Isolation and Biochemical Characterization of Bacillus pumilus Lipases from the Antarctic

( Arifin arild Ranlym ),( Soon Ja Kim ),( Joung Han Yim ),( Antonius Suwanto ),( Hyung Kwoun Kim ) 한국미생물 · 생명공학회 2013 Journal of microbiology and biotechnology Vol.23 No.5

Lipase-producing bacterial strains were isolated from Antarctic soil samples using the tricaprylin agar plate method. Seven strains with relatively strong lipase activities were selected. All of them turned out to be Bacillus pumilus strains by the 16S rRNA gene sequence analysis. Their corresponding lipase genes were cloned, sequenced, and compared. Finally, three different Bacillus pumilus lipases (BPL1, BPL2, and BPL3) were chosen. Their amino acid sequence identities were in the range of 92-98% with the previous Bacillus pumilus lipases. Their optimum temperatures and pHs were measured to be 40oC and pH 9. Lipase BPL1 and lipase BPL2 were stable up to 30oC, whereas lipase BPL3 was stable up to 20oC. Lipase BPL2 was stable within a pH range of 6-10, whereas lipase BPL1 and lipase BPL3 were stable within a pH range of 5-11, showing strong alkaline tolerance. All these lipases exhibited high hydrolytic activity toward pnitrophenyl caprylate (C8). In addition, lipase BPL1 showed high hydrolytic activity toward tributyrin, whereas lipase BPL2 and lipase BPL3 hydrolyzed tricaprylin and castor oil preferentially. These results demonstrated that the three Antarctic Bacillus lipases were alkaliphilic and had a substrate preference toward short- and mediumchain triglycerides. These Antarctic Bacillus lipases might be used in detergent and food industries.

갯벌로부터 분리된 미생물에 의해 생산된 지질 분해 효소의 특성

최충식 ( Choong Sik Choi ),이순열 ( Soon Youl Lee ),이재학 ( Jae Hag Lee ) 한국식품영양학회 2009 韓國食品營養學會誌 Vol.22 No.1

본 연구는 산업적으로 활용할 수 있는 lipase를 개발하기 위해 갯벌에서 분리된 Gelidibacter sp. YH333와 Vibrio sp. YH339에 의해 생산되는 lipase의 특성에 대해 연구하였다. 분리 균주들로부터 세포 외로 방출하는 lipase의 양은 세포수의 증가와 비례하여 급격하게 증가하였다. 분리 균주들에 의해 생산되는 lipase는 대부분 세포 외로 lipase를 방출함으로 lipase가 세포 밖으로 constitutively하게 분비됨을 알 수 있었다. 두 균주에서 생산된 lipase 모두 p-nitrophenyl laulate(C12:0)에서 가장 높은 활성을 보여 주었다. Gelidibacter sp. YH333에서 생산된 lipase가 Vibrio sp. YH339에 의해 생성된 lipase보다 모든 기질에 있어 높은 활성을 보여주고 있다. Gelidibacter sp. YH333에서는 약 50 KDa, 25KDa 등 두 개의 lipase가 확인되었고 Vibrio sp. YH339에서는 약 50 KDa에 해당하는 lipase가 확인되었다. This study was performed to characterize the lipases produced from Gelidibacter sp. YH333 and Vibrio sp. YH339 isolated from mud flats for industrial application of a lipase. Amount of the lipases secreted from the isolated strains was sharply increased in the proportion of increase of number of the cells. The lipases produced from the isolated strains were constitutively secreted from the cells. The lipase activity of Gelidibacter sp. YH333 was higher than that of Vibrio sp. YH339 to p-nitrophenyl esters. The lipases produced from both strains showed the highest activity in p-nitrophenyl laulate among various p-nitrophenyl esters. The molecular weights of the lipases from Gelidibacter sp. YH333 were about 50 KDa and 25 KDa, respectively. Molecular weight of the lipase from Vibrio sp. YH339 was about 50 KDa.

Lipase 유전자의 보존적 영역 탐색

이동근,김철민,김상진,이상현,이재화 한국생명과학회 2003 생명과학회지 Vol.13 No.5

Lipase 유전자들의 보존적 영역을 탐색하기 위해 LED(Lipase Engineering Database)와 COG (Clusters of Orthologous Groups of proteins)를 통하여 각각 132개와 24개의 서열을 얻어 분석하였다. Lipase 유전자의 염기서열은 아주 다양하였고 LED의 각 상동성그룹 (homologous family) 별로 독특한 아미노산 서열이 보존적인 것을 확인 할 수 있었다. COG0657에 속하는 lipase들은 LED의 Moraxella lipase 1 homologous group과 유사한 아미노산 보존적 영역이 있음을 확인하였다. 다양한 반응 조건에 적응하는 혹은 높은 활성을 갖는 새로운 lipase 유전자를 원핵생물에서 탐색하기 위하여 다양한 시도들이 수행되어 왔지만 그들은 모두 배양가능한 미생물에 국한되어 있었다. 배양할 수 없는 세균의 유전자원을 포함하는 메타게놈에 대한 유용성 역시 최근에 널리 인식되고 있다. Lipase 유전자의 다양성으로 보았을 때 메타게놈을 이용한 새로운 lipase 유전자를 찾는 작업도 가능할 것으로 사료되어 lipase유전자 일부를 (222∼713 bp) 증폭시키는 총 10개의 PCR primer를 설계하였으며 그 가능성을 NCBI의 BLAST를 통하여 검증하였다. Lipase 유전자의 염기서열은 아미노산 서열보다 아주 다양하여 비교적 많은 수의 primer set이 필요하였다. 각 primer set의 증폭효율은 각 LED group의 16.7%와 60.0% 사이였고 개별적인 primer set을 이용할 때 보다 3.6배 효율이 높은 것으로 드러났다. Lipase 유전자의 다양성은 설계된 primer set들을 이용하여 새로운 lipase 유전자를 검출할 가능성을 높이는 것으로 사료되었다. For the investigation of conserved regions in lipase genes, 132 and 24 sequences were obtained from LED (Lipase Engineering Database) and COG (Clusters of Orthologous Groups of proteins), respectively. There was high diversity in lipase genes and peculiar amino acid sequences were conserved for each homologous family of LED. Similar conserved amino acid sequences were detected from COG0657 and Moraxella lipase 1 homologous group of LED. Although many studies have attempted to detect new lipase genes in procaryotes, they have been limited culturable bacteria. The importance of metagenome, including DNA from non-culturable bacteria, is known. Due to the high diversity, we assumed it might be possible to detect new lipase gene from metagenome. Due to the high diversity of nucleotide sequences in lipase genes, 10 primer sets were designed. Designed primer sets were inspected in BLAST of NCBI and they could amplify a part of the lipase gene from 222 to 713 bp. They can amplify 16.7%∼60.0% of each lipase homologous group which was 3.6 fold higher than each sets. They might offer a high probability of detecting new lipase genes, owing to high efficiency and the diversity of lipase genes.

Lipase-producing Filamentous Fungi from Non-dairy Creamer Industrial Waste

Triyaswati, Desty,Ilmi, Miftahul The Korean Society for Microbiology and Biotechnol 2020 한국미생물·생명공학회지 Vol.48 No.2

Lipase-producing fungi have been isolated from environments containing lipids. The non-dairy creamer industrial waste has a high amount of lipids so it is a potential source for the isolation of lipase-producing fungi. However, the study of fungi that secrete lipase from this industrial waste has not been reported. The purpose of this study was to obtain lipase-producing filamentous fungi from non-dairy creamer industrial waste. Mineral salt and potato dextrose agar were used as media for the isolation process. The qualitative screening was conducted using phenol red agar medium and the quantitative screening using broth medium containing glucose and olive oil. Isolates producing the highest amounts of lipase were identified with molecular methods. We found that 5 out of 19 isolated filamentous fungi are lipase producers. Further analysis showed that isolate Ms.11 produced the highest amount of lipase compared to others. Based on ITS sequence Ms.11 was identified as Aspergillus aculeatus. The lipase activity in medium containing 1% glucose + 1% olive oil at pH 7.0 and 30℃ after 96 and 120 h of incubation was 5.13 ± 0.30 U/ml and 5.22 ± 0.59 U/ml, respectively. The optimum lipase activity was found at pH 7.0, 30℃ and using methanol or ethanol in the reaction tube. Lipase was more stable at 20-30℃ and maintained 85% of its activity. It was concluded that isolate Ms.11 is a potential source of lipase that catalyzes transesterification reactions. Further studies are required to optimize lipase production to make the strain suitable for industry purposes.

Optimization of Medium for Lipase Production from Zygosaccharomyces mellis SG1.2 Using Statistical Experiment Design

( Marisa Dian Pramitasari ),( Miftahul Ilmi ) 한국미생물 · 생명공학회 2021 한국미생물·생명공학회지 Vol.49 No.3

Lipase (triacylglycerol lipase, EC 3.1.1.3) is an enzyme capable of hydrolyzing triacylglycerol, to produce fatty acids and glycerol and reverse the reaction of triacylglycerol synthesis from fatty acids and glycerol through transesterification. Applications of lipase are quite widespread in the industrial sector, including in the detergent, paper, dairy, and food industries, as well as for biodiesel synthesis. Lipases by yeasts have attracted industrial attention because of their fast production times and high stability. In a previous study, a lipase-producing yeast isolate was identified as Zygosaccharomyces mellis SG1.2 and had a productivity of 24.56 U/mg of biomass. This productivity value has the potential to be a new source of lipase, besides Yarrowia lypolitica which has been known as a lipase producer with a productivity of 0.758 U/mg. Lipase production by Z. mellis SG1.2 needs to be increased by optimizing the production medium. The aims of this study were to determine the significant component of the medium for lipase production and methods to increase lipase production using the optimum medium. The two methods used for the statistical optimization of production medium were Taguchi and RSM (Response Surface Methodology). The data obtained were analyzed using Minitab 18 and SPSS 23 software. The most significant factors which affected lipase productivity were olive oil and peptones. The optimum medium composition consisted of 1.02% olive oil, 2.19% peptone, 0.05% MgSO₄·7H₂O, 0.05% KCl, and 0.2% K₂HPO₄. The optimum medium was able to increase the lipase productivity of Z. mellis SG1.2 to 1.8-fold times the productivity before optimization.

Lipase-producing Filamentous Fungi from Non-dairy Creamer Industrial Waste

Desty Triyaswati,Miftahul Ilmi 한국미생물·생명공학회 2020 한국미생물·생명공학회지 Vol.48 No.2

Lipase-producing fungi have been isolated from environments containing lipids. The non-dairy creamer industrial waste has a high amount of lipids so it is a potential source for the isolation of lipase-producing fungi. However, the study of fungi that secrete lipase from this industrial waste has not been reported. The purpose of this study was to obtain lipase-producing filamentous fungi from non-dairy creamer industrial waste. Mineral salt and potato dextrose agar were used as media for the isolation process. The qualitative screening was conducted using phenol red agar medium and the quantitative screening using broth medium containing glucose and olive oil. Isolates producing the highest amounts of lipase were identified with molecular methods. We found that 5 out of 19 isolated filamentous fungi are lipase producers. Further analysis showed that isolate Ms.11 produced the highest amount of lipase compared to others. Based on ITS sequence Ms.11 was identified as Aspergillus aculeatus. The lipase activity in medium containing 1% glucose + 1% olive oil at pH 7.0 and 30℃ after 96 and 120 h of incubation was 5.13 ± 0.30 U/ml and 5.22 ± 0.59 U/ml, respectively. The optimum lipase activity was found at pH 7.0, 30℃ and using methanol or ethanol in the reaction tube. Lipase was more stable at 20−30℃ and maintained 85% of its activity. It was concluded that isolate Ms.11 is a potential source of lipase that catalyzes transesterification reactions. Further studies are required to optimize lipase production to make the strain suitable for industry purposes.

Lipase Activity in Thermus thermophilus HB8: Purification and Characterization of the Extracellular Enzyme

Eirini Kretza,Christos P. Papaneophytou,Rigini M. Papi,Konstantina Karidi,Costas Kiparissides,Dimitrios A. Kyriakidis 한국생물공학회 2012 Biotechnology and Bioprocess Engineering Vol.17 No.3

In this study, the lipolytic activity of Thermus thermophilus HB8 was examined. The addition of various oils increased the production of extracellular lipolytic activity,while a combination of olive oil and glucose increased both extracellular and intracellular lipolytic activity. The oxygen transfer rate had a significant influence on both biomass and production of extra- or intra-cellular lipolytic activity. The formation of white halos due to the hydrolysis of oleic acid ester (Tween 80) in agar plates containing Nile Blue and the formation of Ca2+-oleate indicated the secretion of lipase. When the cell-free supernatant of cells grown in basal reach medium or the corresponding intracellular extract were electrophoresed under denatured and renatured conditions, using α-naphthyl acetate and Fast Blue RR,major bands at 56 kDa or 62 and 32 kDa were observed,respectively. The 56 kDa extracellular enzyme was partial purified and characterized. Its peak of activity occurred at 80oC and pH 7.0, while the T1/2 was 1 h at 100oC. The Km of the partial purified enzyme was 1 mM and the Vmax was 0.044 U/mL/min when using p-nitrophenyl laurate as substrate. The presence of Ca2+and Hg2+ stimulated lipase activity, whereas Zn2+, Co2+, or EDTA inhibited lipase activity. The highest activity was observed in the presence of coconut oil and p-nitrophenyl laurate (pNPL). Purified lipase was the most stable in the presence of various organic solvents, such as pentanol, chloroform and n-dodecane. Because of the superior thermostability and stability in the presence of organic solvents of T. thermophilus extracellular lipase, this lipase holds great promise for use in industrial applications. In this study, the lipolytic activity of Thermus thermophilus HB8 was examined. The addition of various oils increased the production of extracellular lipolytic activity,while a combination of olive oil and glucose increased both extracellular and intracellular lipolytic activity. The oxygen transfer rate had a significant influence on both biomass and production of extra- or intra-cellular lipolytic activity. The formation of white halos due to the hydrolysis of oleic acid ester (Tween 80) in agar plates containing Nile Blue and the formation of Ca2+-oleate indicated the secretion of lipase. When the cell-free supernatant of cells grown in basal reach medium or the corresponding intracellular extract were electrophoresed under denatured and renatured conditions, using α-naphthyl acetate and Fast Blue RR,major bands at 56 kDa or 62 and 32 kDa were observed,respectively. The 56 kDa extracellular enzyme was partial purified and characterized. Its peak of activity occurred at 80oC and pH 7.0, while the T1/2 was 1 h at 100oC. The Km of the partial purified enzyme was 1 mM and the Vmax was 0.044 U/mL/min when using p-nitrophenyl laurate as substrate. The presence of Ca2+and Hg2+ stimulated lipase activity, whereas Zn2+, Co2+, or EDTA inhibited lipase activity. The highest activity was observed in the presence of coconut oil and p-nitrophenyl laurate (pNPL). Purified lipase was the most stable in the presence of various organic solvents, such as pentanol, chloroform and n-dodecane. Because of the superior thermostability and stability in the presence of organic solvents of T. thermophilus extracellular lipase, this lipase holds great promise for use in industrial applications.

Amberlite에 고정화된 Lipase 제조 및 효소적 Interesterification을 이용한 반응 특성 연구

박소라(So Ra Park),이기택(Ki Teak Lee) 한국식품과학회 2014 한국식품과학회지 Vol.46 No.3

본 연구는 미생물로부터 유래된 5종(AH, AK, AY, PS와 R)의 lipase들을 Amberlite XAD 7에 흡착법으로 고정화 시킨 후 각 immobilized lipase들의 특성을 알아보았다. 고정화 전과 후의 단백질 함량 및 각 free lipase들과 immobilized lipase들을 이용한 interesterification 반응물의 지방산과 TAG 조성을 분석하였다. 또한, immobilized lipase에 있어 중요한 요인인 reusability를 확인하였다. Free lipase의 단백질 함량은 2.22-11.41%로 AH가 가장 높았던 반면, immobilized lipase에서는 AH, PS와 AK가 mg protein/g support이 높았다. 한편, 반응 특성을 알아보기 위해 카놀라유, PEE와 StEE를 기질로 하여 batch type interesterification을 진행하였을 때, free lipase의 경우 free lipase R을 제외한 다른 free lipase들은 반응시간이 1시간에서 72시간으로 증가함에 따라 총 포화지방산 함량이 증가하였으며 그 중 free lipase AH가 반응성이 가장 높았다. 또한, RP-HPLC를 통해 free lipase AK 반응물을 분석한 결과, 반응시간이 길어질수록 카놀라유(0시간)에서 볼 수 있었던 57.49 area%의 OOO가 6.53 area%로 감소하였다. 이는 각 free lipase들이 PEE와 StEE를 효소적 반응에 이용했기 때문이라고 판단된다. 한편, immobilized lipase AY와 R의 경우 반응시간이 1시간에서 48시간으로 증가하여도 카놀라유(0시간)의 총 포화지방산 함량과 큰 차이가 없었으나 immobilized AK의 경우 48시간에서 38.3 area%의 포화지방산 함량으로 가장 높은 활성을 보였다. 또한, 이를 사용하여 continuous type으로 반응하였을 때 유속이 느릴수록 효소와 기질 사이의 접촉 시간이 길어져 반응물의 총 포화지방산 함량이 증가함을 알 수 있었다. Reusability는 immobilized AK, AH와 PS 모두에서 두 번째 반복 반응을 하였을 때, 첫 번째 반응보다 총 포화지방산이 120-196.5% 증가하였다. 그러나 bounding protein 함량이 가장 높았던 immobilized AK는 support에 흡착되었던 free lipase AK의 탈착이 일어나 2번째 반응 후부터 활성이 감소한 반면, immobilized AH와 PS는 활성이 비교적 유지되었다. Immobilized lipases were prepared by physical adsorption using lipase AK, AY, AH, PS and R on Amberlite<SUP>®</SUP>XAD<SUP>®</SUP>7 HP resin. With the immobilized lipases (10%), structured lipid was synthesized by enzymatic interesterification of canola oil, palmitic ethyl ester, and stearic ethyl ester in order to study the reaction characteristics. Among the lipase, the highest protein content was obtained from lipase AH (11.41%) before immobilization, while the highest levels of bound protein was observed from immobilized lipase AK (63.91%). Immobilized lipase AK had the highest interesterification activity (38.3% of total saturated fatty acid). Lipase AK was also used for a continuous reaction in which the slow flow of reactant resulted in increased reaction rate. Reusability of immobilized AK, AH and PS increased at the second reaction (120-196.5%). However, the activity of immobilized AK, which had the highest bound protein content (63.91%) decreased after the third reaction, while the activity of immobilized AH and PS was maintained until the sixth reaction.

Purification, Distribution, and Characterization Activity of Lipase from Oat Seeds (Avena sativa L.)

Jung, Hyuk,Moon, SangJun The Korean Society for Applied Biological Chemistr 2013 Applied Biological Chemistry (Appl Biol Chem) Vol.56 No.6

Plant lipases have been chiefly studied as an esterase for hydrolyzation of triacylglycerol (a true lipase), which supplies energy for seed germination. Lipases are widely distributed in plants, animals, insects, and microorganisms. However, recent studies suggest that plant lipases have physiological functions other than triacylglycerol hydrolysis. In the present study, a plant lipase that has enzyme properties distinct from those of a true lipase was purified and characterized from oat seedlings. The lipase was purified 189-fold to a 0.53% purification ratio with high specific activity (34.656 U/mg). Analysis of the protein by Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a homogenous purified lipase. The lipase had higher enzyme specificity to monoacylglyceride and short chain fatty acids. Synthesis of the lipase was active at an early stage of germination for 6 days and decreased thereafter. Most of the lipase was found in the upper part of the oat seedling excluding the root. Within the young leaves, the lipase is located only in vessels and sieve tubes. However, infection of a pathogen, Pseudomonas syrinae pv. oryzae, elevated the lipase synthesis. In addition, the lipase had an ability to hydrolyze E.coli lipopolysaccharide. These results suggested that oat lipase may play a physiological role in defense against pathogens.

양송이 배지에서 유래한 Lipase 생산균을 이용한 바이오디젤 생산

김현희,김찬겸,한창훈,이찬중,공원식,윤민호 한국버섯학회 2015 한국버섯학회지 Vol.13 No.1

A lipase producing bacterium was isolated from button mushroom bed, which showing high clear zone on agar media containing Tributyrin as the substrate. The strain was identified as Burkholderia cepacia by analysis of 16S rDNA gene sequence. Crude lipase (CL) was partially purified from 70% ammonium sulfate precipitation using the culture filtrate of B. cepacia. Immobilized lipases were prepared by cross-linking method with CL from B. cepacia and Novozyme lipase (NL) onto silanized Silica-gel as support. Residual activitiy of the immobilized CL (ICL) and immobilized NL (INL) was maintained upto 61% and 72%, respectively. Biodiesel (Fatty acid methyl ester, FAME) was recovered by transesterification and methanolysis of Canola oil using NaOH, CL and ICL as the catalysts to compare the composition of fatty acids and the yield of FAME. Total FAME content was NaOH 781 mg L-1, CL 681 mg L-1 and ICL 596 mg L-1, in which the highest levels of FAME was observed to 50% oleic acid (C18:1) and 22% stearic acid (C18:0). In addition, the unsaturated FAME (C18:1, C18:2) decreased, while saturated FAME (C16:0, C18:0) increased according to increasing the reaction times with both CL and ICL, supporting CL possess both transesterification and interesterification activity. When reusability of ICL and INL was estimated by using the continuous reaction of 4 cycles, the activity of ICL and INL was respectively maintained 66% and 79% until the fourth reaction.