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

매미 허물로부터 화학적 처리법으로 키틴을 분리하고 그 물리화학적 특성을 조사하였다. 얻어진 키틴은 백색의 분말 형태였으며 그 수율은 31.7%였다. 매미 키틴은 30℃에서 진한 염산 가수분해 반응으로 올리고당으로 분해되었으며, 반응시간에 따라 키틴의 분해율은 증가하여 24시간에는 44.0%의 분해율을 기록하였다. GlcNAc1-3의 함량 비율은 반응 0.5시간에 66.5%, 반응 12시간에는 84.4%로 증가하였다. 반응 24시간에는 단당 GlcNAc의 함량이 79.4%를 차지하였으며, 4당 이상은 검출되지 않았다. 매미허물 키틴은 게, 새우 키틴과 같은 형태의 FT-IR 스펙트럼 양상이 나타나 α-chitin으로 확인되었고, 아세틸화도는 88.1%로 조사되었다. 매미 허물 키틴의 X-선 회절 분석결과 2θ = 9.4˚, 19.4˚에서 면간격이 9.39 Å와 4.60 Å로 각각 측정되었으며, 결정화도 및 결정크기의 값은 각각 81.9%, 4.6 nm로 α-chitin인 게, 새우 키틴의 특성치와 유사하였다. 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%.

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-, γ-키틴이 천연 재료로부터 분리되었고 이의 특성이 FT-IR spectrophotometer, solid state CP/MAS ^(13)C NMR spectrophotometer, TGA, XRD에 의해 확인되었다. 또한, 본 연구에서 분리한 키틴을 이용하여 서방성 제제로서의 가능성을 알아보기 위하여 약물전달체를 제조하였다. α-, β-, and γ-chitin의 점도평균분자량(Mvis)이 점도계를 이용하여 측정하였으며, 결과로써 각각의 분자량이 701, 612, and 524 kDa임을 확인하였다. FT-IR스펙트럼에서 아마이드 Ⅰ에서의 흡수밴드가 α-키틴에 있어서는 이중선으로, β-키틴에서는 단일선으로 나타났으며 γ-키틴에서는 α-, β-키틴의 중간형태로 나타났음을 확인하였다. Solid state CP/MAS ^(13) NMR 스펙트럼결과에서 α-키틴의 경우 C3과 C5의 피크가 각각 73과 75ppm에서 나타났으며 β-키틴은 74 ppm에서 단일선으로 나타났고, γ-키틴의 경우 C3과 C5의 흡수 피크가 α-키틴과 유사한 형태의 피크를 나타냈었다. X-ray회절 분석에서는 α-키틴의 경우 9.6, 19.6, 21.1, 23.7에서 4개의 결정면을 확인할 수 있었고, β-키틴의 경우 9.1, 20.3에서 2개의 결정면을 나타내었다. 또한 역평행의 구조와 평행의 구조가 혼재되어 있는 γ-키틴의 경우 β-키틴 보다 오히려 α-키틴에 가까운 결정성을 나타내었다. 그러나 400℃까지 가열하였을 경우 5°~35°에서 나타나던 of α-, β- and γ-chitin의 결정면이 사라졌음을 알 수 있었다. DSC 측정결과로써, α-, β-, γ-chitin의 구조적 특성으로 인해 각각 다른 발열 피크를 나타내었으며, 결과로써 α-chitin의 경우 330, β-chitin은 220 그리고 γ-chitin은 300℃임을 확인하였다. 또한, 본 연구에서 제조한 키틴을 사용하여, 약물전달체를 제조하고, 약물의 방출 거동을 살펴 본 결과, β<γ<α순으로 β-키틴이 서방성의 방출 거동을 나타내었다. 이는 분자간 수소결합이 존재하지 않은 β-키틴 분자 내로 약물이 도입되고 또한 이들간의 상호 인력에 의해 약물 방출이 지연되는 것으로 사료되며, 따라서 본 연구에서 제조된 α-β-γ-chitin의 서방성 약물 전달체로써의 가능성을 확인하였다. 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.

Carboxymethyl Chitin의 고유점도에 미치는 pH 및 분자량의 영향

박성민,이근태,PARK Seong-Min,LEE Keun-Tai 한국수산과학회 1995 한국수산과학회지 Vol.28 No.4

수용성 chitin 유도체인 CM-chitin의 식품분야 또는 기타 산업적 이용을 위한 기초자료를 제시할 목적으로 희박용액중에서 CM-chitin의 고유점도에 미치는 pH 및 분자량의 영향을 조사한 결과를 요약하면 다음과 같다. Chitin으로부터 조제한 CM-chitin을 0.1M NaCl 용액에 녹여 $30^{\circ}C$에서 측정한 CM-chitin의 고유점도는 1.23dl/g이었고, 분자량은 15,500이었으며, CM-chitin과 반응한 NaOH의 당량으로부터 계산한 치환도는 0.62이었다. CM-chitin의 고유점도에 대한 pH의 영향은 pH 7.0이하에서는 pH가 낮을수록 고유점도는 급격히 감소하였으며, pH 7.0이 상에서는 pH가 증가하여도 고유점도의 변화는 거의 나타나지 않았다. 초음파 처리 시간에 따른 고유점도와 분자량의 변화는 처리시간 20분까지는 고유점도와 분자량은 각각 3.1dl/g에서 2.55dl/g, 15,500에서 12,600으로 감소하여 감소속도가 빨랐으나 20분 이후부터는 거의 일정하여 1시간 처리 후의 고유점 도와 분자량은 각각 2.5dㅣ/g, 12,370이었다. 용매를 물로 사용하였을 때 CM-chitin의 고유점도와 분자량과의 관계에 있어서 K는 $3.48\times10^{-4}이었고, v는 0.94이었다. Effects of pH and molecular weight on the intrinsic viscosity of carboxymethyl chitin (CM-chitin) in dilute regime were studied. When the prepared CM-chitin was dissolved in 0.1M NaCl solution at $30^{\circ}C$, the intrinsic viscosity, molecular weight and degree of substitution of CM-chitin were 1.23dl/g, 15,500 and 0.62, respectively. The lower intrinsic viscosity $([\eta])$ of CM-chitin was showed at the lower pH than 7.0 and the higher pH (>7.0) did not result in any increase in intrinsic viscosity. Intrinsic viscosity decreased from 3.1dl/g to 2.55d1/g in water at $25^{\circ}C$ and from 15,500 to 12,600 as molecular weight for 20min of sonication treatment. The Mark-Houwink constant K and v of CM-chitin in water at $25^{\circ}C$ were $3.48\times10^-4$ and 0.94, respectively. So intrinsic viscosity could be expressed using molecular weight as followed equation; $[\eta]=3.48\times10^{-4}M^{0.94}$, consistent with random roil behaviour.

저농도 용액에서의 Carboxymethyl Chitin의 사슬배좌와 전해질 거동

박성민,이근태,김상무,PARK Seong-Min,LEE Keun-Tae,KIM Sang-Moo 한국수산과학회 1995 한국수산과학회지 Vol.28 No.4

To elucidate the intrinsic rheological properties of carboxymethyl chitin (CM-chitin) from the shell of red snow cyab (Chinonecetes japonicus), the configuration and polyelectrolyte behavior of CM-chitin in low concentration solution were investigated. Unperturbed dimensions were ranged from $127{\AA}\;to\;113{\AA}$ as root mean square end-to-end distance$(r_0)$, $52{\AA}$ to $46{\AA}$ as radius of gyration$(S_0)$. The intramolecular expansion tarter(a) was not varied with molecular weight and was 2.1. And effective bond length $(b_0)$ was $14.5{\AA}$. In perturbed condition, Flory constant was $2.35\times10^{21}$. When ionic strength were 0.02 and 1.0, intrinsic viscosity were 1.95dl/g and 1,06 dl/g, respectively. These results suggested that CM-chitin is a polyelectrolyte in aqueous media. At infinite ionic strength, intrinsic viscosity was 0.91dl/g. The intrinsic stiffness of CM-chitin backbone was estimated by evaluating the stiffness parameter (B) as 0.11 and agreed well with the results of k-carrageenan. CM-chitin 분자의 고유특성을 규명하기 위하여 물성학적 방법으로 저농도 용액에서의 CM-chitin의 고유특성인 사슬배좌와 전해질 거동에 대하여 조사하였다. 비교란 상태에서 CM-chitin 사슬의 양단간 거리는 $127{\AA}$효에서 $113{\AA}$으로 분자량이 작을수록 양단간 거리는 좁혀지는 경향을 보였다 회전반경의 경우에도 $52{\AA}$에서 $46{\AA}$으로 양단간 거리와 같은 경향을 보였다. 팽창인자는 분자량에 관계없이 거의 일정하게 나타났으며, 결합사슬의 길이는 $14.5{\AA}$이었다. 그리고 교란 상태에서 CM-chitin의 Flory 점도상수는 $2.35\times10^{21}$이었다. CM-chitin의 이온강도에 따른 고유점도의 변화에 있어서 이온강도가 0.02일 때 고유점도는 1.95d1/g이었으며, 이온강도 1.0일 때는 고유점도가 1.06dl/g로 이온강도가 증가함에 따라 고유점도는 감소하는 경향을 보여 CM-chitin이 전해질임이 확인되었다. 그리고 전하를 완전히 제거한 상태에서의 CM-chitin의 고유점도$([\eta]_\infty)$는 0.91dl/g이었다. 한편 사슬의 유연성은 k-carrageenan과 거의 같은 것으로 나타났다.

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

본 연구에서는 천연자원으로부터 α-, β- 및 γ-chitin을 분리하였고, 이를 이용하여 α-, β- 및 γ-chitosan을 제조하였다. 원재료의 화학적 조성과 chitin과 chitosan의 일반성분을 분석하였으며 상대점도측정과 Kina 적정법을 이용하여 점도평균분자량과 탈아세틸화도를 측정하였고 FT-IR spectrophotometer, soild state CP/MAS ^(13)C NMR spectrophotometer 에 의해 α-, β- 및 γ-chitin과 chitosan의 제조를 확인하였다. α-, β- 및 γ-chitin의 각각의 분자량이 701, 612 그리고 524 kDa으로 측정되었으며 α-, β-및 γ-chitosan의 분자량이 603, 607, 329 kDa임을 확인하였다. α-, β-및 γ-chitin의 탈아세틸화도가 21.8%, 3?.3% 그리고 44.7%로 확인되었고 α-, β- 및 γ-chitosan의 탈아세틸화도가 97.1%, 99.2%, 그리고 96.6% 임을 확인하였다. Chitin의 FT-IR 스펙트럼에서 amide I에서의 흡수 밴드가 α-chitin에 있어서는 이중선으로, β-chitin에 있어서는 단일선으로 나타났으며 γ-chitin에서는 α-, β-chitin의 중간형태로 나타났음을 확인하였다. Chitosan의 FT-IR 스펙트럼에서 탈아세틸화 반응에 의해 amide Ⅰ과 amide Ⅱ의 흡수 피크가 현저히 감소하였음을 확인하였다. Chitin의 Solide state CP/MAS ^(13)C NMR 스펙트럼결과에서 α-chitin의 경우 C3과 C5의 피크가 각각 73과 75 ppm에서 나타났으며 β-chitin은 74 ppm에서 단일선으로 나타났고, γ-chitin의 경우 C3과 C5의 흡수피크가 α-chitin과 유사한 형태의 피크를 나타내었다. Chitosan의 Soilde state CP/MAS ^(13) NMR 스펙트럼에서 C1~C6가 잘 나타나 있고, 탈아세틸화 반응에 의해 메틸탄소 및 카르보닐 탄소가 거의 나타나지 않았다. 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

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

S. marcescens PRNK-1로부터 얻어진 키틴분해 조효소와 식용 갈색거저리 유충으로부터 얻어진 콜로이달 키틴의 산업적 활용에 대하여 연구하였다. 식용 갈색거저리로부터 얻어진 키틴의 형태는 게 껍질에서 확인된 α-form 형태임을 X-ray 회절분석법과 FT-IR 분석법을 통하여 확인하였다. 게 껍질과 식용갈색거저리를 이용하여 제조한 콜로이달 키틴을 기질로 하여 S. marcescens PRNK-1로부터 얻어진 키틴분해 조효소와 반응한 결과 식용 갈색거저리 키틴에서 더 높은 함량의 N-아세틸글루코사민을 생성함을 확인하였다. 식용 갈색거저리 유충으로부터 얻어진 콜로이달 키틴이 S. marcescens PRNK-1로부터 얻어진 키틴분해 조효소와 반응하여 N-아세틸글루코사민 생산을 확인함으로써 산업적으로 응용 가능성이 매우 높을 것으로 기대된다. 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.

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.