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Microbiology and Biotechnology Letters

Review(총설)

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Microbial Biotechnology  |  Protein Structure, Function, and Engineering

Microbiol. Biotechnol. Lett. 2023; 51(4): 333-352

https://doi.org/10.48022/mbl.2309.09009

Received: September 20, 2023; Revised: October 28, 2023; Accepted: December 4, 2023

Transaminases for Green Chemistry: Recent Progress and Future Prospects

Shreya Pandya1 and Akshaya Gupte2*

1Department of Microbiology, Natubhai V. Patel College of Pure and Applied Sciences, Vallabh Vidyanagar-388 120, Gujarat, India
2P. G. Department of Biosciences, Sardar Patel University, Satellite Campus, Bakrol, Anand-388315, Gujarat, India

Correspondence to :
Akshaya Gupte,       akshaya_gupte@hotmail.com

Abstract

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Transaminase represents the most important biocatalysts used for the synthesis of chiral amines due to their stereoselectivity. They allow asymmetric synthesis with high yields and enantioselectivity from their corresponding ketones. Due to their environmentally friendly access for the preparation of chiral amines, they have attracted growing attention in recent times. Thus, the production of chiral compounds by transaminase catalysed reactions is considered as an important application in synthetic organic chemistry. Therefore, transaminase is considered to be an important enzyme in the pharmaceutical and chemical industries. ω-Transaminase holds great potential because of its wide substrate specificity thus making it a suitable enzyme to be used at an industrial scale. This review highlights the reaction mechanism, classification, substrate specificity, and biochemical properties. The review also showcases the application of ω-transaminase in organic chemistry with a focus on the production of active pharmaceutical ingredients (APIs).

Keywords: ω-Transaminases, chiral amines, biocatalyst, active pharmaceutical ingredients

Graphical Abstract


Introduction

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Transamination using enzymes has gained significant interest in recent years as an efficacious method for the synthesis of chiral amines. Chiral amines are indispensable and prevalent as a key building block in agrochemicals, fine chemicals, and active pharmaceutical ingredients. Therefore, they have attracted particular attention from synthetic chemists. In this context, there is increasing interest in reactions, including ω-transaminases, which have been identified as a greener and more sustainable method for the production of chiral amines. Various enzymes, including hydrolases, transferases, and oxidoreductases, have been used for the making of chiral amines. One such enzyme is a PLP (Pyridoxal 5-Phosphate)-dependent transaminases, that catalyze the asymmetric amination of a ketone to a corresponding amine. High enantioselectivity is exhibited by this biocatalyst and has a broad substrate tolerance. Therefore, it is widely accepted for the production of optically pure amines.

Transaminase (Tams) was discovered by Needham [1] and Braunstein and Kreitzman [2]. They are ubiquitous and are known to be present in bacteria, fungi, plants, and even in humans, where they have a wide range of clinical implications [3]. They are located in the cytosol and mitochondria and thus play a vital role in the integration of many metabolic processes. They are used to synthesize chiral amines on a commercial scale. They have also been employed in the pharmaceutical, flavor and fragrance, chemical, food, and cosmetics industries [4]. The general mechanism is shown in Fig. 1 which means that the enzyme transfers the amino group from the amino donor and converts it into its corresponding keto acid and the amino acceptor, by accepting the amino group, will convert it into the corresponding amino acid/amine. More than 50% of the drugs on the market are chiral compounds. APIs are used to prepare medicines that have a backbone made of chiral amines and are used to treat several diseases like obesity, cancer, tuberculosis, diabetes, etc. The most popular example is the formation of Sitagliptin an antidiabetic drug produced by asymmetric synthesis, using transaminase with a purity of 99.95%.

Figure 1.General mechanism of Transaminase enzyme.

Biocatalytic processes have been developed using transaminases, which are environmentally benign alternatives to chemo-catalytic methods because they can operate under gentle conditions thus averting the need for flammable metal-organic reagents. These biotransformation reactions allow the formation of several intermediates which are precursors to many pharmaceutically crucial drugs and are also sometimes used as chiral resolving auxiliaries. Biotransformation reactions catalyzed by transaminase confers high stability, high turnover rate, stringent enantioselectivity, and no requirement of external cofactor regeneration and thus becoming an enzyme of interest in both research and pharma industry. This review will focus on the approaches used for classification, applications, engineering, and the novel approaches used in Tam discovery. Highlighting the changing trends during the last two decades. Furthermore, this will also address and explore the benefits and drawbacks of Tam discovery and their applications in industrial biocatalysis.

Active Site Model

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To understand the transamination reaction by transaminase, a detailed knowledge of the active site is necessary. Several ω-transaminases have been isolated from different sources of microbial origin. However, the substrate specificity shown was found to be similar. An active site model of omega transaminase from Vibrio fluvialis JS17 was proposed which was based on substrate specificity and reactivity [5]. Based on the experiments, it was observed that the active site of the enzyme had the presence of 2 pockets i.e., large and small pockets. Both the pockets have specificity for different substrates. The large pocket accepts hydrophobic and carboxylate groups while on the other hand, the S pocket shows steric hindrance for the carboxylate group, as the small pocket does not allow the entry of large and bulky groups. The model suggests that there is a dual recognition of the ω-transaminase enzyme. To check the substrate specificity various amino donors and acceptors were used. They found that the enzyme shows more reactivity towards (S)-enantiomers. Docking studies of Pseudomonas putida with X-ray structure were carried out and it was found that 6 major conserved residues out of 9 contributed to the substrate specificity [6]. Residues like Tyr23, Phe88*, and Tyr152 were important for the hydrophobic type interaction, whereas Arg414 is responsible for accepting the carboxylate group in the large pocket. The presence of residues like Trp60 and Ile 262 created a steric hindrance in the S pocket. Watanabe et al. [7] were the first who describe the crystal structure of ω-Amino acid: pyruvate aminotransferase (ω-APT) from Pseudomonas sp. F-126. The 3D structure gave insights into the active site of the enzyme. Apart from this, various other studies such as substrate docking simulations, Xray structure of ω-Tams from different bacteria, and structures of the engineered enzyme have also been reported by other scientists [8−10].

Mechanism of Transaminase

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Transaminases stick to a prototypical bi-reactant mechanism called a ping-pong bi-bi or double displacement reaction mechanism where the enzyme will first bind to the substrate and has to release the corresponding product before binding to the second substrate (Fig. 2). The transaminase reaction is divided into two steps: (1) Oxidative deamination and (2) Reductive amination. Transaminase is an apoenzyme as it requires the attachment of PLP to become a holoenzyme. The PLP cofactor first binds to the lysine residue in the active center of the enzyme, forming a Schiff's base, an internal aldimine.

Figure 2.Two step mechanism of transaminase.

In the first step- the ε-amino group of the lysine residue is replaced by the amino donor which acts as a substrate. It is a transaldimination reaction that leads to the formation of an external aldimine [11]. The pyridoxal ring acts as an electron sink and allows the conversion of external aldimine to ketamine with a quinoid intermediate via a 1, 3-prototropic shift. Meanwhile, the hydrolysis of ketamine will occur which ultimately leads to the release of keto-product as well as pyridoxamine-5’-phosphate (PMP), a deciding intermediate. In the second step- the reaction only carries forward with the formation of PMP, which is an amino donor in the second half, and the ketones and aldehydes will be the amino acceptors. The amino donor E-PMP (Enzyme-PMP complex) transfers the amino group to the acceptors ultimately leading to the release of corresponding amino acids and also the regeneration of E-PLP (Enzyme-PLP complex) for the next run of reaction.

Classification

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Transaminase enzymes are categorized under various criteria. Based on the position of the transferred amino group, transaminases have been broadly grouped into α-Tams and ω-Tams [12]. α-Transaminase acts only on the α-amino groups and allows only the formation of α-amino acids for which the presence of a carboxyl group in the α-position is required. On the other hand, ω-Tams transfers amino groups from a donor to an acceptor. Here, the amino group transferred will be from a non-α position amino acid or an amino compound with no carboxylic group which leads to the formation of chiral amine and a ketone co-product. Their ability to accept different ketones and aldehydes for the biotransformation reaction makes them a dominant player in the field of biocatalysis [13].

Transaminase can also be classified based on its fold types. The first report of the classification of PLPdependent enzymes was reported by Grishin et al. [14] and was refined by Percudani and Perracchi [15]. Transaminases are part of a major group of PLP-dependent enzymes and are divided into seven major structural groups (fold types). This fold type implies the active site geometry which has transformed over time and is capable of sequestering PLP and also conducting all the reactions. Transaminase has been identified only in Fold Types I and IV (Fig. 3). Transaminase classes I, II, III, and V are all Fold Type I enzymes. Fold Type IV transaminases are (R)-selective.

Figure 3.Classification of PLP enzymes based on fold types.

One more criterion, based on which transaminase enzymes can be classified, is “Chirality”. Many enzymes can distinguish between two optical isomers of a particular substrate and transaminase is one of them. The two isomers are just chemical species that are non-superimposable. They can be designated as (S)- or (R)- as per the CIP (Cahn-Ingold-Prelog) set of rules as shown in Fig. 4.

Figure 4.The clockwise and counter-clockwise arrangement of two amine enantiomers that determine the (R)- or (S)- configuration.

Based on sequence and structure similarity, transaminase can also be categorized into six classes. Class I and II including L-aspartate transaminase and L-alanine transaminase, class III ω-transaminases, class IV Damino acid transaminases and branched-chain transaminases (BCAT), class V are L-serine transaminase, and class VI includes sugar transaminase as described in Table 1 [16, 17].

Table 1 . Classification of various transaminases.

Transaminase classEnzymes namesE.C. No
Class I+IIAspartate transaminase2.6.1.1
Alanine transaminase2.6.1.2
Aromatic transaminase2.6.1.57
Tyrosine transaminase2.6.1.5
Histidine-phosphate transaminase2.6.1.9
Phenylalanine transaminase2.6.1.58
Class IIIAcetyl ornitihine transaminase2.6.1.11
Ornithine transaminase2.6.1.13
omega-amino acid transaminase2.6.1.18
beta-aminocarboxylic acid transaminase-
Gamma aminobutyrate transaminase2.6.1.19
Diaminopelargonate transaminase-
Class IVD-Alanine transaminase2.6.1.21
Branched-chain transaminase2.6.1.42
Class VSerine transaminase2.6.1.51
Phosphoserine transaminase2.6.1.33
Class VITDP-4-amino-4,6-dideoxy-D-glucose transaminase2.6.1.33
L-glutamine:2-deoxy-scyllo-inosose transaminase2.6.1.100
TDP-3-keto-6-D-hexose transaminase2.6.1.89

Transaminase Challenges

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Transaminases are a very good option for the production of optically active chiral amines. They have wide applications and are also used at the industrial level for the production of many crucial drugs. In the case of transaminases, the two major problems faced by the enzyme are the unfavorable equilibrium constant and substrate/product inhibition [18]. Other problems include its poor thermostability and low solubility for different substrates. One of the approaches to shifting the equilibrium constant in the forward direction is to use the amine donors in large amounts, which will lead the reaction further in the direction of the amine product. Isopropyl amine was used as an amine donor in the presence of ATA 113 TAM to convert the 4-acetylbutarate to 6-methyl-2-piperidone [19].

Removal of the co-product is another way of shifting equilibrium. This strategy can be applied through different techniques like working under reduced pressure, i.e, evaporation of co-product and chemical transformation or degradation with the help of additional enzymes (cascade reactions/coupling reactions). The properties of the product amine and other components of the reaction mixture will decide which strategy is best. The other problem is substrate/product inhibition. Initially, the problem was solved by simply increasing the substrate concentration and by the removal of the product. The problem of product inhibition can be resolved with an approach to protein engineering. It can be done in two ways (1) rational design and (2) directed evolution. These two methods can be used in the redesigning of substrate specificity of transaminase. Another problem with transaminases is the solubility of the substrate. Other than aqueous media, organic solvents are also used in the reaction medium. They have certain advantages such as they can decrease the problem of substrate/ product inhibition to a certain level and also resolve the problem of substrate solubility and mass transfer at low boiling points as solvents are easy to evaporate. The presence of the organic solvents does not only lower the enzyme stability but also interferes with the downstream processing. Another problem with transaminase is the substrate specificity of the enzyme, which also favors the equilibrium of the reaction. As the equilibrium constant changes with different substrates, therefore, choosing the right amino donor and acceptor becomes very important and thus can provide the necessary conditions for the asymmetric synthesis of chiral amines.

Uncovering the Novel Transaminases

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The specialty of any biocatalyst, especially when it synthesizes optically pure amines, depends on its specificity towards a substrate. Transaminase shows a wide range of substrate specificity. Exploration of new biocatalysts will allow us to find such new enzymes that have a good substrate specificity as well as good enantioselectivity, which will ultimately lead to the path of new drug molecule synthesis and would further help in the curing of disease or can be used as an active pharmaceutical intermediate.

Fundamentally, there are two approaches for uncovering novel enzymes with unique properties and wide applications (1) gene mining and (2) protein engineering. Gene mining takes a look at the enzymes present in nature through different techniques. Nevertheless, it is time-consuming. On the other hand, protein engineering is more about tailor-making an enzyme either by improving its functional properties or by trimming the chemical synthesis [20]. With the help of these two strategies, transaminases with different substrate specificity from different bacteria with different methods can be identified.

Gene mining

Nature holds a large database and therefore gene mining of enzymes (transaminases) becomes necessary. These new enzymes may possess characteristics that have never been recognized before. Today, with the use of such advanced technology it has become possible to discover new biocatalysts directly either from experimental assays or by the assumption of the protein structures of its amino acid sequences. There are different strategies of gene mining by which different transaminases have been discovered with different methods.

The transaminases have been exploited mainly by [21]. The culture-based technique helped to uncover various transaminase enzymes. Tam from Klebsiella pneumoniae JS2F and Bacillus thuringiensis JS64 have been isolated by enrichment culture technique using 1-phenylethylamine (1-PEA) as the sole source of nitrogen. One of the best examples is V. fluvialis (Vf-TAm), a very well-characterized (S)-selective transaminase enzyme. Most of the things that are known today about transaminases are because of experiments and observations made on Vf-Tam [22]. Enrichment culture techniques have been used repeatedly for the isolation of transaminases. Transaminases from Alcaligenes denitrificans Y2k-2 (Ad-TAm), Mezorhizobium sp. LUK (Mz-TAm), and Bacillus megaterium SC6394 (Bm-TAm) isolated were found to be S-selective [23−25]. Many R-selective transaminases have also been discovered such as ω-Tam from Arthrobacter sp. KNK168 (Arth-TAm). This transaminase was isolated using 3, 4-dimethoxyamphet-amine as a source of nitrogen in the medium [26]. Transaminase enzymes were also discovered using Ramines as the only nitrogen source in the medium. Among the different enzymes identified transaminase from Curtobacterium pusillum, and Microbacterium ginsengisoli were able to use R-amines efficiently but showed less sequence similarity with R-amine transaminase sequence [27]. Transaminases belonged to two different Bacillus spp. namely, Bacillus halotolerans and Bacillus subtilis subsp. stercoris respectively were isolated from petroleum refineries and oil field. They have an acidophilic profile (optimum activity at pH-5, with 70% activity remaining at pH-3), and show tolerance towards organic solvents as well as high functionality at 30% DMSO [28]. Examples of (S)-selective transaminases identified using the activity-guided method are presented in (Table 2).

Table 2 . Transaminase discovered from cultured microorganisms.

Source organisms(S)/(R)CommentsReference
Activity-guided approach
Arthrobacter sp. KNK168(R)Enrichment media used with 3,4-dimethoxyamphetamine as SNS26
Alcaligenes denitrificans Y2k-2(S)Enrichment media with β-amino-n-butyric acid as SNS23
Mesorhizobium sp. LUK(S)Enrichment media substituted with β-amino-3-phenylpropionic acid24
Bacillus megaterium SC6394(S)Enrichment media used with 1-cyclopropylethylamine as SNS25
Pseudomonas fluorescens KNK08-18(S)Enrichment media with 7-methoxy-2-aminotetraline as SNS44
Curtobacterium pusillum
Microbacterium ginsengisoli		(R)Enrichment media used with various (R)-amines27
Pseudomonas putida NBRC14164(S)Enrichment media with 1-PEA as SNS45
Bacillus halotolerans
Bacillus subtilis subsp. stercoris		(S)Enrichment media with 1-PEA as SNS28
Albidovulum sp. SLM16(R, S)Enrichment media with α-MBA as SNS43
Meiothermus sp. PNK-Is4.(S)Enrichment media substituted with β-phenylalanine a46
Sequence homology
Chromobacterium violaceum DSM30191(S)Homologous sequence searching with Vf-Tam29
Caulobacter crescentus(S)Homologous sequence searching with A. denitrificans Tam47
Paracoccus denitrificans(S)Homologous sequence searching with Vf-Tam30
Polaromonas sp. JS666(S)Homologous sequence searching with Mesorhizobium Tam48
Ochrobactrum anthropi(S)Homologous sequence searching with Pd-Tam6
Burkholderia vietnamensis(S)Homologous sequence searching with Vf-Tam50
Halomonas elongata(S)Homologous sequence searching with Vf-Tam51
Bacillus megaterium(S)Homologous sequence searching with EcK12-Tam51
Halomonas sp. CSM-2(S)-53
Thermorudis peleae(S)Homologous sequence searching with Thermomicrobium roseum-Tam54
Key motif-based search
Aspergillus terreus(R)Fungal source32
Nectria haematococca(R)Fungal source33
Actinobacter sp.(R)-56
Chlororflexi bacterium(R)Bacterial source55

SNS = sole nitrogen source

α-MBA /1-PEA = α-methylbenzylamine/1-phenylethylamine

Homologous sequence searching is another technique used to identify new transaminases. In this method just by searching for the sequences that are homologous to those that have been identified by the enrichment media culture technique. By using the sequence of the already identified organism as a template, we can find the desired information of the functional gene from the different databases. Using the archetypal Vf-TAm sequence as a template, ω-TAm from Chromobacterium violaceum (Cv-TAm) was discovered and characterized [29]. Paracoccus denitrificans (Pd-TAm) a novel transaminase was discovered based on sequence homolog to Vf-Tam [30].

Another strategy is using key motif searches. It is a new method to predict the enzyme functions on the basis of key motifs present in the sequences [31]. The method was very effective and helped to identify 17(R)-selective ω-Tam. This is of significance for the prediction of function rather than identifying sequence similarity. Based on this strategy, many R-selective Tam have been identified in databases. The discovery of R-selective transaminases from fungi, including Cordyceps, Nectria, Capronia, and Trichoderma spp. was reported by many scientists [32−35].

Metagenomic mining for enzyme discovery

Metagenomic mining is a new approach that allows the direct isolation of DNA from the environment (e-DNA) and also nullifies the necessity of culturing the organisms at the same time [36]. This metagenomic mining is further bifurcated into two channels (1) sequence-driven metagenomics and (2) functional metagenomics.

Sequence-driven metagenomics. The focus of this approach is the sequence analysis of DNA which was extracted directly from the environment and aims to sequence a particular gene of interest. The sequence analysis involves steps like assembly, binning, gene prediction, and annotations of newly discovered sequences. To get the new sequence, the approach also needs reference sequence information [37]. This approach is very helpful in investigating extreme environments (hot springs metagenome, hypersaline) to identify novel transaminases. Cloning and over-expression of 11 putative tams were obtained from the metagenome of the oral cavities of humans [38]. Transaminases have also been isolated from extreme environments through sequencedriven metagenomics, especially from the hot springs of Iceland and Italy [39]. A sequence-driven approach was used for the successful retrieval of Tams from a domestic drain metagenome [40]. They were able to clone and express successfully 29 sequences in E. coli of the 36 fulllength non-redundant class-III Tam sequences. Another work on metagenomic mining was carried out in the hypersaline environment which yielded a functional Tam [41]. The Tams were obtained using a sequencedriven approach and were found to be S-selective. In nature, S-selective enzymes are in abundance. Hence, there is a need for using new novel approaches and techniques for discovering the R-selective enzymes from various metagenomes.

Functional metagenomics. This technique is used rarely but plays an important role. It involves e-DNA isolation which will be cloned and expressed into vectors. DNA is cut into pieces of the desired size and cloned into a suitable expression vector. Vectors transform the host, which is generally E. coli, which is then screened for their activity. It also generated fosmid libraries for enzyme discovery. (S)-selective Tam can also be discovered using fosmid library generation along with screening using o-xylylene diamine as an amino donor [42]. Of the 10 enzymes characterized by functional metagenomics, interestingly, all were found to be S-selective. However, 4 of these enzymes were able to accept both S and R enantiomers of 2-aminononane. As such, the functional metagenomic approach is a powerful tool that looks set to feature enzyme discovery more in the future. (Table 3) represents different transaminase genes isolated from metagenomic mining.

Table 3 . Transaminase discovered using metagenomics approaches.

Enzymes(S)/(R)CommentsReferences
Sequence-driven approach
pQR1108-pQR1118(S)Metagenome derived from oral cavities of humans38
KMG-TAm4(S)Metagenome derived from Triassic period salt mine53
pQR2188-pQR2191(S)Metagenome derived from DNA isolated from domestic drain40
AT-872, AT-4421 and AT-1132(R)Metagenome derived from Curonian Lagoon58
Functional metagenomics
pRT15-TA(S)Fosmid library generated for screening. Sequence 15% shorter than Vf-Tam and Cv-Tam42
TR1 to TR10(S)Fosmid library Metagenomic derived from 28 geographical distinct environemnt including, chronically polluted marine sediment samples, an acidic beach pool and the genome of Pseudomonas oleovorans57


Protein engineering

In recent years numerous studies involving the protein modification, and engineering of Tams have increased. To make the enzyme more efficient, protein engineering is done to design an advanced enzyme that is better than its wild part. Tailor-made biocatalysts are needed to achieve high activity and stability under nonideal conditions such as pH, temperature, and solvent. Based on the available information about the proteins, there are two options: one is rational design and the other is directed evolution. The knowledge of the protein structure is essential for the rational design to identify which is the most suitable amino acid to be substituted in order to achieve a specific goal (enantio-selectivity, thermal stability, substrate spectrum, tolerance to organic solvents).

Directed evolution describes the process of evolving enzymes in the laboratory by the in vitro imitation of Darwinian evolution. In 2018, Frances Arnold received the Nobel Prize in Chemistry for pioneering the field of directed evolution, highlighting the importance of this methodology for the adaption of proteins and biocatalysts to benefit humankind. Directed evolution involves random mutagenesis and rational design concerned more with sequences and structural analysis [59].

Random mutagenesis. Directed evolution is directly concerned with random mutagenesis. Random mutagenesis is the technique that allows us to speed up this naturally occurring phenomenon of advancing the gene ability through some physical or chemical agents [60]. Here, the gene is selected and its ability is improved through mutagenesis followed by library preparation of the mutants which is further selected or screened (Fig. 5). For example, to improve the substrate specificity of aspartate aminotransferase, some modifications have been done by directed evolution using DNA shuffling and selection in the auxotrophic Escherichia coli strain. The mutant obtained after 5 rounds of selection was screened and found that there was an increase in catalytic efficiency towards the substrate β-branched amino and 2-oxo acids up to 105-fold. The mutant showed a 30-fold decrease in efficiency for native substrate when compared with the wild-type. Out of 113 amino acid substitutions in the mutant version 6 were major and had almost 80−90% contribution to the total effect [61]. The wild type was mesophilic in nature but was later developed into thermostable aminotransferase after several rounds of mutagenesis via error-prone PCR. The activity of the enzyme towards substituted S-aminotetralin was upgraded from 5.9 to 1582.8 IU/g with an overall improvement in product yield due to reduced biocatalyst loading [62]. In another study, it was reported that transaminase from Aspergillus terrus shows low activity in organic solvent which is indeed required for product separation [63]. Therefore, through random mutagenesis along with combinatorial mutation, they try to improve the surface area. Three loops were taken as the site of mutation and then later the mutant T23I/T200K/ P260S (M3) was found to be a potential one. The mutant shows a higher catalytic efficiency for the 1-acetyl naphthalene, more stable in organic solvents, and was much better than its wild type.

Figure 5.Outline of directed evolution experiment [64].

Rational design. Rational design is a top-down approach that involves more computer modeling and techniques, such as site-directed mutagenesis. In this approach, prominence is given to the mastery of protein structure and amino acid interactions from the start. Site-directed mutagenesis has appeared to be valuable in studies, which led to the confirmation of structural and mechanical aspects of many enzymes. Protein engineering by rational design can be used to improve properties of enzymes like enantioselectivity, specificity towards the bulky substrate, and substrate promiscuity. It was reported that the active site of V. fluvialis Tams has the presence of two binding pockets, one large and the other small [65]. The presence of a residue named (W57) in the large pocket of V. fluvialis Tam, causes steric hindrance against binding of substrates. However, the addition of a glycine residue in the large pocket instead of the W57 residue led to the formation of a mutant. The mutant showed an increase in the substrate specificity towards both aromatic and aliphatic amine, up to 30 folds more than the wild type. A simple substitution of glycine increases the range of substrate from aliphatic to aromatic amines for the enzyme. The engineering of small pockets in the active site of the enzyme was investigated in many transaminases. The small pocket present in the active site of V. fluvialis was engineered, which was capable of performing the transamination reaction of α-hydroxyl ketones and aryl-alkyl ketones bearing an alkyl substituent larger than a methyl group [66]. Mutation of the residues in the small pocket was also investigated in Pseaudomonas denitrificans V153A for the alpha-keto acid amino acceptor with a long alkyl side chain. An increase in the activity of the mutant was observed four times more than the wild type. However, the mutant did not possess the specificity toward the alpha-keto acid with a shorter side chain [67]. The β-TA from C. crescentus was also studied to increase the substrate specificity of the enzyme towards the aromatic β-amines with a bulky phenyl ring. To enlarge the space in the active site so that the bulky phenyl rings can accommodate two residues were mutated. The specificity of the 2 mutants (N285A and V227G) was increased by 3 and 2 folds respectively [47]. TA from O. anthropic was also explored to increase its substrate specificity towards the bulky substrate. Residues in both the pockets, large and small present, were tried to mutate. It was observed that the residue named L57 makes the substrate unable to bind to a small pocket by causing steric hindrance [12, 47]. The hindrance was removed by an alanine mutation which enhances the specificity of the mutant version up to 150-fold for the aromatic amines, 5-fold towards the aliphatic substrates, and a 48-fold increase for the α-keto acids. Other than this, the mutation was also caused in the W58 residue of a large pocket to remove the steric hindrance and was replaced with leucine. The mutated one (W58L) has the ability to aminate the bulky acetones from the corresponding amines [68]. Sitagliptin is a well-known compound synthesized by a transaminase which has the presence of two large groups on both sides of its amino groups. The wild-type enzyme ATA-117 shows inertness towards its corresponding ketones i.e, prositagliptin. Although the substrate specificity for the prositagliptin ketone was improved through structure-based engineering. Many rounds of mutation were carried out to develop a desired mutant. At first, the mutations were done in large pockets at 12 sites from which the Mut1 variant was developed with a single S223P. Activity of the enzyme enhanced but no activity of sitagliptin could be determined, then, to accommodate the large trifluorophenyl group, a mutation was caused at 4 sites in the small pocket. Then the Mut1b variant was developed with 3 mutations along with S223P. A minor Tam activity was detected. All the mutations done previously were combined to get the desired result. Eventually, the Mut2 variant was obtained with 6 additional mutations and gave the needed result with an almost 75-fold increase in the activity [9]. Studies were carried out in which a mesophilic Arthrobacter citreus was engineered into a thermostable mutant with 17 mutations after 5 rounds of screening [62]. The best mutant exhibited better activity at 55℃ than the wild-type protein (30℃). Apart from substrate specificity, enantio-selectivity and substrate promiscuity are two other properties that can be upgraded by protein engineering. Substrate promiscuity involves the mutations of residues in the active site of an enzyme which react directly with the functional moiety of substrate rather than reshaping and expansion of the pocket size. Enantio-selectivity can also be improved by protein engineering. Attempts were made to improve the enantio-selectivity of C. violaceum ω-transaminase. Another study reported an increase in enantio-specificity and a change in enantiomeric preference in C. violaceum ω-transaminase [69]. They created a mutant W60C through site-directed mutagenesis. They substituted cysteine in place of tryptophan in the active site. The mutant W60C showed increases in enatio-specificity up to 2-fold for 1-phenylethylamine, 9-fold for 1-aminotetralin, and 15-fold for 2-aminotetralin. They further engineered the enzyme through molecular docking and tried to change the enantiomeric preference. A mutant F88/A231F was created whose enatiopreference was shifted from 3.9 (S) to 63 (R) and is substrate-dependent. Further, the Try60Cys variant of C. violaceum ω-transaminase was studied [70]. The variant shows more specificity towards (S)-1-Phenylethylamine (29-folds) and 4-substituted acetophenone (5-folds) than the wild type. He also compared the wild type and mutant and concluded that the mutant not only shows a higher specificity constant but also follows the Swain-Lupton parameterization. Other studies reported the identification of a hypothetical ω-Tam from a Chloroflexi bacterium based on a motif sequence search [55]. The (R)-selective enzyme was engineered through a site-directed mutagenesis technique to improve its activity for ketones. Using site-directed mutagenesis, the 3 main residues present in the small pocket of the enzymes Y190, Q192, and G288 were mutated. The results obtained after the mutation conclude that the mutation was successful and the mutant Q192G was the potential one with 9.8 times higher activity than its wild type. Random mutagenesis and rational design are two potent methods to improve the enzyme. Both methods have different approaches for the improvement of enzymes but ultimately yield a mutant with better efficiency (Fig. 6).

Figure 6.Comparison of rational design and directed evolution studies.

Screening

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With the use of enrichment culture techniques and protein engineering, several transaminases have been found in recent years. Transaminases, particularly ω-transaminases, have a broad substrate specificity that includes both amino donors and acceptors. This wide range of substrate specificity highlights the enzymés versatility and functionality, making it necessary to determine the enzymés specificity and enantioselectivity before being used commercially. As a result, many high-throughput screening methods have been developed to aid in the selection of an ideal enzyme for a given application at both the industrial and research level.

Screening procedures should meet the following criteria: low cost, efficiency, speed, sensitivity, and applicability. The old traditional procedures rely on HPLC for detection, whereas GC and MS are substantially more timeconsuming. Enrichment cultivation is an effective method to discover transaminases and transaminase mutants since it provides information on specificity, product inhibition, and enantioselectivity [71]. However, the knowledge regarding the enzyme activity and biomass cannot be proportional directly to this method, which shows its limitations [72]. Therefore, various methods have been developed that speed up the identification of enzymes, such as screening of transaminase by the use of α-MBA as an amine donor and acetophenone detection by HPLC or spectrophotometrically (Table 4). The high throughput screening methodology for transaminase using phenol red as a pH indicator with LDH/ GDH (Lactate dehydrogenase/Glucose dehydrogenase) was used for the screening of the ω-Tams enzyme. Here L-alanine was used as an amino donor and LDH removed the pyruvate which was generated and GDH helped in the regeneration of the NADH cofactor [29, 73, 74]. The other two approach involves the use of isopropylamine as an amino donor instead of L-alanine and the use of amino acid dehydrogenase which converts pyruvate back to alanine. One of the most widely used methods is the O-XDA assay [75]. O-XDA undergoes polymerization during the reaction and forms black precipitates.

Table 4 . High-throughput screening assays for transaminase.

Sr. No.SubstrateAdditional ReagentMethod of DetectionEvaluation Type*Liquid or SolidReferences
1AlanineOxidase, pH indicatorColour changeCL74
2α-amino acidCuSO4/MeOHBlue colourEL82
3Alanine and glutamateOxidase, tetrazoliumColour changeCL83
4Alanine and glycineOxidase, HRP, colour indicatorsColour changeCBoth81
5Phenylethylamine (1-PEA)-Absorption at 245 nm with a UV SpectrometerCL84
6ortho-Xylylenediamine-Dark colourCBoth85
7Complex of Cu (II)-Ala instead of alanineCu (II) and BSAFluorescence quenching with a fluorescence spectrometerCL77
8EWG-substituted aromatic amines-Colored productCBoth75
92-Hydroxy ketoneTetrazoliumColour changeEL86
101-(6-Methoxynaphth-2yl) alkylamines-Fluorescence at 450 nm with a fluorescence spectrometerCL87
11Substituted aminotetraline-Colored product exposed to airCL62
122-(4-nitrophenyl) ethan-1-amine-Red colourCBoth88

C : continuous

E : end point

L : liquid

A colorimetric method using CuSO4/MeOH solution was developed which gives a blue color with an amino acid that can be detected at 595 nm [76]. A similar assay for the conversion of alanine to pyruvate was applied to characterize glutamic-pyruvic transaminase. In the assay dissociation of the Cu(II)-L-alanine complex is observed which results in free Cu(II) ions thereby combining with BSA, leading to a fluorescence quench which is monitored at 340 nm [77]. Another colorimetric detection is carried out using amino acid oxidases (AAO) and horseradish peroxidase (HRP) where an alanine is deaminated by AAO to produce hydrogen peroxide (H2O2), which can be further detected using HRP [78-80]. Tetrazolium salts are also considered one of the efficient reagents which assist in detecting 2-hydroxyl ketone. In the presence of 2-hydroxyl ketone, 2, 3, 5-triphenyl tetrazolium chloride (TTC) changes its color from colorless to red and forms a formazan precipitate. Using this assay, the polyamines, a novel class of bifunctional mono- and diamine transaminases were characterized. Another interesting method of detection is based on pH change. Here the pyruvate is reduced by LDH and oxidation of glucose to gluconic acid by GDH, which leads to the change in color of the pH indicator. Glyoxylate is used as an amine acceptor in glycine oxidase-based assay. It is based on a similar principle of transaminase/ amino acid oxidase. The technique has been successfully used for the profiling of the substrate of (R)- and (S)- transaminases [81]. A kinetic assay was developed that measures acetophenone at 300 nm and allows both qualitative and quantitative screening of transaminases [73]. A spectrophotometric method was developed where 2-(4-nitrophenyl) ethan-1-amine was used as a donor reacting with pyruvate as an acceptor and PLP as a co-factor in the presence of Tam enzyme which led to the formation of red precipitates. The reaction was stopped using DMSO as the red precipitates are more soluble in that and retain their red color for a long time. The absorbance measured was at 465 nm [88].

Transaminase in Biocatalytic Applications

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The kinetic resolution, Asymmetric synthesis, and Deracemization are the approaches used for the synthesis of enantiopure amines using ω-transaminase.

Kinetic resolution

Kinetic resolution is one of the straightforward synthesis strategies for the production of chiral compounds and divides the two enantiomers into a racemic mixture (Fig. 7). In this approach, an enantioselective transaminase is used which converts the undesired amine into its corresponding keto product while the slow-reacting desired amine will remain in the reaction unconverted which can then be easily separated [89]. Kinetic resolution faces many drawbacks, such as a maximum theoretical yield of 50% is possible only, low atom efficiency of reactions, more amount of amine acceptors is required, and the ketone products may have an inhibitory effect on the enzyme. To cope with such problems, a kinetic resolution is used in association with deracemization and asymmetric synthesis. To make it more efficient, the transaminase enzyme is coupled with other enzymes.

Figure 7.Synthetic approaches for ω-transaminase mediated amine synthesis: -Kinetic resolution with (S)-ω-transaminase [20].

However, the method of kinetic resolution has been upgraded to increase the yields. A step of racemization is included in it which will take back the undesired product to its starting racemic mixture. This method is now called dynamic kinetic resolution. Four new ω-TAs from Pseudomonas putida NBRC 14164 were identified and characterized for the racemic amines, 3-dihydro-1Hinden-1-amine,4-phenylbutan-2-amine, and amino alcohols such as 2-aminobutan-1-ol, 2-amino-2-phenylethanol. The four enzymes were Pp21050, PpbauA, Pp36420 and PpspuC. All four enzymes showed their specificity toward one or more than one substrate. They were able to convert the substrate into their corresponding ketone and amine or amine alcohol. However, the enzyme PpspuC has the ability to prepare enantiopure (S)-2-amino-2-phenylethanol via kinetic resolution, which was also demonstrated on a 100 ml scale reaction [45]. The kinetic resolution of α-methylbenzylamine using ω-transaminases from Bacillus thuringiensis JS64 was demonstrated. 500 mM α-MBA was successfully resolved to (R) - α-MBA and that, too, was above 95% enantiomeric excess, and 51.3% conversion was achieved [90].

Another stumbling block of kinetic resolution is product inhibition due to ketone-product formation, which can be overcome by shifting the reaction equilibrium towards the product formation by physical extraction/vaporization.

Asymmetric synthesis

In 2007, asymmetric synthesis was considered one of the most desirable reactions by five of the largest pharmaceutical manufacturers worldwide [91]. Asymmetric synthesis and deracemization can generate 100% theoretical yield and thus both methods are preferred at the industrial level [92]. Asymmetric synthesis involves the prochiral molecules as substrate which can be converted into two enantiomers formed in an unequal amount. The product that is formed is generally a racemic mixture but if the reaction is carried out using a particular stereoselective enzyme then only one isomer will form in, more amount over another (Fig. 8). Asymmetric synthesis of alpha methylbenzylamine produced from acetophenone and alanine using crude extract and whole cells enzymes. Here, the ω-Tams and ALS (acetolactate synthase) were co-expressed in recombinant E. coli and then the biotransformation reactions were carried out [93]. Another reaction reported was the asymmetric synthesis of 2-substituted pyrrolidines and piperidines. Among the different transaminases already available, ATA-117-Rd6 was the best (R)-selective Tam, and PjSTA-R6-8 was the best (S)-selective TA for all the substrates. 2-substituted pyrrolidine and piperidines can be produced with ee (Enantiomeric excess) >95% and a yield range from 10% to 90% [94]. Busto et al. [95] demonstrated the synthesis of a key intermediate (R)-2, 3, 4, 9-tetrahydro-1H-carbazol-3-amine, used in the synthesis of the anti-allergic drug Ramatroban. They used different ω-Tams from different sources but amongst all, (R)-Arthrobacter (ArRmut11-ω-TA) was the potential one. They used isopropylamine initially as the amine donor, which was ideal, but produced various side products also and therefore, they eventually shifted to (R)-1-phenylethylamine, which was proved to be the best amine donor with a 96% conversion and 97% of ee. Serinol or 2-amino-1, 3-propanediol, is an amino alcohol. Serinol is chiral in nature as it contains fatty acid chains. It is a pharmaceutically important compound and also a building block in the synthesis of various other chiral compounds. Costa and co-workers used (R)-amine transaminase from Aspergillus fumigatus wild-type (AspFum) [32] variants of the V. fluvialis enzyme [61], wild-type V. fluvialis [96], and amine transaminase-11718 from Codexis [9]. For the preparation of (S)-2-amino-3-hydroxypropyl hexanoate and (R)-2-amino-3-hydroxypropyl hexanoate (Serinol-monoesters) 1-Hexanoyloxy-3-hydroxyacetone and α-phenylethylamine was used as substrate along with PLP, HEPES buffer and enzyme solutions (10 mg/ml of lyophilized enzyme). Among the various amine transaminases used, the wildtype enzyme from Aspergillus fumigatus was a promising one and produced the (R)-amine with a 93% conversion and 99% enantiomeric excess. For (S) - amine, V. fluvialis F85L/V153A variant was a potential one and produced the (S)-amine with 92% conversion and 92% ee [97]. Steroids are a diverse and large class of secondary metabolites necessary for many biological processes and their control. 17-α-amino steroids were found to be of particular interest as non-natural steroids which can be used as an intermediate for the production of steroidal derivatives [98]. The asymmetric synthesis of LHomophenylalanine (L-HPA) was carried out using a recombinant amino acid transaminase (AroAT) from 2-oxo-4-phenylbutyric acid (2-OPBA) and L-aspartate [99]. Further, the synthesis of (2S, R)-2-amino-1, 3, 4-butanetriol) ABT, a chiral amino alcohol, was described using the substrate L-erythrulose and α-MBA. The targeted product was synthesized through an asymmetric synthesis approach and they have concluded in their findings that α-MBA shows toxicity towards the transaminase enzyme therefore they ultimately increased the concentration of L-erythrulose and lessened the concentration of MBA which led to a higher yield of ABT. They have observed a 99% conversion into ABT while there was only a 35% conversion when MBA was used in higher concentration [100]. Another excellent transaminase was from Pseudomonas jessenni (PjTA-R6). It was a mutant used for the asymmetric synthesis of aliphatic amines [101, 102]. They used the mutant to synthesize aliphatic amines and compared their results with the standard transaminases from V. fluvialis (VfTA) and Chromobacterium violecium (CvTA). PjTA-R6 was a potent mutant and synthesized various aliphatic amines and gave good results as compared to the reference transaminase enzymes. The enantioselectivity was also much higher than the VfTA and CvTA and hence it is favorable for amine synthesis.

Figure 8.Asymmetric synthesis.

Deracimization

Deracimization is a process that employs two stereocomplementary ω-Tam. The formation of ≤50% enantiopure amine is done in the primary step of the deracemization process and a ketone co-product is formed by the amination of amine (Fig. 9). Then the asymmetric amination of the ketone is carried out by an enantiocomplementary ω-TA, which leads to the formation of the pure amine with up to 100% yield. Mexiletine is a pharmaceutically important drug that is synthesized through deracemization. It is an orally effective antiarrhythmic drug [103, 104]. Deracemization of amines such as 4-phenylbutan-2-amine, a precursor for the antihypertensive dilevalol; sec-butylamine, 1-methoxy-2-propanamine and 1-cyclohexyl ethylamine, precursors of inhibitors of tumor necrosis factor-α (TNF-α); and 1-phenyl-1-propylamine, which is a precursor of antidepressant agent corticotropin-releasing factor type-1 receptor was also reported [103, 105].

Figure 9.Deracemization of racemic amine using (R) ω-Tam and (S) ω-Tam.

Cascades

Enzymatic cascades are way more advanced as compared to other strategies. They require more than one enzyme for the synthesis of the target compound. ω-transaminase is used in the cascades along with the other enzymes to synthesize the crucial compounds in minimal steps and in less time with high purity and enantioselectivity. The additional benefits these cascades provide are the removal of toxic intermediates and the removal of wastes, minimum use of organic solvents, and thus increased atom efficiency [78, 89]. There are four main types of Cascade reactions: (1) Linear cascades represent consecutive transformations in one pot. The good part of the linear cascade is that we can avoid intermediates which are toxic and unstable and may be explosive. (2) Parallel cascades, are generally used in redox biocatalysis where the second reaction is going simultaneously, coupled with the product formation of the previous reactions. (3) The third type, orthogonal cascades, are almost similar to parallel cascades, often used in shifting the reaction equilibrium towards product formation. (4) The fourth type, is cyclic cascades, which are mostly used for the stereo-inversion processes.

Many such cascades are reported using ω-transaminase. Fuchs reported the synthesis of naftifine, which is an antifungal reagent. Naftifine was synthesized from cinnamic alcohol with an overall yield of 51%. The synthesis was done through a cascade route that employed two enzymes, i.e. galactose oxidase from Fusarium NRRL 2903 and the (S)-selective transaminase from V. fluvialis [106]. Similarly, alcohol dehydrogenase, alanine dehydrogenase, and ω-TA have been employed recently for the conversion of ether alcohol substrates to their corresponding ether amine products in an artificial redox-natural cascade [107]. Other than this, ω-Tams are also a good option for the synthesis of unnatural amino acids. Unnatural L-Homoalanine has been synthesized using two enzymes i.e., Threonine deaminase from E. coli and transaminase from P. denitificans. The unnatural amino acid is formed using the natural amino acid L-threonine as a substrate. Dehydration and deamination of substrate were carried out by threonine deaminase which formed the product 2-oxobutyrate which was further converted to L-homoalanine by transaminase using benzylamine as an amine donor. The benefit of including transaminase in this reaction is that it gives 100% theoretical yield and, also, the equilibrium is always on the side of the amino acid product [45]. The one-pot cascade reaction was reported which involves two major biocatalysts; one is transaminase and the other is monoamine oxidase [98]. For the production of 2, 5-disubstituted pyrrolidines exclusively, a major pharmaceutical compound, two biocatalysts are used in a one-pot cascade reaction. Different strategies were tried for the respective target compound but when the two biocatalysts were used together along with NH3·BH3 for reduction, the target compound formed in one step with a yield of 82% and 99% di stereoselectivity (de). Amino alcohols are also an important class as they can be used to produce amines or some chiral alcohols are also synthesized, which have pharmaceutical importance. Another example of a cascade reported was the production of amino alcohols prepared by cascade reaction using transketolase enzyme and transaminase enzyme. Achiral substrates like propanol and hydroxypyruvate were used to form (3S)-1,3 dihydroxypentane-2-one using transketolase enzyme and then the product formed was further converted by ω-Tams into 2S,3S aminopentane-1,3 diol using isopropylamine as an amine donor [109].

Substrate Specificity

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Substrate specificity of transaminase is one of the major aspects. The substrate specificity of the enzyme will let us know which type of compound or product will form and in what percent yield. If we consider α-Tams, then they only react with α-amino acids and α-keto acids, whereas ω-Tams are much more versatile in this aspect. They can easily accept amino acids, amines, ketones, and aldehydes also. They have a broad substrate spectrum and are therefore more useful at the industrial level. Shin characterized the transaminase from V. fluvialis JS17. The enzyme was tested against the different substrates to determine its substrate specificity. Among the amino donors, the enzyme activity was found to be maximum for arylic chiral amines like (S)-α-MBA, (S)-1-aminoindane, (S)-1-methyl-3-phenylpropylamine. Polyamine and amino acid substrates were inert and only L-alanine showed reactivity. Among the amino acceptors, pyruvate followed by glyoxylate showed good reactivity [110]. The substrate specificity of the transaminase enzyme identified in Nectria haematococca was reported [33]. Seven different substrates were taken to check the substrate specificity of the enzyme. They ultimately found that the Nectria Tam is (R)-selective and shows good activity towards (R)-MBA. Different amino donors and acceptors were tested for the enzyme TPF isolated from Pseudomonas fluorescens KNK08-18. Among the different donors (S)-1-PEA was the best. Afterward, rac-2-heptylamine, rac-3-heptylamine, and L-alanine were good. Among the amino donors, pyruvic acid and glyoxylic acid were found to be the most reactive [42]. A detailed account of substrate specificity for the transaminase isolated from the Chloroflexi bacterium (CbTA) was also given by researchers. The different amino donors were tried out for CbTA and out of all, the enzyme has maximum activity for long-chain aliphatic amines and cyclic amines [55]. For the amino donors, the enzyme shsows higher activity towards 2'-nitroacetophenone, 4'-methoxyacetophenone, benzaldehyde, and 5-nitrosalicylaldehyde. Iwasaki et al. [111] examined the substrate specificity of (R)-Tam with both amino donors and acceptors. He found that the enzyme exhibits maximum activity for aliphatic or acrylic amines like sec-butylamine, 2-amino octane, and methyl benzylamine, which was considered the best amino donor. With respect to amino acceptors, pyruvic was the best among all the carbonyl compound checks.

Conclusion and Future Prospects

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Transaminase has evolved as an interesting enzyme. The enzymes discovered so far have the potential to synthesize different chiral compounds. The enzyme is a good alternative to conventional methods which are long and require both time and money. The transaminases synthesize various pharmaceutically important drugs or their intermediates in a single step. The different methods of discovering the enzyme have now made it easy to explore various transaminase enzymes. The metagenomic methods are very advanced and managed to discover some (R)-transaminases as well as some extremophiles. Along with this, protein engineering has also helped to increase the ability of the enzyme to accept a wide range of substrates so that the production of different amines can be possible. It has developed various new enzymes to study and also improved the enzymatic properties of already existing enzymes.

Rational design and directed evolution also aid in the potential of transaminase to catalyze the reaction which is desirable for pharmaceuticals. The enzyme also doesn’t require a stringent condition and the cofactor of the reaction generates itself, which is a plus point.

Despite all these advantages of transaminase, it has faced some challenges during the synthesis reaction. Poor stability of the product, a narrow range of substrates, and unfavorable equilibrium during the reaction are major challenges for the enzyme. Despite the shortcomings faced by transaminases, the applications are much more and quite interesting, which has brought the attention of the pharma world towards them. The most historic application of transaminase was the development of the anti-diabetic drug sitagliptin through protein engineering. Synthesis of such drugs at the industrial level helped society, the environment, and industries in an excellent way.

Nevertheless, ω-transaminase puts forward several opportunities in the future for the development of any process. Transaminases, especially ω-transaminase because of their wide range of substrate specificity, are most useful in the biotransformation reactions for the synthesis of drug molecules and thus they have been exploited extensively in recent years. However, for the isolation of (R)-Tams and the related biotransformation reaction, there is a need for new and advanced techniques and screening methods. Advanced screening methods are required for easy detection of amines and ketones. Plate assays are not much regarding transaminase enzyme, so that can also be developed. Some focus should also be given to thermophiles and their discovery, which may open the doors of unexpected opportunities for the future. Currently, the focus in the field of chiral amine is to isolate the enzyme with much advanced features that include enantio-selectivity, region-selectivity, and chemo-selectivity, and to accomplish this more importance should be given to protein engineering with site-directed mutagenesis (rational design and directed evolution). The enzyme has potential not only in the production of drug molecules but is also environmentally friendly and a good alternative for organic chemistry.

Author Contributions

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All authors contributed to the review conception and design. Literature review, data collection, and analysis were performed by Shreya Pandya and, Akshaya Gupte. The first draft of the manuscript was written by Shreya Pandya and, Akshaya Gupte and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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