such as an ammonium, calcium, iron, magnesium, potassium or sodium salt.

[0102] Having described the present invention in detail, the present invention is further described in the following examples.

[0103] Example

[0104] The following examples are presented for illustrative purposes only and are not intended to be limiting.

[0105] Example 1: Generation of variant nitrile hydratase and variant nitrile hydratase operons

[0106] The development of the biocatalyst of the present invention includes the screening of multiple variant nitrile hydratase and host cells, the introduction of various genetic modifications into many nitrile hydratase genes, and the evaluation of the performance (stability / activity) of the variants. Nitrile hydratases from several different microbial species have been studied. The methods used by the inventors to improve the biocatalyst include in vivo recombination, site-specific and random mutagenesis, chaperone co-expression, operon optimization, expression plasmid optimization, codon selection optimization, and fermentation optimization.

[0107] These efforts resulted in the isolation of variant nitrile hydratase α and β subunits comprising SEQ ID NO: 2 and SEQ ID NO: 1, respectively. Both variants are derived from the α and β subunits of nitrile hydratase produced by a Pseudonocardia thermophila strain (Pseudonocardia thermophila strain available as deposit DSM 43832). Figure 1 and Figure 2 The specific combinations of mutations contained in these variants are further shown in the sequence alignment of . When co-expressed, it was found that the enzymes produced by these variants exhibited the best combination of enhanced stability and / or activity compared to the parent nitrile hydratase produced endogenously by the thermophilic Pseudonocardia strain.

[0108] Furthermore, the inventors observed that a variant nitrile hydratase comprising an alpha subunit comprising L6T, A19V, F126Y and a beta subunit comprising E108R / A200E / S212Y mutations had the second best combination of enhanced stability and / or activity compared to the parent nitrile hydratase.

[0109] In addition, further to enhance nitrile hydratase expression and nitrile hydratase stability during amide synthesis (when introduced into an exemplary industrial host strain), the operon containing the endogenous nitrile hydratase gene of Pseudonocardia thermophila DSM 43832 was further extensively modified relative to the wild-type nitrile hydratase gene and the operon containing the wild-type nitrile hydratase gene. Specifically, as Figure 3As shown in, the codon usage of the variant nhhA and nhhB genes and the nhhG gene was optimized for Corynebacterium glutamicum (based on the codon usage of highly expressed genes in Corynebacterium glutamicum published in: Eikmanns, B. (1998), "Identification, Sequence Analysis, and Expression of a Corynebacterium glutamicum Gene Cluster Encoding the Three Glycolytic Enzymes Glyceraldehyde-3-Phosphate Dehydrogenase, 3-Phosphoglycerate Kinase, and Triosephosphate Isomerase", Journal of Bacteriology (J Bac) 174, 6076-6086); the nhhBAG operon was improved to include a nucleotide sequence for cloning DSM The 43832 strain nhhBAG operon was modified to remove the amidase gene upstream of nhhB in the endogenous operon; and the operon was modified to include an intergenic ribosome binding site to eliminate overlap, because in the wild-type operon sequence, the TGA stop codon of nhhB overlaps with the ATG start codon of nhhA, and similarly, the TGA stop codon of nhhA overlaps with the GTG start codon of nhhG. A variant operon containing these modifications is contained in SEQ ID NO:7.

[0110] Example 2: Development of novel nitrile hydratase and operon

[0111] The development of the biocatalyst Corynebacterium glutamicum strain of the present invention includes introducing the plasmid comprising the operon described in the aforementioned embodiment into the selected industrial strain. As previously mentioned, the inventor selected Corynebacterium glutamicum MB001 (DE3) as the industrial parent strain, which has been deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ) with strain number 102071. In addition, a detailed description of the strain is published in: Kortmann M. et al. (2015), "A chromosomally encoded T7 RNA polymerase-dependent gene expression system for Corynebacterium glutamicum: construction and comparative evaluation at the single-cell level", Microbial Biotechnology. 8: 253-65.

[0112] The parent strain is selected because it has been used for other industrial processes and because it does not contain the nitrile hydratase gene. The exemplary production strain comprises the nitrile hydratase operon present on the plasmid, approximately 30 copies per cell. However, it is contemplated that by engineering transformation of Corynebacterium glutamicum MB001 (DE3) bacterial strain instead, the nitrile hydratase operon of one or more copies is integrated into the Corynebacterium glutamicum cell genome (that is, in order to improve strain stability and avoid using antibiotic resistance genes and in order to further maximize enzyme output), suitable or further enhanced results can be obtained.

[0113] Additionally, since the use of encapsulated nitrile hydratase from Pseudonocardia thermophila has been described (see, e.g., Martinez et al. (2014), "Acrylamide production using encapsulated nitrile hydratase from Pseudonocardia thermophila in a sol–gel matrix", J Molecular Catalysis B: Enzymatic, 100, 19-24), and its thermostability is reported to be improved compared to the enzyme in solution, the variant nitrile hydratase may alternatively be encapsulated in a sol-gel matrix or lipid vesicles and used in this form as a biocatalyst in the amide synthesis process).

[0114] Example 3: Use of the novel nitrile hydratase biocatalyst for AMD synthesis

[0115] The performance of exemplary biocatalysts comprising different variant nitrile hydratases expressed in different bacteria including the production strains of the Corynebacterium glutamicum nitrile hydratase described in the previous examples was compared for their stability / activity when used during the synthesis of acrylamide (AMD) from acrylonitrile (AN) as described below. These experiments and results are further summarized in the table below.

[0116] In these experiments, desalted water and biocatalyst were mixed and AN feed was started. As shown, in these methods, the entire AN feed was carried out within 3 hours or 1 hour. In these syntheses, the target AMD concentration was 52% or 54%. Due to sampling during the synthesis, the final AMD concentration is usually slightly smaller. If the synthesis is successful, no AN will be detected from the solution at the end of the experiment.

[0117] Biocatalyst X1 has a variant nitrile hydratase derived from Pseudonocardia and subsequently modified to contain specific mutations, and the host is Escherichia coli (E. coli). It converts all AN into AMD at a dose of 5.7 g dry cells / kg 100% AMD. However, separation of the biocatalyst from the ready-made AMD by centrifugation was unsuccessful, so E. coli was abandoned as a host cell.

[0118] As described above, different biocatalysts comprising different variant nitrile hydratases (also derived from Pseudonocardia and subsequently modified to contain specific mutations) were expressed in different bacteria including the production strain producing the Corynebacterium glutamicum nitrile hydratase described in the previous examples and their stability / activity when used during the synthesis of acrylamide (AMD) from acrylonitrile (AN) were compared.

[0119] The experiments and the results obtained therefrom are summarized in the following Tables 1 and 2. In addition, the different biocatalysts identified in the tables and their comparative results when used for AMD synthesis are briefly summarized as follows:

[0120] Biocatalyst X2 Contains a nitrile hydratase from Rhodococcus, and the host is Corynebacterium glutamicum. It only converted about one-third of the added AN, giving a final AMD concentration of 18% at a dose of 6.0 g dry cells / kg 100% AMD. Therefore, development of this combination was stopped.

[0121] Biocatalysts X3, X4 and X5Contains nitrile hydratase from Pseudonocardia, and the host is Corynebacterium glutamicum. They are all biocatalysts from different batches at different stages of development. In the experiment, all 3 forms converted all AN into AMD.

[0122] Biocatalyst X4 After fermentation, it was dried and re-wetted before use in AMD synthesis. The re-wetted biocatalyst was able to convert all AN into AMD.

[0123] Biocatalyst X5 The final form was included, which, as shown in the table, exhibited significantly higher activity per unit of cell dry matter. A dose of only 1.7 g dry cells / kg 100% AMD converted all AN to AMD.

[0124] Biocatalyst R1 Contains nitrile hydratase from Rhodococcus, and the host is Rhodococcus. As shown in the table, in the experiment where all feed AN was added within 1 hour, at the high dose of 6.9 kg dry cells / kg 100% AMD, only about 2 / 3 of the added AN was converted into AMD. The biocatalyst was inactivated by high levels of AN.

[0125] Biocatalyst X6 Contains nitrile hydratase from Pseudonocardia, and the host is Corynebacterium glutamicum. In the experiment where all AN was added within 1 h, all added AN was converted to AMD at a dose of 4.6 kg dry cells / kg 100% AMD in the 21 h experiment. Therefore, this biocatalyst tolerates high concentrations of AN and AMD.

[0126] The results observed from these experiments are summarized in the table below.

[0127] Table 1 Based on the results in Table 1 above, it can be seen that the biocatalyst of the present invention has enhanced stability and activity over extended periods of time (at least 3 hours) when used for AMD synthesis. The results also show that the biocatalyst of the present invention can be used in different forms, such as a dried form. The Sequence Listing on the following page contains exemplary sequences used in the Examples.

[0128] Sequence Listing

[0129] Variant DSM 43832 nhhBβ subunit: Amino acid sequence: SEQ ID NO: 1

[0130] MNGVYDVGGTDGLGPINRPADEPVFRAEWEKVAFAMFPATFRAGFMGLDEFRFGIEQMNPAEYLESPYYWHWIRTYIHHGVRTGKIDLEELERRTQYYRENPDAPLPDHEQKPELIE FVNQAVYGGLPASREVDRPPKFKEGDVVRFSTASPKGHARRARYVRGKTGTVVKHHGAYIYPDTAGNGLGECPEHLYTVRFTEQELWGPEGDPNSSVYYDCWEPYIELVDTKAAAA*

[0131] Variant DSM 43832 nhhA alpha subunit: Amino acid sequence: SEQ ID NO: 2

[0132] MTENITRKSDEEIQKEITVRVKALESMLIEQGILTTSMIDRMAEIYENEVGPHLGAKVVVKAWTDPEFKKRLLADGTEACKELGIGGLQGEDMMWVENTDEVHHVVVCTLCSCYPWPVLGLPPNWYKEPQYRSRVVREPRQLLKEEFGFEVPPSKEIKVWDSSSEMRFVVLPQRPAGTDGWSEEELATLVTRESMIGVEPAKAVA*

[0133] DSM 43832 nhhG activating protein: Amino acid sequence: SEQ ID NO: 3

[0134] MSAEAKVRLKHCPTAEDRAAADALLAQLPGGDRALDRGFDEPWQLRAFALAVAACRAGRFEWKQLQQALISSIGEWERTHDLDDPSWSYYEHFVAALESVLGEEGIVEPEALDERTAEVLANPPNKDHHGPHLEPVAVHPAVRS*

[0135] Variant DSM 43832 nhhB: beta subunit: nucleic acid coding sequence SEQ ID NO: 4

[0136] ATGAACGGCGTTTACGACGTTGGCGGCACCGACGGCCTGGGTCCAATCAACCGCCCAGCAGACGAGCCAGTTTTCCGCGCTGAGTGGGAGAAGGTTGCATTCGCTATGTTCCCAGCAACCTTCCGCGCTGGCTTCATGGGCCTGGACGAGTTCCGCTTCGGCATCGAGCAGATGAACCCAGCAGAGTACCTGGAGTCCCCATACTACTGGCACTGGATCCGCACCTACATCCACCACGGCGTTCGCACCGGCAAGATCGACCTGGAGGAGCTGGAGCGTCGCACCCAGTACTACCGCGAGAACCCAGACGCTCCACTGCCAGACCACGAGCAGAAGCCAGAGCTGATCGAGTTCGTTAACCAGGCAGTTTACGGCGGCCTGCCAGCTTCCCGCGAGGTTGACCGCCCACCAAAGTTCAAGGAAGGCGACGTTGTTCGCTTCTCCACCGCATCCCCAAAGGGTCACGCACGCCGCGCTCGCTACGTTCGCGGCAAGACCGGCACCGTTGTTAAGCACCACGGCGCATACATCTACCCAGACACCGCTGGTAACGGCCTGGGCGAGTGCCCAGAGCACCTGTACACCGTTCGCTTCACCGAGCAGGAGCTGTGGGGTCCAGAGGGCGACCCAAACTCCTCCGTTTACTACGACTGCTGGGAGCCATACATCGAGCTGGTTGACACCAAGGCAGCTGCAGCTTGA

[0137] Variant DSM 43832 nhhA α-subunit: Nucleic acid coding sequence: SEQ ID NO:5

[0138] ATGACCGAGAACATCACCCGCAAGTCCGACGAGGAGATCCAGAAGGAGATCACCGTTCGCGTTAAGGCTCTGGAGTCCATGCTGATCGAGCAGGGCATCCTGACCACCTCTATGATCGACCGCATGGCAGAGATCTACGAGAACGAGGTTGGCCCACACCTGGGCGCTAAGGTTGTTGTTAAGGCATGGACCGACCCAGAGTTCAAGAAGCGCCTGCTGGCTGACGGTACCGAGGCATGCAAGGAGCTGGGTATCGGCGGCCTGCAGGGCGAGGACATGATGTGGGTTGAGAACACCGACGAGGTTCACCACGTTGTTGTTTGCACCCTGTGCTCCTGCTACCCATGGCCAGTTCTGGGCCTGCCACCAAACTGGTACAAGGAGCCACAGTACCGCTCCCGCGTTGTTCGCGAGCCACGCCAGCTGCTGAAGGAAGAGTTCGGCTTCGAGGTTCCACCATCCAAGGAGATCAAGGTTTGGGACTCCTCCTCCGAGATGCGCTTCGTTGTTCTGCCACAGCGCCCAGCTGGTACCGACGGTTGGTCCGAAGAGGAGCTGGCAACCCTGGTTACCCGCGAGTCCATGATCGGCGTTGAGCCAGCAAAGGCTGTTGCCTGA

[0139] Variant DSM 43832 nhhG activator protein: Nucleic acid coding sequence: SEQ ID NO:6

[0140] ATGTCCGCTGAGGCAAAGGTTCGCCTGAAGCACTGCCCAACCGCAGAGGACCGCGCAGCTGCAGACGCACTGCTGGCTCAGCTGCCAGGCGGCGACCGCGCACTGGACCGCGGCTTCGACGAGCCATGGCAGCTGCGCGCTTTCGCACTGGCTGTTGCAGCATGCCGCGCAGGCCGCTTCGAGTGGAAGCAGCTGCAGCAGGCTCTGATCTCCTCCA TCGGCGAGTGGGAGCGCACCCACGACCTGGACGACCCATCCTGGTCCTACTACGAGCACTTCGTTGCTGCACTGGAGTCCGTTCTGGGCGAGGAAGGCATCGTTGAGCCAGAGGCACTGGACGAGGCACCGCAGAGGTTCTGGCTAACCCACCAAACAAGGACCACCACGGCCCACACCTGGAGCCAGTTGCAGTTCACCCAGCTGTTCGCTCCTAA

[0141] Variant DSM 43832 nhhBAG operon (comprising NdeI / XhoI restriction sites for cloning the nhhBAG operon of strain DSM 43832 and lacking the amidase gene present upstream of nhhB in the endogenous operon): SEQ ID NO: 7

[0142]

[0143] Wild-type DSM 43832 nhhBAG operon (lacking the amidase gene present upstream of nhhB in the endogenous operon): SEQ ID NO: 8

[0144]

[0145] Having described exemplary embodiments of the present invention, the invention will be further described in the following claims.

Claims

1. A variant nitrile hydratase comprising (i) an alpha subunit (nhhA) having an amino acid sequence having at least 98%, 99% or 100% sequence identity to SEQ ID NO:2 and (ii) a beta subunit (nhhB) having an amino acid sequence having at least 98%, 99% or 100% sequence identity to SEQ ID NO:1, provided that the alpha subunit comprises one, two or three of the following mutations: L6T, A19V and F126Y, and the beta subunit comprises one or two of the following mutations: E108D and A200E or comprises all three of the following mutations: E108R, A200E and S212Y; The variant nitrile hydratase has enhanced stability and / or activity.

2. The nitrile hydratase according to claim 1, wherein: (i) the α subunit comprises the following mutations: L6T, A19V and F126Y, and the β subunit comprises the following mutations: E108D and A200E or E108R, A200E and S212Y; or (ii) the enzyme subunit is expressed in combination with an nhhG activator protein, optionally comprising an amino acid sequence that is at least 98%, 99% or 100% identical to SEQ ID NO:3; (iii) the nitrile hydratase comprises a soluble enzyme or a cell producing the nitrile hydratase; (iv) the nitrile hydratase is immobilized on a solid support; (v) the nitrile hydratase is encapsulated in, for example, a vesicle, a sol-gel matrix or other material that optionally provides improved thermal stability compared to the enzyme in solution; or (vi) Any combination of (i) to (iv).

3. One or more nucleic acids encoding nitrile hydratase, the nitrile hydratase comprising an α subunit and a β subunit according to claim 1 or 2 or 3 and optionally an activator protein, wherein the nucleic acid encoding one or more of the α subunit, the β subunit and optionally the activator protein is codon-optimized to increase expression in a desired microorganism, the desired microorganism is optionally a yeast, a fungus or a bacterium, or further optionally encoding one or more of the α subunit, the β subunit and optionally the activator protein is codon-optimized to increase expression in Corynebacterium glutamicum ATCC13032 or its derivative MB001 (DE3) deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) with strain number 102071.

4. One or more nucleic acids according to claim 3, wherein (i) the desired microorganism is a bacterium selected from the group consisting of Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Brevibacterium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Arthrobacter, Brevibacterium, Corynebacterium rynebacterium, Microbacterium, Micrococcus, Nocardia, Pseudonocardia, Trichoderma, Myrothecium, Aureobasidium, Candida, Cryptococcus, Debaryomyces, Geotri chum, Hanseniaspora, Kluyveromyces, Pichia, Rhodotorula, Comomonas and Pyrococcus, or the microorganism can be selected from bacteria of the genera Rhodococcus, Pseudomonas, Escherichia and Geobacillus, or from the following species: Rhodococcus rhodochrous, Rhodococcus pyridinovorans, Rhodococcus erythropolis, Rhodococcus equi, Rhodococcus ruber, Rhodococcus opacus, Aspergillus niger.niger), Acidovoraxavenae, Acidovorax facilis, Agrobacterium tumefaciens, Agrobacterium radiobacter, Bacillus subtilis, Bacillus pallidus, Bacillus smithii, Bacillus sp BR449, Bradyrhizobium oligotrophicum, Bradyrhizobium diazoefficiens, Bradyrhizobium japonicum, Burkholderia cenocepacia, Burkholderia gladioli, Escherichia coli coli), Geobacillus sp. RAPc8, Klebsiella oxytoca, Klebsiella pneumonia, Klebsiella variicola, Mesorhizobium ciceri, Mesorhizobium opportunityunistum, Mesorhizobium sp F28, Moraxella, Pantoea endophytica, Pantoea agglomerans, Pseudomonas chlororaphis, Pseudomonas putida, Rhizobium, Rhodopseudomonas palustris, palustris), Serratia liquefaciens, Serratia marcescens, Amycolatopsis, Arthrobacter, Brevibacterium species CH1spCH1), Brevibacterium sp CH2, Brevibacterium sp R312, Brevibacterium imperiale, Corynebacterium nitrilophilus, Corynebacterium pseudodiphteriticum, Corynebacterium glutamicum, Corynebacterium hoffmanii, Microbacterium imperiale, Microbacterium smegmatis, Micrococcus luteus, Nocardia globerula, Nocardia rhodochrous, Pseudonocardia thermophila, Trichoderma, Myrothecium verrucaria), Aureobasidium pullulans, Candida famata, Candida guilliermondii, Candida tropicalis, Cryptococcus flavus, Cryptococcus sp UFMG-Y28, Debaryomyces hanseii, Geotrichum candidum, Geotrichum sp JR1, Hanseniaspora, Kluyveromyces thermotolerans, Pichia kluyveri, Rhodotorula glutinis, Comomonas testosterone, Pyrococcus abyssi), Pyrococcus furiosus, Pyrococcus horikoshii, Brevibacteriumcasei) or Nocardia sp. 163; and optionally the microorganism comprises a bacterium of the species Corynebacterium glutamicum, further optionally the strain Corynebacterium glutamicum ATCC13032 or its derivative strain MB001 (DE3); (ii) the β subunit is encoded by the nucleic acid of SEQ ID NO: 4; (iii) the α subunit is encoded by the nucleic acid of SEQ ID NO: 5; (iv) the activator protein is encoded by the nucleic acid of SEQ ID NO: 6; or (v) Any combination of (i) to (iv).

5. A nitrile hydratase operon comprising a nucleic acid encoding the α subunit (nhhA) and the β subunit (nhhB) and optionally an activator protein (nhhG) of the variant nitrile hydratase according to claim 1 or 2, optionally wherein the nucleic acid is a nucleic acid according to any one of claims 3 to 4.

6. The operon according to claim 5, which is derived from yeast, fungi or bacteria expressing nitrile hydratase, optionally bacteria of the genus Pseudomonas, and further optionally Pseudonocardia thermophila.

7. The operon according to claim 5 or 6, comprising SEQ ID NO:

7.

8. An extrachromosomal sequence, optionally a plasmid comprising the nucleic acid or operon according to any one of claims 3 to 7.

9. A microorganism, optionally a yeast, fungus or bacterium, further optionally an industrial microorganism, which optionally does not endogenously express nitrile hydratase or endogenously expresses nitrile hydratase, the microorganism being engineered to contain a nucleic acid encoding a nitrile hydratase according to claim 1 or 2, or a nucleic acid encoding the nitrile hydratase or an operon or extrachromosomal sequence comprising the nucleic acid, which is optionally according to any one of claims 3 to 8, further optionally wherein one or more of the nucleic acids are contained in one or more extrachromosomal sequences (plasmids) or integrated into the chromosomal DNA of the microorganism.

10. The microorganism according to claim 9, which is a bacterium selected from the following: Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Brevibacterium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Arthrobacter, Brevibacterium, Corynebacterium, Microbacterium, Micrococcus, Nocardia, Pseudonocardia, Trichoderma, Myroculitis, Candida, Cryptococcus, Debaryomyces, Geotrichum, Hansenula, Kluyveromyces Saccharomyces, Pichia, Rhodotorula, Comamonas and Pyrococcus, or bacteria selected from the genera Rhodococcus, Pseudomonas, Escherichia and Geobacillus, or selected from the following species: Rhodococcus rhodochrous, Rhodococcus pyridinophilus, Rhodococcus erythropolis, Rhodococcus equi, Rhodococcus erythropolis, Rhodococcus opacus, Aspergillus niger, Acidovorax avenacum, Acidovorax agile, Agrobacterium tumefaciens, Agrobacterium radiobacterium, Bacillus subtilis, Bacillus pallidum, Bacillus smithii, Bacillus species BR449, oligotrophic bradyrhizobium, efficient nitrogen-fixing bradyrhizobium, soybean bradyrhizobium, Burkholderia cenocepacia, Burkholderia gladiolus, Escherichia coli, Bacillus geobacillus Species RAPc8, Klebsiella oxytoca, Klebsiella pneumoniae, Klebsiella variegata, Mesorhizobium chinense, Mesorhizobium opportunum, Mesorhizobium species F28, Moraxella, Pantoea endophytes, Pantoea agglomerans, Pseudomonas chlororaphis, Pseudomonas putida, Rhizobium, Rhodopseudomonas palustris, Serratia liquefaciens, Serratia marcescens, Amycoloma spp., Arthrobacter, Brevibacterium species CH1, Brevibacterium species CH2, Brevibacterium species R312, Brevibacterium moth, Corynebacterium azotobacterium, Corynebacterium pseudodiphtheroides, Corynebacterium glutamicum, Corynebacterium glutamicum, Corynebacterium hofmannii, Microbacterium moth, Microbacterium smegmatis, Micrococcus luteus, Nosema sphaeroides Cardia, rose-colored Nocardia, thermophilic Pseudonocardia, Trichoderma, Verrucos spores Myrocardium, budding short stalk mold, Candida anonymum, Candida guillimon, Candida tropicalis, Cryptococcus flavus, Cryptococcus species UFMG-Y28, Debaryomyces hansenii, candidum, Geotrichum species JR1, spore Hansenula, thermotolerant Kluyveromyces, Pichia kluyveri, red yeast, Comamonas testosteroni, Pyrococcus abyss, Pyrococcus furiosus, Pyrococcus horikoshi, Brevibacterium casei or Nocardia species 163; and is optionally a bacterium of the species Corynebacterium glutamicum, optionally a strain Corynebacterium glutamicum ATCC13032 or a derivative thereof MB001 (DE3).

11. The microorganism according to claim 9 or 10, which is an industrial microorganism that expresses nitrile hydratase endogenously, and the nucleic acid or operon replaces the endogenous nitrile hydratase gene or the operon comprising the endogenous nitrile hydratase gene.

12. A method for producing an amide compound from a nitrile compound, the method comprising: The nitrile compound is contacted with the nitrile hydratase according to any one of claims 1 to 11 or a microorganism expressing the nitrile hydratase.

13. The method according to claim 12, wherein the amide compound is selected from the group consisting of acrylamide, methacrylamide, acetamide and nicotinamide, preferably acrylamide, and the nitrile compound is selected from the group consisting of acrylonitrile, methacrylonitrile, acetonitrile and 3-cyanopyridine, preferably acrylonitrile.

14. The method of claim 12 or 13, using one or more of: a soluble nitrile hydratase, an encapsulated nitrile hydratase, an immobilized nitrile hydratase, or a whole cell or lysed microbial cell biocatalyst comprising the nitrile hydratase.

15. A method according to claim 12, 13 or 14 using a whole microbial cell biocatalyst which optionally can be freshly produced (i.e. directly from fermentation); stored, for example in frozen form (frozen in wet form); or dried, such as a freeze-dried product or a spray-dried form thereof.

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