Influence of hyaluronidase addition on the production of hyaluronic acid by batch culture of Streptococcus zooepidemicus

Enhancement of hyaluronic acid (HA) production by Streptococcus zooepidemicus through increasing oxygen transfer rate via degradation of HA by hyaluronidase was investigated. Dissolved oxygen (DO) level became a limiting factor for HA production during 8–16 h, and thus hyaluronidase (0.05, 0.10, 0.15, 0.20, 0.25 g/l) was added at 8 h to degrade HA. Oxygen transfer rate coefficient and DO level during 8–16 h increased with increased hyaluronidase concentration. Compared to 5.0 ± 0.1 g/l of the control without hyaluronidase addition, HA production was increased from 5.0 ± 0.1 g/l to 6.0 ± 0.1 g/l when hyaluronidase concentration was 0.15 g/l. Further increase of hyaluronidase concentration had no effect on HA production. The molecular weight of HA decreased with the increased hyaluronidase concentration and decreased to 21 kDa when hyaluronidase concentration was 0.25 g/l from 1300 kDa of the control. The prepared low molecular weight HA (LMW-HA) could function as potential anti-angiogenic substances, antiviral and anti-tumor agents to possibly be used as functional food ingredients.     

Development of a novel method for feeding a mixture of -lactic acid and acetic acid in fed-batch culture of Ralstonia eutropha for poly- -3-hydroxybutyrate production

Fermentative production of poly- -3-hydroxybutyrate [P(3HB)] from a mixture of -lactic acid and acetic acid by Ralstonia eutropha was investigated. For fed-batch culture with cell density, it is necessary to control the concentration of these organic acids in the culture medium below the inhibitory level for cell growth. Therefore, a novel feeding method, termed the computer-controlled pH-stat substrate feeding method, was developed using the rate of increase of the pH (pH-increasing rate) of the culture medium as an indicator for feed control. The pH-increasing rate, which was calculated every minute by a pH meter-linked computer, represented secondary information regarding substrate consumption by cells. When the pH-increasing rate decreased to 5% of the maximum increasing rate, acidic substrate solution was fed into the fermentor until the pH was reduced to 7.00. Using this feeding strategy, the cell concentration and PHA content obtained in 42 h were 75.0 g/l and 73.1% (w/w), respectively, resulting in a high P(3HB) productivity of 1.30 g/l·h.     

Cloning and Sequence Analysis of the estA gene encoding enzyme for producing (R)-β-acetylmercaptoisobutyric acid from Pseudomonas aeruginosa 1001

The estA gene encoding the enzyme that catalyzes the production of (R)-β-acetylmercaptoisobutyric acid from (R,S)-ester from Pseudomonas aeruginosa 1001, was cloned in Escherichia coli and its nucleotide sequence was determined, revealing the presumed open reading frame encoding a polypeptide of 316 amino acid residues (948 nucleotides). The overall A+T and C+G compositions were 32.59% and 67.41%, respectively. The amino acid sequence of the estA gene product showed a significant similarity with that of the triacylglycerol lipase from Psychrobacter immobilis (38% identity), triacylglycerol lipase from Moraxella sp. (36% identity), and two forms of carboxyl esterases from Acinetobacter calcoaceticus (17% and 17% identities). The deduced amino acid sequences have a pentapeptide consensus sequence, G-X-S-X-G, having an active serine residue, and another active site, dipeptides H-G, located at 70–100 amino acids upstream of the G-X-S-X-G consensus sequence.