원재료로부터 분리된 α-, β-, γ-키틴의 물리화학적 특성과 약물 방출

장미경 ( Mi Kyeong Jang ),최창용 ( Chang Yong Choi ),최혜영 ( Hey Young Choi ),김태형 ( Tai Hyoung Kim ),손소희 ( So Hee Son ),장지태 ( Ji Tae Jang ),양현필 ( Hyun Pil Yang ),정특래 ( Teok Rae Jung ),강성구 ( Seong Koo Kang ),나 한국키틴키토산학회 2003 한국키틴키토산학회지 Vol.8 No.1

α-, β-, and y-chitin were isolated from natural resources by chemical method to investigate the crystalline structure of chitin. Their characterization was identified by FT-IR spectrophotometer, solid state CPMAS ^(13)C NMR spectrophotometer, DSC and XRD. A molecular weight (M_(vis)) of α- , β-, and y-chitin were determined by viscometer resulting in 701, 612, and 524 kDa, respectively. And we have prepared the drug carrier according to the α- , β-, and y-chitin and release profile was investigated. At FT-IR spectra, α-, β-chitin showed doublet and singlet at amide I band, respectively, and y-chitin showed intermediate form between α- and β-chitin. From solid state CP/MAS ^(13)C NMR spectra, two signals appeared at around 73 and 75 ppm assigned to C3 and C5 carbon atoms in a-chitin are sharply separated, the signals of C3 and C5 in β-chitin shows singlet at around 74 ppm. In case of y-chitin, two signals show at around 73 and 75 ppm assigned to C3 and C5 carbon atoms. From the X-ray diffraction results, a-chitin was observed four crystalline reflections, shown at 9.6, 19.6, 21.1, and 23.7 by crystalline structure. Also, β-chitin showed two crystalline reflections as indicated at 9.1˚, and 20.3˚ in the crystalline structure spectroscopy. y-chitin with structure of both antiparallel and parallel was close to X-ray diffraction patterns of a-chitin. As the result of DSC, due to difference in structural characteristics, remarkable differences in the exothermic transition for α-, β-, and y-chitin were observed. However, four crystalline reflections observed in the 28 range of 5 - 35˚ were disappeared in case of α-, β- and y-chitin after heating up to 400℃. The exothermic peak in α-, β-, and y-chitin were shows at 330, 220, and 300℃, respectively. The drug carrier were prepared by using α-, β-, and y-chitin, and the behavior of drug release were investigated by PBS 7.4 at 37±0.5℃. As the result, the drug release behavior was order of y-chitin > a-chitin > β-chitin.

Biochemical Characterization of Two Mycerial Chitin Deacetylases from Absidia coerulea CHK-2

Kim, Cheorl-Ho,Shim, Jae-Kyoung 東國大學校 1997 東國論叢 Vol.36 No.-

정상세포의 암화과정에는 세포표면당쇄변화를 수반하는 데, 간암, 난소암, 자궁암 및 폐암에서는 N-acetylglucosamine(GlcNAc) 잔기를 UDP-N-acetylglucosaminyltransferase-III와 0V가 β-1,4- 및 β-1,6-결합으로 전이시키는 것으로 알려져 있다. 따라서, 이들을 저해하는 GlcNAc유도체의 탐색과 개발은 이로 인한 암전이를 억제할 수 있을 것으로 기대되고 있다. 이에, 본 연구에서는 chitin deacetylase(CD)생성진균인 Absidia coerulea CHI-2로부터 2가지 서로 다른 CD효소를 정제하였다. 정제효소CD-1,2는 분자량 65 및 75 kDA을, pI 5.2와 5.7을 각각 나타냈으며 그중 CD-1은 당단백질이 밝혀졌다. O-하이드록실chitin (glycol chitin)을 기질로 할 때, 최적온도와 pH는 각각 약45전후와 약5.0를 나타냈으며 Mn^2+ 이온에 의해 활성화되었다. 본 효소는 chitooligosaccharide와 chitin에 강한 활성을 보였으며 Glocol chitin에 대한 K_m치는 7.96과 16.4mM 그리고 K_cat는 35.4와 32.1/sec였다. 그러나, 단당인 N-acetylglucosamine잔기에 대한 활성이 없으며 N-acetylglucosamine-β-1,4-D-N acetylglucosamine (chitobiose)에 대해서는 약간 활성을 나타내었다. 현재 이들을 이용한 얌전이억제용 당유도체개발을 진행중에 있다. Two different myceliar chitin deacetylase(CD)s, named as CD-1 and CD-2, have been purified from CD-hyperproducing fungus, Absidia coerulea CHK-2. The enzymes efficiently released the acetyl groups of glycol chitin. The purified enzymes had molecular masses of about 65 and 75 kDa on denaturated and natural conditions. The values of pI were 5.2 and 5.7, respectively. The chitin deacetylase CD-1, but not Cd-2, when resolved by SDS-PAGE, were positive for Schiff staining, suggesting that the enzyme CD-1 is glycoprotein. When O-hydroxylated chitin (glycol chitin) was used as a substrate, the enzymes displayed a temperature optimum of around 45-50℃ and optimum pHs 5.5 and 4.5, repectively. The enzymes were stable to incubation from pH 3.0 to pH 6.0 at 4℃ for 24 hr. The presence of chitin protected the enzyme from heat inactivation, the extent depending upon the substrate concentration. The activity of the enzyme was stimulated by Mn^2+ ion, however, inhibited by Fe^3+. the enzyme activities were retained even in 100 mM sodium acetate and they are active on chitooligosaccharides with more than two N-acetylglucosamine residues (chitobiose). However, the enzymes are not active on N-acetylglucosamine. The enzymes had the apparent K_m values of 7.96 and 16.4 mN, and K_cat values of 35.4 /sec and 32.1/sec for glycol chitin, respectively. Abbreviations : CD, chitin deacetylase : SDS, sodium dodecyl sulfate: PAGE, polyacrylamide gel electrophoresis : EDTA, ethylenediamine tetraacetic acid: EGTA, ethylene glocol bis(2-aminoethylether)tetraacetic acid: BSA, bobine serum albumin: FPLC, fast performance liquid chromatography: PMSF, phenylmethyl sulfonyl fluoride.

식용 갈색거저리 유충에서 분리한 키틴으로부터 Serratia marcescens PRNK-1에 의한 N-아세틸글루코사민의 생산

서동준 ( Dong-jun Seo ),문채영 ( Chaeyeong Moon ),송용수 ( Yong-su Song ),최승희 ( Seung-hee Choi ),한연수 ( Yeon Soo Han ),조용훈 ( Yong Hun Jo ),노미영 ( Mi Young Noh ),정우진 ( Woo-jin Jung ) 한국키틴키토산학회 2017 한국키틴키토산학회지 Vol.22 No.3

In this study, crab shell chitin and Tenebrio molitor larva (Mealworm) chitin were characterized by X-ray diffraction and FTIR analysis. From the X-ray diffraction results, α-form chitin was observed four crystalline reflections, shown at 9.4°, 19.3°, 20.8°, and 23.3° in crab shell chitin, and at 9.44°, 19.3°, 20.7°, and 23.3° in mealworm chitin by crystalline structure. From the FT-IR spectra results, α-form chitin showed doublet at amide I band in both crab shell chitin and mealworm chitin. Manufacturing process of colloidal chitin from mealworm was conducted with acid, alkali, and decoloration and then adjusted at pH 6~7 with 70% ethanol. Degradation pattern of colloidal chitin from crab shell and mealworm was investigated after reaction of chitinase-producing bacterium Serratia marcescens PRNK-1 by TLC and HPLC. Production of N-acetyl-glucosamine showed rapidly at 3 hr after reaction with crab shell and mealworm colloidal chitin and crude enzyme of S. marcescens PRNK-1 on TLC plates. N-acetyl-glucosamine was produced by 5,041.3 ppm and 5,319.8 ppm at 3 days in crab shell colloidal chitin and in mealworm colloidal chitin, respectively, after reaction with crude enzyme of S. marcescens PRNK-1. Our results indicate that the colloidal chitin obtained from mealworm could be used as useful industrial resources at production of N-acetyl-glucosamine.

α, β, γ-키틴으로 제조한 키토산의 특성

최혜영 ( Choe Hye Yeong ),김태형 ( Kim Tae Hyeong ),손소희 ( Son So Hui ),공병기 ( Gong Byeong Gi ),최창용 ( Choe Chang Yong ),김동곤 ( Kim Dong Gon ),장미경 ( Jang Mi Gyeong ),노홍균 ( No Hong Gyun ),나재운 ( Na Jae Un ) 한국키틴키토산학회 2004 한국키틴키토산학회지 Vol.9 No.1

α-, β- and γ-chitin were isolated from crab shell, squid pen, beetles cuticles by acid, alkali treatment and α-, β- and γ-chitosan were prepared from α-, β- and γ-chitin. Chemical compositions of raw materials and elemental of chitin and chitosan were analyzed. A weight-average molecular weight and degree of deacetylation (DDA) were determines by viscometry and Kina titration. Its structural characterization was analyzed by FT-IR spectrophotometer and solid state CP/MAS C NMR spectrophotometer. A molecular weight of α-, β- and γ-chitin were determined by viscometer resulting in 701. 612, and 524, kDa, respectively. A molecular weight of α-, β- and γ-chitosan were calculated with 603., 607 and 329 kDa, respectively. The DDA of α-, β- and γ-chitin were 21.8%, 32.3% and 44.7%, respectively. The DDA of α-, β- and γ-chitosan were 97.1%, 99.2% and 96.6%, respectively. At the FT-IR spectra of chitin, α-, β- chitin. And at the FT_IR sectra of chitosan, absorption band of amide I and amide H decreased because of the deacetylation of chitin, where as the absorption band of amine group was newly formed. From solid state CP/MAS C NMR spectra of chitin, two signals appeared at around 73 and 75 ppm assigned to C3 and C5 carbon atoms in α-chitin are sharply resolved, the signals of C3 and C5 in β-chitin shows singlet at around 74 ppm. In case of γ-chitin, two signals show at around 73 and 75 ppm assigned to C3 and C5 carbon atoms. From solid stat CP/MAS C NMR spectra of chitosan, the carbon of the C1-C6 positions were cleared identified and peaks of CH_3 and C=0 decreased significantly because of the deacetylation

매미허물에서 분리한 키틴의 물리화학적 특성

홍성현 ( Seong Hyun Hong ),조경현 ( Gyung Hyun Jo ),주완택 ( Wan Taek Ju ),박노동 ( Ro Dong Park ) 한국키틴키토산학회 2010 한국키틴키토산학회지 Vol.15 No.2

In this study, the physicochemical properties of the chitin isolated from cicadae (Platypleura kaempferi) periostracum was investigated. The prepared chitin was white flake form and the yield was 31.7%. The chitin was degraded into oligosaccharide during acid hydrolysis at 30℃ with concentrated HCl. The hydrolysis rate was 6.8% only after 0.5 h reaction, but increased to 44.0% after 24 h reaction. In distribution of the oligosaccharides in the hydrolyzates, ratio of GlcNAc1-3 content increased from 66.5% after 0.5 h reaction to 84.4% after 12 h reaction. The ratio of monomer GlcNAc content was 79.4% after 24 h reaction and no oligosaccharides bigger than GlcNAc4 was detected. It is found that cicadae periostracum chitin belongs to α-chitin resulting from the FT-IR spectra similar with those of crab and shrimp chitins. In X-ray diffractometer, two peaks were exhibited at 2θ = 9.5˚ (interplanar distance: 9.36Å) and 2θ = 19.4o(4.58Å). The crystallinity index and crystal size of the chitin were determined to be 81.9% and 4.6 nm, respectively, which are similar with those of crab and shrimp chitins, again confirming that cicadae periostracum chitin belongs to α-chitin. The degree of acetylation of cicadae periostracum chitin was calculated to be 88.1%.

매미허물에서 분리한 키틴의 물리화학적 특성

박노동,조경현,주완택,홍성현 한국키틴키토산학회 2010 한국키틴키토산학회지 Vol.15 No.2

In this study, the physicochemical properties of the chitin isolated from cicadae (Platypleura kaempferi) periostracum was investigated. The prepared chitin was white flake form and the yield was 31.7%. The chitin was degraded into oligosaccharide during acid hydrolysis at 30oC with concentrated HCl. The hydrolysis rate was 6.8% only after 0.5 h reaction, but increased to 44.0% after 24 h reaction. In distribution of the oligosaccharides in the hydrolyzates, ratio of GlcNAc1-3 content increased from 66.5% after 0.5 h reaction to 84.4% after 12 h reaction. The ratio of monomer GlcNAc content was 79.4% after 24 h reaction and no oligosaccharides bigger than GlcNAc4 was detected. It is found that cicadae periostracum chitin belongs to α-chitin resulting from the FT-IR spectra similar with those of crab and shrimp chitins. In X-ray diffractometer, two peaks were exhibited at 2θ = 9.5o (interplanar distance : 9.36Å) and 2θ = 19.4o(4.58Å). The crystallinity index and crystal size of the chitin were determined to be 81.9% and 4.6 nm, respectively, which are similar with those of crab and shrimp chitins, again confirming that cicadae periostracum chitin belongs to α-chitin. The degree of acetylation of cicadae periostracum chitin was calculated to be 88.1%.

Fluorescein-5 isothiocyanate conjugated-chitin-binding domain probe (FITC-CBD)-coupled detection of chitin in the peritrophic membrane of Monochamus alternatus (Coleoptera: Cerambycidae)

Khondkar Ehteshamul Kabir,Daizo Koga,Takuma Takanashi,Kotaro Konno 한국응용곤충학회 2012 Journal of Asia-Pacific Entomology Vol.15 No.3

This study investigated the potential of a fluorescein-5 isothiocyanate conjugated-chitin-binding domain (FITC-CBD) probe to detect chitinous materials in the peritrophic membrane (PM) structure of the Japanese pine sawyer beetle, Monochamus alternatus. Results through direct observations indicated that the fluorescent probe specifically bound to chitin, and specifically labeled the chitinous materials in the PM. Applying the probe was easy, as fluorescence was stable, and the excitation maximum of the structures stained with the probe was within a range covered by most existing fluorescence microscopes. These advantages make fluorescence staining an easy method of choice for specifically visualizing the chitin-rich internal structures of insects. Additionally, this simple approach might have the added advantage of revealing chitinolysis events in vivo and in vitro. This study investigated the potential of a fluorescein-5 isothiocyanate conjugated-chitin-binding domain (FITC-CBD) probe to detect chitinous materials in the peritrophic membrane (PM) structure of the Japanese pine sawyer beetle, Monochamus alternatus. Results through direct observations indicated that the fluorescent probe specifically bound to chitin, and specifically labeled the chitinous materials in the PM. Applying the probe was easy, as fluorescence was stable, and the excitation maximum of the structures stained with the probe was within a range covered by most existing fluorescence microscopes. These advantages make fluorescence staining an easy method of choice for specifically visualizing the chitin-rich internal structures of insects. Additionally, this simple approach might have the added advantage of revealing chitinolysis events in vivo and in vitro.

Properties of Crude Enzymes Obtained from Three Chitinolytic Bacteria Cultured in Different Chitin Substrates

( Gwang Rok Ryu ),( Seong-gyu An ),( Yong-su Song ),( Woo-jin Jung ) 한국키틴키토산학회 2024 한국키틴키토산학회지 Vol.29 No.1

Chitin is the second most abundant polysaccharide after cellulose, and the enzyme that can decompose chitin is chitinase, which has high industrial value. In this study, the characteristics of chitinase produced by three chitinolytic bacterial isolates, Jeongeupia naejangsanensis GR-1, Paenibacillus gorillae GR-2, and Paenibacillus chitinolyticus GR-3, were investigated according to three chitin substrates: crab shell colloidal chitin (CC), Korean blockish cicada colloidal chitin (KC), and mealworm colloidal chitin (MC). The determination of the protein content and chitinase activity showed low values for two chitin substrates, excluding CC. The chitinase expression patterns of the three bacteria were investigated through SDS-PAGE, native-PAGE, and gradient (5%-12%) native-PAGE. The results confirmed that the J. naejangsanensis GR-1 expressed a different pattern of chitinase in KC. Through TLC using colloidal chitin as a substrate, it was found that J. naejangsanensis GR-1 produced endo- and exo-type chitinase, whereas P. gorillae GR-2 and P. chitinolyticus GR-3 mainly produced endo-type chitinase. API analysis confirmed that the three bacteria were positive for N-acetyl-D-glucosamine, a monosaccharide of chitin. Given the current lack of research investigating the chitinase of these three strains, the findings from this study have potential value for the industrial application of chitinases.

Separation and Property of Chitinase Extracted from Rape Seeds by Chitin Affinity Columns with Three Different Regenerated Chitins

( Yong Su Song ),( Dong Jun Seo ),( Woo Jin Jung ) 한국키틴키토산학회 2010 한국키틴키토산학회지 Vol.15 No.4

The chitinases of rape seeds were separated by chitin affinity columns regenerated from three different molecular chitosans of 20 cPs, 276 cPs and 1,200 cPs. The chitinase of rape seeds occurred in the order of Ch1, Ch2, and Ch3 on SDS-PAGE. The protein contents recovered were 3.95, 1.59, and 1.90% from chitin affinity columns of 20, 276, and 1200 cPs, while the respective chitinase activities were 47.2, 80.9, and 60.2%. The hydrolysis products of seed chitinase observed on TLC plates primarily consisted of dimers and trimers after 12 hrs of incubation with swollen chitin, while they were primarily dimers and monomers on TLC plates after 12 hrs of incubation with chitin-oligomers. In conclusion, the highest recovery rate of the chitinase activity from rape was obtained using a chitin affinity column filled with 276 cPs chitosan, and the chitinase was a very specific enzyme for the selective production of GlcNAc monomers and dimers.

Chitin으로부터 다양한 Chitosan의 제조와 특성

조형재,황성규,이기창,이한섭,김판기 한국식품위생안전성학회 1998 한국식품위생안전성학회지 Vol.13 No.1

수산계 폐기물로부터 chitin 유도체의 다양한 응용에도 불구하고 chitin의 상업적 이용은 적절한 용매의 부재와 화학적 저항성으로 인하여 제한적으로 이용되었다. 그러므로 Mima의 방법을 응요하여 NaOH 농도, 반응시간, 온도 등을 조절하여 탈아세틸화반응에 의한 다양한 점도가 다른 chitosan을 제조하였으며, 2종의 가교제를 이용하여 가교결합에 의한 결정성을 증가시킨 가교 chitosan을 제조하였다. 제조한 점도가 다른 chitosan과 가교 chitosan유도체를 다양한 분석기기를 이용하여 측정하였다. chitosan을 제조시 반응시간을 높이거나 반응온도를 높이면 탈아세틸화는 높아지나 분자사슬의 크기, 즉 점도와 분자량은 감소하였다. 반응온도, 반응시간과 알칼리농도에 따라 활용분야에 맞는 chitosan을 제조할 수 있다. Chitin is known as biodegradable natural polymer. But, in spite of various application of chitin from waste marine sources, commercial use of chitin has been limited due to highly resistance to chemicals and the absense of proper solvents. herefore, we studied that another viscosity chitosan were prepared from chitin which were deacetylated under various concentration of NaOH solution, reaction time and temperature by the application of Mima's method. The major parameters for these manufacturing methods were found to be concentration of alkali solution, reaction time and temperature etc. Besides, we studied that various chitosan derivatives were prepared from chitin by crosslinkage with epichlorohydrin and 1, 3-dichloro-propanol. The effects of these parameters on another viscosity (molecular weight) chitosan and crosslinked chitosan dervatives were investigated by various analysis apparatus. SEM analysis showed that both chitin and chitosan had a particle shaped morphology and another molecular weight chitosan according to the particle size was much smaller than that of chitin.