臺灣博碩士論文加值系統

地衣芽孢桿菌(Bacillus licheniformis)和枯草桿菌(Bacillus subtilis)為革蘭氏陽性（gram-positive）、兼性厭氧（facultative anaerobe）之產孢桿菌。廣泛應用於工業生產蛋白酶，並經FDA（美國食品暨藥物管理局）認可為GRAS（generally recognized as safe）級之宿主。由於地衣芽孢桿菌及枯草桿菌具有分泌蛋白質之能力，使其成為工業上具發展潛力之蛋白質分泌宿主。幾丁聚醣為幾丁質經由乙醯化而獲得之產物。使用幾丁聚醣酶來分解幾丁聚醣，可產生具有多種生理活性之幾丁寡醣。幾丁寡醣之生理活性有抑制細菌和真菌生長、具抗腫瘤活性以及在高等植物中誘導抗菌素產生。
在先前研究過程中，本實驗室已建立地衣芽孢桿菌及枯草桿菌之持續表現系統。於本研究中，我們建立了地衣芽孢桿菌和枯草桿菌之分泌及細胞表層展示系統來表現枯草桿菌之幾丁聚醣酶基因。
實驗中使用了3個不同表現元件，包括ECSveg、SECS-3、SEC-6-lacO，於地衣芽孢桿菌及枯草桿菌中進行測試，結果顯示此3個表現元件均可被地衣芽孢桿菌及枯草桿菌之RNA polymerase所辨識，而表現元件SECS-6-lacO的表現效率高於ECSveg及SECS-3。本論文建立了分泌表現載體pSL-CSPCSN及pSL-YSPCSN，經轉形於地衣芽孢桿菌和枯草桿菌中後，可順利表現幾丁聚醣酶。在活性方面，地衣芽孢桿菌和枯草桿菌中帶有幾丁聚醣酶訊息胜肽之表現載體pSL-CSPCSN所表現之重組幾丁聚醣酶酵素活性高於帶有鹼性彈性蛋白酶訊息胜肽之載體pSL-YSPCSN。實驗中亦建立了地衣芽孢桿菌誘導表現系統，其含有可表現lacI基因之載體pBL1，於此誘導表現系統中可誘導表現含SECS-6-lacO表現元件之載體。本實驗建構了細胞表層展示載體pSLB-CSPCSN及pVEGB-CSPCSN，pSLB-CSPCSN上帶有SECS-6-lacO表現元件，可順利轉形於地衣芽孢桿菌誘導表現系統B. licheniformis (pBL1)及枯草桿菌誘導表現系統B. subtilis (pBL1)中，經誘導可表現細胞表層展示蛋白質融合之重組幾丁聚醣酶；而pVEGB-CSPCSN載體則無法順利轉形成功。在地衣芽孢桿菌及枯草桿菌誘導表現系統中，可藉由蛋白質電泳及西方墨點法來偵測融合有細胞表層展示蛋白質之重組幾丁聚醣酶的表現。以1 mM及0.5 mM之IPTG來比較不同誘導濃度對其表現量之影響，結果並無明顯之差異。而細胞表層蛋白表現後所位於細胞之部位可藉由蛋白質電泳及西方墨點法來偵測，結果發現經表現後，細胞表層蛋白可鍵結於細胞壁上，經由全菌體活性測試後可發現其具有酵素活性。

Bacillus licheniformis and Bacillus subtilis are gram-positive, facultative anaerobic endospore-forming bacteria. They have been used for industrial production of several proteases and are classified as a GRAS (generally recognized as safe) microorganisms by FDA. Some properties of B. licheniformis and B. subtilis such as the capability to secret protein have led these strains to be attractive hosts for secretory production of heterologous proteins. Chitosan is obtained by the deacetylation of chitin. Enzymatic hydrolysis of chitosan by using chitosanase could be used for preparation of chitosan oligosaccharides which have received growing attention because they perform a variety of biological activities, such as inhibiting the growth of bacteria and fungi, exerting antitumor activity, acting as immunopotential effectors, and inducing the phytoalexin product in higher plants.
A constitutive expression system for B. licheniformis and B. subtilis has been established in our laboratory. In this study, we have developed secretory and cell surface display system for expression of chitosanase gene from B. subtilis in B. licheniformis and B. subtilis.
Three expression control sequences including ECSveg, SECS-3, SECS-6-lacO were used as expression signals for B. licheniformis and B. subtilis. The results showed that all three expression signals could be recognized by B. licheniformis and B. subtilis RNA polymerase. The expression efficiency of SECS-6-lacO was higher than that of ECSveg and SECS-3. The secretory expression vectors including pSL-CSPCSN and pSL-YSPCSN were constructed and transformed into B. licheniformis and B. subtilis and could be expressed recombinant chitosanase. The activity of recombinant chitosanase expressed by pSL-CSPCSN is higher than that expressed by pSL-YSPCSN in B. licheniformis and B. subtilis. An inducible expression system based on SECS-6-lacO and lacI-containing plasmid pBL1 was established for B. subtilis in previous research and for B. licheniformis in this study. The cell surface display expression plasmids including pSLB-CSPCSN and pVEGB-CSPCSN were constructed and plasmid pSLB-CSPCSN, but not pVEGB-CSPCSN, could be transformed into B. licheniformis (pBL1) and B. subtilis (pBL1). Expression of cell wall binding protein fused chitosanase in B. licheniformis (pBL1) and B. subtilis (pBL1) was detected by SDS-PAGE and western blot. The expression level of different induction concentration by 1 mM and 0.5 mM IPTG is similar. The location of cell wall binding protein fused chitosanase in the cell fractions was detected by SDS-PAGE and western blot. The result showed that the cell wall binding protein fused chitosanase could be displayed on the cell wall, and the enzyme activity can be detected by whole cell activity test.

中文摘要-------------------------------------------------------------------------------------------------------------------------i
英文摘要------------------------------------------------------------------------------------------------------------------------ii
壹、前言-------------------------------------------------------------------------------------------------------------------------1
一、芽孢桿菌屬-----------------------------------------------------------------------------------------------------------------1
（一）地衣芽孢桿菌（Bacillus licheniformis）之簡介----------------------------------------------------------------------------1
（二）枯草桿菌（Bacillus subtilis）之簡介-------------------------------------------------------------------------------------2
二、幾丁質、幾丁聚醣與幾丁聚醣酶-----------------------------------------------------------------------------------------------2
（一）幾丁質與幾丁聚醣--------------------------------------------------------------------------------------------------------2
（二）幾丁聚醣酶--------------------------------------------------------------------------------------------------------------4
三、蛋白質表現途徑-------------------------------------------------------------------------------------------------------------4
（一）分泌途徑----------------------------------------------------------------------------------------------------------------5
（二）細胞表層展示------------------------------------------------------------------------------------------------------------6
（三）細胞壁結合蛋白質--------------------------------------------------------------------------------------------------------7
貳、實驗目的---------------------------------------------------------------------------------------------------------------------9
參、實驗策略--------------------------------------------------------------------------------------------------------------------10
肆、材料與方法------------------------------------------------------------------------------------------------------------------11
一、菌種、質體及培養條件------------------------------------------------------------------------------------------------------11
（一）菌種-------------------------------------------------------------------------------------------------------------------11
（二）質體-------------------------------------------------------------------------------------------------------------------11
二、藥品、試劑----------------------------------------------------------------------------------------------------------------12
三、質體DNA之抽取-------------------------------------------------------------------------------------------------------------13
（一）大腸桿菌質體抽取------------------------------------------------------------------------------------------------------ 13
（二）枯草桿菌質體抽取-------------------------------------------------------------------------------------------------------13
（三）地衣芽孢桿菌質體抽取---------------------------------------------------------------------------------------------------14
四、染色體DNA之抽取-----------------------------------------------------------------------------------------------------------14
（一）枯草桿菌染色體DNA之抽取------------------------------------------------------------------------------------------------14
（二）地衣芽孢桿菌染色體DNA之抽取--------------------------------------------------------------------------------------------15
五、聚合酶連鎖反應及回收------------------------------------------------------------------------------------------------------15
（一）幾丁聚醣酶基因之增幅---------------------------------------------------------------------------------------------------15
（二）細胞壁結合蛋白質基因cwlC之增幅-----------------------------------------------------------------------------------------16
（三）聚合酶連鎖反應產物之回收-----------------------------------------------------------------------------------------------16
六、DNA電泳、剪切、回收、黏合-------------------------------------------------------------------------------------------------16
（一）DNA電泳----------------------------------------------------------------------------------------------------------------16
（二）以限制酶剪切DNA--------------------------------------------------------------------------------------------------------16
（三）回收限制酶剪切之DNA----------------------------------------------------------------------------------------------------17
七、電勝任細胞之製備與電轉形條件----------------------------------------------------------------------------------------------17
（一）大腸桿菌---------------------------------------------------------------------------------------------------------------17
（二）地衣芽孢桿菌-----------------------------------------------------------------------------------------------------------17
（三）枯草桿菌---------------------------------------------------------------------------------------------------------------18
八、Colony PCR篩選轉形株------------------------------------------------------------------------------------------------------18
九、表現載體構築--------------------------------------------------------------------------------------------------------------19
（一）大腸桿菌胞內誘導表現幾丁聚醣酶載體pET-CSN之構築------------------------------------------------------------------------19
（二）地衣芽孢桿菌及枯草桿菌分泌表現載體pSL-CSPCSN之構築---------------------------------------------------------------------19
（三）地衣芽胞桿菌及枯草桿菌分泌表現載體pSL-YSPCSN之構築---------------------------------------------------------------------19
（四）地衣芽孢桿菌及枯草桿菌表層展示中間載體pSL-CWB之構築--------------------------------------------------------------------19
（五）地衣芽孢桿菌及枯草桿菌表層展示載體pSLB-CSPCSN之構築--------------------------------------------------------------------19
（六）地衣芽孢桿菌及枯草桿菌表層展示載體pVEGB-CSPCSN之構築-------------------------------------------------------------------19
十、重組質體剪切確認及定序----------------------------------------------------------------------------------------------------19
十一、Gycine-SDS PAGE --------------------------------------------------------------------------------------------------------20
十二、西方轉漬法 -------------------------------------------------------------------------------------------------------------20
十三、大腸桿菌胞內幾丁聚醣酶純化與濃縮----------------------------------------------------------------------------------------20
十四、抗體之製備--------------------------------------------------------------------------------------------------------------21
十五、不同表現元件於地衣芽孢桿菌中活性之比較----------------------------------------------------------------------------------21
十六、不同IPTG誘導濃度對啟動子於地衣芽孢桿菌中表現之比較----------------------------------------------------------------------22
十七、枯草桿菌及地衣芽孢桿菌之細胞劃分----------------------------------------------------------------------------------------22
（一）地衣芽胞桿菌-----------------------------------------------------------------------------------------------------------22
（二）枯草桿菌---------------------------------------------------------------------------------------------------------------22
十八、幾丁聚醣活性測定--------------------------------------------------------------------------------------------------------23
（一）平板培養基測試法-------------------------------------------------------------------------------------------------------23
（二）酵素反應測定-----------------------------------------------------------------------------------------------------------23
伍、結果與討論------------------------------------------------------------------------------------------------------------------24
一、大腸桿菌表現載體之構築----------------------------------------------------------------------------------------------------24
二、地衣芽孢桿菌及枯草桿菌分泌表現載體之構築----------------------------------------------------------------------------------24
（一）分泌載體pSL-CSPCSN之構築-----------------------------------------------------------------------------------------------24
（二）分泌載體pSL-YSPCSN之構築-----------------------------------------------------------------------------------------------25
三、地衣芽孢桿菌及枯草桿菌表層展示載體之構築----------------------------------------------------------------------------------25
（一）表層展示中間載體pSL-CWB之構築------------------------------------------------------------------------------------------25
（二）表層展示載體pSLB-CSPCSN之構築------------------------------------------------------------------------------------------26
（三）表層展示載體pVEGB-CSPCSN之構築-----------------------------------------------------------------------------------------26
四、地衣芽孢桿菌雙質體系統之建立與枯草桿菌雙質體系統之應用--------------------------------------------------------------------27
五、不同表現訊號於地衣芽孢桿菌之比較 -----------------------------------------------------------------------------------------27
六、幾丁聚醣酶於地衣芽孢桿菌及枯草桿菌之分泌表現------------------------------------------------------------------------------27
（一）地衣芽孢桿菌之分泌表現-------------------------------------------------------------------------------------------------28
（二）枯草芽孢桿菌之分泌表現-------------------------------------------------------------------------------------------------28
七、幾丁聚醣酶於地衣芽孢桿菌及枯草桿菌之誘導表現------------------------------------------------------------------------------28
（一）不同誘導濃度於地衣芽孢桿菌之生長、表層展示表現---------------------------------------------------------------------------29
（二）不同誘導濃度於枯草桿菌之生長、表層展示表現-------------------------------------------------------------------------------29
八、以細胞劃分偵測帶有細胞壁結合蛋白質之幾丁聚醣酶表現與存在部位--------------------------------------------------------------29
（一）地衣芽孢桿菌細胞劃分---------------------------------------------------------------------------------------------------29
（二）枯草桿菌細胞劃分-------------------------------------------------------------------------------------------------------30
九、活性測定------------------------------------------------------------------------------------------------------------------30
（一）地衣芽孢桿菌分泌表現活性測定-------------------------------------------------------------------------------------------30
（二）枯草桿菌分泌活性測定---------------------------------------------------------------------------------------------------31
（三）地衣芽孢桿菌及枯草桿菌表層展示之幾丁聚醣酶活性測定---------------------------------------------------------------------31
陸、結論------------------------------------------------------------------------------------------------------------------------32
柒、參考文獻--------------------------------------------------------------------------------------------------------------------64
捌、附錄------------------------------------------------------------------------------------------------------------------------70



表目錄
頁次
表一、B. licheniformis中蛋白質純化、選殖或表現至其他宿主之實例------------------------------------------------------------------33
表二、一些Bacillus sp.來源的幾丁聚醣酶之特性 -----------------------------------------------------------------------------------34




圖目錄
頁次
圖一、(A)幾丁質與(B)幾丁聚糖之結構----------------------------------------------------------------------------------------------35
圖二、(A) Streptomyces N174幾丁聚糖酶之結構 (B) Bacillus circulans幾丁聚糖酶之結構----------------------------------------------36
圖三、Bacillus subtilisDB104幾丁聚醣酶結構預測 ---------------------------------------------------------------------------------37
圖四、微生物之細胞表層展示------------------------------------------------------------------------------------------------------38
圖五、枯草桿菌之分泌途徑--------------------------------------------------------------------------------------------------------39
圖六、訊息胜肽 (signal peptide) 之典型結構--------------------------------------------------------------------------------------40
圖七、枯草桿菌之穿膜機制--------------------------------------------------------------------------------------------------------41
圖八、細胞表層展示系統於 (A) 革蘭氏陰性菌及 (B) 革蘭氏陽性菌--------------------------------------------------------------------42
圖九、革蘭氏陽性菌不同型式的細胞表層蛋白----------------------------------------------------------------------------------------43
圖十、CwlC的domain結構----------------------------------------------------------------------------------------------------------44
圖十一、pET-CSN構築流程圖-------------------------------------------------------------------------------------------------------45
圖十二、pSL-CSPCSN構築流程圖----------------------------------------------------------------------------------------------------46
圖十三、pSL-YSPCSN構築流程圖----------------------------------------------------------------------------------------------------47
圖十四、pSL-CWB構築流程圖-------------------------------------------------------------------------------------------------------48
圖十五、pSLB-CSPCSN構築流程圖---------------------------------------------------------------------------------------------------49
圖十六、pVEGB-CSPCSN構築流程圖--------------------------------------------------------------------------------------------------50
圖十七、不同表現元件於地衣芽孢桿菌中GFP+活性之比較------------------------------------------------------------------------------51
圖十八、利用蛋白質電泳觀察地衣芽孢桿菌分泌表現幾丁聚醣酶之情形------------------------------------------------------------------52
圖十九、利用蛋白質電泳觀察枯草桿菌分泌表現幾丁聚醣酶之情形----------------------------------------------------------------------53
圖二十、以西方墨點法偵測幾丁聚醣酶於地衣芽孢桿菌(A)及枯草桿菌(B)24小時之分泌表現情形--------------------------------------------54
圖二十一、不同誘導濃度表現幾丁聚醣酶對地衣芽孢桿菌及枯草桿菌生長之影響----------------------------------------------------------55
圖二十二、利用蛋白質電泳觀察地衣芽孢桿菌表層展示表現幾丁聚醣酶------------------------------------------------------------------56
圖二十三、利用蛋白質電泳觀察地衣芽孢桿菌胞外表現幾丁聚醣酶之情形----------------------------------------------------------------57
圖二十四、以西方墨點法偵測幾丁聚醣酶於地衣芽孢桿菌誘導24小時(A)及枯草桿菌誘導10小時(B)之表現情形--------------------------------58
圖二十五、利用蛋白質電泳觀察枯草桿菌表現表層展示幾丁聚醣酶之情形----------------------------------------------------------------59
圖二十六、利用蛋白質電泳觀察枯草桿菌胞外表現表層展示幾丁聚醣酶之情形------------------------------------------------------------60
圖二十七、利用西方墨漬法偵測地衣芽孢桿菌細胞劃分結果----------------------------------------------------------------------------61
圖二十八、利用西方墨漬法偵測枯草桿菌細胞劃分結果--------------------------------------------------------------------------------62
圖二十九、地衣芽孢桿菌(A)及枯草桿菌(B)分泌表現之幾丁聚醣酶活性------------------------------------------------------------------63

王志鵬。2001年。一、枯草桿菌Bacillus subtilis DB104電轉形效率之增進。二、設計、合成第一型抗凍蛋白基因及其在枯草桿菌、大腸桿菌中之表現。國立中興大學食品科學系碩士論文。

洪碧霙。2003年。蜡蚧輪枝菌幾丁聚醣酶之特性分析及應用研究。朝陽科技大學應用化學系碩士論文。

彭宣融。2006年。開發重組乳酸鏈球菌口服疫苗。國立中興大學食品暨應用生物科技學系碩士論文。

黃德琳。1999年。黴菌中幾丁聚醣酵素之篩選、純化與性質的研究。國立交通大學應用化學系碩士論文。

戴君如。2003年。Bacillus subtilis群之分類近況。食品工業。35（7）：42-53。

羅秋梅。2005年。建立地衣芽孢桿菌表現系統以應用於靈芝免疫調節蛋白質生產。國立中興大學食品科學系碩士論文。

Adachi, W., Sakihama, Y., Shimizu, S., Sunami, T., Fukazawa, T., Suzuki, M.,
Yatsunami, R., Nakamura, S. and Takenaka. A. 2004. Crystal Structure of Family
GH-8 Chitosanase with Subclass II Specificity from Bacillus sp. K17. J. Mol. Biol.
343:785–795.

Akita, M., Sasaki, S., Matsuyama, S. and Mizushima, S. 1990. SecA interacts with secretory proteins by recognizing the positive charge at the amino terminus of the signal peptide in Escherichia coli. J Biol Chem, 265: 8164-9.

Bernkop-Schnurch, A. and Krajicek, M.E. 1998. Mucoadhesive polymers as platforms for peroral peptide delivery and absorption: synthesis and evaluation of different chitosan-EDTA conjugates. J Control Release, 50: 215-23.

Birnboim, H.C. and Doly, J. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res, 7: 1513-23.

Boucher, I., Dupuy, A., Vidal, P., Neugebauer, W.A. and Brzezinski, R. 1992. Purification and characterization of a chitosanase from Streptomyces N174. Appl Microbiol Biotechnol, 38: 188-193.

Brockmeier, U., Caspers, M., Freudl, R., Jockwer, A., Noll, T. and Eggert, T. 2006. Systematic screening of all signal peptides from Bacillus subtilis: a powerful strategy in optimizing heterologous protein secretion in Gram-positive bacteria. J Mol Biol, 362: 393-402.

Choi, Y.J., Kim, E.J., Piao, Z., Yun, Y.C. and Shin, Y.C. 2004. Purification and characterization of chitosanase from Bacillus sp. strain KCTC 0377BP and its application for the production of chitosan oligosaccharides. Appl Environ Microbiol, 70: 4522-31.

Declerck, N., Machius, M., Wiegand, G., Huber, R. and Gaillardin, C. 2000. Probing structural determinants specifying high thermostability in Bacillus licheniformis alpha-amylase. J Mol Biol, 301: 1041-57.

Desvaux, M., Dumas, E., Chafsey, I. and Hebraud, M. 2006. Protein cell surface display in Gram-positive bacteria: from single protein to macromolecular protein structure. FEMS Microbiol Lett, 256: 1-15.

Deuerling, E., Mogk, A., Richter, C., Purucker, M. and Schumann, W. 1997. The ftsH gene of Bacillus subtilis is involved in major cellular processes such as sporulation, stress adaptation and secretion. Mol Microbiol, 23: 921-33.

Fu, L.L., Xu, Z.R., Li, W.F., Shuai, J.B., Lu, P. and Hu, C.X. 2007. Protein secretion pathways in Bacillus subtilis: implication for optimization of heterologous protein secretion. Biotechnol Adv, 25: 1-12.

Fukamizo, T., Honda, Y., Goto, S., Boucher, I. and Brzezinski, R. 1995. Reaction mechanism of chitosanase from Streptomyces sp. N174. Biochem J, 311 ( Pt 2): 377-83.

Fukamizo, T., Ohkawa, T., Ikeda, Y. and Goto, S. 1994. Specificity of chitosanase from Bacillus pumilus. Biochim Biophys Acta, 1205: 183-8.

Gupta, R., Beg, Q.K. and Lorenz, P. 2002. Bacterial alkaline proteases: molecular approaches and industrial applications. Appl Microbiol Biotechnol, 59: 15-32.

Harwood, C.R. and Cutting, S.M. 1990. Molecular Biological Methods for Bacillus. John Wiley & Sons, West Sussex, England.

Izume, M., Nagae, S., Kawagishi, H., Mitsutomi, M. and Ohtakara, A. 1992. Action pattern of Bacillus sp. no. 7-M chitosanase on partially N-acetylated chitosan. Biosci Biotechnol Biochem, 56: 448-53.

Jensen, K.K. and Hulett, F.M. 1989. Protoplast transformation of Bacillus licheniformis MC14. J Gen Microbiol, 135: 2283-7.

Jo, Y.Y., Jo, K.J., Jin, Y.L., Kim, K.Y., Shim, J.H., Kim, Y.W. and Park, R.D. 2003. Characterization and kinetics of 45 kDa chitosanase from Bacillus sp. P16. Biosci Biotechnol Biochem, 67: 1875-82.

Kim, K.W., Thomas, R.L., Lee, C. and Park, H.J. 2003. Antimicrobial activity of native chitosan, degraded chitosan, and O-carboxymethylated chitosan. J Food Prot, 66: 1495-8.

Kobayashi, G., Fujii, K., Serizawa, M., Yamamoto, H. and Sekiguchi, J. 2002. Simultaneous display of bacterial and fungal lipases on the cell surface of Bacillus subtilis. J Biosci Bioeng, 93: 15-9.

Kuroda, A., Asami, Y. and Sekiguchi, J. 1993. Molecular cloning of a sporulation-specific cell wall hydrolase gene of Bacillus subtilis. J Bacteriol, 175: 6260-8.

Kuroda, A. and Sekiguchi, J. 1991. Molecular cloning and sequencing of a major Bacillus subtilis autolysin gene. J Bacteriol, 173: 7304-12.

Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-5.

Lee, S.Y., Choi, J.H. and Xu, Z. 2003. Microbial cell-surface display. Trends Biotechnol, 21: 45-52.

Lee, Y. C., Cho, Y. S., Choi, Y. L. 2006. Cloning, purification, and characterization
of chitosanase from Bacillus sp. DAU101. Appl Microbiol Biotechnol. 273:
113–121.

Mandelstam, J. and Waites, W.M. 1968. Sporulation in Bacillus subtilis. The role of exoprotease. Biochem J, 109: 793-801.

Masson, J., Boucher, I., Neugebauer, W., Ramotar, D. and Brzezinski, R. 1995. A new chitosanase gene from a Nocardioides sp. is a third member of glycosyl hydrolase family 46 MICROBIOLOGY+, 141: 2629-2635.

Merchante, R., Pooley, H.M. and Karamata, D. 1995. A periplasm in Bacillus subtilis. J Bacteriol, 177: 6176-83.

Miller, G.L. 1959. Use of DinitrosaIicyIic Acid Reagent for Determination of Reducing Sugar. ANAL CHEM, 31: 426-428.

Miller, J.F. 1994. Bacterial transformation by electroporation. Method Enzymol, 235: 375-85.

Mishima, M., Shida, T., Yabuki, K., Kato, K., Sekiguchi, J. and Kojima, C. 2005. Solution structure of the peptidoglycan binding domain of Bacillus subtilis cell wall lytic enzyme CwlC: characterization of the sporulation-related repeats by NMR. Biochemistry, 44: 10153-63.

Rivas, L.A., Parro, V., Moreno-Paz, M. and Mellado, R.P. 2000. The Bacillus subtilis 168 csn gene encodes a chitosanase with similar properties to a streptomyces enzyme. Microbiology, 146 ( Pt 11): 2929-36.

Saito, J., Kita, A., Higuchi, Y., Nagata, Y., Ando, A. and Miki, K. 1999. Crystal structure of chitosanase from Bacillus circulans MH-K1 at 1.6-A resolution and its substrate recognition mechanism. J Biol Chem, 274: 30818-25.

Schallmey, M., Singh, A. and Ward, O.P. 2004. Developments in the use of Bacillus species for industrial production. Can J Microbiol, 50: 1-17.

Shida, T., Hattori, H., Ise, F. and Sekiguchi, J. 2001. Mutational analysis of catalytic sites of the cell wall lytic N-acetylmuramoyl-L-alanine amidases CwlC and CwlV. J Biol Chem, 276: 28140-6.

Shih, I.L. and Van, Y.T. 2001. The production of poly-(gamma-glutamic acid) from microorganisms and its various applications. Bioresour Technol, 79: 207-25.

Smith, G.P. 1985. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science, 228: 1315-7.

Su, C. X., Wang, D.G., Yao, L. M. and Yu, Z. L. 2006. Purification, Characterization, and Gene Cloning of a Chitosanase from Bacillus Species Strain S65. J. Agric. Food
Chem. 54: 4208-14.

Tanabe, T., Morinaga, K., Fukamizo, T. and Mitsutomi, M. 2003. Novel chitosanase from Streptomyces griseus HUT 6037 with transglycosylation activity. Biosci Biotechnol Biochem, 67: 354-64.

Tjalsma, H., Bolhuis, A., Jongbloed, J.D., Bron, S. and van Dijl, J.M. 2000. Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome. Microbiol Mol Biol Rev, 64: 515-47.

Uchida, Y. and Ohtakara, A. 1988. Chitosanase from Bacillus Species. Method Enzymol, 161: 501-505.

Waites, W.M., Kay, D., Dawes, I.W., Wood, D.A., Warren, S.C. and Mandelstam, J. 1970. Sporulation in Bacillus subtilis. Correlation of biochemical events with morphological changes in asporogenous mutants. Biochem J, 118: 667-76.

Wang, J.J., Greenhut, W.B. and Shih, J.C. 2005. Development of an asporogenic Bacillus licheniformis for the production of keratinase. J Appl Microbiol, 98: 761-7.

Westers, L., Westers, H. and Quax, W.J. 2004. Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. Biochim Biophys Acta, 1694: 299-310.

Wang, J.P., Yeh, C.M., Tsai, Y.C. 2006. Improved Subtilisin YaB Production in Bacillus subtilis Using Engineered Synthetic Expression Control Sequences. J Agric Food Chem, 54(25)：9405-9410.

Yoon, H. G., Kim, H. Y., Lim, Y. H., Kim, H. K., Shin, D. H., Hong, B. S. and Cho,
H. Y. 2000. Thermostable Chitosanase from Bacillus sp. Strain CK4: Cloning and
Expression of the Gene and Characterization of the Enzyme. Appl. Environ. 66(9) 3727-34.

Young, D.H. and Kauss, H. 1983. Release of Calcium from Suspension-Cultured Glycine max Cells by Chitosan, Other Polycations, and Polyamines in Relation to Effects on Membrane Permeability. Plant Physiol, 73: 698-702.