Sarintip Sooksai

In Vivo Regulation of Alcohol Dehydrogenase and Lactate Dehydrogenase in Rhizopus Oryzae to Improve l-Lactic Acid Fermentation

Rhizopus oryzae is becoming more important due to its ability to produce an optically pure l-lactic acid. However, fermentation by Rhizopus usually suffers from low yield because of production of ethanol as a byproduct. Limiting ethanol production in living immobilized R. oryzae by inhibition of alcohol dehydrogenase (ADH) was observed in shake flask fermentation. The effects of ADH inhibitors added into the medium on the regulation of ADH and lactate dehydrogenase (LDH) as well as the production of cell biomass, lactic acid, and ethanol were elucidated. 1,2-diazole and 2,2,2-trifluroethanol were found to be the effective inhibitors used in this study. The highest lactic acid yield of 0.47 g/g glucose was obtained when 0.01 mM 2,2,2-trifluoroethanol was present during the production phase of the pregrown R. oryzae. This represents about 38% increase in yield as compared with that from the simple glucose fermentation. Fungal metabolism was suppressed when iodoacetic acid, N-ethylmaleimide, 4,4′-dithiodipyridine, or 4-hydroxymercury benzoic acid were present. Dramatic increase in ADH and LDH activities but slight change in product yields might be explained by the inhibitors controlling enzyme activities at the pyruvate branch point. This showed that in living R. oryzae, the inhibitors regulated the flux through the related pathways.

An extracellular lipase from the endophytic fungi Fusarium oxysporum isolated from the Thai medicinal plant, Croton oblongifolius Roxb.

From 65 endophytic fungal isolates, ten were found to produce extracellular lipase activity, with Fusarium oxysporum isolate PTM7, isolated from the leaves of Croton oblongifolius Roxb. (Plao yai), yielding the highest level. The lipase activity in the basal culture medium of PTM7 was highest with 1% (v/v) olive oil, 1% (w/v) peptone and 0.5% (w/v) sodium nitrate as the carbon, organic and inorganic nitrogen sources, respectively. A 37.4 kDa lipase was enriched with 41.4-fold to apparent homogeneity from PTM7 culture media using 80% saturation ammonium sulfate precipitation, DEAE-cellulose anion exchange and Superdex-75 gel filtration chromatography, but at a final yield of only 2.21%. The enriched lipase showed optimal activity at pH 8 and 30 o

1,2-Diazole and 2,2,2-Trifluoroethanol and Their Regulatory Effects on Ethanol and Lactic Acid Formation in the Living Culture of Rhizopus oryzae

In heterofermentation of Rhizopus oryzae, ethanol is the major byproduct which reduces the production of a desired product, an optically pure l-lactic acid. To improve lactic acid production, regulating the alcohol fermentative pathway to limit ethanol production has been done by various techniques. In vitro study on alcohol dehydrogenase (ADH) inhibition in several organisms showed that 1,2-diazole and 2,2,2-trifluoroethanol were competitively bound at the active sites that eventually limited ethanol production. In this study, 1,2-diazole and 2,2,2-trifluoroethanol were present during fermentation of R. oryzae. It was found that both 1,2-diazole and 2,2,2-trifluoroethanol not only strongly affected ethanol formation but they also indirectly regulated lactate production as observed by the decreasing affinity for glucose flux toward lactate and ethanol production. The increase in both ethanol and lactate formation rates revealed 1,2-diazole and 2,2,2-trifluoroethanol not only regulated the reversible redox reaction by ADH, but they also caused the dynamic change in the conversion of all metabolites in the living R. oryzae in order to maintain the balanced flux for cellular growth and maintenance.

Construction and sequencing analysis of scFv antibody fragment derived from monoclonal antibody against norfloxacin (Nor155)

Norfloxacin belongs to the group of fluoroquinolone antibiotics which has been approved for treatment in animals. However, its residues in animal products can pose adverse side effects to consumer. Therefore, detection of the residue in different food matrices must be concerned. In this study, a single chain variable fragment (scFv) that recognizes norfloxacin antibiotic was constructed. The cDNA was synthesized from total RNA of hybridoma cells against norfloxacin. Genes encoding VH and VL regions of monoclonal antibody against norfloxacin (Nor155) were amplified and size of VH and VL fragments was 402 bp and 363 bp, respectively. The scFv of Nor155 was constructed by an addition of (Gly4Ser)3 as a linker between VH and VL regions and subcloned into pPICZαA, an expression vector of Pichia pastoris. The sequence of scFv Nor155 (GenBank No. AJG06891.1) was confirmed by sequencing analysis. The complementarity determining regions (CDR) I, II, and III of VH and VL were specified by Kabat method. The obtained recombinant plasmid will be useful for production of scFv antibody against norfloxacin in P. pastoris and further engineer scFv antibody against fluoroquinolone antibiotics.

Expression, purification and biological activity of monomeric insulin precursors from methylotrophic yeasts

The methylotrophic yeasts Pichia pastoris and Hansenula polymorpha have been used for the production of recombinant monomeric insulin precursor (MIP). Recombinant plasmids with one, two and four cassettes of the MIP gene have been successfully constructed in the pPICZαA expression vector to study the effects of gene copy number on MIP production. The MIP protein can be detected by dot-blot analysis from the culture broth of P. pastoris KM71H 24 h after placement in MMH induction medium. The secretion levels of MIP protein in culture broth at 72 h after induction indicated that P. pastoris KM71H with one cassette of the MIP gene had highest MIP protein levels (4.19 ± 0.96 mg L-1). The transcription levels of the MIP gene increased proportionately with copy number. However, the amount of secreted MIP protein showed no correlation. The MIP molecular mass was 5756.951 Da, as confirmed by typical MALDI-TOF mass spectrometry. The MIP protein in culture broth was purified by two steps purification including SP Sepharose Fast Flow chromatography followed by ultrafiltration (10 kDa MW cutoff). The percentage of MIP recovery after the two-step purification was 70%, with a single band in a native-PAGE. The biological activity of tryptic hydrolyzed MIP was determined via the expression of the glucose transporter 4 gene (GLUT4) in H9c2 (2-1) cell line by RT-qPCR, and the results demonstrated that the MIP protein can induce glucose uptake and upregulation of GLUT4 mRNA transcription at 3 h and that this activity was related to Humalog® insulin.

Molecular Cloning and Comparative Analysis of Variable Regions of Monoclonal Antibody against Enrofloxacin Clone 48

Enrofloxacin is fluoroquinolone antibiotic which prohibited approved for treatment in animals. However, their residues in animal products can pose adverse side effects to consumer. Therefore, the maximum residue limit of these drugs has been enforced in many countries. In this study, the cDNA encoding VH and VL genes was amplified from monoclonal antibody which was specific to enrofloxacin clone 48, cloned and sequenced. The obtained sequences were compared in the NCBI databases by using blastp program. The results found that VH nucleotide was composed of about 399 bps and theirs deduced amino acids showed 80-85% degree identities to the Ig superfamily group. A totally 356 bps of VL nucleotide was found and showed the degree of identities of 97-100% with an immunoglobulin kappa light chain. Moreover, the CDR I, CDR II and CDR III of the VH and VL sequences were specified. The results indicated that the highest degree of VH sequence homology (highest relationship) found in accession no. B26471 with 80% homology. However, 100% of VL sequences were found similarly to accession no. AHJ10945.1 than the others. The obtained results provided the useful and important information for the further recombinant antibody construction and production against enrofloxacin antibiotic.