Effects of oxygen on invertase expression in continuous culture of recombinant Saccharomyces cerevisiae containing the SUC2 gene

The yeast SUC2 gene, cloned on a multicopy plasmid pRB58, was used to study the effect of oxygen on the invertase expression of the recombinant Saccharomyces cerevisiae. Glucose repression was not the only factor affecting the invertase expression. The results obtained from the single-stage continuous cultures under microaerobic conditions showed that invertase expression was also strongly dependent on oxygen availability, and moving from anaerobic to aerobic conditions led to a five-fold increase in specific invertase activity. However, the cell yields under anaerobic conditions were quite low compared to those under aerobic conditions. These opposite effects of oxygen on cell growth and gene expression offer a strategy for maximizing invertase productivity by a two-stage continuous culture. The first stage was operated at a low level of glucose, around 100 mg/l, under aerobic conditions in order to obtain a high yield of yeast biomass, and the second stage maintained anaerobic conditions with residual glucose levels of 50 mg/l to derepress and fully induce invertase expression. The two-stage continuous culture resulted in a 2.5-fold increase in invertase productivity over that of a single-stage continuous culture. Received: 28 July 1998 / Received revision: 22 September 1998 / Accepted: 7 November 1998     

Methionine biosynthesis and its regulation in Corynebacterium glutamicum: parallel pathways of transsulfuration and direct sulfhydrylation

There are two alternative pathways leading to methionine synthesis in microorganisms: The transsulfuration pathway involves cystathionine as the intermediate and utilizes cysteine as the sulfur source, but the direct sulfhydrylation pathway bypasses cystathionine and uses inorganic sulfur instead. While most microorganisms synthesize methionine via either one of these pathways, Corynebacterium glutamicum utilizes both pathways, which appear to be fully functional. In C. glutamicum, each pathway is catalyzed by independent enzymes and is tightly regulated by methionine. Although the physiological significance of parallel pathways remains to be elucidated, their presence suggests metabolic flexibility and efficient adaptation of the organism to its environment.     

Native-feather degradation by Fervidobacterium islandicum AW-1, a newly isolated keratinase-producing thermophilic anaerobe

A native-feather-degrading thermophilic anaerobe was isolated from a geothermal hot stream in Indonesia. Isolate AW-1, identified as a member of the species Fervidobacterium islandicum, was shown to degrade native feathers (0.8%, w/v) completely at 70 degrees C and pH 7 with a maximum specific growth rate (0.14 h(-1)) in Thermotoga- Fervidobacterium(TF) medium. After 24 h of culture, feather degradation led to an increase in free amino acids such as histidine, cysteine and lysine. Moreover, nutritionally essential amino acids such as tryptophan and methionine, which are rare in feather keratin, were also produced as microbial metabolites. A homomultimeric membrane-bound keratinolytic protease (>200 kDa; 97 kDa subunits) was purified from a cell extract of F. islandicum AW-1. The enzyme exhibited activity toward casein and soluble keratin optimally at 100 degrees C and pH 9, and had a half-life of 90 min at 100 degrees C. The enzyme showed higher specific activity for the keratinous substrates than other proteases and catalyzed the cleavage of peptide bonds more rapidly following the reduction of disulfide bridges in feather keratin by 10 mM dithiothreitol. Therefore, the enzyme from F. islandicum AW-1 is a novel, thermostable keratinolytic serine protease.     

Effect of atherosclerosis on lysosomal cholesterol esterase activity in rabbit aorta.

Radiolabeled cholesteryl oleate, when incorporated into phospholipid vesicles, was hydrolyzed at acid pH by an enzyme present in rabbit aortic homogenates. In contrast, cholesteryl oleate presented as an acetone dispersion was not effectively hydrolyzed at acid pH under identical conditions. Using the vesicle preparation as substrate, a sensitive assay system for the acid hydrolase was developed in which hydrolysis was proportional to protein concentration and incubation time, and was independent of substrate concentration. The physical state of the vesicles was apparently not altered by the assay conditions, and no hydrolysis of the vesicle-associated phospholipid was detected. Acid cholesterol esterase activity in atherosclerotic aortic tissue was 2.5-fold greater than that of control tissue, and even greater increases were observed in the activities of other lysosomal enzymes (N-acetyl-beta-d-glucosaminidase and beta-glucuronidase). Glucose-6-phosphatase activity was also increased in aortas from cholesterol-fed animals while 5' nucleotidase activity remained unchanged. Labeled triolein also was incorporated into phospholipid vesicles and was hydrolyzed by an acid lipase in aortic tissue. Similarities between triolein and cholesteryl oleate hydrolysis existed with respect to pH optimum and the effect of cholesterol feeding on activity, suggesting that a single enzyme may hydrolyze both lipids.     

Octamer binding protein-1 is involved in inhibition of inducible nitric oxide synthase expression by exogenous nitric oxide in murine liver cells

Nitric oxide (NO) has diverse effects on immune responses and hepatic functions. In BNL CL.2 cells, the murine embryonic liver cells, inducible nitric oxide synthase (iNOS) mRNA expression appeared after 3 h of treatment with IFN-gamma and LPS. Interestingly, mRNA and protein expression of iNOS was down-regulated by sodium nitroprusside (SNP) and diethylamine dinitric oxide in a time- and dose-dependent manner, but not by H2O2. TNF-alpha gene expression was also dramatically reduced by SNP, but IL-6 gene expression was inhibited much less. IFN-gamma and LPS-induced chloramphenicol acetyltransferase activity of iNOS promoter constructs was inhibited by SNP. Electrophoretic mobility shift assay showed that SNP inhibited IFN-gamma plus LPS-induced Oct-1 binding activity, and the inhibition was reversed by DTT. Mutation in the Oct-1 site completely abolished iNOS promoter activity. In addition, supershift assay and Southwestern analysis demonstrated that the Oct-1 binding activity was inhibited by SNP. Taken together, these results indicate that NO suppresses IFN-gamma plus LPS-induced iNOS expression, and that Oct-1 is an important element in this process.     

Production of a platelet aggregation inhibitor, salmosin, by high cell density fermentation of recombinant Escherichia coli

Optimal conditions for a high cell density fermentation were investigated in a recombinant Escherichia coli producing salmosin, a platelet aggregation inhibitor. The optimized carbon and nitrogen sources were glycerol 10 g/l, yeast extract 30 g/l, and bacto-tryptone 10 g/l, yielding the dry cell weight (DCW) of 10.61 g/l in a 500 ml flask culture. The late-stage induction with 1% L-arabinose in a 5 l jar fermentor showed the highest DCW of 65.70 g/l after 27 h of the fed-batch fermentation. Around 2,200 mg/l of the protein was expressed as an inclusion body that was then refolded to obtain the active salmosin of 96 mg/l. We also confirmed the inhibitory activity against platelet aggregation of the active salmosin from the high cell density fermentation.     

One step immobilization of peptides and proteins by using modified parylene with formyl groups

One-step immobilization method for peptides and proteins is developed by using modified parylene film with formyl groups which is suitable for microplate-based immunoassay and SPR biosensor application. The immobilization of peptides and proteins is achieved through the covalent bonding of the formyl group with the primary amine groups of peptides and proteins, which no additional activation step is required. In this work, the immobilization efficiency of parylene-H is estimated in comparison with parylene-A and physical adsorption, using biotinylated-cyclic citrullinated peptide (biotinylated-CCP), human chorionic gonadotropin (hCG) and horseradish peroxidase (HRP) as model proteins. The applicability of parylene-H film to SPR biosensor is demonstrated by estimating the detection range and sensitivity of SPR biosensor at various thicknesses. The immobilization efficiency of parylene-H film for SPR biosensor was compared with physical adsorption by using HRP as a model protein.     

Production of D-tagatose at high temperatures using immobilized Escherichia coli cells expressing L-arabinose isomerase from Thermotoga neapolitana

Escherichia coli cells expressing l-arabinose isomerase from Thermotoga neapolitana (TNAI) were immobilized in calcium alginate beads. The resulting cell reactor (2.4 U, t _1/2 = 43 days at 70°C) in a continuous recycling mode at 70°C produced 49 and 38 g d-tagatose/l from 180 and 90 g d-galactose/l, respectively, within 12 h.