scholarly journals Identification, Cloning, and Characterization of a Novel Ketoreductase from the Cyanobacterium Synechococcus sp. Strain PCC 7942

2008 ◽  
Vol 74 (21) ◽  
pp. 6697-6702 ◽  
Author(s):  
Kathrin H�lsch ◽  
Jan Havel ◽  
Martin Haslbeck ◽  
Dirk Weuster-Botz
Keyword(s):  
Specific Activity ◽  
Acyl Carrier Protein ◽  
Asymmetric Reduction ◽  
Enantiomeric Excess ◽  
High Specific Activity ◽  
Lactobacillus Brevis ◽  
Ph Optimum ◽  
Synechococcus Sp ◽  
Prochiral Ketones ◽  
Pcc 7942

ABSTRACT A new ketoreductase useful for asymmetric synthesis of chiral alcohols was identified in the cyanobacterium Synechococcus sp. strain PCC 7942. Mass spectrometry of trypsin-digested peptides identified the protein as 3-ketoacyl-[acyl-carrier-protein] reductase (KR) (EC 1.1.1.100). The gene, referred to as fabG, was cloned, functionally expressed in Escherichia coli, and subsequently purified to homogeneity. The enzyme displayed a temperature optimum at 44�C and a broad pH optimum between pH 7 and pH 9. The NADPH-dependent KR was able to asymmetrically reduce a variety of prochiral ketones with good to excellent enantioselectivities (>99.8%). The KR showed particular high specific activity for asymmetric reduction of ethyl 4-chloroacetoacetate (38.29 � 2.15 U mg−1) and 2′,3′,4′,5′,6′-pentafluoroacetophenone (8.57 � 0.49 U mg−1) to the corresponding (S)-alcohols. In comparison with an established industrial enzyme like the alcohol dehydrogenase from Lactobacillus brevis, the KR showed seven-times-higher activity toward 2′,3′,4′,5′,6′-pentafluoroacetophenone, with a remarkably higher enantiomeric excess (>99.8% [S] versus 43.3% [S]).

Asymmetric reduction of enones with Synechococcus sp. PCC 7942

2004 ◽  
Vol 15 (11) ◽  
pp. 1677-1679 ◽  
Author(s):  
Kei Shimoda ◽  
Naoji Kubota ◽  
Hiroki Hamada ◽  
Misato Kaji ◽  
Toshifumi Hirata
Keyword(s):  
Asymmetric Reduction ◽  
Synechococcus Sp ◽  
Pcc 7942

scholarly journals Arabinoxylan Oligosaccharide Hydrolysis by Family 43 and 51 Glycosidases from Lactobacillus brevis DSM 20054

2013 ◽  
Vol 79 (21) ◽  
pp. 6747-6754 ◽  
Author(s):  
Herbert Michlmayr ◽  
Johannes Hell ◽  
Cindy Lorenz ◽  
Stefan Böhmdorfer ◽  
Thomas Rosenau ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Functional Food ◽  
Specific Activity ◽  
High Specific Activity ◽  
Lactobacillus Brevis ◽  
Glycoside Hydrolases ◽  
Content Type ◽  
Food And Feed ◽  
Oligosaccharide Hydrolysis

ABSTRACTDue to their potential prebiotic properties, arabinoxylan-derived oligosaccharides [(A)XOS] are of great interest as functional food and feed ingredients. While the (A)XOS metabolism ofBifidobacteriaceaehas been extensively studied, information regarding lactic acid bacteria (LAB) is still limited in this context. The aim of the present study was to fill this important gap by characterizing candidate (A)XOS hydrolyzing glycoside hydrolases (GHs) identified in the genome ofLactobacillus brevisDSM 20054. Two putative GH family 43 xylosidases (XynB1 and XynB2) and a GH family 43 arabinofuranosidase (Abf3) were heterologously expressed and characterized. While the function of XynB1 remains unclear, XynB2 could efficiently hydrolyze xylooligosaccharides. Abf3 displayed high specific activity for arabinobiose but could not release arabinose from an (A)XOS preparation. However, two previously reported GH 51 arabinofuranosidases fromLb. breviswere able to specifically remove α-1,3-linked arabinofuranosyl residues from arabino-xylooligosaccharides (AXHm3 specificity). These results imply thatLb. brevisis at least genetically equipped with functional enzymes in order to hydrolyze the depolymerization products of (arabino)xylans and arabinans. The distribution of related genes inLactobacillalesgenomes indicates that GH 43 and, especially, GH 51 glycosidase genes are rare among LAB and mainly occur in obligately heterofermentativeLactobacillusspp.,Pediococcusspp., members of theLeuconostoc/Weissellabranch, andEnterococcusspp. Apart from the prebiotic viewpoint, this information also adds new perspectives on the carbohydrate (i.e., pentose-oligomer) metabolism of LAB species involved in the fermentation of hemicellulose-containing substrates.

scholarly journals Discovery and molecular and biocatalytic properties of hydroxynitrile lyase from an invasive millipede,Chamberlinius hualienensis

2015 ◽  
Vol 112 (34) ◽  
pp. 10605-10610 ◽  
Author(s):  
Mohammad Dadashipour ◽  
Yuko Ishida ◽  
Kazunori Yamamoto ◽  
Yasuhisa Asano
Keyword(s):  
Specific Activity ◽  
Enantiomeric Excess ◽  
High Specific Activity ◽  
Potassium Cyanide ◽  
Aromatic Aldehydes ◽  
Building Blocks ◽  
Industrial Biotechnology ◽  
Hydroxynitrile Lyase ◽  
Wide Range ◽  
Very High

Hydroxynitrile lyase (HNL) catalyzes the degradation of cyanohydrins and causes the release of hydrogen cyanide (cyanogenesis). HNL can enantioselectively produce cyanohydrins, which are valuable building blocks for the synthesis of fine chemicals and pharmaceuticals, and is used as an important biocatalyst in industrial biotechnology. Currently, HNLs are isolated from plants and bacteria. Because industrial biotechnology requires more efficient and stable enzymes for sustainable development, we must continuously explore other potential enzyme sources for the desired HNLs. Despite the abundance of cyanogenic millipedes in the world, there has been no precise study of the HNLs from these arthropods. Here we report the isolation of HNL from the cyanide-emitting invasive millipedeChamberlinius hualienensis, along with its molecular properties and application in biocatalysis. The purified enzyme displays a very high specific activity in the synthesis of mandelonitrile. It is a glycosylated homodimer protein and shows no apparent sequence identity or homology with proteins in the known databases. It shows biocatalytic activity for the condensation of various aromatic aldehydes with potassium cyanide to produce cyanohydrins and has high stability over a wide range of temperatures and pH values. It catalyzes the synthesis of (R)-mandelonitrile from benzaldehyde with a 99% enantiomeric excess, without using any organic solvents. Arthropod fauna comprise 80% of terrestrial animals. We propose that these animals can be valuable resources for exploring not only HNLs but also diverse, efficient, and stable biocatalysts in industrial biotechnology.

A novel ene-reductase from Synechococcus sp. PCC 7942 for the asymmetric reduction of alkenes

2012 ◽  
Vol 47 (12) ◽  
pp. 1988-1997 ◽  
Author(s):  
Yilei Fu ◽  
Kathrin Hoelsch ◽  
Dirk Weuster-Botz
Keyword(s):  
Asymmetric Reduction ◽  
Synechococcus Sp ◽  
Pcc 7942

Asymmetric Reduction of Enones with Synechococcus sp. PCC 7942.

ChemInform ◽  
2004 ◽  
Vol 35 (42) ◽  
Author(s):  
Kei Shimoda ◽  
Naoji Kubota ◽  
Hiroki Hamada ◽  
Misato Kaji ◽  
Toshifumi Hirata
Keyword(s):  
Asymmetric Reduction ◽  
Synechococcus Sp ◽  
Pcc 7942

scholarly journals The purification and properties of a ribonucleoenzyme, o-diphenol oxidase, from potatoes

1970 ◽  
Vol 118 (1) ◽  
pp. 15-23 ◽  
Author(s):  
K. Balasingam ◽  
W. Ferdinand
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Disc Electrophoresis ◽  
Ph Optimum ◽  
Molecular Weights ◽  
Major Form ◽  
Purification And Properties ◽  
Minimal Molecular Weight

1. o-Diphenol oxidase was isolated from potato tubers by a new approach that avoids the browning due to autoxidation. 2. There are at least three forms of the enzyme, of different molecular weights. The major form, of highest molecular weight, was separated from the others in good yield and with high specific activity by gel filtration through Bio-Gel P-300. 3. The major form is homogeneous by disc electrophoresis but regenerates small amounts of the species of lower molecular weight, as shown by rechromatography on Bio-Gel P-300. 4. There is an equal amount of RNA and protein by weight in the fully active enzyme. The RNA cannot be removed without loss of activity, and is not attacked by ribonuclease. 5. The pH optimum of the enzyme is at pH5.0 when assayed with 4-methylcatechol as substrate. It is ten times more active with this substrate than with chlorogenic acid or catechol. The enzyme is fully active in 4m-urea. 6. A minimal molecular weight of 36000 is indicated by copper content and amino acid analysis of the protein component of the enzyme. 7. The protein contains five half-cystinyl residues per 36000 daltons, a value similar to that found in o-diphenol oxidase from mushrooms. It also contains tyrosine residues although, when pure, it does not turn brown by autoxidation.

Characterization of a lysosomal proteinase purified from haploid cells of Physarum flavicomum undergoing encystment

1983 ◽  
Vol 61 (5) ◽  
pp. 1357-1366 ◽  
Author(s):  
Henry R. Henney Jr. ◽  
Hiltrud U. White
Keyword(s):  
Specific Activity ◽  
High Specific Activity ◽  
Acid Proteinase ◽  
Thiol Groups ◽  
Ph Optimum ◽  
Golgi Bodies ◽  
Cell Extracts ◽  
Thiol Reagent ◽  
Lysosomal Proteinase ◽  
Ph Range

Haploid cells of Physarum flavicomum differentiating to dormant microcysts exhibited prominent Golgi bodies, lysosomes, and autophagic vacuoles digesting intracellular materials. Total intracellular acid proteinase activities, as well as enzyme specific activity, increased during the encystment process. Lysosomes, isolated by fractionation of cell extracts on sucrose density gradients, were identified by their ultrastructural characteristics as well as their content of the following enzymes of high specific activity: acid proteinase, acid β-galactosidase, acid phosphatase, and β-glucuronidase. The lysosomal acid proteinase was chromatographically purified, and its molecular weight was estimated to be 32 000. The proteinase was most stable in the pH range of 2–3 which similarly corresponds to its pH optimum using hemoglobin and Azocasein, respectively, as substrates. Its isoelectric point was about 2. The enzyme exhibited little activity and was unstable at pH 7 and above. The rate of activity of the proteinase was maximal at 55 °C, and good stability of the enzyme was noted up to 45 °C. The proteinase required a thiol reagent for stability. Pepstatin, which specifically affects acid proteinases, inhibited the enzyme. Also, compounds reactive with enzyme thiol groups were highly effective inhibitors of the proteinase. The lysosomal enzyme is an acid (carboxyl) proteinase with essential thiol groups.

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