• Microbiology and Biotechnology Letters
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• v.17 no.3
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• pp.263-268
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• 1989

To maximize the performance of recombinant cell fermentation process through optimizing environmental conditions, the production of invertase from recombinant Saccharomyces cerebisiae Containing SUC2 gene was studied as a model. The recombinant cells showed biphasic growth on glucose. Since the promoter of the SUC2 is regulated by the concentration of glucose in the medium, expression of invertase by recombinant yeast began when the glucose concentration decreased in a range of 0.25-0.4 g/L during the batch culture. Plasmid segregation occured frequently during glucose fermentation, and infrequently during ethanol oxidation. A rapid appearance of invertase activity with glucose was observed under nonaerated condition, and the maximum specific invertase activity was about 1.5 times as high as under aerobic condition, In fed batch culture, when n low level of glucose was continuously supplied to the tormentor after the time of glucose depletion during growth phase, specific and total invertase activity increased about 1.7 and 2.9 fold, respectively, in a batch culture.

• Journal of Life Science
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• v.14 no.3
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• pp.429-434
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• 2004

Levansucrase gene (levU) from Zymomonas mobilis was subcloned downstream of GALl promoter in pYES 2.0 and pYInu-AT [GALl0 promoter＋exoinulinase signal sequence of Kluyveromyces marxianus], resulting pYES-levU and pYInu-levU, respectively. The two expression plasmids were introduced into an invertase-deficient strain, Saccharomyces cerevisiae SEY2102, and then transformants showing high activity of levansucrase were selected. When each yeast transformants was cultivated in medium containing galactose, the extracellular and intracellular activities of levansucrase reached about 7.17 U/㎖ with the strain harboring pYES-levU and 6.61 U/㎖ with the strain harboring pYInu-levU. It was found that about 50% of levansucrase were detected in the medium and periplasmic space, and exoinulinase signal sequence didn't enhance the secretion efficiency. Furthermore, the recombinant levansucrase expressed in yeast seems to be produced as a hyper-glycosylated form.

• Microbiology and Biotechnology Letters
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• v.32 no.3
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• pp.206-211
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• 2004

Levansucrase gene(lscA) from Pseudomonas aurantiaca was subcloned downstream of GAL1 promoter in pYES 2.0 and pYInu-AT [GAL10 promoter ＋ exoinulinase signal sequence of Kluyveromyces marxianus], resulting pYES-lscA and p YInu-lscA, respectively. The two expression plasmids were introduced into an invertase-deficient strain, Sacchromayces cerevisiae SEY2102, and transformants with high activity of levansucrase were selected. When each yeast transform ants was cultivated in medium containing galactose, the extracellular and intracellular activities of levansucrase reached about 8.62 U/ml with the strain harboring pYES-lscA and 5.43 U/ml with the strain harboring pYInu-lscA. The levansucrase activity of 80% was detected in the periplasmic space and cytoplasm. The levansucrase activity in the medium of SEY2102/pYInu-lscA was 0.87 U/ml whereas that of SEY2102/pYES-lscA was 0.47 U/ml, which implying the exoinulinase signal sequence didn't enhance the secretion efficiency of levansucrase. Furthermore, the recombinant levansucrase expressed in yeast seems to be produced as a hyper-glycosylated form.

• 한국생물공학회:학술대회논문집
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• 2000.04a
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• pp.478-481
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• 2000

The INU2 gene encoding an endoinulinase of Aspergillus ficuum was expressed by the Kluyveromyces marxianus INU1 promoter in a SUC2-deleted Saccharomyces cerevisiae to produce the endoinulinase free of an exoinulinase and an extracellular invertase in the culture medium. When inulin was included in the medium, a recombinant yeast strain produced the sufficient amount of the enzyme to make a halo around its colony. An expression of endoinulinase was dependent on the culture temperature and shaking. The highest expression of endoinulinase was observed at $30^{\circ}C$, and 150rpm.

• Journal of agriculture & life science
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• v.45 no.6
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• pp.201-212
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• 2011

The alkaline protease gene was cloned from a halo-tolerant alkalophilic Bacillus clausii I-52 isolated from the heavily polluted tidal mud flat of West Sea in Inchon Korea, which produced a strong extracellular alkaline protease (BCAP). Based on the full genome sequence of Bacillus subtilis, PCR primers were designed to allow for the amplification and cloning of the intact pro-BCAP gene including promoter region. The full-length gene consists of 1,143 bp and encodes 381 amino acids, which includes 29 residues of a putative signal peptide and an additional 77-amino-acid propeptide at its N-terminus. The mature BCAP deduced from the nucleotide sequence consists of 275 amino acids with a N-terminal amino acid of Ala, and a relative molecular weight and pI value was 27698.7 Da and 6.3, respectively. The amino acid sequence shares the highest similarity (99%) to the nattokinase precursor from B. subtilis and subtilisin E precursor from B. subtilis BSn5. The substrate specificity indicated that the recombinant BCAP could hydrolyze efficiently the synthetic substrate, N-Suc-Ala-Ala-Pro-Phe-pNA,and did not hydrolyze the substrates with basic amino acids at the P1 site. The recombinant BCAP was strongly inhibited by typical serine protease inhibitor, PMSF, indicating that BCAP is a member of the serine proteases.

• Journal of Microbiology
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• v.34 no.4
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• pp.334-340
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• 1996

The enzyme, cis,cis-muconate lactonizing enzyme has been proposed to play a key role in the $\beta$-ketoadipate pathway of benzoate degradation. A 3.2-kb EcoRI fragment termed as pRSU2, isolated from a Pseudomonas cepacia genomic library was able to complement the catB defective mutant. Several relevant restriction enzyme sites were determined within the cloned fragment. In Pseudomonas putida SUC2 carrying pRSU2, the enzyme activity was relatively higher than those of the induced or partially induced state of wild type P. putida PRS2000. It was probably due to higher expression of P. cepacia catB in P. putida PRS2000. It was probably due to higher expression of P. cepacia catB in P. putida. One possible interpretation of these results is that the catB promoter in P. cepacia is recognized within P. putida, resulting in the almost same expression level.

• Microbiology and Biotechnology Letters
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• v.25 no.3
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• pp.258-265
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• 1997

The effects of medium pH and culture temperature on the expression and secretion of inulinase and invertase were investigated with recombinant Saccharomyces cerevisiae cells. These cells were obtained by transformation of 2$\mu$-based plasmids pYI10 and pYS10 which contain Kluyveromyces marxianus inulinase gene (INU1A) and S. cerevisiae invertase gene (SUC2), respectively, in the downstream of GAL1 promoter. The expression level and localization of inulinase and invertase were not affected significantly by the initial medium pH: secretion efficiencies of inulinase and invertase into the medium were about 90% and 60%, respectively, in the pH ranges of 4.0 to 6.5. However, the expression and secretion of both enzymes were strongly dependent on the culture temperature. The highest expression (7.7 units/mL) and secretion (6.7 units/mL) of inulinase were observed at 28$\circ$C and 30$\circ$C. As a consequence of decreased localization of inulinase in the periplasmic space, the secretion efficiency increased from 68% at 20$\circ$C, to 95% at 35$\circ$C,. The total expression level and secretion efficiency of invertase increased from 19 units/mL and 55% at 20$\circ$C to 25 units/mL and 68% at 35$\circ$C, respectively. Irrespective of the culture temperature, the invertase activity in the cellular fraction (periplasmic space and cytoplasmic fractions) was kept constant at around 33-45%.

• Journal of Microbiology and Biotechnology
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• v.11 no.6
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• pp.1115-1119
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• 2001

Eight Escherichia coli cells with aerobic growth deflects were isolated by the insertion of ${\lambda}placMu53$, a hybrid bacteriophage of ${\lambda}$ and Mu, which created transcriptional fusion to lacZY. Two of these mutants, CLIO and CLl2, were irradiated with UV to obtain specialized transducing phages. The phages that took out the neighboring chromosomal DNA of the related gene responsible for deflective aerobic growth were identified. The in vivo cloned chromosomal sequence revealed that the mutated gene of CLIO was located at min 34.5 on the Escherichia coli linkage map and 1,599,515 on the physical map. The physical map indicated that there were 7 cistrons in the operon. We named this operon oxg10. The promoter sequence of oxg10 exhibited a possible binding site far SoxS, a transcriptional regulator that activates the transcription of various SoxRS regulon genes. Transferring the oxg10:: ${\lambda}placMu53$ mutation into the wild-type strain, RZ4500, resulted in the inhibition of normal aerobic growth, while the salute mutation in strain MO inhibited aerobic cell growth completely. The full operon sequences of oxg10 were cloned from the Excherichia coli genomic library. The mutated gene of CLl2 was identified to be a sucA gene encoding the ${\alpha}$-ketoglutarate dehydrogenase El component in the TCA cycle.

• Journal of Microbiology and Biotechnology
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• v.13 no.6
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• pp.976-983
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• 2003

Seven Escherichia coli cells defective with aerobic growth were isolated by the insertion of ${\lambda}placMu53$, a hybrid bacteriophage of ${\lambda}$ and Mu, which created a transcriptional fusion to lacZY. These insertion mutant cells were tested on an XG ($5-bromo-4-chloro-3-indolyl-{\beta}-D-galactopyranoside$) medium for anaerobic expression of lacZ by fusion to a promoter. The chromosomal DNA from these strains were digested by EcoRI, and the EcoRI fragments that contained the fused gene and lacZ sequence were identified by Southern hybridization, using lacZ containing plasmid as a probe. The EcoRI fragment from each strain was cloned and sequenced. The sequence data were compared with the GenBank database. The mutated gene of three strains, CYT4, CYT5, and OS11, was found to be identical, and it was nrdAB that encoded ribonucleoside diphosphate reductase. The gene nrdAB was at min 50.5 on the Escherichia coli linkage map and 2,348,084 on the physical map, and is involved in hemAe-related reduction-oxidation reaction. OS6 and OS14 mutant strains had insertion at min 8.3 and the mutated gene was hemB. The hemB encodes 5-aminolevulinate dehydratase or porphobilinogen synthase. The OS3 mutant had insertion in cydB at min 16.6. The cydAB encodes cytochrome d oxidase. In the case of OS1, the fusion was made with sucA, the E1 component of ${\alpha}-ketoglutarate$ dehydrogenase.