Characterization of the second form of NADH-cytochrome b5 reductase gene from arachidonic acid-producing fungus Mortierella alpina 1S-4

The second type of cytochrome b5 reductase (Cb5R-II) gene was characterized in the arachidonic acid-producing fungus Mortierella alpina 1S-4. Its cDNA (897 bp) and predicted amino acid (298 aa) sequences show more than 70% similarity to the previously isolated first type of Cb5R. Highly conserved exon-intron organization suggests that the two genes evolved from the duplication of a common ancestral gene. Cb5R-II has a flavin-binding domain at its highly hydrophobic N-terminal and an NADH-binding domain at the C-terminal. In comparison with Cb5R genes from other sources, high homology (46-54%) was found for yeast and plant genes. Phylogenetic analysis revealed that microbial and plant Cb5R genes represent a gene family evolved from one prototype and are different from mammalian Cb5R genes.     

Major Peptides from Amaranth (Amaranthus cruentus) Protein Inhibit HMG-CoA Reductase Activity

The objective of this study was to identify the major peptides generated by the in vitro hydrolysis of Amaranthus cruentus protein and to verify the effect of these peptides on the activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), a key enzyme in cholesterol biosynthesis. A protein isolate was prepared, and an enzymatic hydrolysis that simulated the in vivo digestion of the protein was performed. After hydrolysis, the peptide mixture was filtered through a 3 kDa membrane. The peptide profile of this mixture was determined by reversed phase high performance chromatography (RP-HPLC), and the peptide identification was performed by LC-ESI MS/MS. Three major peptides under 3 kDa were detected, corresponding to more than 90% of the peptides of similar size produced by enzymatic hydrolysis. The sequences identified were GGV, IVG or LVG and VGVI or VGVL. These peptides had not yet been described for amaranth protein nor are they present in known sequences of amaranth grain protein, except LVG, which can be found in amaranth α‑amylase. Their ability to inhibit the activity of HMG-CoA reductase was determined, and we found that the sequences GGV, IVG, and VGVL, significantly inhibited this enzyme, suggesting a possible hypocholesterolemic effect.     

Characterization of FabG and FabI of the Streptomyces coelicolor Dissociated Fatty Acid Synthase

Streptomyces coelicolor produces fatty acids for both primary metabolism and for biosynthesis of the secondary metabolite undecylprodiginine. The first and last reductive steps during the chain elongation cycle of fatty acid biosynthesis are catalyzed by FabG and FabI. The S. coelicolor genome sequence has one fabI gene (SCO1814) and three likely fabG genes (SCO1815, SCO1345, and SCO1846). We report the expression, purification, and characterization of the corresponding gene products. Kinetic analyses revealed that all three FabGs and FabI are capable of utilizing both straight and branched‐chain β‐ketoacyl‐NAC and enoyl‐NAC substrates, respectively. Furthermore, only SCO1345 differentiates between ACPs from both biosynthetic pathways. The data presented provide the first experimental evidence that SCO1815, SCO1346, and SCO1814 have the catalytic capability to process intermediates in both fatty acid and undecylprodiginine biosynthesis.     

Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae

The aldehyde reductase gene ARI1 is a recently characterized member of an intermediate subfamily within the short‐chain dehydrogenase/reductase (SDR) superfamily that clarified mechanisms of in situ detoxification of 2‐furaldehyde and 5‐hydroxymethyl‐2‐furaldehyde by Saccharomyces cerevisiae. Uncharacterized open reading frames (ORFs) are common among tolerant candidate genes identified for lignocellulose‐to‐advanced biofuels conversion. This study presents partially purified proteins of two ORFs, YDR541C and YGL039W, and direct enzyme assay evidence against aldehyde‐inhibitory compounds commonly encountered during lignocellulosic biomass fermentation processes. Each of the partially purified proteins encoded by these ORFs showed a molecular mass of approximately 38 kDa, similar to Ari1p, a protein encoded by aldehyde reductase gene. Both proteins demonstrated strong aldehyde reduction activities toward 14 aldehyde substrates, with high levels of reduction activity for Ydr541cp toward both aromatic and aliphatic aldehydes. While Ydr541cp was observed to have a significantly higher specific enzyme activity at 20 U/mg using co‐factor NADPH, Ygl039wp displayed a NADH preference at 25 U/mg in reduction of butylaldehyde. Amino acid sequence analysis identified a characteristic catalytic triad, Ser, Tyr and Lys; a conserved catalytic motif of Tyr–X–X–X–Lys; and a cofactor‐binding sequence motif, Gly–X–X–Gly–X–X–Ala, near the N‐terminus that are shared by Ydr541cp, Ygl039wp, Yol151wp/GRE2 and Ari1p. Findings of aldehyde reductase genes contribute to the yeast gene annotation and aids development of the next‐generation biocatalyst for advanced biofuels production. Copyright © 2015 John Wiley & Sons, Ltd.     

COLOUR REACTION SCREENING OF CLONES WITH DIACETYL AND ACETOIN REDUCTASE ACTIVITIES

A genomic library of the chromosomal DNA of a brewing strain (Saccharomyces carlsbergensis) has been made in a Escherichia coli HB101 strain as host. A coloured test allowing one to isolate recombined clones possessing diacetyl (DR) and acetoin (AR) reductase activities is described. This test allowed one to detect 3 Escherichia coli clones having both activities (DR and AR) from among 3000 E. coli strains constituting the genomic library of Saccharomyces carlsbergensis in E. coli.     

Influence of curcuma (Curcuma xanthorrhiza Roxb) on lipid metabolism in rabbits

The efficacy of curcuma (Curcuma xanthorrhiza) in lowering blood plasma lipid was evaluated in rabbits. Forty male New Zealand white rabbits (2.3 kg) were divided into four equal groups and offered isoatherogenic diets with no curcuma, low curcuma (2 g kg^?1), medium curcuma (3 g kg^?1) and high curcuma (4 g kg^?1) for 120 days. Faeces and feed samples were collected. Body weight was measured weekly. Blood samples at 1, 2 and 4 months and liver samples at the last blood collection were taken. Curcuma did not influence feed, protein and fat consumption and protein excretion (P > 0.05) but significantly (P < 0.05) increased fat excretion. Cholesterol concentration was decreased by 46.6, 56.4 and 63.2% and HDL concentration was decreased by 9.9, 14.5 and 21.9% at 2, 3 and 4 g kg^?1 curcuma respectively. Curcuma significantly (P < 0.05) decreased LDL concentration and significantly (P < 0.01) decreased triglyceride concentration by 20.4, 28.5 and 29.5% at 2, 3 and 4 g kg^?1 respectively. HMG‐CoA reductase inhibitor was significantly (P < 0.05) increased by curcuma. Glucose was significantly (P < 0.05) reduced by 6.2, 7.6 and 18.0% at 2, 3 and 4 g kg^?1 curcuma respectively. Lipid peroxidation was prevented at 3 and 4 g kg^?1 curcuma. The enhanced fat excretion could have been mediated through an acceleration of lipid metabolism from extrahepatic tissues to the liver, which would increase the excretion of cholesterol via the bile and into the faeces. Since this is central to lipid metabolism, curcuma has potential as a phytotherapeutic agent under atherosclerosis and cardiovascular disease conditions. © 2002 Society of Chemical Industry     

Favorable effect of very low initial KLa value on xylitol production from xylose by a self-isolated strain of Pichia guilliermondii

Xylitol production from xylose by a self-isolated furfural and 5-hydroxymethyl furfural assimilating Pichia guilliermondii was studied under oxygen limitation. An extremely low initial volumetric oxygen transfer coefficient (0.075 h^− 1) was found most favorable to the xylitol production with yield of 0.61 g g^− 1. Related enzymes activities were also investigated and discussed.     

蜡样芽孢杆菌Bacillus cereus LJ01中亚硝酸盐还原酶的基因克隆、表达和纯化

利用克隆表达技术,对一种蜡样芽孢杆菌Bacillus cereus LJ01的亚硝酸盐还原酶（nitrite reductase,NiR）基因进行克隆、原核表达,利用Ni柱亲和层析和DEAE Sepharose Fast Flow交换层析对表达的重组NiR进行纯化,分析重组酶的性质。研究结果显示,该NiR含有一个1?623?bp的开放阅读框,编码540?个氨基酸序列,重组NiR的分子质量约为67?ku;该酶中同时存在铁离子和铜离子,且含量分别为51.0?mg/kg和184.5?mg/kg;圆二色谱结果显示α-螺旋结构在重组NiR中所占比例最大。