• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.229-229
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• 2003

• Microbiology and Biotechnology Letters
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• v.47 no.4
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• pp.546-554
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• 2019

This study, for the first time, reports the functional expression of lipase B derived from the yeast Candida antarctica (CALB) in Corynebacterium strain using the Escherichia coli plasmid PK18. The CALB gene fragment encoding a 317-amino-acid protein was successfully obtained from the total RNA of C. antarctica. CALB was readily produced in the Corynebacterium strain without the use of induction methods described in previous studies. This demonstrated the extracellular production of CALB in the Corynebacterium strain. CALB produced in the Corynebacterium MB001 strain transformed with pEC-CALB recombinant plasmid exhibited maximum extracellular enzymatic activity and high substrate affinity. The optimal pH and temperature for the hydrolysis of 4-nitrophenyl laurate by CALB were 9.0 and 40℃, respectively. The enzyme was stable at pH 10.7 in the glycine-KOH buffer and functioned as an alkaline lipase. The CALB activity was inhibited in the presence of high concentration of Mg²⁺, which indicated that CALB is not a metalloenzyme. These properties are key for the industrial application of the enzyme.

• Journal of Microbiology and Biotechnology
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• v.15 no.2
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• pp.296-301
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• 2005

The effect of aqueous methanol on the catalytic properties of porcine pancreatic lipase has been investigated. The k$_{CAT}$, values for the hydrolysis of N$^{alpha}$-benzyloxycarbonyl-L­lysine p-nitrophenyl ester at 0$^{circ}$C increased in a linear manner with increasing methanol concentration. However, the K$_{M}$ values were not influenced at methanol concentrations lower than $30\%$ and then began to increase at higher concentrations in an exponential fashion. Based on product analysis, the increase in k$_{CAT}$, with increasing methanol concentration can be accounted for by nucleophilic competition of methanol for the acyl enzyme intermediate, indicating that the rate-limiting step of the porcine pancreatic lipase-catalyzed reaction is deacylation under current experimental conditions. The exponential increase in K$_{M}$ at methanol concentrations higher than $30\%$ is attributed to the hydrophobic partitioning effect on substrate binding. There was no loss of lipase activity over a 4 h period in $60\%$ methanol concentration at pH$^{circ}$ 5.5 and 0$^{circ}$C. By monitoring the intrinsic fluorescence and absorbance, no evidence for structural changes by methanol was observed.

• Proceedings of the Korea Technical Association of the Pulp and Paper Industry Conference
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• 2006.06a
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• pp.129-136
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• 2006

Although cleaner and cheaper deinking of ONP could be performed at the neutral or low alkaline condition excessive loss from froth-flotation is unavoidable and so reduction of alkali or caustic soda dosage sacrifices recycling yield. Now the new trade-off regarding alkali dosage versus flotation yield is urgently required in order to set the optimized neutral or low alkaline deinking process of ONP. Lipase from Thermomyces Lanuginosus has an effect on desizing and deacetylation reaction and it could be applied to the stock of pre flotation secondary stage in order to reduce the flotation reject without the sacrifice of optical properties of flotation accepts. Instead of inorganic base, lipase could be applied as a biochemical catalyst for the selective modification of valuable hydrophobic particles in deinking stock, for example cellulose fines and inorganic fillers covered by hydrophobic additives or contaminants. When the enzymatic hydrolysis of ester bond could be made on the surface of hydrophobic particulates, unwanted float of fine particles could be prevented. Now the enhancement of flotation selectivity or the modification of the hydrophobicity of deinking stock is expected to be promoted by the enzymatic pre treatment. And the reduction of recycling cost with the saves of raw material, recovered paper would be possible as a result.

• Journal of Microbiology and Biotechnology
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• v.13 no.1
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• pp.57-63
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• 2003

Using fungal (Fusarium solani f. pisi) and bacterial (Pseudomonas mendocina) cutinases, the initial hydrolysis rate of p-nitrophenyl esters was systematically estimated for a wide range of enzyme and substrate concentrations using a 96-well microplate reader. Both cutinases exhibited a high substrate specificity; i.e. a high hydrolytic activity on p-nitrophenyl butyrate (PNB), yet extremely low activity on p-nitrophenyl palmitate (PNP). When compared to the hydrolysis of PNB and PNP by other hydrolases [lipases and esterases derived from different microbial sources, such as bacteria (Pseudomonas cepacia, Psedomonas furescens, Baciilus stearothermophilus), molds (Aspeillus niger, mucor miehei), and yeasts (Candida rugosa, Candida cylindracea)], the above substrate specificity would seem to be a unique characteristic of cutinases. Secondly, the hydrolytic activity of the cutinases on PNB appeared much faster than that of the other hydrolytic enzymes mentioned above. Furthermore, the current study proved that even when the cutinases were mixed with large amounts of other hydrolases (lipases or esterases), the Initial hydrolysis rate of PNB was determined only by the cutinase concentration for each PNB concentration. This property of cutinase activity would seem to result from a higher accessibility to the substrate PNB, compared with the other hydrolytic enzymes. Accordingly, these distinct properties of cutinases may be very useful in the rapid and easy isolation of various natural cutinases with different microbial sources, each of which may provide a novel industrial application with a specific enzymatic function.

• Journal of Microbiology and Biotechnology
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• v.18 no.4
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• pp.735-741
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• 2008

In our previous study, a surfactant-coated Candida rugosa lipase immobilized in microemulsion-based organogels was exploited for the synthesis of ethyl isovalerate. In the present study, we are focusing on the effective reuse of lipase immobilized in microemulsion-based organogels (MBGs) in terms of retainment of the catalytic activity. As water is one of the co-products in esterification reactions, the removal of water becomes a priority to allow the reaction to work in the forward direction and to prevent back hydrolysis. Taking this fact into consideration, the lipase-containing microemulsion-based organogels were given pretreatment and/or several intermittent treatments with dry reverse micellar solution of AOT in organic solvent during repeated cycles of ester synthesis. The pretreated MBGs with dry reverse micellar solution exhibited lower water content and higher initial rates of esterification in comparison with untreated freshly prepared MBGs. The esterification efficiency of untreated MBGs started decreasing after 5 cycles of reuse and was almost completely lost by the end of the $8^{th}$ cycle. In contrast, pretreated MBGs exhibited a gradual decrease in esterification efficiency after 5 cycles and retained about 80% of the initial activity at the end of the $8^{th}$ cycle. The intermittent treatment of MBGs after every 3 cycles resulted in enhanced reusability of immobilized lipase for up to 9 cycles without significant loss in esterification activity, after which it resulted in a slow decrease in activity with about 27% lower activity at the end of the $12^{th}$ cycle. Furthermore, the treatment conditions such as concentration of AOT in liquid dessicant and time of treatment were optimized with respect to our system. The granulated MBGs proved to be better in terms of initial esterification rates (1.2-fold) as compared with the pelleted MBGs.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.97-104
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• 2004

Water-sludge bacteria were screened to find a lipase enantioselectively hydrolyzing itraconazole precursor, which is well known as the starting material of antifungal drug agents. A bacterial strain was isolated and identified as Acinetobacter junii SY-01. After the strain was cultivated, the enzyme was purified 39.4-fold using ultrafiltration and gel filtration through a Sephadex G-100 chromatographic column and the activity yield was 34.9%. The molecular weight of the enzyme was about 40 kDa, as measured by SDS-PAGE, and the optimum pH was 7.0- 9.0 and stable at pH 6.0- 9.0. The optimum temperature was 45- $5^{\circ}C$, and 73% of the enzymes activity remained after incubation at 70% for 1 h. Enzyme activity was enhanced by gall powder, sodium deoxycholate, a cationic detergent Tween 80, and a non-ionic detergent Triton X-100, but was markedly inhibited by metal ions such as $Hg^{2＋},Cu^{2＋},Ni^{2＋}/,Ca^{2＋}$, and an anionic-surfactant sodium dodecylsulfate. The $K_{m}$ values for (R)- and (S)-enantiomers of the itraconazole precursor were 0.385 and 21.83 mM, respectively, and the $V_{max} values ($\mu$Mㆍmin^{-1}.)$ were 6.73 and 6.49, respectively. The acetyl group among the different acyl moieties of itraconazole precursor showed the highest enantioselectivity for the hydrolysis by the Acinetobacter junii SY-01 lipase, and the lipase from Acinetobacter junii SY-01 displayed better enantioselectivity than that of commercially available lipases and esterases.

• Journal of Microbiology and Biotechnology
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• v.9 no.6
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• pp.773-777
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• 1999

Scale-up experiments for hydrolysis of beef tallow, fat, and palm kernel with lipase derived from Candida cylindracea were carried out in 1-1, 100-1, and 10,000-1 reactors. The optimum agitation speed for the hydrolysis of the 1-1 reactor was investigated and found to be 350rpm, and this was a basis for the scale-up of agitation speed. The hydrolysis system in this work was the oil-water system in which the hydrolysis seems to process a heterogeneous reaction. An emulsion condition was the most important factor for determining the reaction rate of hydrolysis. Therefore, the scale-up of agitation speed was performed by using the power n = 1/3 in an equation of the rules of thumb method. The geometrical similarity for scaling-up turned out to be unsatisfactory in this study. Thus, the working volume per one agitator was used for the scale-up. In the case of scale-up from a 1-1 reactor to a 100-1 reactor, the hydrolysis of palm kernel was very much scaled-up by initiating the rules of thumb method. However, the hydrolysis of fat and beef tallow in a 100-1 reactor was a little higher than that of the 1-1 reactor because of the difference of geometrical similarity. The scale-up of hydrolysis from the 100-1 reactor to the 10,000-1 reactor was improved compared to that of the 1-1 to 100-1 reactor. The present results indicated that the scale-up of hydrolysis in the oil-water system by the rules of thumb method was more satisfactory under the condition of geometrical similarity. Even in the case where geometrical similarity was not satisfactory, the working volume per one agitator could be used for the scale-up of a heterogeneous enzyme reaction.

• Applied Biological Chemistry
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• v.37 no.4
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• pp.272-276
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• 1994

The dried cinnamon was extracted with enzymes, salts, sugars and additives in order to find the most effective extraction material. Enzymatic hydrolysis of cinnamon suspension with cellulase, hemicellulase, pectinase, ${\beta}-1.4-glucosidase$, tannase and lipase showed a little increase of their cinnamic aldehyde contents. Solid yield, antioxidant activity and degree of browning were increased in hemicellulase treatment. Acid and alkali extraction of cinnamon showed a some increase in solid yields and antioxidant activity was increased by addition of glucose and Na-ascorbate. Cinnamic aldehyde contents and degree of browning were increased in extraction with Na-citrate addition.

• Microbiology and Biotechnology Letters
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• v.33 no.1
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• pp.29-34
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• 2005

Lipase-producing bacteria (S3) were isolated from intertidal flat at Saemanguem. A isolated strain was identified as Psychrobacter species by physiological and fermentational characterization as well as 16S rRNA analysis. The strain was then named as Psychrobacter sp. S3. P. sp. S3 grew most rapidly at $30^{\circ}C$, but grew well even at $10^{\circ}C$ and its lipase activity was most high when cultivated at $20^{\circ}C$. Lipase S3 had optimum temperature of $30^{\circ}C$ for the hydrolysis of p-nitrophenyl caproate and had more than $80^{\circ}C$ activity even at $10^{\circ}C$. The activation energy was calculated to be 1.5 kcal/mol, which showed that it was a typical cold-adapted enzyme. It was an alkaline enzyme with optimum pH of $9.0\~9.5$. It could hydrolyze various length of triglycerides. Among them, it hydrolyzed most rapidly $C_4,\;C_{14},\; C_{16}-length$ triglycerides. When added to tributyrin-agarose gel, lipase S3 hydrolyzed tributyrin most rapidly at 30 and $40^{\circ}C$, but it could hydrolyze well even at $4^{\circ}C$.