• KSBB Journal
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• 제14권6호
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• pp.696-701
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• 1999

본 연구에서는 lipase를 이용해서 ricinoleic acid를 대량생산하기 위해 피마자유의 완전가수분해 조건을 찾고자 하였다. 널리 알려진 lipase CR, lipase CC, lipase PP를 대상으로 피마자유의 가수분해의 가능성을 시험하고, 유기용매를 사용함으로써 가수분해도를 향상시킬 수 있음을 확인하였다. 일반적으로 lipase의 활성을 감소시키는 극성용매의 경우 피마자유의 가수분해에 있어서도 효소의 활성을 감소시켰고, 물과 섞이지 않는 hydrophobic solvent가 피마자유의 가수분해도를 크게 증가시키는 것을 확인하였다. 본 연구에서는 isopropyl ether의 효과가 가장 크며, 조건에 따라 가수분해도를 두 배 이상 증가시킨다는 것을 확인하였다. 그리고 유기용매를 사용함으로써 pH의 영향을 바꾸거나 감소시킬 수 있다는 사실도 확인하였다. 용매와 물의 부피비에 의해서 가수분해가 영향을 받는다는 사실과 특히 유기용매보다는 물의 양에 절대적으로 영향받는다는 사실을 발견하였다. 하지만, 물과 유기용매의 부피비와 함께 lipase와 피마자유의 무게비도 매우 중요하다는 것을 확인하였다. 30$^{\circ}C$에서 isopropyl ether를 사용할 경우 무게 비로 2 wt%일 때는 약 82%, 4 wt% 이상의 lipase CC나 lipase CR을 사용하면 피마자유가 완전히 가수분해되는 사실을 발견하였다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제7권6호
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• pp.367-370
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• 2002

Candida cylindracea lipase was immobilized by adsorption on acid washed glass beads. It was observed that protein loading of the support depends on the size of the particle, with smaller particle containing higher amount of protein per unit weight. Initial reaction rate linearly varied up to enzyme concentration of 17.25 U/mL. Amount of free fatty acids produced was linearly proportional up to the enzyme loading of 1650 $\mu$g/g of bead. Achievement of chemical equilibrium took longer time in the case of less protein loading. Degree of hydrolysis was found to decrease in second and third consecutive batch operations on repeated use of immobilized lipase.

• KSBB Journal
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• 제15권1호
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• pp.72-75
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• 2000

Candida cylindracea 유래의 효소 Lipase-OF 360,000을 사용하여 물고기 기름을 구성하고 있는 각 지방산의 가수분해 특성을 규명하여 보았다. 물고기 기름을 구성하고 있는 다양한 지방산중 $C_{14.0}$, $C_{16.0}$ 및 $C_{18.0}$의 포화지방산과 이중결합이 하나인 $C_{16.1}$, $C_{18.1}$(n-7), $C_{18.1}$(n-9), $C_{20.1}$ 및 $C_{22.1}$의 불포화 지방산은 $\omega$-3 다중불포화지방산에 비하여 쉽게 가수분해되었다. $\omega$-3다중불포화지방산중 탄소수는 동일하나 불포화도가 상이할 경우 불포화도가 낮은 지방산이 불포화도가 높은 지방산보다 쉽게 가수분해되는 특성을 나타내었으며 불포화도는 동일하나 탄소수가 다른 경우 탄소수가 적은 지방산이 탄소수가 많은 지방산보다 쉽게 가수분해되었다. $\omega$-3 다중불포화지방산중 가수분해 반응 후 모노-, 디- 및 트리-클리세라이드 혼합물에 가장 많이 농축되는 지방산은 DHA로 물고기 기름을 구성하는 총 $\omega$-3 지방산의 31.87%에서 가수분해반응 120시간 후에는 글리세라이드 혼합물을 구성하는 총 $\omega$-3 지방산의 51.89%까지 증가하였다.

• Journal of Microbiology and Biotechnology
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• 제13권1호
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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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• 제9권6호
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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.

• 한국응용과학기술학회지
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• 제18권3호
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• pp.214-232
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• 2001

CTA ester bonds in TG molecules were not attacked by pancreatic lipase and lipases produced by microbes such as Candida cylindracea, Chromobacterium viscosum, Geotricum candidium, Pseudomonas fluorescens, Rhizophus delemar, R. arrhizus and Mucor miehei. An aliquot of total TG of all the seed oils and each TG fraction of the oils collected from HPLC runs were deuterated prior to partial hydrolysis with Grignard reagent, because CTA molecule was destroyed with treatment of Grignard reagent. Deuterated TG (dTG) was hydrolyzed partially to a mixture of deuterated diacylglycerols (dDG), which were subsequently reacted with (S)-(+)-1-(1-naphthyl)ethyl isocyanate to derivatize into dDG-NEUs. Purified dDG-NEUs were resolved into 1, 3-, 1, 2- and 2, 3-dDG-NEU on silica columns in tandem of HPLC using a solvent of 0.4% propan-1-o1 (containing 2% water)-hexane. An aliquot of each dDG-NEU fraction was hydrolyzed and (fatty acid-PFB ester). These derivatives showed a diagnostic carboxylate ion, $(M-1)^{-}$, as parent peak and a minor peak at m/z 196 $(PFB-CH_{3})^{-}$ on NICI mass spectra. In the mass spectra of the fatty acid-PFB esters of dTGs derived from the seed oils of T. kilirowii and M. charantia, peaks at m/z 285, 287, 289 and 317 were observed, which corresponded to $(M-1)^{-}$ of deuterized oleic acid ($d_{2}-C_{18:0}$), linoleic acid ($d_{4}-C_{18:0}$), punicic acid ($d_{6}-C_{18:0}$) and eicosamonoenoic acid ($d_{2}-C_{20:0}$), respectively. Fatty acid compositions of deuterized total TG of each oil measured by relative intensities of $(M-1)^-$ ion peaks were similar with those of intact TG of the oils by GLC. The composition of fatty acid-PFB esters of total dTG derived from the seed oils of T. kilirowii are as follows; $C_{16:0}$, 4.6 mole % (4.8 mole %, intact TG by GLC), $C_{18:0}$, 3.0 mole % (3.1 mole %), $d_{2}C_{18:0}$, 11.9 mole % (12.5 mole %, sum of $C_{18:1{\omega}9}$ and $C_{18:1{\omega}7}$), $d_{4}-C_{18:0}$, 39.3 mole % (38.9 mole %, sum of $C_{18:2{\omega}6}$ and its isomer), $d_{6}-C_{18:0}$, 41.1 mole % (40.5 mole %, sum of $C_{18:3\;9c,11t,13c}$, $C_{18:3\;9c,11t,13r}$ and $C_{18:3\;9t,11t,13c}$), $d_{2}-C_{20:0}$, 0.1 mole % (0.2 mole % of $C_{20:1{\omega}9}$). In total dTG derived from the seed oils of M. charantia, the fatty acid components are $C_{16:0}$, 1.5 mole % (1.8 mole %, intact TG by GLC), $C_{18:0}$, 12.0 mole % (12.3 mole %), $d_{2}-C_{18:0}$, 16.9 mole % (17.4 mole %, sum of $C_{18:1{\omega}9}$), $d_{4}-C_{18:0}$, 11.0 mole % (10.6 mole %, sum of $C_{18:2{\omega}6}$), $d_{6}-C_{18:0}$, 58.6 mole % (57.5 mole %, sum of $C_{18:3\;9c,11t,13t}$ and $C_{18:3\;9c,11t,13c}$). In the case of Aleurites fordii, $C_{16:0}$; 2.2 mole % (2.4 mole %, intact TG by GLC), $C_{18:0}$; 1.7 mole % (1.7 mole %), $d_{2}-C_{18:0}$; 5.5 mole % (5.4 mole %, sum of $C_{18:1{\omega}9}$), $d_{4}-C_{18:0}$ ; 8.3 mole % (8.5 mole %, sum of $C_{18:2{\omega}6}$), $d_{6}-C_{18:0}$; 82.0 mole % (81.2 mole %, sum of $C_{18:3\;9c,11t,13t}$ and $C_{18:3 9c,11t,13c})$. In the stereospecific analysis of fatty acid distribution in the TG species of the seed oils of T. kilirowii, $C_{18:3\;9c,11t,13r}$ and $C_{18:2{\omega}6}$ were mainly located at sn-2 and sn-3 position, while saturated acids were usually present at sn-1 position. And the major molecular species of $(C_{18:2{\omega}6})(C_{18:3\;9c,11t,13c})_{2}$ and $(C_{18:1{\omega}9})(C_{18:2{\omega}6})(C_{18:3\;9c,11t,13c})$ were predominantly composed of the stereoisomer of $sn-1-C_{18:2{\omega}6}$, $sn-2-C_{18:3\;9c,11t,13c}$, $sn-3-C_{18:3\;9c,11t,13c}$, and $sn-1-C_{18:1{\omega}9}$, $sn-2-C_{18:2{\omega}6}$, $sn-3-C_{18:3\;9c,11t,13c}$, respectively, and the minor TG species of $(C_{18:2{\omega}6})_{2}(C_{18:3\;9c,11t,13c})$ and $ (C_{16:0})(C_{18:3\;9c,11t,13c})_{2}$ mainly comprised the stereoisomer of $sn-1-C_{18:2{\omega}6}$, $sn-2-C_{18:2{\omega}6}$, $sn-3-C_{18:3\;9c,11t,13c}$ and $sn-1-C_{16:0}$, $sn-2-C_{18:3\;9c,11t,13c}$, $sn-3-C_{18:3\;9c,11t,13c}$. The TG of the seed oils of Momordica charantia showed that most of CTA, $C_{18:3\;9c,11t,13r}$, occurred at sn-3 position, and $C_{18:2{\omega}6}$ was concentrated at sn-1 and sn-2 compared to sn-3. Main TG species of $(C_{18:1{\omega}9})(C_{18:3\;9c,11t,13t})_{2}$ and $(C_{18:0})(C_{18:3\;9c,11t,13t})_{2}$ were consisted of the stereoisomer of $sn-1-C_{18:1{\omega}9}$, $sn-2-C_{18:3\;9c,11t,13t}$, $sn-3-C_{18:3\;9c,11t,13t}$ and $sn-1-C_{18:0}$, $sn-2-C_{18:3\;9c,11t,13t}$, $sn-3-C_{18:3\;9c,11t,13t}$, respectively, and minor TG species of $(C_{18:2{\omega}6})(C_{18:3\;9c,11t,13c})_{2}$ and $(C_{18:1{\omega}9})(C_{18:2{\omega}6})(C_{18:3\;9c,11t,13c})$ contained mostly $sn-1-C_{18:2{\omega6}$, $sn-2-C_{18:3\;9c,11t,13t}$, $sn-3-C_{18:3\;9c,11t,13t}$ and $sn-1-C_{18:1{\omega}9}$, $sn-2-C_{18:2{\omega}6}$, $sn-3-C_{18:3\;9c,11t,13t}$. The TG fraction of the seed oils of Aleurites fordii was mostly occupied with simple TG species of $(C_{18:3\;9c,11t,13t})_{3}$, along with minor species of $(C_{18:2{\omega}6})(C_{18:3\;9c,11t,13t})_{2}$, $(C_{18:1{\omega}9})(C_{18:3\;9c,11t,13t})_{2}$ and $(C_{16:0})(C_{18:3\;9c,11t,13t})$. The sterospecific species of $sn-1-C_{18:2{\omega}6}$, $sn-2-C_{18:3\;9c,11t,13t}$, sn-3-C_{18:3\;9c,11t,13t}$, $sn-1-C_{18:1{\omega}9}$, $sn-2-C_{18:3\;9c,11t,13t}$, $sn-3-C_{18:3\;9c,11t,13t}$ and $sn-1-C_{16;0}$, $sn-2-C_{18:3\;9c,11t,13t}$, $sn-3-C_{18:3\;9c,11t,13t}$ are the main stereoisomers for the species of $(C_{18:2{\omega}6})(C_{18:3\;9c,11t,13t})_2$, $(C_{18:1{\omega}9})(C_{18:3\;9c,11t,13t})_{2}$ and $(C_{16:0})(C_{18:3\;9c,11t,13t})$, respectively.