Hydroxynitrile lyases in stereoselective catalysis

(R)- as well as (S)-cyanohydrins are now easily available as a result of the excellent accessibility, the relatively high stability and the easy handling of hydroxynitrile lyases (HNLs). The optimization of reaction conditions (solvent, temperature, and using site-directed mutagenesis, etc.) has enabled HNL-catalyzed preparations of optically active cyanohydrins on a technical scale. The enantioselectivity of chiral metal-complex-catalyzed additions of trimethylsilyl cyanide to aldehydes has been improved, but is, by far, not yet competitive with the HNL-catalyzed reactions.     

Hydroxynitrile Lyases from Prunus Seeds in the Preparation of Cyanohydrins

The hydroxynitrile lyase (HNL) activity of nine defatted Prunus seeds was compared for catalyzing the addition of HCN to aromatic, heteroaromatic and α,β-unsaturated aldehydes. Although the conversion and enantiomeric excess (ee) of the corresponding cyanohydrins were both influenced by the HNL source and the chemical structure of the aldehyde, Prunus HNLs were all suitable for the enantioselective preparation of cyanohydrins.     

Crosslinked enzyme aggregates of hydroxynitrile lyase partially purified from Prunus dulcis seeds and its application for the synthesis of enantiopure cyanohydrins

Hydroxynitrile lyases are powerful catalysts in the synthesis of enantiopure cyanohydrins which are key synthons in the preparations of a variety of important chemicals. The response surface methodology including three‐factor and three‐level Box–Behnken design was applied to optimize immobilization of hydroxynitrile lyase purified partially from Prunus dulcis seeds as crosslinked enzyme aggregates (PdHNL‐CLEAs). The quadratic model was developed for predicting the response and its adequacy was validated with the analysis of variance test. The optimized immobilization parameters were initial glutaraldehyde concentration, ammonium sulfate saturation concentration, and crosslinking time, and the response was relative activity of PdHNL‐CLEA. The optimal conditions were determined as initial glutaraldehyde concentration of 25% w/v, ammonium sulfate saturation concentration of 43% w/v, and crosslinking time of 18 h. The preparations of PdHNL‐CLEA were examined for the synthesis of (R)‐mandelonitrile, (R)‐2‐chloromandelonitrile, (R)‐3,4‐dihydroxymandelonitrile, (R)‐2‐hydroxy‐4‐phenyl butyronitrile, (R)‐4‐bromomandelonitrile, (R)‐4‐fluoromandelonitrile, and (R)‐4‐nitromandelonitrile from their corresponding aldehydes and hydrocyanic acid. After 96‐h reaction time, the yield–enantiomeric excess values (%) were 100?99, 100?21, 100?99, 83?91, 100?99, 100?72, and 100?14%, respectively, for (R)‐mandelonitrile, (R)‐2‐chloromandelonitrile, (R)‐3,4‐dihydroxymandelonitrile, (R)‐2‐hydroxy‐4‐phenyl butyronitrile, (R)‐4‐bromomandelonitrile, (R)‐4‐fluoromandelonitrile, and (R)‐4‐nitromandelonitrile. The results show that PdHNL‐CLEA offers a promising potential for the preparation of enantiopure (R)‐mandelonitrile, (R)‐3,4‐dihydroxymandelonitrile, (R)‐2‐hydroxy‐4‐phenyl butyronitrile, and (R)‐4‐bromomandelonitrile with a high yield and enantiopurity. © 2014 American Institute of Chemical Engineers Biotechnol. Prog, 30:818–827, 2014     

Aboriginal preparation ofCycas seeds in Australia1

The seeds of cycad plants are a toxic food used by many Aboriginal groups in northern Australia. Acute symptoms produced after consumption of untreated Cycas seeds are due to azoxyglycosides, especially cycasin, although the toxic dose depends on the animal species tested. There are three traditional methods used to treat these seeds: brief leaching in water; prolonged leaching in water; and aging. Aboriginal people living at Donydji outstation in northeast Arnhem Land, most regularly consume aged seeds ofCycas angulata R.Br. Analyses of fresh seeds and seeds prepared at Donydji and in the laboratory indicate that cycasin is effectively removed by all the traditional preparation techniques, although each technique has an end product with different storage and handling properties. The social implications of processing need further elaboration, but these techniques have a long history and archaeological remains of seeds in Australia may date back to the Pleistocene.     

Characterization of cold-active pectate lyases from psychrophilic Mrakia frigida

AIMS: This study was conducted to determine optimal conditions for pectate lyase (PL) production by two psychrophilic yeast strains and to compare the properties of the cold-active enzymes using mesophilic PL as reference enzyme. METHODS AND RESULTS: Two psychrophilic yeasts isolated from remote geographical locations (European Alps, north Siberia) produced extracellular cold-active PL. Both strains were identified as Mrakia frigida by analysis of ITS and large subunit (LSU) rRNA sequences. Maximum enzyme production occurred at a cultivation temperature of 1 or 5 degrees C. The apparent optimum for enzyme activity was observed at 30 degrees C and pH 8.5-9. The enzymes were thermolabile, but were resistant to repeated freezing and thawing. CONCLUSION: We describe for the first time alkaline PL-producing representatives of the yeast species M. frigida. The two strains produce cold-active PL with similar properties, but have a different enzyme production pattern. SIGNIFICANCE AND IMPACT OF THE STUDY: The enzymes described in this study could be useful for a wide range of applications, such as low-temperature pretreatment of wastewater containing pectic substances.     

中国木薯醇腈酶在汉逊酵母中的表达

α-醇腈酶(α-hydroxynitrilelyase, HNL)是手性醇腈化合物生物合成十分有用的工具, 能够催化羰基化合物和HCN立体选择性的加工形成手性醇腈化合物。应用PCR扩增得到HNL基因, 连接到pMD18-T 中进行测序, 然后通过EcoRⅠ和NotⅠ将其连接到汉逊酵母表达载体pHMOXGαA中。通过在含有4 mg/mL的G418的YPD培养基上进行筛选获得阳性重组子, 用0.5%的甲醇诱导96 h。酶活测定和SDS-PAGE分析显示HNL在汉逊酵母NCYC495(leu1.1)中得到正确表达, 每毫升发酵液中获得2.015 U的醇腈酶。     

The C-S lyases of higher plants: preparation and properties of homogeneous alliin lyase from garlic (Allium sativum)

Alliin lyase from garlic (Allium sativum) has been purified to homogeneity. The purification procedure involves the use of affinity chromatography on concanavalin A-Sepharose 4B. Addition of polyvinylpolypyrrolidone to the homogenizing medium greatly improves the specific activity of the extract. The enzyme is a glycoprotein as seen by its ability to bind to concanavalin A-Sepharose 4B and by its positive periodic acid-Schiff base stain. It has a carbohydrate content of 5.5%. Km values for this enzyme were estimated to be 5.7 mM for S-ethyl-L-cysteine sulfoxide and 3.3 mM for S-allyl-L-cysteine sulfoxide. The molecular weight of this garlic enzyme, as determined by gel filtration, was found to be 85,000; the molecule consists of two equal subunits of Mr 42,000. The amino acid content was found to be similar to that reported previously for onion alliin lyase, although there is twice as much tryptophan in the garlic alliin lyase as in the onion enzyme. By both chemical and spectral methods the enzyme was found to have two molecules of pyridoxal 5-phosphate per enzyme molecule, suggesting one per subunit. There are significant differences in the nature of these findings from those previously reported from this laboratory for the onion enzyme. Studies are in progress to compare further the alliin lyases from garlic and onion.     

An optimized preparation method to obtain high-quality RNA from dry sunflower seeds

In an attempt to isolate high-quality, intact total RNA from sunflower (Helianthus annuus) seeds for investigation of the molecular mechanisms of mutations, we tested various procedures, using kits, including RNAiso Plus, RNAiso Plus+RNAiso-mate for Plant Tissue, Trizol, and the Qi method, but no high-quality total RNA of high integrity was obtained with any of these methods, probably due to the high content of polyphenols, polysaccharides, and secondary metabolites in mature sunflower seeds. Modifications were made to the Qi method. To avoid polyphenol oxidation, frozen dry seeds free of the seedcase were ground in a mortar with an equal amount of PVP30, and the fine ground powder was transferred to an extraction buffer with 2% PVP30 (w/v), 5% β-mercaptoethanol (v/v) and LiCl (8 M). A sample homogenate was extracted with chloroform prior to acidic phenol-chloroform extraction. The total RNA was precipitated with 1/4 volume of NaAc and 2 volumes of absolute ethanol to prevent contamination by polysaccharides. The yield of total RNA was 29.95 μg/100 mg husked dry seeds; the ratios of A260/A230 and A260/A280 were 2.44 and 2.09, respectively. Electrophoretic analysis clearly showed 28S and 18S ribosomal RNA bands. Using the extracted RNA, a fragment of the actin gene was successfully expressed by RT-PCR. This modified protocol is suitable for isolating high-quality total RNA from sunflower seeds for molecular research.     

Hydroxynitrile lyase from Hevea brasiliensis: Molecular characterization and mechanism of enzyme catalysis

(S)-Hydroxynitrile lyase (Hnl) from the tropical rubber tree Hevea brasiliensis is a 29 kDa single chain protein that catalyses the breakdown or formation of a C(SINGLE BOND)C bond by reversible addition of hydrocyanic acid to aldehydes or ketones. The primary sequence of Hnl has no significant homology to known proteins. Detailed homology investigations employing PROFILESEARCH and secondary structure prediction algorithms suggest that Hnl is a member of the α/β hydrolase fold protein family and contains a catalytic triad as functional residues for catalysis. The significance of the predicted catalytic residues was tested and confirmed by site-directed mutagenesis and expression of mutant and wild-type proteins in the yeast, Saccharomyces cerevisiae. Based on these data we suggest a mechanistic model for the (S)-cyanohydrin synthesis catalyzed by hydroxynitrile lyase from Hevea brasiliensis. Proteins 27:438–449, 1997. © 1997 Wiley-Liss, Inc.     

Large-scale preparation of a lectin from sunn hemp seeds (Crotalaria juncea)

A procedure for large-scale preparation of a lectin from Crotalaria juncea seeds is described. The method involve fractionation by pH- and ammonium sulfate precipitation followed by biospecific affinity chromatography. The adsorbent used for the affinity chromatography was prepared by coupling galactose to Sepharose 6B activated with divinylsulfone. A comparison of different apparatus and techniques involved in the preparation is discussed. The yield and quality of the lectin prepared at a large scale were comparable with laboratory-scale preparation. From 50 kg Crotalaria juncea beans, 14.4 g Crotalaria lectin were obtained.     

Purification and characterization of heparin lyases from Flavobacterium heparinum.

Heparin lyase I has been purified from Flavobacterium heparinum and has been partially characterized (Yang, V. C., Linhardt, R. J., Berstein, H., Cooney, C. L., and Langer, R. (1985) J. Biol. Chem. 260, 1849-1857). There has been no report of the purification of the other polysaccharide lyases from this organism. Although all three of these heparin/heparan sulfate lyases are widely used, with the exception of heparin lyase I, there is no information on their purity or their physical and kinetic characteristics. The absence of pure heparin lyases and a lack of understanding of the optimal catalytic conditions and substrate specificity has stood in the way of the use of these enzymes as reagents for the specific depolymerization of heparin and heparan sulfate into oligosaccharides for structure and activity studies. This paper describes a single, reproducible scheme to simultaneously purify all three of the heparin lyases from F. heparinum to apparent homogeneity. Heparin lyase I (heparinase, EC 4.2.2.7), heparin lyase II (no EC number), and heparin lyase III (heparitinase, EC 4.2.2.8) have molecular weights (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and isoelectric points (by isoelectric focusing) of M(r) 42,800, pI 9.1-9.2, M(r) 84,100, pI 8.9-9.1, M(r) 70,800, pI 9.9-10.1, respectively. Their amino acid analyses and peptide maps demonstrate that while these proteins are different gene products they are closely related. The kinetic properties of the heparin lyases have been determined as well as the conditions to optimize their activity and stability. These data should improve the application of these important enzymes in the study of heparin and heparan sulfate.     

Isolation and characterization of two chondroitin lyases from Bacteroides thetaiotaomicron.

Two chondroitin lyases were isolated from the colon anaerobe Bacteroides thetaiotaomicron. Both enzymes had similar molecular weights (104,000 and 108,000) and similar isoelectric points (8.0 and 7.9, respectively). Both enzymes were active against chondroitin sulfates A, B, and C and unsulfated polysaccharides, such as chondroitin and hyaluronic acid, although one of the enzymes was twice as active against chondroitin as the other enzyme. Both had similar Km values for chondroitin sulfates A and C (40 to 70 micrograms/ml) and for chondroitin (300 to 400 micrograms/ml). Neither enzyme could degrade the highly sulfated mucopolysaccharide heparin, but heparin was a potent inhibitor of the activity of both enzymes. Although enzymes I and II were similar in many respects, a comparison of peptides resulting from partial digestion with N-chlorosuccinimide or papain demonstrated that the two proteins are not related.     

Enzymatic hydrolysis of cyanohydrins with recombinant nitrile hydratase and amidase from hodococcus erythropolis

Nitrile hydratase and amidase from Rhodococcus erythropolis CIMB11540 were both cloned and expressed in Escherichia coli. Crude cell free extracts were used for the hydrolysis of different aromatic cyanohydrins. Nitrile hydratase expression was increased up to 5-fold by redesign of the expression cassette. The recombinant enzymes were successfully used for the conversion of several cyanohydrins to the corresponding alpha-hydroxy amides and acids while retaining enantiopurity.     

香菇基因组中L-半胱氨酸亚砜裂解酶同源蛋白的生物信息学分析

L-半胱氨酸亚砜裂解酶（L-cysteine sulfoxide lyase,C-S lyase）是香菇中含硫风味物质生物合成途径的关键酶之一。本文基于6个不同香菇菌株的全基因组测序数据,挖掘了24个潜在的香菇L-半胱氨酸亚砜裂解酶（Lentinula edodes C-S lyase,Lecsl）同源基因,对其编码蛋白的生理生化特性、信号肽、跨膜结构域、转录活性、分子进化、保守基序和蛋白三级结构等方面进行了分析。结果发现,这24个香菇Lecsl同源蛋白含有相同的蛋白结构域（IPR015424和IPR000192）,都属于L-半胱氨酸脱巯基酶家族（PTHR43092:SF2）,都不含信号肽和跨膜结构,但它们的蛋白稳定性有所不同。对24个Lecsl同源蛋白进行聚类分析发现,其中的11个组成了新的进化分支,这一分支的Lecsl同源蛋白在香菇的菌丝体或子实体中有转录活性,且含有蒜酶和L-半胱氨酸脱硫酶的保守基序19,推测这一分支的Lecsl同源蛋白在香菇中具有催化产生含硫风味物质和内源性甲醛的活性。进一步分析发现,这一分支又分为两个亚支,其中一支包含已发现的Lecsl/LE01_CSL1,并且在香菇的菌丝体和子实体阶段都有转录活性;另一个亚支上的C-S lyase同源蛋白仅在菌丝体中有转录活性,推测这两个亚支的L-半胱氨酸亚砜裂解酶分别在香菇生长发育的不同阶段发挥催化作用。通过三维结构的解析,阐明了Lecsl中保守基序19亦是使蒜酶产生催化活性的关键结构域,并且利用分子动力学模拟的方法,预测保守基序19中的Asn3、Gln5和Ser6是香菇C-S lyase产生催化活性的关键氨基酸残基。     

The occurrence of nitrate reductase in leaves of prunus species

Nitrate reductase was found in leaves of apricot Prunus armeniaca, sour cherry P. cerasus, sweet cherry P. avium, and plum P. domestica, but not in peach P. persica, from trees grown in sand culture receiving a nitrate containing nutrient solution. Nitrate was found in the leaves of all species. Nitrate and nitrate reductase were found in leaves of field-grown apricot, sour cherry, and plum trees. The enzyme-extracting medium contained insoluble polyvinylpyrrolidone, and including dithiothreitol or mercaptobenzothiazole did not improve enzyme recovery. Inclusion of cherry leaf extract diminished, and peach leaf extract abolished, recovery of nitrate reductase from oat tissue. Low molecular weight phenols liberated during extraction were probably responsible for inactivation of the enzyme. The enzyme from apricot was two to three times as active as from the other species. Both nicotine adenine diphosphopyridine nucleotide and flavin mononucleotide were effective electron donors. The enzyme was readily induced in apricot leaves by 10 mm nitrate supplied through the leaf petiole.     

Sequencing and heterologous expression of an epimerase and two lyases from iminodisuccinate-degrading bacteria

Recently, degradation of all existing epimers of the complexing agent iminodisuccinate (IDS) in the bacterial strain Agrobacterium tumefaciens BY6 was proven to depend on an epimerase and a C-N lyase (Cokesa et al., Appl. Environ. Microbiol. 70:3941-3947, 2004). In the bacterial strain Ralstonia sp. strain SLRS7, a corresponding C-N lyase is responsible for the initial degradation step (Cokesa et al., Biodegradation 15:229-239, 2004). The ite gene, encoding the IDS-transforming epimerase, and the genes icl(B) and icl(S), encoding the IDS-converting BY6-lyase and SLRS7-lyase, respectively, were cloned and sequenced. The epimerase gene encodes a protein with a predicted subunit molecular mass of 47.6 kDa. The highest degree of epimerase amino acid sequence identities was found with proteins of unknown function, indicating a novel protein. For the lyases, the deduced amino acid sequences show high similarity to enzymes of the fumarase II family. A classification into a new subfamily within the enzyme family is proposed. The subunit molecular masses of the lyases were calculated to be 54.4 and 54.7 kDa, respectively. In Agrobacterium tumefaciens BY6, the ite gene was on an approximately 180-kb circular plasmid, whereas the icl(B) gene was chromosomal like the corresponding icl(S) gene in Ralstonia sp. strain SLRS7. Heterologous expression in Escherichia coli and subsequent purification revealed recombinant enzymes with in vitro activity similar to that of the corresponding enzymes from the wild-type strains.