Bacillus cereus TKU030 係以烏賊軟骨為唯一碳/氮源，篩選自淡江大學
校園土壤之幾丁聚醣酶、幾丁質酶及蛋白酶之生產菌，本研究探討B. cereus
TKU030 發酵烏賊軟骨生產幾丁聚醣酶之較適培養條件，所得發酵上清液經硫
酸銨沉澱、Sephacryl S-100 膠體層析、DEAE-Sepharose 離子交換層析以及Sephacryl S-100 再膠體層析等步驟，純化出一種幾丁聚醣酶 (CHS)。CHS
之分子量經SDS-PAGE 測為43 kDa；最適反應溫度、最適反應pH、熱安定
性以及pH 安定性分別為40oC、pH 4、30-40oC 以及pH 3-7。於抑制劑方面，
CHS 活性會受Mn2+、Cu2+、Fe2+ 及含硼化合物TKUPSP017 所抑制。
  
  TKUPSP017 濃度於0.05% (w/v) 時的抑制率達100%，抑制效果會隨
CHS 與TKUPSP017 預反應時間的增長而有所提升。此外，抑制劑結構對於
CHS 酵素活性的影響，以及添加TKUPSP017 於培養基對於B. cereus
TKU030 生長之影響亦有探討。CHS 被抑制不會影響TKU030 的生長，隨著
TKUPSP017 濃度提高，TKU030 生長狀況反而越好。

The chitosanase, chitinase and proease-producing strain
TKU030 was isolated from soil in Tamkang University with squid pen powder as the sole carbon/nitrogen source and identified as Bacillus cereus. A chitosanase (CHS) was purified from the culture supernatant by ammonium sulfate precipitation, Sephacryl S-100 gel chromatography, DEAE- Sepharose column chromatography and Sephacryl S-100 rechromatography . The molecular mass of CHS determined by SDS-PAGE was approximately 43 kDa. The optimum temperature, optimum pH, thermal stability, and pH stability of CHS were 40oC, pH 4, 30-40oC and pH 3-7, respectively.The activity of CHS was inhibited by Mn2+, Cu2+, Fe2+ and a boron-containing compound of TKUPSP017.

  The inhibition of CHS activity reached 100% at 0.05% (w/v) of TKUPSP017. The inhibitory activity increased with the
preincubating time of TKUPSP017 and CHS. The effects of
inhibitor structures on enzyme activity and growth of strain TKU030 in media supplemented with TKUPSP017 were also studied.Growth of strain TKU030 was not influenced by inhibiting CHS activity but enhanced by increasing TKUPSP017 concentration.

中文摘要....................................................I
英文摘要...................................................II
目錄.....................................................…IV
圖目錄.....................................................IX
表目錄....................................................XII

第一章 緒論 ................................................1
第二章 文獻回顧 .............................................2
2.1 幾丁質與幾丁聚醣..........................................2
2.2 N-乙醯幾丁寡醣與幾丁寡醣 ..................................5
2.3 幾丁質酶與幾丁聚醣酶 .....................................5
2.4 蛋白酶 .................................................6
2.5 Bacillus cereus 之簡介 .................................7
第三章 材料與方法 ............................................9
3.1 實驗菌株 ...............................................9
3.2 實驗材料 ...............................................9
3.3 實驗儀器 ..............................................10
3.4 酵素生產菌株之篩選 ......................................11
3.5 幾丁質酶活性測定 ........................................12
3.6 幾丁聚醣酶活性測定 ......................................12
3.7 蛋白酶活性測定 .........................................13
3.8 較適培養條件之探討 ......................................13
3.8.1 培養液體積 ..........................................13
3.8.2 碳/氮源濃度與培養時間 .................................14
3.9 酵素之分離純化 .........................................14
3.9.1 粗酵素液之製備 .......................................14
3.9.2 陰離子交換層析 .......................................15
3.9.3 膠體過濾層析 .........................................15
3.10 蛋白質電泳分析 ........................................16
3.11 酵素之特性分析 ........................................16
3.11.1 酵素最適反應溫度 .....................................16
3.11.2 酵素熱安定性 ........................................16
3.11.3 酵素最適反應pH ......................................17
3.11.4 酵素pH 安定性 ......................................17
3.11.5 金屬離子及化學藥品對酵素活性之影響 ......................17
3.11.6 界面活性劑對酵素活性之影響 ............................18
3.11.7 酵素之基質特異性 ....................................18
3.12 還原糖量之測定 ........................................19
3.13 總糖量之測定 (H2SO4-Phenol 法) ........................19
3.14 幾丁聚醣酶水解基質及寡醣分析 .............................20
3.14.1 基質之水解 .........................................20
3.14.2 N-乙醯幾丁寡醣之製備 .................................20
3.14.3 N-乙醯幾丁寡醣之組成分析 ..............................21
第四章 結果與討論 ...........................................22
4.1 幾丁聚醣酶、幾丁質酶及蛋白酶生產菌之篩選與鑑定 ...............22
4.2 酵素較適生產條件探討 ....................................22
4.2.1 培養液體.............................................23
4.2.2 不同碳/氮源濃度 ......................................23
4.2.3 較適培養條件探討結果 ..................................24
4.3 幾丁質酶及幾丁聚醣酶之純化 ...............................24
4.3.1 粗酵素液之製備 .......................................24
4.3.2 離子交換樹脂層析 ......................................25
4.3.3 膠體過濾層析 .........................................26
4.3.4 綜合結果 ............................................26
4.4 幾丁聚醣酶分子量測定 ....................................28
4.4.1 SDS-PAGE ..........................................28
4.4.2 幾丁聚醣酶生態質譜鑑定 (peptide mass mapping) ..........28
4.5 幾丁質酶與幾丁聚醣酶之特異性分析 ...........................29
4.5.1 幾丁質酶與幾丁聚醣酶之最適反應溫度及熱安定性 ...............29
4.5.2 幾丁質酶與幾丁聚醣酶之最適pH 值及酸鹼安定性 ...............29
4.5.3 金屬離子及化學藥品對幾丁質酶與幾丁聚醣酶活性之影響...........30
4.5.4 界面活性劑對幾丁質酶與幾丁聚醣酶活性之影響 ................31
4.6 B. cereus TKU030 發酵烏賊軟骨所得幾丁寡醣組成分析 ..........31
4.7 水解基質之探討 .........................................32
4.7.1 總糖與還原糖含量之分析 .................................32
4.7.2 利用HPLC 分析幾丁寡醣組成 .............................32
4.8 B. cereus TKU030 幾丁聚醣酶之抑制劑探討 ..................33

第五章 結論 ...............................................68
第六章 參考文獻 ............................................69

圖目錄
圖 2.1 纖維素、幾丁質及幾丁聚醣之結構 ......................... 3
圖 4.1 16S rDNA 部分鹼基序列分析及API 試驗結果 .............. 36
圖 4.2 培養液體積對B. cereus TKU030 生產幾丁質酶、幾丁聚醣
酶及蛋白酶之影響 ...........................................37
圖 4.3 不同碳/氮源對B. cereus TKU030 生產幾丁質酶之影響 .......38
圖 4.4 不同碳/氮源對B. cereus TKU030 生產幾丁聚醣酶之影響.......39
圖 4.5 不同碳/氮源對B. cereus TKU030 生產蛋白酶之影響 .........40
圖 4.6 B. cereus TKU030 幾丁質與幾丁聚醣酶之Sephacryl
S-100 層析圖譜 ............................................41
圖 4.7 B. cereus TKU030 幾丁質與幾丁聚醣酶DEAE-Sepharose
CL-6B 層析圖譜 ............................................41
圖 4.8 B. cereus TKU030 幾丁聚醣酶之Sephacryl S-100 層析圖
譜 .......................................................42
圖 4.9 B. cereus TKU030 幾丁聚醣酶SDS-PAGE 之分子量分析.......43
圖 4.10 溫度對B. cereus TKU030 幾丁質酶 (A) 及幾丁聚醣酶
(B)之討論 .................................................44
圖 4.11 酸鹼度對B. cereus TKU030 幾丁質酶 (A) 及幾丁聚醣酶
(B) 之討論 ................................................45
圖 4.12 銅離子對幾丁質酶活性的影響 ............................46
圖 4.13 幾丁寡糖之HPLC 組成分析圖 (A) 標準品 (B) B. cereus
TKU030 發酵1% SPP 所得上清液 ...............................47
圖 4.14 WSC 經B. cereus TKU030 粗酵素液水解不同時間所得總
糖及還原糖含量 .............................................48
圖 4.15 WSC 經B. cereus TKU030 粗酵素液水解不同時間所得
幾丁寡醣之HPLC 分析圖 ..................................... 49
圖 4.16 WSC 經B. cereus TKU030 粗酵素液水解不同時間所得幾
丁寡醣之HPLC 分析圖 ....................................... 50
圖 4.17 抑制劑與幾丁聚醣酶預反應時間對酵素活性抑制率之影響........ 51
圖 4.18 幾丁聚醣培養基加入抑制劑TKUPSP017 對B. cereus
TKU030 生長之影響 ........................................ 51
圖 4.19 添加0.05 %TKUPSP017 於培養基對B. cereus TKU030
生長之影響 ................................................52
圖 4.20 添加幾丁聚醣酶抑制劑對B. cereus TKU030 生長之影響.......53

表目錄
表 2.1 幾丁質與幾丁聚醣之應用 .................................4
表 4.1 B. cereus TKU030 生產酵素之較適培養條件 .............. 54
表 4.2 不同細菌源幾丁質酶/蛋白酶之酵素活性比較 ..................54
表 4.3 不同細菌源之幾丁質酶與幾丁聚醣酶特性比較 .................55
表 4.4 B. cereus TKU030 幾丁質酶純化總表 (I) ................58
表 4.5 B. cereus TKU030 幾丁質酶純化總表 (II) ...............58
表 4.6 B. cereus TKU030 幾丁聚醣酶純化總表 (I) ..............59
表 4.7 B. cereus TKU030 幾丁聚醣酶純化總表 (II) .............59
表 4.8 各種化學品對幾丁質酶、幾丁聚醣酶和蛋白酶活性之影響..........60
表 4.9 界面活性劑對幾丁質酶及幾丁聚醣酶之影響 ...................61
表 4.10 Bacillus sp. TKU030 幾丁質酶及幾丁聚醣酶之基質特異性....63
表 4.12 抑制劑對B. cereus TKU030 幾丁聚醣酶之抑制效果 .........64
表 4.13 含硼化合物對Bacillus cereus TKU030 幾丁聚醣酶之影響
(%) ......................................................64
表 4.14 抑制劑TKUPSP017, TKUPSP074 及TKUPSP114 對其
他細菌生產幾丁聚醣酶之抑制活性 ............................... 65
表 4.15 含硼化合物對B. cereus TKU030 幾丁聚醣酶活性之抑制率 ... 66

Adrangi S, Faramarzi MA, Shahverdi AR, andSepehrizadeh Z
(2010) Purification and characterization of two extracellular endochitinases fromMassilia timonae. Carbohydrate Research, 345:402-407.

Bernfeld P (1955) Amylase, α and β. Methods in Enzymology,
1:179-158.

Chang WT, Chen YC, and Jao CL (2007) Antifungal activity and
enhancement of plant growth by Bacillus cereus grown on
shellfish chitin wastes. Bioresource Technology, 98:1224-1230.

Chiang CL, Chang CT, and Sung HY (2003) Purification and
properties of chitosanase from a mutant of Bacillus subtilis
IMR-NK1. Enzyme and Microbial Technology, 32: 260–267.

Chui VWD, Mok KW, Ng CY, Luong BP, and Ma KK (1996)
Removeal and recovery cooper (II), chromium (III), and nickel (II)
from solution using crude shrimp chitin packed in small columns. Environment International, 22:463-468.

Da Silva Airesa R, Steindorffb AS, Ramadab MHS, De Siqueirab
SJL, and Ulhoab CJ (2012) Biochemical characterization of a 27 kDa 1,3-β-D-glucanase from Trichoderma asperellum induced by cell wall of Rhizoctonia solani. Carbohydrate Polymers, 87:1219–1223.

Dembitsky VM, Al Quntar AAA, and Srebnik M (2011) Natural and Synthetic small boron-containing molecules as potential
inhibitors of bacterial and fungal quorum sensing. Chemical
Reviews, 111:209-237.

Dobois M, Gilles KA, Hamilton JK, Rebers PA, and Smith F (1956)Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28:350-356
 
Gao XA, Ju WT, Jung WJ, and Park RD (2008) Purification and
characterization of chitosanasefrom Bacillus cereusD-11.
Carbohydrate Polymers, 72:513–520.

Ghaouth AE, Arul J, Grenier J, and Asselin A (1992) Effect of chitosan and other polyions on chitin deacetylase in Rhizopus stolonifer. Experimental Mycology, 16:173-177.

Han Y, Yang B, Zhang F, Miao X, and Li Z (2009) Characterization of antifungal chitinase from MarineStreptomyces sp. DA11 associated with SouthChina sea sponge Craniella australiensis. Marine Biotechnology, 11:132–140.

Hartl L, Zach S, Seidl-Seiboth V (2012) Fungal chitinases: diversity,
mechanistic propertiesand biotechnological potential. Applied Microbiology and Biotechnology, 93:533–543.

Harish Prashanth KV and Tharanathan RN (2007) Chitin/chitosan: modifications and their unlimited application potential. Trends in Food Science and Technology, 18:117-131.

He H, Silo-Suh LA, Handelsman J, and Clardy J (1994)
Zwittermicin A, an antifungal and plant protection agent from Bacillus cereus. Teyrahedron Letters, 35:2499-2502.
Hirano S (1999) Chitin and chitosan as novel biotechnological materials. Polymer International, 48:732-734.

Hsu SK, Chung YC, Chung CT, and Sung HY (2012) Purification
and characterization of two chitosanase isoforms from
the sheaths of bamboo shoots. Journal of Agricultural and Food Chemistry, 60:649-657.
I
dris HA, Labuschagne N, and Korsten L (2007)Screening
rhizobacteria for biological control of Fusarium root and crown rot of sorghum in Ethiopia. Biological Control, 40:97-106.

Imoto T and Yagishita K (1971) A simple activity measurement by lysozyme.Agricultural and Biological Chemistry, 35:1154-1156.

Jankiewicz U, Brzezinska MS, and Saks E (2012) Identification and characterization of a chitinase of Stenotrophomonas maltophilia, a bacterium that is antagonistic towards fungal phytopathogens.
Bioscience and Bioengineering, 113:30-35.

Laemmli UK (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature, 227:680-685.
Li YC, Sun XJ, Bi Y, Ge YH, and Wang Y (2009) Antifungal activity of chitosan on Fusarium sulphureum in pelation to dry rot of potato tuber. Agricultural Sciences in China, 8:597-604.

Liu W, Wang X, Wu L, Chen M, Tu C, Luo Y, and Christie P (2012) Isolation, identification and characterization of Bacillus amyloliquefaciensBZ-6, a bacterial isolate for enhancing oil recovery from oily sludge. Chemosphere, 87:1105-1110.

Liu D, Cai J, Xie CC, and Chen YH (2010) Purification and partial characterization of a 36-kDa chitinase from Bacillus thuringiensis subsp. colmeri, and its biocontrol potential. Enzyme and Microbial Technology, 46: 252–256.

Lee YS, Yoo JS, Chung SY, and Lee YC (2006) Cloning,
purification, and characterization of chitosanasefrom Bacillus sp. DAU101. Applied Microbiology and Biotechnology, 73: 113–121.

Lee YS, Park IH, YooJS, Chung SY, Lee YC, Cho YS, Ahn SC, Kim CM, and Choi YL (2007) Cloning, purification, and
characterization of chitinase from Bacillus sp. DAU101.
Bioresource Technology, 98: 2734–2741.

Liang TW, Kuo YH, Wu PC, Wang CL, Dzung NA, and Wang SL
(2010) Purification and characterization of a chitosanase and a protease by conversion of shrimp shell wastes fermented by Serratia marcescens subsp. Sakuensis TKU019. Journal of the Chinese Chemical Society, 57:857-863.

Ma WC, Lien TS, Wu ST, Yu ST, and Too JR (2007) Screening of a chitinase-producing strain and characterization of its chitinases. Science and Engineering Technology, 3:25-34.

Meng X, Yang L,Kennedy JF, and TianS (2010) Effects of chitosan and oligochitosan on growth of two fungal pathogensand physiological properties in pear fruit. Carbohydrate Polymers, 81:70–75.

Miller GL (1959) Use of dinitrosalicylic acid reagent for
determination of reducting sugar. Analytical Chemistry,
31:426-428.

Nguyen HA, Nguyen TH, Nguyen TT, Peterbauer CK, Mathiesen G,
and Haltrich D (2012) Chitinase from Bacillus
licheniformisDSM13: Expression in Lactobacillus plantarum
WCFS1 and biochemical characterization. Protein Expression
and Purification, 81:166–174.

Park JK, Chung MJ,Choi HN, and Park YI (2011) Effects of the
molecular weight and the degree of deacetylation of chitosan
oligosaccharides on antitumor activity. Molecular Sciences,
12:266-277.

Pagnoncelli MGB, De Araujo NK,Da Silva NMP, De Assis CF,
Rodrigues S,and De Macedo GR (2010) Chitosanase production
by Paenibacillusehimensis and its application for chitosan
hydrolysis.BrazilianArchivesof Biology and Technology,
53:1461-1468.

Ravi Kumar MNV (2000) A review of chitin and chitosan
applications. Reactive and Functional Polymers, 46:1-27.
Rabeeth M, Anitha A, and Srikanth G (2011) Purification of an antifungal endochitinase from a potential biocontrol agent Streptomyces griseus. Biological Sciences, 14:788-797.

Radwan MA, Farrag SAA, Abu-Elamayem MM, Ahmed NS (2012)
Extraction, characterization, and nematicidal activityof chitin and chitosan derived from shrimp shell wastes. Biology and Fertiity of Soils, 48:463-468.

Read TD, Akmal A, Bishop Lilly K, Chen PE, Cook C, Kiley MP,
Lentz S, Mateczun A, Nagarajan N, Nolan N, Osborne BI, Pop M, Sozhamannan S, Stewart AC, Sulakvelidze A, Thomason B,
Willner K, and Zwick ME (2009) Annotation of the Bacillus
thuringiensis BGSC 4BD1 genome. Biological Defense Research
Directorate.

Reginster JY, Deroisy R, Rovati LC, Lee RL, Lejeune E, Bruyere O, Giacovelli G, Henrotin Y, Dacre JE, and Gossett C (2001) Longterm effects of glucosamine sulphate on osteoarthritis progression:a randomised, placebo-controlled clinical trial. Lancet, 357:251-256.

Reyes-Ramirez A, Escudero-Abarca BI, Aguilar-Uscanga G,
Hayward-Jones PM, and Eleazar Barbozacorona J (2004)
Antifungal activity of Bacillus thuringiensis chitinase and its potential for the biocontrol of phytopathogenic fungi in soybean seeds. Food Microbiology and Safety, 69:131-134.

Schuttelkopf AW, Gros L, Blair DE, Frearson JA, van Aalten DMF, Gilbert IH (2010) Acetazolamide-based fungal chitinase inhibitors. Bioorganic & Medicinal Chemistry, 18: 8334–8340.

Shahidi F, Arachchi JKV, and Jeon YJ (1999) Food applications of chitin and chitosans. Trends in Food Science and Technology, 10:37-51.

Su C, Wang D, Yaol, and Yu Z (2006) Purification, characterization, and gene cloning of a chitosanase from Bacillus species strain S65. Journal of Agricultural and Food Chemistry, 54:4208-4214.

Varasteh F, Arzani K,Barzegar M, Zamani Z (2012) Changes in
anthocyanins in arils of chitosan-coated pomegranate(Punica
granatum L. cv. Rabbab-e-Neyriz) fruit during cold storage. Food Chemistry, 130: 267–272.

Wang SL, Kao TY, Wang CL, Yen YH, Chern KM, and Chen YH
(2006) A solvent stable metalloprotease produced by Bacillus sp. TKU004 and its application in the deproteinization of squid pen for β-chitin preparation. Enzyme and Microbial Technology, 39:724-731.

Wang SL, Lin HT, Liang TW, Chen YJ,Yen YH, and Guo SP (2008a) Reclamation of chitinous materials by bromelain for the preparationof antitumor and antifungal materials. Bioresource Technology, 99:4386-4393.

Wang SL and Yeh PY (2008b) Purification and characterization of a chitosanase from a nattokinase producing strain Bacillus subtilis TKU007. Process Biochemistry, 43:132–138.

Wang SL, Peng JH, Liang TW, and Liu KC (2008c) Purification and characterization of a chitosanase from Serratia
marcescensTKU011. Carbohydrate Research, 343:1316–1323.

Wang SL, Chen SJ, and Wang CL (2008d) Purification and
characterization of chitinases and chitosanasesfrom a new
species strain Pseudomonas sp. TKU015 using shrimp shells as
a substrate. Carbohydrate Research, 343:1171–1179.

Wang SL, Chen TR, Liang TW, Wu PC (2009a) Conversion and
degradation of shellfish wastes by Bacillus cereusTKU018
fermentation for the production of chitosanases and bioactive materials. Biochemical Engineering Journal, 48:111–117.

Wang SL, Chao CH, Liang TW and Chen CC (2009b) Purification
and characterization of protease and chitinasefrom Bacillus
cereusTKU006 and conversion of marine wastes by these
enzymes. Marine Biotechnology, 11:334–344.

Wang SL, Lin CL, Liang TW, Liu KC, and Kuo YH (2009c)
Conversion of squid pen by Serratia ureilytica TKU013 for the production of enzymes and antioxidants. Bioresource
Technology, 100 :316–323.

Wang SL, Liou JY, Liang TW, and Liu KC (2009d) Conversion of
squid pen by using Serratia sp. TKU020 fermentation for the
production of enzymes, antioxidants, and N-acetyl
chitooligosaccharides. Process Biochemistry, 44:854-861.

Wang SL, Chang TJ, Liang TW (2010a) Conversion and
degradation of shellfish wastes by Serratia sp. TKU016
fermentation for the production of enzymes and bioactive
materials. Biodegradation, 21:321–333.

Wang SL, Hsu WH, and Liang TW (2010b) Conversion of squid pen by Pseudomonas aeruginosa K187 fermentation for the
production of N-acetyl chitooligosaccharides and biofertilizers. Carbohydrate Research, 345:880-885.

Wang SL, Li JY, Liang TW, Hsieg JL, and Tseng WN (2010c)
Conversion of shrimp shell by using Serratia sp. TKU017
fermentation for the production of enzymes and antioxidants.
Journal of Microbiology and Biotechnology, 20:117-126.

Wang SL, Lin BS, Liang TW, Wand CL, Wu PC, and Liu JR (2010d) Purification and characterization of chitinase from a new species strain, Pseudomonas sp. TKU008. Journal of Microbiology and Biotechnology, 20:1001-1005.

Wang SL, Liang TW, and Yen YH (2011) Bioconversion of
chitin-containing wastes for the production of enzymes and
bioactive materials. Carbohydrate Polymers, 84:732–742.

Wang J, Zhou W, Yuan H, and Wang Y (2008) Characterization of a novel fungal chitosanase Csn2 from Gongronella sp. JG.
Carbohydrate Research, 343:2583–2588.

Wang S, Shao B, Fu H, and Rao P (2009) Isolation of a
thermostable legume chitinase and studyon the antifungal
activity. Biotechnologically Relevant Enzymes and Proteins,
85:313–321.

Wee YJ, Reddy LV, Yoon SD, and Ryu HW (2011) Purification and enzymatic properties of theextracellular and
constitutivechitosanaseproduced by Bacillus subtilis
RKY3.Society of Chemical Industry, 86:757-762.

Wu Y, Wang Y, Luo G, and Dai Y (2009) In situ perparation of
magnetic Fe3O4-chitosan nanoparticles for lipase immobilization by cross-linking and oxidation in aqueous solution. Bioresource Techonology, 100:3459-3464.

Xu W, Yang S, Bhadury P, He J, He M, Gao L, Hu D, Song B (2011) Synthesis and bioactivity of novel sulfone derivatives containing 2,4-dichlorophenylsubstituted 1,3,4-oxadiazole/thiadiazole moiety as chitinase inhibitors. Pesticide Biochemistry and Physiology, 101:6–15

Yan R, DingD, Guan W, Hou J, and Li M (2008)Control of grey
mould rot of loquat with chitinase expressed inPichia pastoris. Crop Protection, 27:1312–1317.

Zakariassen H, Klemetsen L, Sakuda S, Vaaje-Kolstad G,
VarumKM, Sorlie M, and Eijsink VGH (2010) Effect of enzyme
processivity on the efficacy of a competitive chitinase inhibitor. Carbohydrate Polymers, 82:779–785.