Immobilized Baliospermum montanum hydroxynitrile lyase catalyzed synthesis of chiral cyanohydrins

Hydroxynitrile lyase (HNL) catalyzed enantioselective CC bond formation is an efficient approach to synthesize chiral cyanohydrins which are important building blocks in the synthesis of a number of fine chemicals, agrochemicals and pharmaceuticals. Immobilization of HNL is known to provide robustness, reusability and in some cases also enhances activity and selectivity.We optimized the preparation of immobilization of Baliospermium montanum HNL (BmHNL) by cross linking enzyme aggregate (CLEA) method and characterized it by SEM. Optimization of biocatalytic parameters was performed to obtain highest % conversion and ee of (S)-mandelonitrile from benzaldehyde using CLEA-BmHNL. The optimized reaction parameters were: 20 min of reaction time, 7 U of CLEA-BmHNL, 1.2 mM substrate, and 300 mM citrate buffer pH 4.2, that synthesized (S)-mandelonitrile in ∼99% ee and ∼60% conversion. Addition of organic solvent in CLEA-BmHNL biocatalysis did not improve in % ee or conversion of product unlike other CLEA-HNLs. CLEA-BmHNL could be successfully reused for eight consecutive cycles without loss of conversion or product formation and five cycles with a little loss in enantioselectivity. Eleven different chiral cyanohydrins were synthesized under optimal biocatalytic conditions in up to 99% ee and 59% conversion, however the % conversion and ee varied for different products. CLEA-BmHNL has improved the enantioselectivity of (S)-mandelonitrile synthesis compared to the use of purified BmHNL. Nine aldehydes not tested earlier with BmHNL were converted into their corresponding (S)-cyanohydrins for the first time using CLEA-BmHNL. Among the eleven (S)-cyanohydrins syntheses reported here, eight of them have not been synthesized by any CLEA-HNL. Overall, this study showed preparation, characterization of a stable, robust and recyclable biocatalyst i.e. CLEA-BmHNL and its biocatalytic application in the synthesis of different (S)-aromatic cyanohydrins.     

Characterization of cross-linked immobilized lipase from thermophilic mould Thermomyces lanuginosa using glutaraldehyde

Cross-linked enzyme aggregates (CLEAs) have emerged as an interesting biocatalyst design for immobilization. Using this approach, a 1,3 regiospecific, alkaline and thermostable lipase from Thermomyces lanuginosa was immobilized. Efficient cross-linking was observed when ammonium sulphate was used as precipitant along with a two fold increase in activity in presence of SDS. The TEM and SEM microphotographs of the CLEAs formed reveal that the enzyme aggregates are larger in size as compared to the free lipase due to the cross-linking of enzyme aggregates with glutaraldehyde. The stability and reusability of the CLEA with respect to olive oil hydrolysis was evaluated. The CLEA showed more than 90% residual activity even after 10 cycles of repeated use.     

Oral ingestion of mannose alters the expression level of deaminoneuraminic acid (KDN) in mouse organs

Deaminoneuraminic acid (KDN) is a unique member of the sialic acid family. We previously demonstrated that free KDN is synthesized de novo from mannose as its precursor sugar in trout testis, and that the amount of intracellular KDN increases in mouse B16 melanoma cells cultured in mannose-rich media [Angata et al. (1999) J. Biol. Chem. 274, 22949–56; Angata et al. (1999) Biochem. Biophys. Res. Commun. 261, 326–31]. In the present study, we first demonstrated a mannose-induced increase in intracellular KDN in various cultured mouse and human cell lines. These results led us to examine whether KDN expression in mouse organs is altered by exogenously administered mannose. Under normal feeding conditions, intracellular free KDN was present at very low levels (19–48 pmol/mg protein) in liver, spleen, and lung, and was not detected in kidney or brain. Oral ingestion of mannose, both short-term (90 min) and long-term (2 wk), resulted in an increase of intracellular KDN up to 60–81 pmol/mg protein in spleen and lung and 6.9–18 pmol/mg protein in kidney and brain; however, no change was observed in liver. The level of KDN in organs appears not to be determined only by the KDN 9-phosphate synthase activity, but might also be affected by other enzymes that utilize mannose 6-phosphate as a substrate as well as the enzymes that breakdown KDN, like KDN-pyruvate lyase. In blood, the detectable amount of free KDN did not change on oral ingestion of mannose. These findings indicate that mannose in the diet affects KDN metabolism in various organs, and provide clues to the mechanism of altered KDN expression in some tumor cells and aged organs.     

KDN (Deaminated neuraminic acid): Dreamful past and exciting future of the newest member of the sialic acid family

KDN is an abbreviation for 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid, and its natural occurrence was revealed in 1986 by a research group including the present authors. Since sialic acid was used as a synonym for N-acylneuraminic acid at that time, there was an argument if this deaminated neuraminic acid belongs to the family of sialic acids. In this review, we describe the 20 years history of studies on KDN (KDNology), through which KDN has established its position as a distinct member of the sialic acid family. These studies have clarified that: (1) KDN occurs widely among vertebrates and bacteria similar to the occurrence of the more common sialic acid, N-acetylneuraminic acid (Neu5Ac), but its abundant occurrence in animals is limited to lower vertebrates. (2) KDN is found in almost all types of glycoconjugates, including glycolipids, glycoproteins and capsular polysaccharides. (3) KDN residues are linked to almost all glycan structures in place of Neu5Ac. All linkage types known for Neu5Ac; α2,3-, α2,4-, α2,6-, and α2,8- are also found for KDN. (4) KDN is biosynthesized de novo using mannose as a precursor sugar, which is activated to CMP-KDN and transferred to acceptor sugar residues. These reactions are catalyzed by enzymes, some of which preferably recognize KDN, but many others prefer Neu5Ac to KDN. In addition to these basic findings, elevated expression of KDN was found in fetal human red blood cells compared with adult red blood cells, and ovarian tumor tissues compared with normal controls. KDNase, an enzyme which specifically cleaves KDN-linkages, was discovered in a bacterium and monoclonal antibodies that specifically recognize KDN residues in KDNα2,3-Gal- and KDNα2,8-KDN-linkages have been developed. These have been used for identification of KDN-containing molecules. Based on past basic studies and variety of findings, future perspective of KDNology is presented.     

A new design strategy with stochastic optimization on the preparation of magnetite cross-linked tyrosinase aggregates (MCLTA)

In this study, a new design strategy with a systematic optimization process is proposed for the preparation of magnetite cross-linked tyrosinase aggregates (MCLTA) by using the concentration of magnetite nanoparticle, glutaraldehyde and tyrosinase enzyme as design variables. A comprehensive study on multiple non-linear neuro-regression analysis has been performed as a compelling alternative to the insufficient approaches on modeling-design-optimization of MCLTA. For this aim, the experimental process has been modeled with 13 candidate functional structures by using a hybrid method to test the accuracy of their predictions. R²_training, R²_testing values, and boundedness of the functions have been checked to reveal the realistic ones. Then four different design approaches in terms of three distinct scenarios have been used to optimize the process. The results show that, all models define the process well, depending on R²_training. However, only five and nine models are appropriate based on R²_testing for the first use activity and residual activity, respectively. On the other hand, depending on to be a realistic value, model TON best describes the "first use activity," while the best one is FONT for residual activity. It is also concluded that the scenario types and selection of constraints for design variables affect the optimization results.     

Identification and partial characterization of soluble and membrane-bound KDN(deaminoneuraminic acid)-glycoproteins in human ovarian teratocarcinoma PA-1, and enhanced expression of free and bound KDN in cells cultured in mannose-rich media

KDN (Deaminoneuraminic acid, or deaminated neuraminic acid) is a minor but biosynthetically independent member of the sialic acid. Human occurrence of KDN has already been established, although its level is so little that it is often undetectable by conventional sialic acid analysis. Elevated expression of KDN in fetal cord blood cells and some malignant tumor cells have been reported. However, in mammalian cells and tissues KDN mostly occurs as the free sugar and little occurred conjugated to glycolipids and/or glycoproteins. A positive correlation between the ratio of free KDN/free Neu5Ac in ovarian adenocarcinomas and the stage of malignancy has been noted for diagnostic use. We hypothesized that elevated expression of KDN in mammalian systems may be closely related to elevated activities of enzymes involved in the formation of sialoglycoconjugates and/or aberrant supply of the precursor sugar, mannose, used in the biosynthesis of KDN. In this study we used human ovarian teratocarcinoma cells PA-1 to further analyze KDN expression in human cells. Major findings reported in this paper are, (i) a 30 kDa KDN-glycoprotein immunostainable with monoclonal antibody, mAb.kdn3G, (specific for the KDNα2 → 3Galβ1→ epitope) and sensitive to KDNase was identified in the membrane fraction of the cell: (ii) a 49 kDa KDN-glycoprotein that is not reactive with mAb.kdn3G but is sensitive to KDNase was identified in the soluble fraction: and (iii) PA-1 cells showed unique response to mannose added to the growth medium in that the levels of both free and bound forms of KDN are elevated. This is the first report on the identification of mammalian KDN-glycoproteins by chemical and biochemical methods.     

New stabilized FastPrep-CLEAs for sialic acid synthesis

N-acetyl-d-neuraminic acid aldolase, a key enzyme in the biotechnological production of N-acetyl-d-neuraminic acid (sialic acid) from N-acetyl-d-mannosamine and pyruvate, was immobilized as cross-linked enzyme aggregates (CLEAs) by precipitation with 90% ammonium sulfate and crosslinking with 1% glutaraldehyde. Because dispersion in a reciprocating disruptor (FastPrep) was only able to recover 40% of the activity, improved CLEAs were then prepared by co-aggregation of the enzyme with 10 mg/mL bovine serum albumin followed by a sodium borohydride treatment and final disruption by FastPrep (FastPrep-CLEAs). This produced a twofold increase in activity up to 86%, which is a 30% more than that reported for this aldolase in cross-linked inclusion bodies (CLIBs). In addition, these FastPrep-CLEAs presented remarkable biotechnological features for Neu5Ac synthesis, including, good activity and stability at alkaline pHs, a high K_M for ManNAc (lower for pyruvate) and good operational stability. These results reinforce the practicability of using FastPrep-CLEAs in biocatalysis, thus reducing production costs and favoring reusability.     

CLEAs of Candida antarctica lipase B (CALB) with a bovine serum albumin (BSA) cofeeder core: Study of their catalytic activity

Highly active CALB cross-linked enzyme aggregates (CLEAs) were synthesized using a layered methodology based on the synthesis of a cross-linked protein cofeeder core over which an external layer of lipase was later cross-linked. The layered CALB CLEAs were characterized in terms of their catalytic activity in three different test reactions: esterification of oleic acid and ethanol in absence of solvents, esterification of oleic acid and heptanol in organic medium, and hydrolysis of triolein in emulsioned medium. The impact of the cross-linker/protein mass ratio on CLEAs activity, and its evolution with storage time were evaluated in the solventless synthesis of ethyloleate. The amount of cross-linker used showed to be a key parameter for the evolution of the catalytic activity of CLEAs during storage. Under the best conditions found, hyperactivated CALB CLEAs with up to 188% of recovered activity in ethyl oleate synthesis were obtained. In terms of hydrolytic activity mature layered CALB CLEAs showed a retained activity of 68%. The assay of dried mature layered CALB CLEAs in heptyl oleate synthesis showed catalytic activities much higher than the one exhibited by free CALB, reaching 1 h-fatty acid conversions of 14% and 2%, respectively. The high catalytic activity shown by layered CALB CLEAs, suggests that they are an interesting alternative specially for the catalysis of fatty acid esterifications in both organic and solventless medium.     

Biosynthesis of KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid). Identification and characterization of a KDN-9-phosphate synthetase activity from trout testis.

Although the deaminoneuraminic acid or KDN glycotope (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) is expressed in glycoconjugates that range in evolutionary diversity from bacteria to man, there is little information as to how this novel sugar is synthesized. Accordingly, biosynthetic studies were initiated in trout testis, an organ rich in KDN, to determine how this sialic acid is formed. These studies have shown that the pathway consists of the following three sequential reactions: 1) Man + ATP --> Man-6-P + ADP; 2) Man-6-P + PEP --> KDN-9-P + P(i); 3) KDN-9-P --> KDN + P(i). Reaction 1, catalyzed by a hexokinase, is the 6-O-phosphorylation of mannose to form D-mannose 6-phosphate (Man-6-P). Reaction 2, catalyzed by KDN-9-phosphate (KDN-9-P) synthetase, condenses Man-6-P and phosphoenolpyruvate (PEP) to form KDN-9-P. Reaction 3, catalyzed by a phosphatase, is the dephosphorylation of KDN-9-P to yield free KDN. It is not known if a kinase specific for Man (Reaction 1) and a phosphatase specific for KDN-9-P (Reaction 3) may exist in tissues actively synthesizing KDN. In this study, the KDN-9-P synthetase, an enzyme that has not been previously described, was identified as at least one key enzyme that is specific for the KDN biosynthetic pathway. This enzyme was purified 50-fold from rainbow trout testis and characterized. The molecular weight of the enzyme was estimated to be about 80,000, and activity was maximum at neutral pH in the presence of Mn(2+). N-Acetylneuraminic acid 9-phosphate (Neu5Ac-9-P) synthetase, which catalyzes the condensation of N-acetyl-D-mannosamine 6-phosphate and phosphoenol-pyruvate to produce Neu5Ac-9-P, was co-purified with the KDN-9-P synthetase. Substrate competition experiments revealed, however, that syntheses of KDN-9-P and Neu5Ac-9-P were catalyzed by two separate synthetase activities. The significance of these studies takes on added importance with the recent discovery that the level of free KDN is elevated in human fetal cord but not matched adult red blood cells and in ovarian cancer cells (Inoue, S., Lin, S-L., Chang, T., Wu, S-H., Yao, C-W., Chu, T-Y., Troy, F. A., II, and Inoue, Y. (1998) J. Biol. Chem. 273, 27199-27204). This unexpected finding emphasizes the need to understand more fully the role that free KDN and KDN-glycoconjugates may play in normal hematopoiesis and malignancy.     

脱氨神经氨酸及其在肿瘤中表达的研究进展

脱氨神经氨酸(2-keto-3-deoxy-D-glycero-D-galacto-nononic acid,KDN)是唾液酸家族中的三种核心成员之一。KDN单体主要由甘露糖作为前体糖合成得到,KDN大量存在于低等脊椎动物和细菌中,而在哺乳动物中的表达量却很低。近期,有研究报道,KDN在人类肿瘤中高表达,并且会随着肿瘤恶性程度的增高呈正相关增长,因此,推测KDN可能是某些肿瘤的肿瘤标示物。本文介绍了KDN的结构及其生物合成,重点综述了KDN在生物体内及肿瘤中的表达等研究现状,为以后深入研究KDN奠定了良好的基础。     

Synthesis of the nucleoside analogues of 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (KDN).

Several mono- and di-saccharide nucleoside analogues of 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (KDN, 1) were synthesized under Vorbrüggen, Williamson and Koenigs-Knorr reaction conditions. The stereochemistry at the anomeric position of these compounds were elucidated by means of NMR and acid catalyzed hydrolysis.     

Biotransformation of nitriles to amides using soluble and immobilized nitrile hydratase from Rhodococcus erythropolis A4

A semi-purified nitrile hydratase from Rhodococcus erythropolis A4 was applied to biotransformations of 3-oxonitriles 1a–4a, 3-hydroxy-2-methylenenitriles 5a–7a, 4-hydroxy-2-methylenenitriles 8a–9a, 3-hydroxynitriles 10a–12a and 3-acyloxynitrile 13a into amides 1b–13b. Cross-linked enzyme aggregates (CLEAs) with nitrile hydratase and amidase activities (88% and 77% of the initial activities, respectively) were prepared from cell-free extract of this microorganism and used for nitrile hydration in presence of ammonium sulfate, which selectively inhibited amidase activity. The genes nha1 and nha2 coding for and β subunits of nitrile hydratase were cloned and sequenced.     

Quantitative characteristic of the catalytic properties and microstructure of cross-linked enzyme aggregates of penicillin acylase

The microstructure and the catalytic properties of cross-linked enzyme aggregates (CLEA) of penicillin acylase (PA) obtained under different conditions were investigated. The period of time left between the enzyme precipitation and the cross-linking step was found to influence the structural organization of the resulting enzyme preparation. Confocal fluorescent microscopy of the so-called “fresh” and “mature” CLEAs PA allowed to estimate the “characteristic” diameter of CLEA PA particles, which appeared to be about 1.6 μm, and revealed that the “mature” type was composed of relatively big particles as compared to the “fresh” type. Complementary kinetic studies showed that the “mature” CLEA PA were more effective in both hydrolytic and synthetic reactions. It was suggested that the aggregate size might regulate the extent of covalent modification of PA and thereby influence the catalytic properties of CLEA.     

Synthesis of neoglycoconjugates containing deaminated neuraminic acid (KDN) using rat liver alpha2,6-sialyltransferase

2-Keto-3-deoxy-D- glycero -D- galacto -nononic acid (KDN) was introduced into asialotransferrin and N -acetyllactosamine (LacNAc) from CMP-KDN by using rat liver Galbeta1-->4GlcNAc alpha2, 6- sialyltransferase to form KDN-transferrin and KDN-LacNAc. These structures contain terminal KDNalpha2-->6Gal-residues, a glycotope that has not yet been described in natural glycoconjugates. KDN was transferred to all four Gal residues in asialotransferrin by this enzyme. The incorporation efficiency of KDN from CMP-KDN into asialotransferrin was about half that of Neu5Ac from CMP-Neu5Ac, based on the V max/ K m values for these donor substrates, 0.0527 min-1and 0.119 min-1, respectively. The KDNalpha2-->6Gal linkage was resistant to exosialidase treatment, in contrast to the sensitivity of the Neu5Acalpha2-->6Gal linkage. Interestingly, Sambucus sieboldiana agglutinin (SSA) was shown to prefer KDN-transferrin to the corresponding Neu5Ac-transferrin, as estimated by slot-blot analysis. The use of an alpha2,6-sialyltransferase to synthesize neoglycoproteins containing KDN has not been previously reported. Their facile synthesis using CMP-KDN and sialyltransferases with different specificities offers new possibilities to study the function of neo-KDN- glycoconjugates, and to explore their use in glycotechnology.      

Improving low-temperature activity of Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase

Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase (SacKdgA) displays optimal activity at 95 °C and is studied as a model enzyme for aldol condensation reactions. For application of SacKdgA at lower temperatures, a library of randomly generated mutants was screened for improved synthesis of 2-keto-3-deoxygluconate from pyruvate and glyceraldehyde at the suboptimal temperature of 50 °C. The single mutant SacKdgA-V193A displayed a threefold increase in activity compared with wild type SacKdgA. The increased specific activity at 40–60 °C of this mutant was observed, not only for the condensation of pyruvate with glyceraldehyde, but also for several unnatural acceptor aldehydes. The optimal temperature for activity of SacKdgA-V193A was lower than for the wild type enzyme, but enzymatic stability of the mutant was similar to that of the wild type, indicating that activity and stability were uncoupled. Valine193 has Van der Waals interactions with Lysine153, which covalently binds the substrate during catalysis. The mutation V193A introduced space close to this essential residue, and the increased activity of the mutant presumably resulted from increased flexibility of Lysine153. The increased activity of SacKdgA-V193A with unaffected stability demonstrates the potential for optimizing extremely thermostable aldolases for synthesis reactions at moderate temperatures.     

Structure and function of Mycobacterium smegmatis 7-keto-8-aminopelargonic acid (KAPA) synthase

The biotin biosynthesis pathway is an attractive target for development of novel drugs against mycobacterial pathogens, however there are as yet no suitable inhibitors that target this pathway in mycobacteria. 7-Keto-8-aminopelargonic acid synthase (KAPA synthase, BioF) is the enzyme which catalyzes the first committed step of the biotin synthesis pathway, but both its structure and function in mycobacteria remain unresolved. Here we present the crystal structure of Mycobacterium smegmatis BioF (MsBioF). The structure reveals an incomplete dimer, and the active site organization is similar to, but distinct from Escherichia coli 8-amino-7-oxononanoate synthase (EcAONS), the E. coli homologue of BioF. To investigate the influence of structural characteristics on the function of MsBioF, we deleted bioF in M. smegmatis and confirmed that BioF is required for growth in the absence of exogenous biotin. Based on structural and mutagenesis studies, we confirmed that pyridoxal 5′-phosphate (PLP) binding site residues His129, Lys235 and His200 are essential for MsBioF activity in vivo and residue Glu171 plays an important, but not essential role in MsBioF activity. The N-terminus (residues 1–37) is also essential for MsBioF activity in vivo. The structure and function of MsBioF reported here provides further insights for developing new anti-tuberculosis inhibitors aimed at the biotin synthesis pathway.     

酶固化技术最新研究进展

酶的本质是一种具有催化功能的蛋白质,能影响化学反应。然而,与传统的天然酶分子比较,固化酶相对更为脆弱,而传统的有机或无机催化剂其活性则比较固定。固化酶对于优化产业生产过程非常重要,近几十年来已开发出多种新型固化酶。本文在回顾酶固定化技术最新发展的同时。着重将其最新技术分别从吸附于载体,诱惑侦查及交联等三个方面进行综述。     

酶固化技术最新研究进展(英文)

酶的本质是一种具有催化功能的蛋白质,能影响化学反应。然而,与传统的天然酶分子比较,固化酶相对更为脆弱,而传统的有机或无机催化剂其活性则比较固定。固化酶对于优化产业生产过程非常重要,近几十年来已开发出多种新型固化酶。本文在回顾酶固定化技术最新发展的同时。着重将其最新技术分别从吸附于载体,诱惑侦查及交联等三个方面进行综述。     

Improved isolation of glucuronan from algae and the production of glucuronic acid oligosaccharides using a glucuronan lyase

A new source for the production of bioactive glucuronic acid oligosaccharides (GlcUAOs) from the depolymerization of green seaweed Ulva lactuca glucuronan (Algal glucuronan) has been investigated. Algal glucuronan purification was optimized by the acidic precipitation method which allowed us to separate the polysaccharide mixture extracted from the cell wall of Ulva lactuca using hot water containing sodium oxalate. A series of the GlcUAOs were obtained by enzyme degradation of algal glucuronan with a glucuronan lyase (GL) isolated from Trichoderma strain. The putative bioactive GlcUAOs generated were then purified by size-exclusion chromatography in gram quantity and characterized by ¹H/¹³C NMR spectroscopy and ESI-Q/TOF-mass spectrometry.