Appl Microbiol Biotechnol DOI 10.1007/s00253-015-6507-3
MINI-REVIEW
Distribution, industrial applications, and enzymatic synthesis of D-amino acids Xiuzhen Gao & Qinyuan Ma & Hailiang Zhu
Received: 17 January 2015 / Revised: 22 February 2015 / Accepted: 23 February 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract D-Amino acids exist widely in microbes, plants, animals, and food and can be applied in pharmaceutical, food, and cosmetics. Because of their widespread applications in industry, D-amino acids have recently received more and more attention. Enzymes including D-hydantoinase, N-acyl-D-amino acid amidohydrolase, D-amino acid amidase, D-aminopeptidase, D-peptidase, L-amino acid oxidase, D-amino acid aminotransferase, and D-amino acid dehydrogenase can be used for D-amino acids synthesis by kinetic resolution or asymmetric amination. In this review, the distribution, industrial applications, and enzymatic synthesis methods are summarized. And, among all the current enzymatic methods, D-amino acid dehydrogenase method not only produces D-amino acid by a one-step reaction but also takes environment and atom economics into consideration; therefore, it is deserved to be paid more attention. Keywords D-Amino acids . Enzymatic synthesis . Asymmetric amination . D-Amino acid dehydrogenase
widely in nature, for example, the cell walls of Gram-positive bacteria and some peptide antibiotics contain D-AAs. The presence of D-AAs prevents digestions by proteolytic enzymes. Because of this, D-AAs have become important intermediates in pharmaceutical, food, and other various industries. Therefore, production of D-AAs is of great interest. D-AAs (Fig. 1) can be produced by chemical synthesis, fermentation, and enzymatic transformation. Chemical synthesis yields D-AAs by chiral resolution of DL-amino acids or by asymmetric synthesis from chiral or prochiral starting materials. Because of racemization of D-AAs, high cost and low yields are the major disadvantages of chemical methods. Modified microorganisms without functional D-AA deaminases could be used for D-AA production (Fotheringham et al. 1998), but because of complex enzyme systems and metabolic networks, it is difficult to achieve high optical purity and productivity. Enzymatic methods can yield D-AAs with high optical purity, high productivity, and green process and are therefore ideal for the industrial manufacturing of DAAs. In this review, we introduce and compare all of the enzymatic methods used for D-AA production.
Introduction It is well known that proteins typically incorporate only Lamino acids into their sequences. For many years, few D-amino acids (D-AA) were found, and therefore, D-AAs were considered as unnatural amino acids. With the development of analytical technologies, D-AAs were found to be distributed X. Gao (*) : H. Zhu School of Life Science, Shandong University of Technology, Zibo 255049, China e-mail: [email protected] Q. Ma Technology Center, Weifang Ensign Industry Co., Ltd, Changle 262499, China
Distributions of D-AAs D-AAs
in microbes
The most known distribution of D-AAs is in extracellular or periplasmic polymeric biomolecules of bacterial cell walls, including peptidoglycan, teichoic acids, and poly-γglutamate (Bodanszky and Perlman 1969; Radkov and Moe 2014) (Fig. 2). The presence of D-AAs in Gram-positive bacterial walls prevents digestion by proteolytic enzymes (Yang et al. 2003). D-AAs have appeared in peptide antibiotics synthesized by bacteria and fungi, including gramicidin D (D-Leu and D-Val)
Appl Microbiol Biotechnol Table 1
Fig. 1 Chemical structure of natural D-AA. R group is variant
(Heijenoort 2001), gramicidin S (D-Phe) (Burkhart et al. 1999), polymyxins (D-Phe, D-Leu, D-Ser) (Martín et al. 1999), penicillin G (D-Val) (Katz and Demain 1977), cephalosporin (D-Ala) (Katz and Demain 1977), and actinomycins (D-Val, D-allo-Ile) (Reich 1963). D-AAs
in animals
D-AA
Type
Species
Physical meaning
D-Asp
Protein Protein Peptide Peptide Peptide Peptide Peptide Free
Human, cattle, mouse, rat Human Frog Frog Snail Snail Spider Human, mouse, rat
Aging Aging Opioid peptides Opioid peptides Neuropeptides Neuropeptides Neuropeptides Development
D-Ser D-Ala D-Met D-Phe D-Asn D-Ser D-Asp, D-Ser
in plants and animals
D-Ala
is the most widely reported D-AA in plants, for example, Helianthus annuus and D-Trp derivatives are the second most widely reported in plants (Robinson 1976). D-AAs are present as free or conjugated forms in the C 3 , C 4 , or crassulacean acid metabolism of plants, such as D-Ala, DSer, D-Leu, and conjugated D-amino-n-butyric acid (Vranova et al. 2012). The percentage of D-AAs in various plant parts (seeds, fruits, leaves, etc.) was around 1.5 % of the total amino acid pool (Vranova et al. 2012). Research studies have demonstrated that plants can absorb D-AAs from soil through plant roots (Hopkins and Ferguson 1994; Svennerstam et al. 2007). D-AAs also have been found in mammalian and human tissues in the form of free amino acids, and within peptides and proteins. The physiological functions of many D-AAs have been recently determined (Table 1) (Fujii 2002). D-AAs
D-AAs
in foods
D-AAs,
such as D-Ala, D-Asp, and D-Glu, are present in the raw milk of ruminants (cows, goats, and sheep) and in many
Fig. 2 Bacterial peptidoglycan containing D-AAs (Fujii 2002). a DAP-type peptidoglycan. b Lystype peptidoglycan
fermented foods such as cheese, wine, yogurt, and fish products. These D-AAs may be derived from the raw materials used to make food or microorganisms during fermentation (Abe et al. 1999; Brückner and Hausch 1989; Friedman 1999; Kato et al. 2011).
Industrial applications of D-AAs Pharmaceutical industry It is well known that D-AAs are incorporated or assimilated slower than the L-isomers, so the half-life of antibiotics and drugs containing D-AAs is longer. Additionally, recent research reported that D-AAs can enhance the activity of antimicrobials against biofilms of clinical wound. Within wound, biofilms are the main existence form of wound microorganisms and associate with chronic infections (Sanchez et al. 2014). The discovery is another strong and powerful evidence that D-AAs are of great value in pharmaceutical synthesis.
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Synthesis of semi-synthetic antibiotics D-AAs
are commonly used in semi-synthetic antibiotics synthesis, including traditional and new penicillin core and cephalosporin core antibiotics (Fig. 3). For example, using D-Asp and amoxicillin as raw materials, aspoxicillin can be chemically synthesized (Zhang et al. 2013). Aspoxicillin is popular because of its long half-life and low serum protein-binding characteristics.
inhibitor with (R)-5,5,5-trifluronorvaline as key building block (Gillman et al. 2010; Hanson et al. 2013). Free D-AAs are of great medicinal value. D-Phe alone can treat pain, depression, and Parkinson’s disease (Heller 1982). D-Pro derivatives can be used for the treatment of diseases such as Alzheimer’s disease, diabetes mellitus, familial amyloid polyneuropathy, scrapie, and Kreuzfeld-Jacob disease (Hertel et al. 2000). Food industry
The manufacturing of pharmaceutical drugs and intermediates Compounds and peptides containing D-AAs are very potent in disease therapy. D-Stereoisomer replacement of His and Cys in the Pro-His-Ser-Cys-Asn peptide (ATN-161) showed as a highly potent inhibitor of invasion and lung colonization in human prostate cancer (Veine et al. 2014). D-AA-based peptide inhibitors interfere with the aggregation of amyloid-β peptides, which are major components of the plaque found in the brains of Alzheimer’s disease patients (Kumar and Sim 2014). D-AA-containing peptides can also inhibit or reduce HIV and atherosclerosis (Navab et al. 2002; Welch et al. 2007). Besides peptides containing D-AAs, compounds synthesized from D-AAs could decrease the amyloid-β levels at concentrations, for example BMS-708163, a γ-secretase Fig. 3 Traditional and new core penicillin and cephalosporin semi-synthetic antibiotics containing D-AAs. The cores of antibiotics are in the dashed boxes. D-PG D-phenylglycine, D-pHPG D-para-hydrophenylglycine
In the food industry, D-AAs could be used for synthesizing intense sweeteners with higher potency than that lacking DAAs. Alitame, a L-Asp-D-Ala dipeptide sweetener, is about ten times more potent (2000× sucrose) than Aspartame (LAsp-L-Phe dipeptide) (Walters 1995). These sweeteners containing D-AAs are not only highly potent but they also have low calorie. As such, they could be used in food products for people who suffer from diabetes (Grenby 1991). Besides, DAAs can also be used as food flavor agents, food additives, nutrition additives, and so on. Cosmetics D-AAs
could improve the skin quality. D-Asp has both an antioxidant and collagen production-promoting effect. D-Glu
Appl Microbiol Biotechnol
can recover barriers and reduce wrinkle formation and skin roughness. D-Ser has the ability to reduce ultraviolet damage. Because of these effects on skin quality, D-AAs of any type, as long as they were D-forms, have been added into an oil-inwater type emulsion skin cosmetics in Japan (Omura and Furukawara 2014).
Enzymatic synthesis for D-AAs The enzymatic synthesis of D-AAs could be performed by hydrolases, oxidoreductases, and D-amino acid aminotransferases. Available hydrolases include D-hydantoinase coupled with N-carbamoyl-D-amino acid amidohydrolase, N-acyl-Damino acid amidohydrolase, D-amino acid amidase, D-aminopeptidase, and D-peptidase. L-Amino acid oxidase and D-amino acid dehydrogenase are the most common oxidoreductases. Considering starting materials, enzymatic methods for the production of D-AAs could be divided into three categories: DL-amino acids, such as DL-amino acids and N-acyl-DLamino acid, synthetic intermediates, such as DL-hydantoin, DLamino acid amides, and prochiral substrates, such as α-keto acids and L-amino acids. In this review, enzymatic methods for D-AA production were classified as follows based on the enzymes involved.
addition to the other D-AAs including D-Trp, D-Phe, D-Val, D-Ala, and D-Met (Bommarius et al. 1998). Although this method has been applied successfully for DAA production in industry, there are three main disadvantages of the method. First, the types of D-AAs made using this synthetic method are limited, second, the enzyme activities of natural D-hydantoinase are often low, and third, the substrates of D-hydantoinase possess low solubility. Some efforts have been made to overcome these disadvantages: (a) optimization of the growth and enzymatic reactions of wild strains such as improving the activity of D-hydantoinase from Ochrobactrum anthropi strain 245 up to 10-fold by considering pH and additions of inducer, ammonium, phosphate, heavy metals, and other ions when setting up reactions (Lee and Kim 1998; Pozo et al. 2002), (b) coexpressing D -hydantoinase with D carbamoylase in Eschericia coli, while deleting the enzymes related to D-AA degradation, such as D-amino acid dehydrogenase and D-serine dehydratase (Grifantini et al. 1998; Turner et al. 2004), and (c) improving enzyme catalysis properties by directed evolution such as enhancing the thermostability of D-hydantoinase by DNA shuffling to make the substrate more soluble (Kim et al. 2000). D-Stereospecific
amidohydrolase
D-Stereospecific D-Hydantoinase
and N-carbamoyl-D-amino acid
amidohydrolase D-Hydantoinase
(EC 3.5.2.2) coupled with N-carbamoyl-Damino acid amidohydrolase (D-carbamoylase, EC 3.5.1.77) is an efficient pathway for producing D-AAs in industry. Production of nonnatural amino acids using this method results in a yield of several thousand tons of product per year. This enzymatic catalysis process utilizes DL-5-substituted hydantoins as starting material, which can be obtained by chemical synthesis, and contains two steps: DL-5-Substituted hydantoins were first hydrolyzed into carbamoyl derivatives by D-hydantoinase and then into corresponding D-AAs by the decarbamoylation of D -carbamoylase (Fig. 4). D Hydantoinase and D-carbamoylase were distributed widely in microorganisms. In some microorganisms, the coding genes of the two enzymes are in cluster, which make it simple to isolate enzymes for the process (Hils et al. 2001). The most significant nonnatural amino acid produced by this method are D-phenylglycine and D-p-hydroxyphenylglycine (Fig. 3), in Fig. 4 Enzymatic route for the production of D-phydroxyphenylglycine from DL-hydroxyphenylhydantoin
amidohydrolases, such as N-acyl-D-amino acid amidohydrolase, D-amino acid amidase, D-aminopeptidase, and D-peptidase, could be used for kinetic resolution of racemic amino acid amides to yield D -AAs. The amidohydrolases start from amino acid amides which are inexpensive intermediates from chemical synthesis. Because these hydrolases only react with D-enantiomers, the theoretical yield is 50 %. If coupled with L-AA amide racemase, the racemic amides could be hydrolyzed to D-AA with a theoretical yield of 100 % (Asano and Yamaguchi 2005). The details of the D-stereospecific amidohydrolase classes are as follows.
N-Acyl-D-amino acid amidohydrolase N-Acyl-D-amino acid amidohydrolase (D-amidohydrolase, EC 3.4.4.14) catalyzes the hydrolysis of N-acyl-D-amino acid to its corresponding D-AA and fatty acid (Wakayama and Moriguchi 2001). This is the most useful and convenient method to produce D-AAs. As shown in Fig. 5, this enzymatic method starts with a DL-amino acid mixture or the N-acyl-DLamino acid directly. D -Stereospecific amidohydrolase
Appl Microbiol Biotechnol Fig. 5 Commercial process of Nacyl-D-amino acid amidohydrolase catalysis for the production of D-amino acid
hydrolyzes the N-acyl-D-amino acid to produce its corresponding D-AA, and N-acyl-D-amino acid is left to be used again for chemical racemization. D-AAs produced by this method include D-Ala, D-Arg, D-Asp, D-Glu, and D-Leu (Wakayama et al. 2003). D-Amidohydrolases are only found in limited microorganisms, and their source determines their substrate specificities (Yano et al. 2011). Many methods, including improving the producer bacteria, optimizing culture conditions, recovering enzymes from cell lysate, and protein engineering, have been used to optimize the enzyme for use on large-scale industrial applications (Hsu et al. 2002; Wakayama et al. 2003; Yano et al. 2011). For example, the residues interacting with the side chain of the substrate were mutated to change the substrate specificity of Alcaligenes D-amidohydrolase.
D-Amino
acid amidase
D-Amino
acid amidase (EC 3.5.1.x) catalyzes the stereospecific hydrolysis of D-amino acid amide to yield D-AA and ammonia. A series of D-amino acid amidases has been isolated and characterized from microbial sources. D-Amino acid amidase from O. anthropi SV3 was the first strict D-stereospecific member of this family, which was found in 1989. It acted on D-amino acid amides containing aromatic or hydrophobic side chains, such as D-phenylalaninamide, D-tyrosinamide, and Dleucinamide (Komeda and Asano 2000). The enzyme has been modified by error-prone PCR to improve the thermostability and catalytic activity, and it has been crystalized (Komeda et al. 2003; Okazaki et al. 2007; Okazaki et al. 2008). D-Amino acid amidases active toward other D-amino acid amides have been discovered, such as D-methioninamide, D-glutaminamide, and D-lysinamide (Komeda and Asano 2008). Except amino acid amides as starting materials, DLtert-leucine nitrile was used as starting material to produce D-tert-Leu by coupling nitrile hydratase from Rhodococcus
Fig. 6 Production of D-amino acids by the kinetic resolution of L-amino acid oxidase
erythropolis and D-amino acid amidase from Variovorax paradoxus (Brandão et al. 2004). D-Aminopeptidase D-Aminopeptidase
(EC 3.4.11.19) catalyzes the hydrolysis of from the N-terminus of peptides; D-Ala is most commonly catalyzed. Asano et al. isolated and characterized the Daminopeptidase from O. anthropi SCRC C1-38, which could act on peptide substrates and amino acid amides to give D-Ala (Asano et al. 1989). However, peptide substrates are preferred other than amino acid amides. By coupling with the αamino-ε-caprolactam racemase, a series of D-AAs could be yield from L-amino acid amide with 100 % yield and >99 % enantiomeric excess (Asano et al. 2009). D-AAs
Alkaline D-peptidase Alkaline D-peptidase (EC 3.4.11.-) is a D-stereospecific endopeptidase that acts on peptides composed of aromatic D-AAs. It hydrolyzes the carboxy-terminal peptide of the D-configuration of the amino acid. The alkaline D-peptidase from Bacillus cereus is active toward (DPhe)3 and (D-Phe)4 to yield D-Phe and (D-Phe)2 (Asano et al. 1996). L-Amino
acid oxidase
L-Amino acid oxidase (L-AAO, EC
1.4.3.2) is an enzyme that catalyzes the deamination of L-amino acids with strict stereospecificity and, therefore, provides a simple method for producing D-AAs from a racemic mixture by kinetic resolution (Fig. 6). The L-AAO from Rhodococcus sp. AIU Z-35-1 was used to produce a wide variety of D-AAs because of its broad substrate specificity (Isobe et al. 2010). Compared to Dhydantoinases and D-aminoacylases, this process can produce
Appl Microbiol Biotechnol Fig. 7 The multienzyme system of D-amino acid transaminase for the production of D-phenylalanine and D-tyrosine from phenylpyruvate and hydroxyphenylpyruvate. The bolded compounds are the starting materials
D-Glu, D-Arg, and D-homoserine.
Although L-AAO has broad substrate specificity and can produce more D-AAs, the theoretical yield is only 50 % at maximum.
D-Amino
acid aminotransferase
D-Amino
acid aminotransferase (D-AAT, EC2.6.1.21) catalyzes the transfer of the amino group from D-alanine onto αketoglutarate to yield D-glutamate. In fact, other D-amino acids could also be produced by D-AATs. For example, by using the D-AAT from Lactobacillus salivarius as the biocatalyst, all the keto acids, including α-ketobutyrate, glyoxylate, indole-3-pyruvate, α-ketovalerate, 3-methyl-2-ketobutyrate, and 4hydroxypenylpyruvate, could be aminated with higher activities than α-ketoglutarate to produce their corresponding DAAs (Kobayashi et al. 2013). Hee-Sung Bea et al. applied the thermostable D-AAT from Bacillus sp. YM-1 coupling with glutamate racemase, glutamate dehydrogenase, and formate dehydrogenase on the production of D-Phe and D-Tyr Fig. 8 Reaction scheme for the gram scale synthesis of Dcyclohexylalanine by mesoDAPDH mutant BC621
from phenylpyruvate and hydroxyphenylpyruvate, respectively (Fig. 7) (Bae et al. 1999). As shown in Fig. 7, α-keto acids produced from D-AAs may be aminated by D-AAT. Therefore, special techniques are needed to drive the transamination reaction to completion. Here, L-aspartate is normally used as the donor, the oxaloacetate formed can spontaneously decarboxylate and thus drive the reaction. D-Amino
acid dehydrogenase
The last class involved in D-AAs synthesis is D-amino acid dehydrogenase ( D -AADH, EC 1.4.99.1). D -AADH is a NADPH-dependent oxidoreductase, which can catalyze the asymmetric reductive amination of α-keto acids to form their corresponding D-AAs. Native D-AADHs are limited for D-AA production because of two reasons. First, D-AADH is not as widespread in nature as L-AADH. Second, D-AADHs that have been discovered are usually membrane-bound proteins,
Appl Microbiol Biotechnol
which limit the potential application of these enzymes in the industry. Recently, we exploited a meso-diaminopilemate dehydrogenase (meso-DAPDH, EC 1.4.1.16) from Symbiobacterium thermophilum IAM14863 to catalyze the amination reactions toward a series of α-keto acids (Gao et al. 2012). Of all the substrates investigated, pyruvic acid was the favorable compound. Meso-DAPDH is a special D-AADH which is a key enzyme in the lysine biosynthetic pathway. The meso-DAPDH from S. thermophilum (StDAPDH) is the first native enzyme found in the family which could be used for D-amino acid production. Thinking of the weak activities of StDAPDH toward bulk substrates such as phenylpyruvic acid, sitesaturation mutagenesis and site-combination mutagenesis were performed to improve its specific activity toward phenylpyruvic acid. The mutant, H227V, had a 37.1-fold improvement in specific activity compared to the wild type (Gao et al. 2013). Although other meso-DAPDHs from other origins are not active in D-AA synthesis, using protein engineering, active mutants with high D-stereospecificities and D-AADH activities have been obtained. For example, the mutant BC621, derived from the meso-DAPDH of Corynebacterium glutamicum ATCC13032, had high activity toward cyclohexylpyruvate with strict D-seterospcificity (Fig. 8) (Vedha-Peters et al. 2006). Recently, meso-DAPDHs have become more interesting targets of study for development and application (Akita et al. 2014; Hanson et al. 2013).
Concluding remarks Compared to L-AAs, D-AAs are not abundant in nature, but because of the ease of synthesis and their activity in many processes, they can be applied in pharmaceutical industry, food industry, and cosmetics. In 2011, the Global Industry Analyst, Inc., released a global research report on the market of D-AAs predicting that the global market for D-AAs will reach 3.7 billion dollars by 2017 (http://www.strategyr.com/ D_Amino_Acids_Market_Report.asp). The applications of DAAs in the pharmaceutical industry for the synthesis of pharmaceutical drugs, intermediates and semisynthetic antibiotics, and their potential as research tools in drug discovery and immune response are the major drives of this increase in revenue. Therefore, finding new, efficient, and environmentally friendly synthesis methods for D-AAs are necessary. Among all the aforementioned methods, utilizing the D-AADH method is the most attractive. It can produce DAAs by a one-step reaction with 100 % maximum theoretical yield. Additionally, the only by-product of the reaction is water, and because of this, the environment is protected and atom economics have been taken into consideration. Therefore, more focuses should be placed on the D-AADH method.
Efforts may be made from two aspects: (a) exploiting more with high activities by gene mining, protein engineering, and so on; and (b) resolving the supply of cofactor with low cost such as searching for efficient and low-cost cofactor cycle system, changing the enzyme from NADPHdependent to NADH-dependent.
D-AADHs
Acknowledgments This work was financially supported by the National Science Foundation of China (grant no. 21402109).
References Abe H, Park J, Fukumoto Y, Fujita E, Tanaka T, Washio T, Otsuka S, Shimizu T, Watanabe K (1999) Occurrence of D-amino acids in fish sauces and other fermented fish products. Fish Sci 65:637–641 Akita H, Suzuki H, Doi K, Ohshima T (2014) Efficient synthesis of Dbranched-chain amino acids and their labeled compounds with stable isotopes using D-amino acid dehydrogenase. Appl Microbiol Biotechnol 98:1135–1143 Asano Y, Yamaguchi S (2005) Dynamic kinetic resolution of amino acid amide catalyzed by D-aminopeptidase and α-amino-ε-caprolactam racemase. J Am Chem Soc 127:7696–7697 Asano Y, Nakazawa A, Kato Y, Kondo K (1989) Properties of a novel Dstereospecific aminopeptidase from Ochrobactrum anthropi. J Biol Chem 264:14233–14239 Asano Y, Ito H, Dairi T, Kato Y (1996) An alkaline D-stereospecific endopeptidase with β-lactamase activity from Bacillus cereus. J Biol Chem 271:30256–30262 Asano Y, Komeda H, Flickinger MC (2009) D-Aminopeptidase and alkaline D-peptidase. In: Encyclopedia of Industrial Biotechnology, 10.1002/9780470054581.eib251. John Wiley & Sons, Inc. doi:10. 1002/9780470054581.eib251 Bae H-S, Lee S-G, Hong S-P, Kwak M-S, Esaki N, Soda K, Sung M-H (1999) Production of aromatic D-amino acids from α-keto acids and ammonia by coupling of four enzyme reactions. J Mol Catal B Enzym 6:241–247 Bodanszky M, Perlman D (1969) Peptide antibiotics. Science 163:352– 358 Bommarius AS, Schwarm M, Drauz K (1998) Biocatalysis to amino acidbased chiral pharmaceuticals-examples and perspectives. J Mol Catal B Enzym 5:1–11 Brandão PFB, Verseck S, Syldatk C (2004) Bioconversion of D,L-tertleucine nitrile to D-tert-leucine by recombinant cells expressing nitrile hydratase and D-selective amidase. Eng Life Sci 4:547–556 Brückner H, Hausch M (1989) Gas chromatographic detection of D-amino acids as common constituents of fermented foods. Chromatographia 28:487–492 Burkhart BM, Gassman RM, Langs DA, Pangborn WA, Duax WL, Pletnev V (1999) Gramicidin D-conformation, dynamics and membrane ion transport. Pept Sci 51:129–144 Fotheringham, Taylor PPAH, Ton JLP (1998) Preparation of D-amino acids by direct fermentative means. United States Patent 5728555 Friedman M (1999) Chemistry, nutrition, and microbiology of D-amino acids. J Agric Food Chem 47:3457–3479 Fujii N (2002) D-Amino acids in living higher organisms. Orig Life Evol Biosph 32:103–127 Gao X, Chen X, Liu W, Feng J, Wu Q, Hua L, Zhu D (2012) A novel meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum: overexpression, characterization, and potential for D-amino acid synthesis. Appl Environ Microbiol 78:8595–8600
Appl Microbiol Biotechnol Gao X, Huang F, Feng J, Chen X, Zhang H, Wang Z, Wu Q, Zhu D (2013) Engineering the meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum by site saturation mutagenesis for D-phenylalanine synthesis. Appl Environ Microbiol 79:5078–5081 Gillman KW, Starrett JE, Parker MF, Xie K, Bronson JJ, Marcin LR, McElhone KE, Bergstrom CP, Mate RA, Williams R, Meredith JE, Burton CR, Barten DM, Toyn JH, Roberts SB, Lentz KA, Houston JG, Zaczek R, Albright CF, Decicco CP, Macor JE, Olson RE (2010) Discovery and evaluation of BMS-708163, a potent, selective and orally bioavailable γ-secretase inhibitor. Med Chem Lett 1:120–124 Grenby TH (1991) Intense sweeteners for the food industry: an overview. Trends Food Sci Technol 2:2–6 Grifantini R, Galli G, Carpani G, Pratesi C, Frascotti G, Grandi G (1998) Efficient conversion of 5-substituted hydantoins to D-α-amino acids using recombinant Escherichia coli strains. Microbiology 144:947– 954 Hanson RL, Johnston RM, Goldberg SL, Parker WL, Goswami A (2013) Enzymatic preparation of an R-amino acid intermediate for a γsecretase inhibitor. Org Process Res Dev 17:693–700 Heijenoort JV (2001) Recent advances in the formation of the bacterial peptidoglycan monomer unit. Nat Prod Rep 18:503–519 Heller B (1982) D-phenylalanine treatment. Unites States Patent 4355044 Hertel C, Hoffmann T, Jakob-Roetne R, Norcross RD (2000) For treatment of diseases associated with amyloidosis, such as alzheimer’s disease, diabetes mellitus, familial amyloid polyneuropathy, scrapie, and kreuzfeld-jacob disease. United States Patent 6103910 Hils M, Münch P, Altenbuchner J, Syldatk C, Mattes R (2001) Cloning and characterization of genes from Agrobacterium sp. IP I-671 involved in hydantoin degradation. Appl Microbiol Biotechnol 57: 680–688 Hopkins DW, Ferguson KE (1994) Substrate induced respiration in soil amended with different amino acid isomers. Appl Soil Ecol 1:75–81 Hsu C-S, Lai W-L, Chang W-W, Liaw S-H, Tsai Y-C (2002) Structuralbased mutational analysis of D-aminoacylase from Alcaligenes faecalis DA1. Protein Sci 11:2545–2550 Isobe K, Tamauchi H, Fuhshuku K-i, Nagasawa S, Asano Y (2010) A simple enzymatic method for production of a wide variety of Damino acids using L-amino acid oxidase from Rhodococcus sp. AIU Z-35-1. Enzyme research 2010 Kato S, Ishihara T, Hemmi H, Kobayashi H, Yoshimura T (2011) Alterations in D-amino acid concentrations and microbial community structures during the fermentation of red and white wines. J Biosci Bioeng 111:104–108 Katz E, Demain AL (1977) The peptide antibiotics of Bacillus: chemistry, biogenesis, and possible functions. Bacteriol Rev 41:449–474 Kim GJ, Cheon YH, Kim H-S (2000) Directed evolution of a novel Ncarbamoylase/D-hydantoinase fusion enzyme for functional expression with enhanced stability. Biotechnol Bioeng 68:211–217 Kobayashi J, Shimizu Y, Mutaguchi Y, Doi K, Ohshima T (2013) Characterization of D -amino acid aminotransferase from Lactobacillus salivarius. J Mol Catal B Enzym 94:15–22 Komeda H, Asano Y (2000) Gene cloning, nucleotide sequencing, and purification and characterization of the D-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3. Eur J Biochem 267: 2028–2035 Komeda H, Asano Y (2008) A novel D-stereoselective amino acid amidase from Brevibacterium iodinum: gene cloning, expression and characterization. Enzyme Microb Technol 43:276–283 Komeda H, Ishikawa N, Asano Y (2003) Enhancement of the thermostability and catalytic activity of D-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3 by directed evolution. J Mol Catal B Enzym 21:283–290 Kumar J, Sim V (2014) D-Amino acid-based peptide inhibitors as early or preventative therapy in alzheimer disease. Prion 8:119–124
Lee D-C, Kim H-S (1998) Optimization of a heterogeneous reaction system for the production of optically active D-amino acids using thermostable D-hydantoinase. Biotechnol Bioeng 60:729–738 Martín J, Casqueiro J, Kosalková K, Marcos A, Gutiérrez AFFS (1999) Penicillin and cephalosporin biosynthesis: mechanism of carbon catabolite regulation of penicillin production. Antonie Leeuwenhoek 75:21–31 Navab M, Anantharamaiah GM, Hama S, Garber DW, Chaddha M, Hough G, Lallone R, Fogelman AM (2002) Oral administration of an Apo A-I mimetic peptide synthesized from D-amino acids dramatically reduces atherosclerosis in mice independent of plasma cholesterol. Circulation 105:290–292 Okazaki S, Suzuki A, Komeda H, Yamaguchi S, Asano Y, Yamane T (2007) Crystal structure and functional characterization of a D-stereospecific amino acid amidase from Ochrobactrum anthropi SV3, a new member of the penicillin-recognizing proteins. J Mol Biol 368:79–91 Okazaki S, Suzuki A, Mizushima T, Komeda H, Asano Y, Yamane T (2008) Structures of D-amino acid amidase complexed with L-phenylalanine and with L-phenylalanine amide: insight into the D-stereospecificity of D-amino acid amidase from Ochrobactrum anthropi SV3. Acta Crystallogr D 64:331–334 Omura T, Furukawara T (2014) Oil-in-water type emulsion skin cosmetic. United States Patent 8607750B2 Pozo C, Rodelas B, de la Escalera S, González-López J (2002) d, lHydantoinase activity of an Ochrobactrum anthropi strain. J Appl Microbiol 92:1028–1034 Radkov A, Moe L (2014) Bacterial synthesis of D-amino acids. Appl Microbiol Biotechnol 98:1–12 Reich E (1963) Biochemistry of actinomycins. Cancer Res 23:1428– 1441 Robinson T (1976) D-Amino acids in higher plants. Life Sci 19:1097– 1102 Sanchez CJ, Akers KS, Romano DR, Woodbury RL, Hardy SK, Murray CK, Wenke JC (2014) D-Amino acids enhance the activity of antimicrobials against biofilms of clinical wound isolates of Staphylococcus aureus and Pseudomonas aeruginosa. Antimicrob Agents Chemother 58:4353–4361 Svennerstam H, Ganeteg U, Bellini C, Näsholm T (2007) Comprehensive screening of Arabidopsis mutants suggests the lysine histidine transporter 1 to be involved in plant uptake of amino acids. Plant Physiol 143:1853–1860 Turner RJ, Aikens J, Royer S, DeFilippi L, Yap A, Holzle D, Somers N, Fotheringham IG (2004) D-Amino acid tolerant hosts for Dhydantoinase whole cell biocatalysts. Eng Life Sci 4:517–520 Vedha-Peters K, Gunawardana M, Rozzell JD, Novick SJ (2006) Creation of a broad-range and highly stereoselective D-amino acid dehydrogenase for the one-step synthesis of D-amino acids. J Am Chem Soc 128:10923–10929 Veine D, Yao H, Stafford D, Fay K, Livant D (2014) A D-amino acid containing peptide as a potent, noncovalent inhibitor of α5β1 integrin in human prostate cancer invasion and lung colonization. Clin Exp Metastasis 31:379–393 Vranova V, Zahradnickova H, Janous D, Skene K, Matharu A, Rejsek K, Formanek P (2012) The significance of D-amino acids in soil, fate and utilization by microbes and plants: review and identification of knowledge gaps. Plant Soil 354:21–39 Wakayama M, Moriguchi M (2001) Comparative biochemistry of bacterial N-acyl-D-amino acid amidohydrolase. J Mol Catal B Enzym 12: 15–25 Wakayama M, Yoshimune K, Hirose Y, Moriguchi M (2003) Production of D-amino acids by N-acyl-D-amino acid amidohydrolase and its structure and function. J Mol Catal B Enzym 23:71–85 Walters DE (1995) Using models to understand and design sweeteners. J Chem Educ 72:680–683
Appl Microbiol Biotechnol Welch BD, VanDemark AP, Heroux A, Hill CP, Kay MS (2007) Potent Dpeptide inhibitors of HIV-1 entry. Proc Natl Acad Sci U S A 104: 16828–16833 Yang H, Zheng G, Peng X, Qiang B, Yuan J (2003) D-Amino acids and DTyr-tRNATyr deacylase: stereospecificity of the translation machine revisited. FEBS Lett 552:95–98
Yano S, Haruta H, Ikeda T, Kikuchi T, Murakami M, Moriguchi M, Wakayama M (2011) Engineering the substrate specificity of AlcaligenesD-aminoacylase useful for the production of D-amino acids by optical resolution. J Chromatogr B 879:3247–3252 Zhang J, Xu Y, Li R (2013) Preparation method of aspoxicillin. China Patent 103333180A
MINI-REVIEW
Distribution, industrial applications, and enzymatic synthesis of D-amino acids Xiuzhen Gao & Qinyuan Ma & Hailiang Zhu
Received: 17 January 2015 / Revised: 22 February 2015 / Accepted: 23 February 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract D-Amino acids exist widely in microbes, plants, animals, and food and can be applied in pharmaceutical, food, and cosmetics. Because of their widespread applications in industry, D-amino acids have recently received more and more attention. Enzymes including D-hydantoinase, N-acyl-D-amino acid amidohydrolase, D-amino acid amidase, D-aminopeptidase, D-peptidase, L-amino acid oxidase, D-amino acid aminotransferase, and D-amino acid dehydrogenase can be used for D-amino acids synthesis by kinetic resolution or asymmetric amination. In this review, the distribution, industrial applications, and enzymatic synthesis methods are summarized. And, among all the current enzymatic methods, D-amino acid dehydrogenase method not only produces D-amino acid by a one-step reaction but also takes environment and atom economics into consideration; therefore, it is deserved to be paid more attention. Keywords D-Amino acids . Enzymatic synthesis . Asymmetric amination . D-Amino acid dehydrogenase
widely in nature, for example, the cell walls of Gram-positive bacteria and some peptide antibiotics contain D-AAs. The presence of D-AAs prevents digestions by proteolytic enzymes. Because of this, D-AAs have become important intermediates in pharmaceutical, food, and other various industries. Therefore, production of D-AAs is of great interest. D-AAs (Fig. 1) can be produced by chemical synthesis, fermentation, and enzymatic transformation. Chemical synthesis yields D-AAs by chiral resolution of DL-amino acids or by asymmetric synthesis from chiral or prochiral starting materials. Because of racemization of D-AAs, high cost and low yields are the major disadvantages of chemical methods. Modified microorganisms without functional D-AA deaminases could be used for D-AA production (Fotheringham et al. 1998), but because of complex enzyme systems and metabolic networks, it is difficult to achieve high optical purity and productivity. Enzymatic methods can yield D-AAs with high optical purity, high productivity, and green process and are therefore ideal for the industrial manufacturing of DAAs. In this review, we introduce and compare all of the enzymatic methods used for D-AA production.
Introduction It is well known that proteins typically incorporate only Lamino acids into their sequences. For many years, few D-amino acids (D-AA) were found, and therefore, D-AAs were considered as unnatural amino acids. With the development of analytical technologies, D-AAs were found to be distributed X. Gao (*) : H. Zhu School of Life Science, Shandong University of Technology, Zibo 255049, China e-mail: [email protected] Q. Ma Technology Center, Weifang Ensign Industry Co., Ltd, Changle 262499, China
Distributions of D-AAs D-AAs
in microbes
The most known distribution of D-AAs is in extracellular or periplasmic polymeric biomolecules of bacterial cell walls, including peptidoglycan, teichoic acids, and poly-γglutamate (Bodanszky and Perlman 1969; Radkov and Moe 2014) (Fig. 2). The presence of D-AAs in Gram-positive bacterial walls prevents digestion by proteolytic enzymes (Yang et al. 2003). D-AAs have appeared in peptide antibiotics synthesized by bacteria and fungi, including gramicidin D (D-Leu and D-Val)
Appl Microbiol Biotechnol Table 1
Fig. 1 Chemical structure of natural D-AA. R group is variant
(Heijenoort 2001), gramicidin S (D-Phe) (Burkhart et al. 1999), polymyxins (D-Phe, D-Leu, D-Ser) (Martín et al. 1999), penicillin G (D-Val) (Katz and Demain 1977), cephalosporin (D-Ala) (Katz and Demain 1977), and actinomycins (D-Val, D-allo-Ile) (Reich 1963). D-AAs
in animals
D-AA
Type
Species
Physical meaning
D-Asp
Protein Protein Peptide Peptide Peptide Peptide Peptide Free
Human, cattle, mouse, rat Human Frog Frog Snail Snail Spider Human, mouse, rat
Aging Aging Opioid peptides Opioid peptides Neuropeptides Neuropeptides Neuropeptides Development
D-Ser D-Ala D-Met D-Phe D-Asn D-Ser D-Asp, D-Ser
in plants and animals
D-Ala
is the most widely reported D-AA in plants, for example, Helianthus annuus and D-Trp derivatives are the second most widely reported in plants (Robinson 1976). D-AAs are present as free or conjugated forms in the C 3 , C 4 , or crassulacean acid metabolism of plants, such as D-Ala, DSer, D-Leu, and conjugated D-amino-n-butyric acid (Vranova et al. 2012). The percentage of D-AAs in various plant parts (seeds, fruits, leaves, etc.) was around 1.5 % of the total amino acid pool (Vranova et al. 2012). Research studies have demonstrated that plants can absorb D-AAs from soil through plant roots (Hopkins and Ferguson 1994; Svennerstam et al. 2007). D-AAs also have been found in mammalian and human tissues in the form of free amino acids, and within peptides and proteins. The physiological functions of many D-AAs have been recently determined (Table 1) (Fujii 2002). D-AAs
D-AAs
in foods
D-AAs,
such as D-Ala, D-Asp, and D-Glu, are present in the raw milk of ruminants (cows, goats, and sheep) and in many
Fig. 2 Bacterial peptidoglycan containing D-AAs (Fujii 2002). a DAP-type peptidoglycan. b Lystype peptidoglycan
fermented foods such as cheese, wine, yogurt, and fish products. These D-AAs may be derived from the raw materials used to make food or microorganisms during fermentation (Abe et al. 1999; Brückner and Hausch 1989; Friedman 1999; Kato et al. 2011).
Industrial applications of D-AAs Pharmaceutical industry It is well known that D-AAs are incorporated or assimilated slower than the L-isomers, so the half-life of antibiotics and drugs containing D-AAs is longer. Additionally, recent research reported that D-AAs can enhance the activity of antimicrobials against biofilms of clinical wound. Within wound, biofilms are the main existence form of wound microorganisms and associate with chronic infections (Sanchez et al. 2014). The discovery is another strong and powerful evidence that D-AAs are of great value in pharmaceutical synthesis.
Appl Microbiol Biotechnol
Synthesis of semi-synthetic antibiotics D-AAs
are commonly used in semi-synthetic antibiotics synthesis, including traditional and new penicillin core and cephalosporin core antibiotics (Fig. 3). For example, using D-Asp and amoxicillin as raw materials, aspoxicillin can be chemically synthesized (Zhang et al. 2013). Aspoxicillin is popular because of its long half-life and low serum protein-binding characteristics.
inhibitor with (R)-5,5,5-trifluronorvaline as key building block (Gillman et al. 2010; Hanson et al. 2013). Free D-AAs are of great medicinal value. D-Phe alone can treat pain, depression, and Parkinson’s disease (Heller 1982). D-Pro derivatives can be used for the treatment of diseases such as Alzheimer’s disease, diabetes mellitus, familial amyloid polyneuropathy, scrapie, and Kreuzfeld-Jacob disease (Hertel et al. 2000). Food industry
The manufacturing of pharmaceutical drugs and intermediates Compounds and peptides containing D-AAs are very potent in disease therapy. D-Stereoisomer replacement of His and Cys in the Pro-His-Ser-Cys-Asn peptide (ATN-161) showed as a highly potent inhibitor of invasion and lung colonization in human prostate cancer (Veine et al. 2014). D-AA-based peptide inhibitors interfere with the aggregation of amyloid-β peptides, which are major components of the plaque found in the brains of Alzheimer’s disease patients (Kumar and Sim 2014). D-AA-containing peptides can also inhibit or reduce HIV and atherosclerosis (Navab et al. 2002; Welch et al. 2007). Besides peptides containing D-AAs, compounds synthesized from D-AAs could decrease the amyloid-β levels at concentrations, for example BMS-708163, a γ-secretase Fig. 3 Traditional and new core penicillin and cephalosporin semi-synthetic antibiotics containing D-AAs. The cores of antibiotics are in the dashed boxes. D-PG D-phenylglycine, D-pHPG D-para-hydrophenylglycine
In the food industry, D-AAs could be used for synthesizing intense sweeteners with higher potency than that lacking DAAs. Alitame, a L-Asp-D-Ala dipeptide sweetener, is about ten times more potent (2000× sucrose) than Aspartame (LAsp-L-Phe dipeptide) (Walters 1995). These sweeteners containing D-AAs are not only highly potent but they also have low calorie. As such, they could be used in food products for people who suffer from diabetes (Grenby 1991). Besides, DAAs can also be used as food flavor agents, food additives, nutrition additives, and so on. Cosmetics D-AAs
could improve the skin quality. D-Asp has both an antioxidant and collagen production-promoting effect. D-Glu
Appl Microbiol Biotechnol
can recover barriers and reduce wrinkle formation and skin roughness. D-Ser has the ability to reduce ultraviolet damage. Because of these effects on skin quality, D-AAs of any type, as long as they were D-forms, have been added into an oil-inwater type emulsion skin cosmetics in Japan (Omura and Furukawara 2014).
Enzymatic synthesis for D-AAs The enzymatic synthesis of D-AAs could be performed by hydrolases, oxidoreductases, and D-amino acid aminotransferases. Available hydrolases include D-hydantoinase coupled with N-carbamoyl-D-amino acid amidohydrolase, N-acyl-Damino acid amidohydrolase, D-amino acid amidase, D-aminopeptidase, and D-peptidase. L-Amino acid oxidase and D-amino acid dehydrogenase are the most common oxidoreductases. Considering starting materials, enzymatic methods for the production of D-AAs could be divided into three categories: DL-amino acids, such as DL-amino acids and N-acyl-DLamino acid, synthetic intermediates, such as DL-hydantoin, DLamino acid amides, and prochiral substrates, such as α-keto acids and L-amino acids. In this review, enzymatic methods for D-AA production were classified as follows based on the enzymes involved.
addition to the other D-AAs including D-Trp, D-Phe, D-Val, D-Ala, and D-Met (Bommarius et al. 1998). Although this method has been applied successfully for DAA production in industry, there are three main disadvantages of the method. First, the types of D-AAs made using this synthetic method are limited, second, the enzyme activities of natural D-hydantoinase are often low, and third, the substrates of D-hydantoinase possess low solubility. Some efforts have been made to overcome these disadvantages: (a) optimization of the growth and enzymatic reactions of wild strains such as improving the activity of D-hydantoinase from Ochrobactrum anthropi strain 245 up to 10-fold by considering pH and additions of inducer, ammonium, phosphate, heavy metals, and other ions when setting up reactions (Lee and Kim 1998; Pozo et al. 2002), (b) coexpressing D -hydantoinase with D carbamoylase in Eschericia coli, while deleting the enzymes related to D-AA degradation, such as D-amino acid dehydrogenase and D-serine dehydratase (Grifantini et al. 1998; Turner et al. 2004), and (c) improving enzyme catalysis properties by directed evolution such as enhancing the thermostability of D-hydantoinase by DNA shuffling to make the substrate more soluble (Kim et al. 2000). D-Stereospecific
amidohydrolase
D-Stereospecific D-Hydantoinase
and N-carbamoyl-D-amino acid
amidohydrolase D-Hydantoinase
(EC 3.5.2.2) coupled with N-carbamoyl-Damino acid amidohydrolase (D-carbamoylase, EC 3.5.1.77) is an efficient pathway for producing D-AAs in industry. Production of nonnatural amino acids using this method results in a yield of several thousand tons of product per year. This enzymatic catalysis process utilizes DL-5-substituted hydantoins as starting material, which can be obtained by chemical synthesis, and contains two steps: DL-5-Substituted hydantoins were first hydrolyzed into carbamoyl derivatives by D-hydantoinase and then into corresponding D-AAs by the decarbamoylation of D -carbamoylase (Fig. 4). D Hydantoinase and D-carbamoylase were distributed widely in microorganisms. In some microorganisms, the coding genes of the two enzymes are in cluster, which make it simple to isolate enzymes for the process (Hils et al. 2001). The most significant nonnatural amino acid produced by this method are D-phenylglycine and D-p-hydroxyphenylglycine (Fig. 3), in Fig. 4 Enzymatic route for the production of D-phydroxyphenylglycine from DL-hydroxyphenylhydantoin
amidohydrolases, such as N-acyl-D-amino acid amidohydrolase, D-amino acid amidase, D-aminopeptidase, and D-peptidase, could be used for kinetic resolution of racemic amino acid amides to yield D -AAs. The amidohydrolases start from amino acid amides which are inexpensive intermediates from chemical synthesis. Because these hydrolases only react with D-enantiomers, the theoretical yield is 50 %. If coupled with L-AA amide racemase, the racemic amides could be hydrolyzed to D-AA with a theoretical yield of 100 % (Asano and Yamaguchi 2005). The details of the D-stereospecific amidohydrolase classes are as follows.
N-Acyl-D-amino acid amidohydrolase N-Acyl-D-amino acid amidohydrolase (D-amidohydrolase, EC 3.4.4.14) catalyzes the hydrolysis of N-acyl-D-amino acid to its corresponding D-AA and fatty acid (Wakayama and Moriguchi 2001). This is the most useful and convenient method to produce D-AAs. As shown in Fig. 5, this enzymatic method starts with a DL-amino acid mixture or the N-acyl-DLamino acid directly. D -Stereospecific amidohydrolase
Appl Microbiol Biotechnol Fig. 5 Commercial process of Nacyl-D-amino acid amidohydrolase catalysis for the production of D-amino acid
hydrolyzes the N-acyl-D-amino acid to produce its corresponding D-AA, and N-acyl-D-amino acid is left to be used again for chemical racemization. D-AAs produced by this method include D-Ala, D-Arg, D-Asp, D-Glu, and D-Leu (Wakayama et al. 2003). D-Amidohydrolases are only found in limited microorganisms, and their source determines their substrate specificities (Yano et al. 2011). Many methods, including improving the producer bacteria, optimizing culture conditions, recovering enzymes from cell lysate, and protein engineering, have been used to optimize the enzyme for use on large-scale industrial applications (Hsu et al. 2002; Wakayama et al. 2003; Yano et al. 2011). For example, the residues interacting with the side chain of the substrate were mutated to change the substrate specificity of Alcaligenes D-amidohydrolase.
D-Amino
acid amidase
D-Amino
acid amidase (EC 3.5.1.x) catalyzes the stereospecific hydrolysis of D-amino acid amide to yield D-AA and ammonia. A series of D-amino acid amidases has been isolated and characterized from microbial sources. D-Amino acid amidase from O. anthropi SV3 was the first strict D-stereospecific member of this family, which was found in 1989. It acted on D-amino acid amides containing aromatic or hydrophobic side chains, such as D-phenylalaninamide, D-tyrosinamide, and Dleucinamide (Komeda and Asano 2000). The enzyme has been modified by error-prone PCR to improve the thermostability and catalytic activity, and it has been crystalized (Komeda et al. 2003; Okazaki et al. 2007; Okazaki et al. 2008). D-Amino acid amidases active toward other D-amino acid amides have been discovered, such as D-methioninamide, D-glutaminamide, and D-lysinamide (Komeda and Asano 2008). Except amino acid amides as starting materials, DLtert-leucine nitrile was used as starting material to produce D-tert-Leu by coupling nitrile hydratase from Rhodococcus
Fig. 6 Production of D-amino acids by the kinetic resolution of L-amino acid oxidase
erythropolis and D-amino acid amidase from Variovorax paradoxus (Brandão et al. 2004). D-Aminopeptidase D-Aminopeptidase
(EC 3.4.11.19) catalyzes the hydrolysis of from the N-terminus of peptides; D-Ala is most commonly catalyzed. Asano et al. isolated and characterized the Daminopeptidase from O. anthropi SCRC C1-38, which could act on peptide substrates and amino acid amides to give D-Ala (Asano et al. 1989). However, peptide substrates are preferred other than amino acid amides. By coupling with the αamino-ε-caprolactam racemase, a series of D-AAs could be yield from L-amino acid amide with 100 % yield and >99 % enantiomeric excess (Asano et al. 2009). D-AAs
Alkaline D-peptidase Alkaline D-peptidase (EC 3.4.11.-) is a D-stereospecific endopeptidase that acts on peptides composed of aromatic D-AAs. It hydrolyzes the carboxy-terminal peptide of the D-configuration of the amino acid. The alkaline D-peptidase from Bacillus cereus is active toward (DPhe)3 and (D-Phe)4 to yield D-Phe and (D-Phe)2 (Asano et al. 1996). L-Amino
acid oxidase
L-Amino acid oxidase (L-AAO, EC
1.4.3.2) is an enzyme that catalyzes the deamination of L-amino acids with strict stereospecificity and, therefore, provides a simple method for producing D-AAs from a racemic mixture by kinetic resolution (Fig. 6). The L-AAO from Rhodococcus sp. AIU Z-35-1 was used to produce a wide variety of D-AAs because of its broad substrate specificity (Isobe et al. 2010). Compared to Dhydantoinases and D-aminoacylases, this process can produce
Appl Microbiol Biotechnol Fig. 7 The multienzyme system of D-amino acid transaminase for the production of D-phenylalanine and D-tyrosine from phenylpyruvate and hydroxyphenylpyruvate. The bolded compounds are the starting materials
D-Glu, D-Arg, and D-homoserine.
Although L-AAO has broad substrate specificity and can produce more D-AAs, the theoretical yield is only 50 % at maximum.
D-Amino
acid aminotransferase
D-Amino
acid aminotransferase (D-AAT, EC2.6.1.21) catalyzes the transfer of the amino group from D-alanine onto αketoglutarate to yield D-glutamate. In fact, other D-amino acids could also be produced by D-AATs. For example, by using the D-AAT from Lactobacillus salivarius as the biocatalyst, all the keto acids, including α-ketobutyrate, glyoxylate, indole-3-pyruvate, α-ketovalerate, 3-methyl-2-ketobutyrate, and 4hydroxypenylpyruvate, could be aminated with higher activities than α-ketoglutarate to produce their corresponding DAAs (Kobayashi et al. 2013). Hee-Sung Bea et al. applied the thermostable D-AAT from Bacillus sp. YM-1 coupling with glutamate racemase, glutamate dehydrogenase, and formate dehydrogenase on the production of D-Phe and D-Tyr Fig. 8 Reaction scheme for the gram scale synthesis of Dcyclohexylalanine by mesoDAPDH mutant BC621
from phenylpyruvate and hydroxyphenylpyruvate, respectively (Fig. 7) (Bae et al. 1999). As shown in Fig. 7, α-keto acids produced from D-AAs may be aminated by D-AAT. Therefore, special techniques are needed to drive the transamination reaction to completion. Here, L-aspartate is normally used as the donor, the oxaloacetate formed can spontaneously decarboxylate and thus drive the reaction. D-Amino
acid dehydrogenase
The last class involved in D-AAs synthesis is D-amino acid dehydrogenase ( D -AADH, EC 1.4.99.1). D -AADH is a NADPH-dependent oxidoreductase, which can catalyze the asymmetric reductive amination of α-keto acids to form their corresponding D-AAs. Native D-AADHs are limited for D-AA production because of two reasons. First, D-AADH is not as widespread in nature as L-AADH. Second, D-AADHs that have been discovered are usually membrane-bound proteins,
Appl Microbiol Biotechnol
which limit the potential application of these enzymes in the industry. Recently, we exploited a meso-diaminopilemate dehydrogenase (meso-DAPDH, EC 1.4.1.16) from Symbiobacterium thermophilum IAM14863 to catalyze the amination reactions toward a series of α-keto acids (Gao et al. 2012). Of all the substrates investigated, pyruvic acid was the favorable compound. Meso-DAPDH is a special D-AADH which is a key enzyme in the lysine biosynthetic pathway. The meso-DAPDH from S. thermophilum (StDAPDH) is the first native enzyme found in the family which could be used for D-amino acid production. Thinking of the weak activities of StDAPDH toward bulk substrates such as phenylpyruvic acid, sitesaturation mutagenesis and site-combination mutagenesis were performed to improve its specific activity toward phenylpyruvic acid. The mutant, H227V, had a 37.1-fold improvement in specific activity compared to the wild type (Gao et al. 2013). Although other meso-DAPDHs from other origins are not active in D-AA synthesis, using protein engineering, active mutants with high D-stereospecificities and D-AADH activities have been obtained. For example, the mutant BC621, derived from the meso-DAPDH of Corynebacterium glutamicum ATCC13032, had high activity toward cyclohexylpyruvate with strict D-seterospcificity (Fig. 8) (Vedha-Peters et al. 2006). Recently, meso-DAPDHs have become more interesting targets of study for development and application (Akita et al. 2014; Hanson et al. 2013).
Concluding remarks Compared to L-AAs, D-AAs are not abundant in nature, but because of the ease of synthesis and their activity in many processes, they can be applied in pharmaceutical industry, food industry, and cosmetics. In 2011, the Global Industry Analyst, Inc., released a global research report on the market of D-AAs predicting that the global market for D-AAs will reach 3.7 billion dollars by 2017 (http://www.strategyr.com/ D_Amino_Acids_Market_Report.asp). The applications of DAAs in the pharmaceutical industry for the synthesis of pharmaceutical drugs, intermediates and semisynthetic antibiotics, and their potential as research tools in drug discovery and immune response are the major drives of this increase in revenue. Therefore, finding new, efficient, and environmentally friendly synthesis methods for D-AAs are necessary. Among all the aforementioned methods, utilizing the D-AADH method is the most attractive. It can produce DAAs by a one-step reaction with 100 % maximum theoretical yield. Additionally, the only by-product of the reaction is water, and because of this, the environment is protected and atom economics have been taken into consideration. Therefore, more focuses should be placed on the D-AADH method.
Efforts may be made from two aspects: (a) exploiting more with high activities by gene mining, protein engineering, and so on; and (b) resolving the supply of cofactor with low cost such as searching for efficient and low-cost cofactor cycle system, changing the enzyme from NADPHdependent to NADH-dependent.
D-AADHs
Acknowledgments This work was financially supported by the National Science Foundation of China (grant no. 21402109).
References Abe H, Park J, Fukumoto Y, Fujita E, Tanaka T, Washio T, Otsuka S, Shimizu T, Watanabe K (1999) Occurrence of D-amino acids in fish sauces and other fermented fish products. Fish Sci 65:637–641 Akita H, Suzuki H, Doi K, Ohshima T (2014) Efficient synthesis of Dbranched-chain amino acids and their labeled compounds with stable isotopes using D-amino acid dehydrogenase. Appl Microbiol Biotechnol 98:1135–1143 Asano Y, Yamaguchi S (2005) Dynamic kinetic resolution of amino acid amide catalyzed by D-aminopeptidase and α-amino-ε-caprolactam racemase. J Am Chem Soc 127:7696–7697 Asano Y, Nakazawa A, Kato Y, Kondo K (1989) Properties of a novel Dstereospecific aminopeptidase from Ochrobactrum anthropi. J Biol Chem 264:14233–14239 Asano Y, Ito H, Dairi T, Kato Y (1996) An alkaline D-stereospecific endopeptidase with β-lactamase activity from Bacillus cereus. J Biol Chem 271:30256–30262 Asano Y, Komeda H, Flickinger MC (2009) D-Aminopeptidase and alkaline D-peptidase. In: Encyclopedia of Industrial Biotechnology, 10.1002/9780470054581.eib251. John Wiley & Sons, Inc. doi:10. 1002/9780470054581.eib251 Bae H-S, Lee S-G, Hong S-P, Kwak M-S, Esaki N, Soda K, Sung M-H (1999) Production of aromatic D-amino acids from α-keto acids and ammonia by coupling of four enzyme reactions. J Mol Catal B Enzym 6:241–247 Bodanszky M, Perlman D (1969) Peptide antibiotics. Science 163:352– 358 Bommarius AS, Schwarm M, Drauz K (1998) Biocatalysis to amino acidbased chiral pharmaceuticals-examples and perspectives. J Mol Catal B Enzym 5:1–11 Brandão PFB, Verseck S, Syldatk C (2004) Bioconversion of D,L-tertleucine nitrile to D-tert-leucine by recombinant cells expressing nitrile hydratase and D-selective amidase. Eng Life Sci 4:547–556 Brückner H, Hausch M (1989) Gas chromatographic detection of D-amino acids as common constituents of fermented foods. Chromatographia 28:487–492 Burkhart BM, Gassman RM, Langs DA, Pangborn WA, Duax WL, Pletnev V (1999) Gramicidin D-conformation, dynamics and membrane ion transport. Pept Sci 51:129–144 Fotheringham, Taylor PPAH, Ton JLP (1998) Preparation of D-amino acids by direct fermentative means. United States Patent 5728555 Friedman M (1999) Chemistry, nutrition, and microbiology of D-amino acids. J Agric Food Chem 47:3457–3479 Fujii N (2002) D-Amino acids in living higher organisms. Orig Life Evol Biosph 32:103–127 Gao X, Chen X, Liu W, Feng J, Wu Q, Hua L, Zhu D (2012) A novel meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum: overexpression, characterization, and potential for D-amino acid synthesis. Appl Environ Microbiol 78:8595–8600
Appl Microbiol Biotechnol Gao X, Huang F, Feng J, Chen X, Zhang H, Wang Z, Wu Q, Zhu D (2013) Engineering the meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum by site saturation mutagenesis for D-phenylalanine synthesis. Appl Environ Microbiol 79:5078–5081 Gillman KW, Starrett JE, Parker MF, Xie K, Bronson JJ, Marcin LR, McElhone KE, Bergstrom CP, Mate RA, Williams R, Meredith JE, Burton CR, Barten DM, Toyn JH, Roberts SB, Lentz KA, Houston JG, Zaczek R, Albright CF, Decicco CP, Macor JE, Olson RE (2010) Discovery and evaluation of BMS-708163, a potent, selective and orally bioavailable γ-secretase inhibitor. Med Chem Lett 1:120–124 Grenby TH (1991) Intense sweeteners for the food industry: an overview. Trends Food Sci Technol 2:2–6 Grifantini R, Galli G, Carpani G, Pratesi C, Frascotti G, Grandi G (1998) Efficient conversion of 5-substituted hydantoins to D-α-amino acids using recombinant Escherichia coli strains. Microbiology 144:947– 954 Hanson RL, Johnston RM, Goldberg SL, Parker WL, Goswami A (2013) Enzymatic preparation of an R-amino acid intermediate for a γsecretase inhibitor. Org Process Res Dev 17:693–700 Heijenoort JV (2001) Recent advances in the formation of the bacterial peptidoglycan monomer unit. Nat Prod Rep 18:503–519 Heller B (1982) D-phenylalanine treatment. Unites States Patent 4355044 Hertel C, Hoffmann T, Jakob-Roetne R, Norcross RD (2000) For treatment of diseases associated with amyloidosis, such as alzheimer’s disease, diabetes mellitus, familial amyloid polyneuropathy, scrapie, and kreuzfeld-jacob disease. United States Patent 6103910 Hils M, Münch P, Altenbuchner J, Syldatk C, Mattes R (2001) Cloning and characterization of genes from Agrobacterium sp. IP I-671 involved in hydantoin degradation. Appl Microbiol Biotechnol 57: 680–688 Hopkins DW, Ferguson KE (1994) Substrate induced respiration in soil amended with different amino acid isomers. Appl Soil Ecol 1:75–81 Hsu C-S, Lai W-L, Chang W-W, Liaw S-H, Tsai Y-C (2002) Structuralbased mutational analysis of D-aminoacylase from Alcaligenes faecalis DA1. Protein Sci 11:2545–2550 Isobe K, Tamauchi H, Fuhshuku K-i, Nagasawa S, Asano Y (2010) A simple enzymatic method for production of a wide variety of Damino acids using L-amino acid oxidase from Rhodococcus sp. AIU Z-35-1. Enzyme research 2010 Kato S, Ishihara T, Hemmi H, Kobayashi H, Yoshimura T (2011) Alterations in D-amino acid concentrations and microbial community structures during the fermentation of red and white wines. J Biosci Bioeng 111:104–108 Katz E, Demain AL (1977) The peptide antibiotics of Bacillus: chemistry, biogenesis, and possible functions. Bacteriol Rev 41:449–474 Kim GJ, Cheon YH, Kim H-S (2000) Directed evolution of a novel Ncarbamoylase/D-hydantoinase fusion enzyme for functional expression with enhanced stability. Biotechnol Bioeng 68:211–217 Kobayashi J, Shimizu Y, Mutaguchi Y, Doi K, Ohshima T (2013) Characterization of D -amino acid aminotransferase from Lactobacillus salivarius. J Mol Catal B Enzym 94:15–22 Komeda H, Asano Y (2000) Gene cloning, nucleotide sequencing, and purification and characterization of the D-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3. Eur J Biochem 267: 2028–2035 Komeda H, Asano Y (2008) A novel D-stereoselective amino acid amidase from Brevibacterium iodinum: gene cloning, expression and characterization. Enzyme Microb Technol 43:276–283 Komeda H, Ishikawa N, Asano Y (2003) Enhancement of the thermostability and catalytic activity of D-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3 by directed evolution. J Mol Catal B Enzym 21:283–290 Kumar J, Sim V (2014) D-Amino acid-based peptide inhibitors as early or preventative therapy in alzheimer disease. Prion 8:119–124
Lee D-C, Kim H-S (1998) Optimization of a heterogeneous reaction system for the production of optically active D-amino acids using thermostable D-hydantoinase. Biotechnol Bioeng 60:729–738 Martín J, Casqueiro J, Kosalková K, Marcos A, Gutiérrez AFFS (1999) Penicillin and cephalosporin biosynthesis: mechanism of carbon catabolite regulation of penicillin production. Antonie Leeuwenhoek 75:21–31 Navab M, Anantharamaiah GM, Hama S, Garber DW, Chaddha M, Hough G, Lallone R, Fogelman AM (2002) Oral administration of an Apo A-I mimetic peptide synthesized from D-amino acids dramatically reduces atherosclerosis in mice independent of plasma cholesterol. Circulation 105:290–292 Okazaki S, Suzuki A, Komeda H, Yamaguchi S, Asano Y, Yamane T (2007) Crystal structure and functional characterization of a D-stereospecific amino acid amidase from Ochrobactrum anthropi SV3, a new member of the penicillin-recognizing proteins. J Mol Biol 368:79–91 Okazaki S, Suzuki A, Mizushima T, Komeda H, Asano Y, Yamane T (2008) Structures of D-amino acid amidase complexed with L-phenylalanine and with L-phenylalanine amide: insight into the D-stereospecificity of D-amino acid amidase from Ochrobactrum anthropi SV3. Acta Crystallogr D 64:331–334 Omura T, Furukawara T (2014) Oil-in-water type emulsion skin cosmetic. United States Patent 8607750B2 Pozo C, Rodelas B, de la Escalera S, González-López J (2002) d, lHydantoinase activity of an Ochrobactrum anthropi strain. J Appl Microbiol 92:1028–1034 Radkov A, Moe L (2014) Bacterial synthesis of D-amino acids. Appl Microbiol Biotechnol 98:1–12 Reich E (1963) Biochemistry of actinomycins. Cancer Res 23:1428– 1441 Robinson T (1976) D-Amino acids in higher plants. Life Sci 19:1097– 1102 Sanchez CJ, Akers KS, Romano DR, Woodbury RL, Hardy SK, Murray CK, Wenke JC (2014) D-Amino acids enhance the activity of antimicrobials against biofilms of clinical wound isolates of Staphylococcus aureus and Pseudomonas aeruginosa. Antimicrob Agents Chemother 58:4353–4361 Svennerstam H, Ganeteg U, Bellini C, Näsholm T (2007) Comprehensive screening of Arabidopsis mutants suggests the lysine histidine transporter 1 to be involved in plant uptake of amino acids. Plant Physiol 143:1853–1860 Turner RJ, Aikens J, Royer S, DeFilippi L, Yap A, Holzle D, Somers N, Fotheringham IG (2004) D-Amino acid tolerant hosts for Dhydantoinase whole cell biocatalysts. Eng Life Sci 4:517–520 Vedha-Peters K, Gunawardana M, Rozzell JD, Novick SJ (2006) Creation of a broad-range and highly stereoselective D-amino acid dehydrogenase for the one-step synthesis of D-amino acids. J Am Chem Soc 128:10923–10929 Veine D, Yao H, Stafford D, Fay K, Livant D (2014) A D-amino acid containing peptide as a potent, noncovalent inhibitor of α5β1 integrin in human prostate cancer invasion and lung colonization. Clin Exp Metastasis 31:379–393 Vranova V, Zahradnickova H, Janous D, Skene K, Matharu A, Rejsek K, Formanek P (2012) The significance of D-amino acids in soil, fate and utilization by microbes and plants: review and identification of knowledge gaps. Plant Soil 354:21–39 Wakayama M, Moriguchi M (2001) Comparative biochemistry of bacterial N-acyl-D-amino acid amidohydrolase. J Mol Catal B Enzym 12: 15–25 Wakayama M, Yoshimune K, Hirose Y, Moriguchi M (2003) Production of D-amino acids by N-acyl-D-amino acid amidohydrolase and its structure and function. J Mol Catal B Enzym 23:71–85 Walters DE (1995) Using models to understand and design sweeteners. J Chem Educ 72:680–683
Appl Microbiol Biotechnol Welch BD, VanDemark AP, Heroux A, Hill CP, Kay MS (2007) Potent Dpeptide inhibitors of HIV-1 entry. Proc Natl Acad Sci U S A 104: 16828–16833 Yang H, Zheng G, Peng X, Qiang B, Yuan J (2003) D-Amino acids and DTyr-tRNATyr deacylase: stereospecificity of the translation machine revisited. FEBS Lett 552:95–98
Yano S, Haruta H, Ikeda T, Kikuchi T, Murakami M, Moriguchi M, Wakayama M (2011) Engineering the substrate specificity of AlcaligenesD-aminoacylase useful for the production of D-amino acids by optical resolution. J Chromatogr B 879:3247–3252 Zhang J, Xu Y, Li R (2013) Preparation method of aspoxicillin. China Patent 103333180A
