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Discovery and Molecular and Biocatalytic Properties of Hydroxynitrile Lyase from an Invasive Millipede, Chamberlinius Hualienensis

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Discovery and Molecular and Biocatalytic Properties of Hydroxynitrile Lyase from an Invasive Millipede, Chamberlinius Hualienensis Discovery and molecular and biocatalytic properties of hydroxynitrile lyase from an invasive millipede, Chamberlinius hualienensis Mohammad Dadashipoura,b,1, Yuko Ishidaa,b,1, Kazunori Yamamotoa,b, and Yasuhisa Asanoa,b,2 aBiotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan; and bAsano Active Enzyme Molecule Project, Exploratory Research for Advanced Technology, Japan Science and Technology Agency, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved July 8, 2015 (received for review April 29, 2015) Hydroxynitrile lyase (HNL) catalyzes the degradation of cyanohy- of mandelonitrile using emulsin from the almond, Prunus amyg- drins and causes the release of hydrogen cyanide (cyanogenesis). dalus, as an HNL source (PaHNL), has been demonstrated (8). HNL can enantioselectively produce cyanohydrins, which are valuable Versatile HNLs, homologous to FAD-dependent oxidoreductase, + building blocks for the synthesis of fine chemicals and pharmaceu- α/β-hydrolase, carboxypeptidase, a Zn2 -dependent alcohol de- + ticals, and is used as an important biocatalyst in industrial bio- hydrogenase, and a Mn2 -dependent cupin structure have all been technology. Currently, HNLs are isolated from plants and bacteria. identified from plants and bacteria (9–12). Because PaHNL Because industrial biotechnology requires more efficient and stable shows the highest specific activity in the synthesis of mandelonitrile enzymes for sustainable development, we must continuously explore among known HNLs (13), along with a wide substrate specificity other potential enzyme sources for the desired HNLs. Despite the and high stability, it serves as a valuable catalyst in industrial bio- abundance of cyanogenic millipedes in the world, there has been no technology. Although many scientists have identified HNLs in the precise study of the HNLs from these arthropods. Here we report the last two decades (14, 15), further screening for better enzyme(s) isolation of HNL from the cyanide-emitting invasive millipede Cham- with high reaction rates and stabilities from unexplored bio- berlinius hualienensis, along with its molecular properties and appli- resources is necessary to enrich the current tool box for sus- cation in biocatalysis. The purified enzyme displays a very high tainable industrial development. specific activity in the synthesis of mandelonitrile. It is a glycosylated The millipede Chamberlinius hualienensis (16) (Movie S1), homodimer protein and shows no apparent sequence identity or originally from Hualien, Taiwan, invaded Okinawa Island, Japan homology with proteins in the known databases. It shows biocata- in 1983 and has been expanding its habitat in Kyushu, Japan. lytic activity for the condensation of various aromatic aldehydes with Large swarms of the millipedes enter houses and sometimes potassium cyanide to produce cyanohydrins and has high stability cause train delays (17). The animal secretes mandelonitrile, over a wide range of temperatures and pH values. It catalyzes the benzaldehyde, and hazardous hydrogen cyanide as defense chem- R synthesis of ( )-mandelonitrile from benzaldehyde with a 99% enan- icals, as previously reported for polydesmid millipede species (18, tiomeric excess, without using any organic solvents. Arthropod fauna 19). In the millipede species Apheloria corrugate, a predicted comprise 80% of terrestrial animals. We propose that these animals enzyme is thought to play a role in the decomposition of man- can be valuable resources for exploring not only HNLs but also di- delonitrile and the release of hydrogen cyanide and benzaldehyde verse, efficient, and stable biocatalysts in industrial biotechnology. millipede hydroxynitrile lyase | bioresource exploration | biocatalysis | Significance white biotechnology | arthropod Hydroxynitrile lyase (HNL) has been isolated from plants and CHEMISTRY bacteria and is a valuable tool in the chiral-specific synthesis of nzymes are very specific and efficient catalysts. They are able cyanohydrins, which are important building blocks of fine to catalyze reactions without extreme conditions, such as high E chemicals and pharmaceuticals. To discover more efficient and temperature and pressure, that are often required in chemical stable HNLs, we focused on the invasive cyanogenic millipede synthetic processes. Industrial biotechnologies using these bio- as a bioresource. The HNL identified from the millipede showed catalysts have the advantages of higher reaction rates, efficiencies, not only the highest specific activity toward benzaldehyde and enantioselectivities. This technology also reduces energy among known HNLs, including the almond HNL in industrial consumption and decreases the production of hazardous chem- use, along with wide temperature and pH stabilities, but also BIOCHEMISTRY ical waste. Conventional industrial chemistry is gradually being high enantioselectivity in the synthesis of various cyanohy- replaced by industrial biotechnology. For example, nitrile hydra- drins. These properties make it suitable as an industrial biocat- tase is used for the synthesis of acrylamide from acrylonitrile. This alyst. Arthropods are likely to be valuable sources of potential strategy produces more than 600,000 tons of acrylamide world- biocatalysts for the next generation of industrial biotechnology. wide every year (1). Hydroxynitrile lyase (HNL) is found as a molecular component Author contributions: M.D., Y.I., K.Y., and Y.A. designed research; M.D., Y.I., and K.Y. in plant defense systems and plays a role in the decomposition of performed research; M.D., Y.I., K.Y., and Y.A. analyzed data; and M.D., Y.I., K.Y., and Y.A. stored cyanogenic glycosides into hydrogen cyanide and aldehydes wrote the paper. (2). The enzyme’s reverse activity is used industrially in the enan- The authors declare no conflict of interest. tioselective production of cyanohydrins. Cyanohydrins are valuable This article is a PNAS Direct Submission. building blocks for the synthesis of fine chemicals, pharmaceuticals, Freely available online through the PNAS open access option. and agrochemicals such as denopamine (β1-adrenergic receptor Data deposition: The cDNA sequence reported in this paper has been deposited in the agonist) (3, 4), clopidogrel (platelet aggregation inhibitor) (5), py- DNA Data Bank of Japan (accession no. LC004755). rethroids (insecticides) (6), and (R)-pantolactone [starting com- 1M.D. and Y.I. contributed equally to this work. pound for synthesis of (R)-pantothenic acid, (R)-panthenol, and 2To whom correspondence should be addressed. Email: [email protected]. (R)-pantetheine] (7). The enantioselective condensation of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. benzaldehyde with hydrogen cyanide in the asymmetric synthesis 1073/pnas.1508311112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1508311112 PNAS | August 25, 2015 | vol. 112 | no. 34 | 10605–10610 Downloaded by guest on September 30, 2021 (20). If the enzyme derived from C. hualienensis is an HNL, the properties and inhibitor susceptibility, and demonstrate its substrate large population of invasive millipedes could be used as an im- specificity, kinetic parameters, and gene expression. We describe portant resource for HNL purification. Here we identify HNL from the potential of the millipede HNL to be widely used as a bio- the invasive cyanogenic millipede, characterize its physicochemical catalyst for the enantioselective synthesis of cyanohydrins, building Fig. 1. Characterization of ChuaHNL. (A) Cleavage reaction of racemic mandelonitrile by ChuaHNL. Production of benzaldehyde in 100 mM citrate buffer, pH 5.5, at 22 °C was measured by monitoring the absorbance at 280 nm. Solid and dotted lines indicate absorbance with and without the enzyme, re- spectively. (B) Synthesis of (R)-mandelonitrile from 50 mM benzaldehyde in 400 mM citrate buffer, pH 4.2, at 22 °C. ChuaHNL specifically produces (R)-mandelonitrile, whereas the chemical reaction produces a scant amount of (R)- and (S)-mandelonitrile. Production of mandelonitrile was measured by monitoring the absorbance at 254 nm using an HPLC equipped with a chiral column. Retention times of (R)- and (S)-mandelonitrile were 11.4 min and 14.3 min, respectively. (Inset) Magnified view in the range of 10–15 min. Solid and dotted lines indicate the synthetic reaction of mandelonitrile from benzal- dehyde and potassium cyanide with and without ChuaHNL, respectively. (C) Temperature stability. Enzyme samples were incubated at each temperature for 1 h and assayed for the remaining HNL activity (as shown in B). (D) Optimum temperature. Enzyme samples were assayed at each temperature (as shown in B). (E) pH stability. Enzyme samples were incubated at each temperature for 1 h and assayed for the remaining HNL activity (as shown in B). ●, enzymatic activity in a citrate-phosphate buffer; □, enzymatic activity in a glycine-sodium hydroxide buffer. (F) Optimum pH. Enzyme samples were assayed at each pH value (as shown in B). Values are the mean ± SD; n = 3. 10606 | www.pnas.org/cgi/doi/10.1073/pnas.1508311112 Dadashipour et al. Downloaded by guest on September 30, 2021 Table 1. Purification of ChuaHNL from the millipede C. hualienensis Specific Yield, Purification − Purification step Activity, U* Protein, mg activity, U·mg 1 % fold Body crude homogenate 26,600 4,390 6.10 100 1.00 Saturated
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