Mutation strategies for obtaining chitooligosaccharides with longer chains by transglycosylation reaction of family GH18 chitinase

Enhancing the transglycosylation (TG) activity of glycoside hydrolases does not always result in the production of oligosaccharides with longer chains, because the TG products are often decomposed into shorter oligosaccharides. Here, we investigated the mutation strategies for obtaining chitooligosaccharides with longer chains by means of TG reaction catalyzed by family GH18 chitinase A from Vibrio harveyi (VhChiA). HPLC analysis of the TG products from incubation of chitooligosaccharide substrates, GlcNAc_n, with several mutant VhChiAs suggested that mutant W570G (mutation of Trp570 to Gly) and mutant D392N (mutation of Asp392 to Asn) significantly enhanced TG activity, but the TG products were immediately hydrolyzed into shorter GlcNAc_n. On the other hand, the TG products obtained from mutants D313A and D313N (mutations of Asp313 to Ala and Asn, respectively) were not further hydrolyzed, leading to the accumulation of oligosaccharides with longer chains. The data obtained from the mutant VhChiAs suggested that mutations of Asp313, the middle aspartic acid residue of the DxDxE catalytic motif, to Ala and Asn are most effective for obtaining chitooligosaccharides with longer chains.     

In vitro anticancer activity of loquat tea by inducing apoptosis in human leukemia cells

Fresh loquat leaves have been used as folk health herb in Asian countries for long time, although the evidence supporting their functions is still minimal. This study aimed to clarify the chemopreventive effect of loquat tea extract (LTE) by investigating the inhibition on proliferation, and underlying mechanisms in human promyelocytic leukemia cells (HL-60). LTE inhibited proliferation of HL-60 in a dose-dependent manner. Molecular data showed that the isolated fraction of LTE induced apoptosis of HL-60 as characterized by DNA fragmentation; activation of caspase-3, -8, and -9; and inactivation of poly(ADP)ribose polymerase. Moreover, LTE fraction increased the ratio of pro-apoptotic Bcl-2-associated X protein (Bax)/anti-apoptotic myeloid cell leukemia 1 (Mcl-1) that caused mitochondrial membrane potential loss and cytochrome c released to cytosol. Thus, our data indicate that LTE might induce apoptosis in HL-60 cells through a mitochondrial dysfunction pathway. These findings enhance our understanding for chemopreventive function of loquat tea.     

Purification,cDNA cloning,and characterization of LysM-containing plant chitinase from horsetail (Equisetum arvense)

Chitinase-A (EaChiA), molecular mass 36 kDa, was purified from the vegetative stems of a horsetail (Equisetum arvense) using a series of column chromatography. The N-terminal amino acid sequence of EaChiA was similar to the lysin motif (LysM). A cDNA encoding EaChiA was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1320 nucleotides and encoded an open reading frame of 361 amino acid residues. The deduced amino acid sequence indicated that EaChiA is composed of a N-terminal LysM domain and a C-terminal plant class IIIb chitinase catalytic domain, belonging to the glycoside hydrolase family 18, linked by proline-rich regions. EaChiA has strong chitin-binding activity, however, no antifungal activity. This is the first report of a chitinase from Equisetopsida, a class of fern plants, and the second report of a LysM-containing chitinase from a plant.     

Formation of long-chain N-vanillyl-acylamides from plant oils

Long-chain N-vanillyl-acylamides (LCNVAs) were generated from plant oils and vanillylamine (VA) by nucleophilic amidation without any catalytic reagents. The resulting LCNVAs varied according to the fatty acid composition of the plant oil used. Therefore, the LCNVAs contained in Capsicum oleoresins were products that were spontaneously generated from the oleoresin during storage.     

Properties of Crystalline 3β-Hydroxysteroid Oxidase of Brevibacterium sterolicum

3β-Hydroxysteroid oxidase (3β-hydroxysteroid: oxygen oxidoreductase, EC 1.1.3.6.) from the culture supernatant of Brevibacterium sterolicum ATCC 21387 has a molecular weight of 32,500 and an isoelectric point of 8.9. The enzyme contained 258 amino acid residues and the composition revealed a distinctive feature of a relatively high amount of proline and the absence of alanine and tryptophan. The crystalline enzyme exhibited an absorption spectrum characteristic of a flavoprotein with absorption maxima at 280, 390, and 470 nm with a shoulder at 490 nm. Anaerobic addition of dehydro-epi-androsterone as well as sodium dithionite to the enzyme produced a disappearance of the peaks at 390 and 470 nm. The flavin moiety of the enzyme was isolated and identified as flavin adenine dinucleotide, 1 mole of which was found per mole of protein. The enzyme is sulfhydryl dependent and was inactivated by silver and mercury compounds. Analysis of the enzyme protein by atomic absorption spectrophotometry failed to detect any significant quantity of heavy metals.

Various 3β-hydroxysteroids were oxidized and the relative rates of the oxidation were cholesterol, 100; dehydro-epi-androsterone, 41; pregnenolone, 22; and β-sitosterol, 20. The oxidation product of cholesterol by the enzyme was crystallized and identified as 4-cholesten-3-one by melting point, elementary analysis, optical rotation, UV, IR and NMR spectra. The oxidation of cholesterol proceeded as follows:

The enzyme would be used for some analytical and preparative purposes in the field of steroid chemistry, e.g., microdetermination of cholesterol in serum.     

Isolation and Crystallization of Extracellular 3β-Hydroxysteroid Oxidase of Brevibacterium sterolicum nov. sp.

Among about 500 strains tested, a newly isolated soil bacterium, Brevibacterium sterolicum nov. sp. KY 3463 (ATCC 21387) showed the highest potency in production of 3β-hydroxysteroid oxidase in the culture fluid.

The 3β-hydroxysteroid oxidase was purified from the culture filtrate by a procedure involving ammonium sulfate fractionation, DEAE-cellulose and hydroxyapatite column chromatographies and Sephadex G–75 gel filtration. Crystals of the enzyme were obtained from solutions of the purified preparation by the addition of ammonium sulfate. The crystals appeared as fine rods, with a bright yellow color.

The enzyme is homogeneous by disc gel electrophoresis and ultracentrifugation. Sedimentation velocity yields a value of . It exhibits a typical flavoprotein spectrum of absorption maxima at 280, 390, and 470 mμ.     

Studies on Abscisic Acid

Oxidation of methvl 2-trans-β-ionylideneacetate with X-bromosuccinimide afforded methyl 2-cis and trans-3′-hydroxy-β-ionylideneacetates. NaBH₄ reduction of methyl 2-cis-3′-keto-β-ionylideneacetate and ethyl 4′-keto-α-ionylideneacetate gave methyl 2-cis-3′-hydroxy-β-ionylideneacetate and ethyl 4′-hydroxy-α-ionyiideneacetate respectively. Further, methyl 4′-methoxy-epoxy-α-ionylideneacetate was prepared by epoxidation of methyl 4′-methoxy-α-ionylideneacetate. And then methyl 4′-hydroxy-l′, 2′-dihydro-β-ionylideneacetate was synthesized from ethyl 4-keto-α-cyclogeranate. Growth inhibitory activities of the above compounds on rich seedlings were examined.     

Microbial Resolution of Racemic Carvomenthols

By selected microorganisms dl-carvomenthyl acetate, dl-isocarvomenthyl acetate and dl-neo isocarvomenthyl acetate were asymmetrically hydrolyzed to l-carvomenthol with d-carvomenthyl acetate, l-isocarvomenthol with d-isocarvomenthyl acetate and d-neo isocarvomenthol with l-neo isocarvomenthyl acetate respectively; dl-neo carvomenthyl acetate was not hydrolyzed.     

Studies on Abscisic Acid

Methyl α-ionylideneacetates were oxidized with selenium dioxide to a mixture of methyl 3′-keto-β-ionylideneacetates and a small amount of methyl 4′-keto-α-ionylidene-acetates followed by treatment with active manganese dioxide. By a similar oxidation methyl 3′-keto-β-ionylideneacetates were prepared from methyl β-ionylidene acetates. Methyl 4′-keto-α-ionylideneacetates were obtained by oxidation of methyl α-ionylideneacetates with tert-butyl chromate. Dehydrobromination of methyl bromoionylideneacetate, obtained by bromination of methyl 2-trans-α-ionylideneacetate with N-bromosuccinimide, gave a mixture of methyl 2-trans-dehydro-β-ionylideneacetate and methyl 2-cis-dehydro-β-ionylideneacetate. The growth inhibitory activities of these sesquiterpene carboxylic acids and keto esters on rice seedlings were tested.