摘要
β-葡聚糖酶是重要的工业用酶,可有效消除谷物β-葡聚糖在酿造和饲料工业中产生的负面影响。国内对β-1,3-1,4-葡聚糖酶的研究还处于初级阶段,天然菌株的产酶量及酶的性能满足不了实际应用。然而,分子生物学、基因工程方法的应用为其提供了契机。现在对β-1,3-1,4-葡聚糖酶的研究主要集中在构建基因工程菌株来提高酶的产量和性能方面。本研究应用工业生物技术进行了一系列的探索,旨在找到一种可持续发展的,可用于产业化、规模化的生产β-1,3-1,4-葡聚糖酶的方法。主要研究内容包括:
1.发酵罐流加培养重组大肠杆菌JMl09-pLF3生产β-1,3-1,4-葡聚糖酶
在原有优化培养基基础上,优化了发酵工艺,在7 L发酵罐上进行了分批补料发酵产酶研究。结果表明,在12～32 h之间向发酵罐内恒速流加双倍浓缩的培养基氮源,对细胞生长和产酶有极大的促进作用。最大酶活增长到1680 U/mL,比间歇发酵提高了231.40%;最大细胞密度为7.67 g/L,是间歇发酵3.44倍。
2.高效分泌表达β-1,3-1,4-葡聚糖酶重组大肠杆菌的构建
将热稳定性较好的杂合β-1,3-1,4-葡聚糖酶表达基因(bgl)插入质粒pET-22b(+),并插入kil-Km分泌盒,得到了新的β-葡聚糖酶表达质粒pET-22-bgl-(kil-Km)。将重组质粒转化大肠杆菌E coli JM109和BL21(DE3)。通过SDS-PAGE证明了含有质粒pET-22-bgl-(kil-Km)的重组菌株具有目的蛋白的高效分泌表达的功能。
3.响应面法优化重组E.coli BL21(DE3)-pET-22-bgl-(kil-Km)产酶培养基
在TB培养基基础上,通过单因素实验选择乳糖为最佳碳源,酪蛋白胨为最佳氮源。在单因素优化基础上采用Box-Behnken设计法和RSM响应面分析法考察主要影响因素乳糖浓度、酪蛋白胨浓度和碳氮比三因素的的交互作用。确定了重组E.coli BL21(DE3)-pET-22-bgl-(kil-Km)产酶的优化培养基配方为(g/L):酪蛋白胨33.39,乳糖8.03,甘油9.15,NaCl 10.0,KH_2PO_4 2.31,K_2HPO_4 12.54。利用优化培养基在37℃、150 rpm条件下,摇瓶培养32.5 h,酶活达到1242.94 U/mL,比初始培养基提高了3.3倍。
4.重组酶的分离纯化和酶学性质表征
利用Ni~(2+)与6×His标签特异性结合的性质利用亲和层析纯化重组酶,该方法将目的蛋白纯化了7.69倍,纯化后重组酶的活力为1706.2 U/mL,比活11261.6U/mg,酶活回收率达到86.44%。通过SDS-PAGE分析,可知带有6个His标签的β-葡聚糖酶分子量为26 kDa左右。重组β-1,3-1,4-葡聚糖酶的酶学性质为:最适反应温度和耐热温度均为50℃,最适反应pH在6.0～7.0之间,碱性条件下稳定性较高。
β-1,3-1,4-glucanase is a very important industrial enzyme,whose major application is to eliminate the negative effect ofβ-glucan on brewing and feed industry.In the domestic market so far,the production ofβ-1,3-1,4-glucanase by screened natural strains can not meet the growing industrial need for this enzyme due to low enzyme activity,poor stability and less investigation on this enzyme.Progress in molecular biology and genetic engineering allows recombinant proteins such as enzymes to be produced in bacteria,i.e.,E.coli and other microorganisms in large-scale.Thus this study mainly focused on the construction and use of recombinant E.coli strains to enhance the extracellular production and quality of the enzyme.For this purpose, different cultivation technologies were used to improve the extracellular production ofβ-1,3-1,4-glucanase by recombinant E.coli for sustainable industrial application.The main research work includes:
1.Fed-batch cultivation of recombinant E.coli JM109-pLF3 for the extracellular production ofβ-1,3-1,4-glucanase
The fed-batch fermentation was carried out in a 7 L conventional bioreactor based on the optimized medium as well as optimized cultivation conditions.The results indicated that feeding two-fold concentrated nitrogen source of the optimized medium during the cultivation period of 12-32 h could significantly enhance the biomass and enzyme activity.The highest enzyme activity and biomass reached up to 1680 U/mL and 7.67 g/L,which was 2.31 and 2.44 folds higher than those obtained in the batch fermentation at the same cultivation conditions,respectively.
2.Construction of recombinant E.coli for high level secreted expression ofβ-1,3-1,4-glucanase
The thermo-stable hybrid bgl gene encodingβ-1,3-1,4-glucanase and kil-Km secretion cassette were inserted into the plasmid pET-22b(+),yielding pET-22-bgl-(kil-Km),a newβ-1,3-1,4-glucanase expression plasmid.The recombinant plasmid was then transformed into E.coli JM109 and BL21(DE3), respectively.The IPTG induction could not enhance the enzyme production.The ability of pET-22-bgl-(kil-Km) to express extracellular target protein with high level was proved by SDS-PAGE.
3.Optimization of medium components by Response Surface Methodology for high level enzyme production
Started with the TB medium,through one-factor-at-a-time experiments,lactose and casein peptone were found to be the most suitable carbon and nitrogen sources,for enzyme production by recombinant E.coli BL21(DE3)(pET-22-bgl-(kil-Km), respectively.The statistical analyses of the effect of the carbon and nitrogen source as well as C:N ratio in the medium on enzyme production,by employing RSM and the Box-Behnken design indicated that the optimized medium should contain(g/L): casein 33.39,lactose 8.03,glycerol 9.15,NaCl 10.0,KH_2PO_4 2.31 and K_2HPO_4 12.54. By shake flask cultivation of the recombinant cells on the optimized medium at a temperature of 37℃and a rotation speed of 150 rpm,the enzyme activity of 1242.94 U/mL was achieved after 32.5 h of incubation,which was approximately 3.3 times higher than that obtained on the TB medium at the same conditions.
4.Purification of the recombinant enzyme and characterization of enzyme properties Affinity chromatography was applied to purify the recombinant protein with 6×his tags specific covalent with Ni~(2+) column.The recovery rate was 86.44%.The molecular weight of the target enzyme with 6×his tag was about 26 kDa,in consistent with SDS-PAGE result.The enzyme activity and specific enzyme activity reached 1706.2 U/mL and 11261.6 U/mg with a separation factor of 7.69 after purification, respectively.The optimum temperature and pH for enzyme activity was 50℃and 6.0 -7.0.The enzyme was more stable under base conditions.
引文
Akiba,S.,Yamamoto,K.,Kumagai,H.Transglycosylation activity of the endo-beta-1,4-glucanase from Aspergillus niger IFO31125 and its application[J].J.Biosci.Bioeng.,1999,(87):576-580.
Akita,M.,Kayatama,K.,Hatada,Y.,Ito,S.,Horikoshi,K.A novel beta-glucanase gene from Bacillus halodurans C-125[J].FEMS.Microbiol.Lett.,2005,(248):9-15.
Akiyama,T.,Kaku,H.,Shibuya,N.Purification and properties of a basic endo-1,3-beta-glucanase from rice(Oryza sativa L.)[J].Plant.Cell.Physiol.,1996,(37):702-705.
Baird,S.D.,Johnson,D.A.,Seligy,V.L.Molecular cloning,expression,and characterization of endo-beta-1,4-glucanase genes from Bacillus polymyxa and Bacillus circulans[J].J Bacteriol.,1990,(172):1576-1586.
Bauer,K.A.,Ben-Bassat,A.,Dawson,M.,de,l.P.,V,Neway,J.O.Improved expression of human interleukin-2 in high-cell-density fermentor cultures of Escherichia coli K-12 by a phosphotransacetylase mutant[J].Appl.Environ.Microbiol.,56,1296-1302.
Beckmann,L.,Simon,O.,Vahjen,W.,(2006).Isolation and identification of mixed linked beta -glucan degrading bacteria in the intestine of broiler chickens and partial characterization of respective 1,3-1,4-beta -glucanase activities[J].J.Basic.Microbiol.,1990,(46):175-185.
Belghith,Hafedh,Ellouz-Chaabouni,Semia,Gargouri Ali,(2001).Biostoning ofclenims by Penicillium occitanis(Pol 6) cellulases[J].J.Biotech.,89(2-3),253-262.
Borriss,R.,Buettner,K.,Maentsaelae,P.Structure of the beta-1,3-1,4-glucanase gene of Bacillus macerans:homologies to other beta-glucanases[J].Mol.Gen.Genet.,1990,(222):278-283.
Borriss,R.,Olsen,O.,Thomsen,K.K.,yon,W.D.Hybrid bacillus endo-(1-3,1-4)-beta-glucanases:construction of recombinant genes and molecular properties of the gene products[J].Carlsberg.Res.Commun.,1989,(54):41-54.
Brenes,A.,Smith,M.,Guenter,W.,Marquardt,R.R.Effect of enzyme supplementation on the performance and digestive tract size of broiler chickens fed wheat- and barley-based diets[J].Poult.Sci.,1993,(72):1731-1739.
Castro,O.L.,Bach,E.E.Increased production of beta-1,3 glucanase and proteins in Bipolaris sorokiniana pathosystem treated using commercial xanthan gum[J].Plant.Physiol.Biochem.,2004,(42):165-169.
Chen,J.L.,Tsai,L.C.,Wen,T.N.,Tang,J.B.,Yuan,H.S.,Shyur,L.F.Directed mutagenesis of apecific active site residues on Fibrobacter succinogenes 1,3-1,4-beta -D-glucanase significantly affects catalysis and enzyme structural stability[J].J.Biol.Chem.,2001,(276):17895-17901.
Courtois,J.E.[Use of a filtration enzyme with dominant beta-glucanase activity produced by
Disporotrichum dimorphosporum, in beer brewing [J]. Bull. Acad. Natl. Med., 1987, (171): 693-694.
Distel, D. L., Morrill, W., Laren-Toussaint, N., Franks, D., Waterbury, J. Teredinibacter turnerae gen.nov., sp. nov., a dinitrogen-fixing, cellulolytic, endosymbiotic gamma-proteobacterium isolated from the gills of wood-boring molluscs (Bivalvia: Teredinidae) [J]. Int. J. Syst. Evol. Microbiol.,2002, (52):2261-2269.
Dubendorff, J. W., Studier, F. W. Creation of a T7 autogene. Cloning and expression of the gene for bacteriophage T7 RNA polymerase under control of its cognate promoter [J]. J. Mol.Biol.,1991, (219):61-68.
Ekinci, M. S., McCrae, S. I., Flint, H. J. Isolation and overexpression of a gene encoding an extracellular beta-(1,3-1,4)-glucanase from Streptococcus bovis JB1[J]. Appl. Environ. Microbiol.,1997, (63): 3752-3756.
Erfle, J. D., Teather, R. M., Wood, P. J., Irvin, J. E. Purification and properties of a 1,3-1,4-beta-D-glucanase (lichenase, 1,3-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli [J]. Biochem. J., 1988, (255): 833-841.
Fang, Z. F., Liu, Z. L., Dai, J. J., Qian, H. Y., Qi, Z. L., Ma, L. B., Peng, J. Effects of enzyme addition on the nutritive value of broiler diets containing hulled or dehulled Chinese double-low rapeseed meals [J]. J. Anim. Physiol. Anim., 2008, Nutr.(Ber1).
Fleming, A. B., Tangney, M., Jorgensen, P. L., Diderichsen, B., Priest, F. G Extracellular enzyme synthesis in a sporulation-deficient strain of Bacillus licheniformis [J]. Appl. Environ. Microbiol., 1995,(61): 3775-3780.
Francesch, M., Bernard, K., McNab, J. M. Comparison of two direct bioassays using 3-week-old broilers to measure the metabolisable energy of diets containing cereals high in fibre: differences between true and apparent metabolisable energy values [J]. Br. Poult. Sci., 2002, (43): 580-587.
Genta, F. A., Terra, W. R., Ferreira, C. Action pattern, specificity, lytic activities, and physiological role of five digestive beta-glucanases isolated from Periplaneta Americana [J]. Insect. Biochem. Mol. Biol.,2003,(33): 1085-1097.
Goodey, A. R. The production of heterologous plasma proteins [J]. Trends Biotechnol.,1993, (11) 430-433.
Helmuth, R., Schroeter, A. Molecular typing methods for S. enteritidis [J]. Int. J. Food. Microbiol.,1994, (21): 69-77.
Henry, R. J. A comparison of the non-startch carbohydrates in cereal grains [J]. J. Sci. Food Agric,1985,(36): 1243-1253.
Irvin, J. E., Teather, R. M. Cloning and expression of a Bacteroides succinogenes mixed-linkage beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase) gene in Escherichia coli [J]. Appl.Environ.Microbiol., 1988, (54): 2672-2676.
Iyo, A. H., Forsberg, C. W. A cold-active glucanase from the ruminal bacterium Fibrobacter succinogenes S85 [J]. Appl. Environ. Microbiol., 1999, (65): 995-998.
Jensen, E. B., Carlsen, S. Production of recombinant human growth hormone in Escherichia coli: Expression of different precursors and physiological effects of glucose, acetate, and salts [J].Biotechnol. Bioeng., 1990, (36): 1-11.
Ji, R, Casper, D. P., Brown, P. K., Spangler, D. A., Haydon, K. D., Pettigrew, J. E. Effects of dietary supplementation of an enzyme blend on the ileal and fecal digestibility of nutrients in growing pigs [J].J .Anim. Sci., 2008, (86): 1533-1543.
Kim, J. Y. Overproduction and secretion of Bacillus circulans endo-beta-1,3-1,4-glucanase gene (bg1BC1) in B. subtilis and B. megaterium [J]. Biotechnol. Lett., 2003, (25): 1445-1449.
Kim, K. H., Kim, Y. O., Ko, B. S., Youn, H. J., Lee, D. S. Over-expression of the gene (bg1BC1) from Bacillus circulans encoding an endo-beta-(1-->3),(1-->4)-glucanase useful for the preparation of oligosaccharides from barley beta-glucan [J]. Biotechnol. Lett., 2004, (26): 1749-1755.
Kim, S. A., Cheng, K. J., Liu, J. H. A variant of Orpinomyces joyonii 1,3-1,4-beta-glucanase with increased thermal stability obtained by random mutagenesis and screening [J]. Biosci. Biotechnol.Biochem., 2004, (66): 171-174.
Kleman, G L., Chalmers, J. J., Luli, G W., Strohl, W. R. A predictive and feedback control algorithm maintains a constant glucose concentration in fed-batch fermentations [J]. Appl. Environ. Microbiol.,1991, (57): 910-917.
Lee, S. Y, Chang, H. N. Generation of bacteriophage lambda lysogens by electroporation [J].Biotechniques, 1994, (16): 206-208.
Liu, J. R., Yu, B., Zhao, X., Cheng, K. J. Coexpression of rumen microbial beta-glucanase and xylanase genes in Lactobacillus reuteri [J]. Appl. Microbiol. Biotechnol., 2007, (77): 117-124.
MacDonald, H. L., Neway, J. O. Effects of medium quality on the expression of human interleukin-2 at high cell density in fermentor cultures of Escherichia coli K-12 [J]. Appl. Environ. Microbiol., 1990,(56): 640-645.
Markl, H., Zenneck, C., Dubach, A. C., Ogbonna, J. C. Cultivation of Escherichia coli to high cell densities in a dialysis reactor [J]. Appl. Microbiol. Biotechnol., 1993, (39): 48-52.
Miksch, G., Kleist, S., Friehs, K., Flaschel, E. Overexpression of the phytase from Escherichia coli and its extracellular production in bioreactors [J]. Appl. Microbiol. Biotechnol., 2002, (59): 685-694.
Miksch, G., Neitzel, R., Fiedler, E., Friehs, K., Flaschel, E. Extracellular production of a hybrid beta-glucanase from Bacillus by Escherichia coli under different cultivation conditions in shaking cultures and bioreactors [J]. Appl. Microbiol. Biotechnol., 1997, (47): 120-126.
Miyanishi, N., Inaba, Y., Okuma, H., Imada, C., Watanabe, E. Amperometric determination of laminarin using immobilized beta-1,3-glucanase [J]. Biosens. Bioelectron., 2004, (19) 557-562.
Mizutani, S., Mori, H., Shimizu, S., Sakaguchi, K., Kobayashi, T. Effect of amino acid supplement on cell yield and gene product in Escherichia coli harboring plasmid [J]. Biotechnol. Bioeng.,1986, (28):204-209.
Monreal, J., De, U. F., Villanueva, J. R. Lytic action of beta-(1-3)-glucanase on yeast cells [J]. J.Bacteriol., 1967, (94): 241-244.
Murphy, N., McConnell, D. J., Cantwell, B. A. The DNA sequence of the gene and genetic control sites for the excreted B. subtilis enzyme beta-glucanase [J]. Nucleic Acids Res., 1984, (12): 5355-5367.
Nakashima, K., Azuma, J. Distribution and properties of endo-beta-1,4-glucanase from a lower termite, Coptotermes formosanus (Shiraki) [J]. Biosci. Biotechnol. Biochem., 2000, (64): 1500-1506.
Olsen, O., Borriss, R., Simon, O., Thomsen, K. K. Hybrid Bacillus (1-3,1-4)-beta-glucanases: engineering thermostable enzymes by construction of hybrid genes [J]. Mol. Gen. Genet., 1991, (225):177-185.
Olsen, O., Thomsen, K. K. Processing and secretion of barley (1-3,1-4)-beta-glucanase in yeast[J]. Carlsberg. Res. Commun., 1989, (54): 29-39.
Organization for Economic Cooperation and Development. Biotechnology for Clean Industrial Products and Processes:Towards Industrial Sustainability [C].2004.
Paalme, T., Tiisma, K., Kahru, A., Vanatalu, K., Vilu, R., (1990b). Glucose-Limited fed-batch cultivation of Escherichia coli with computer-controlled fixed growth rate [J]. Biotechnol. Bioeng. 35,312-319.
Pauly, M., Andersen, L. N., Kauppinen, S., Kofod, L. V., York, W. S., Albersheim, P., Darvill, A. A xyloglucan-specific endo-beta-1,4-glucanase from Aspergillus aculeatus: expression cloning in yeast,purification and characterization of the recombinant enzyme [J]. Glycobiology, 1999, (9): 93-100.
Planas, A. Bacterial 1,3-1,4-beta-glucanases: structure, function and protein engineering[J].Biochim.Biophys.Acta [J]., 2000, (1543): 361-382.
Planas, A., Juncosa, M., Lloberas, J., Querol, E. Essential catalytic role of Glu134 in endo-beta-1,3-1,4-D-glucan 4-glucanohydrolase from B. licheniformis as determined by site-directed mutagenesis [J]. FEBS. Lett., 1992, (308): 141-145.
Politz, O., Simon, O., Olsen, O., Borriss, R. Determinants for the enhanced thermostability of hybrid (1-3,1-4)-beta-glucanases [J]. Eur.J. Biochem., 1993, (216): 829-834.
Pugsley, A. P. The complete general secretory pathway in gram-negative bacteria [J]. Microbiol.Rev., 1993, (57): 50-108.
Qiao, J., Dong, B., Li, Y., Zhang, B., Cao, Y. Cloning of a beta-1,3-1,4-Glucanase Gene from Bacillussubtilis MA139 and its Functional Expression in Escherichia coli [K]. Appl. Biochem. Biotechnol., 2008, (3): 67-74.
Randolph, T. W., Marison, I. W., Martens, D. E., von, S. U. Calorimetric control of fed-batch fermentations [J]. Biotechnol.Bioeng., 1990, (36): 678-684.
Ratanakhanokchai, K., Kyu, K. L., Tanticharoen, M. Purification and properties of a xylan-binding endoxylanase from Alkaliphilic bacillus sp. strain K-1[J]. Appl.Environ.Microbiol., 1999, (65):694-697.
Reese, E. T., Maguire, A. Surfactants as stimulants of enzyme production by microorganisms [J].Appl.Microbiol., 1969, (17): 242-245.
Riesenberg, D. High-cell-density cultivation of Escherichia coli [J]. Curr.Opin.Biotechnol., 1991, (2):380-384.
Riesenberg, D., Schulz, V., Knorre, W. A., Pohl, H. D., Korz, D., Sanders, E. A., Ross, A., Deckwer, W.D. High cell density cultivation of Escherichia coli at controlled specific growth rate [J]. J. Biotechnol.,1991,(20): 17-27.
Rose, S. H., van Zyl, W. H. Constitutive expression of the Trichoderma reesei beta-1,4-xylanase gene (xyn2) and the beta-1,4-endoglucanase gene (egl) in Aspergillus niger in molasses and defined glucose media [J]. Appl.Microbiol.Biotechnol., 2002, (58): 461-468.
Royal Belgian Academy Council of Applied Science. Industrial Biotechnology and Sustainable Chemistry [C]. 2004.
Rozzell, J. D. Commercial scale biocatalysis: myths and realities [J]. Bioorg.Med.Chem., 1999, (7):2253-2261.
Sa-Pereira, P., Paveia, H., Costa-Ferreira, M., ires-Barros, M. A new look at xylanases: an overview of purification strategies [J]. Mol.Biotechnol., 2003, (24): 257-281.
Schimming, S., Schwarz, W. H., Staudenbauer, W. L. Properties of a thermoactive beta-1,3-1,4-glucanase (lichenase) from Clostridium thermocellum expressed in Escherichia coli [J]. Biochem Biophys.Res.Commun., 1991, (177): 447-452.
Schimming, S., Schwarz, W. H., Staudenbauer, W. L. Structure of the Clostridium thermocellum gene licB and the encoded beta-1,3-1,4-glucanase. A catalytic region homologous to Bacillus lichenases joined to the reiterated domain of clostridial cellulases [J]. Eur. J. Biochem., 1992, (204): 13-19.
Shibui, T., Munakata, K., Matsumoto, R., Ohta, K., Matsushima, R., Morimoto, Y., Nagahari, K.High-level production and secretion of a mouse-human chimeric Fab fragment with specificity to human carcino embryonic antigen in Escherichia coli [J]. Appl. Microbiol. Biotechnol., 1993, (38):770-775.
Strandberg, L., Enfors, S. O. Factors influencing inclusion body formation in the production of a fused protein in Escherichia coli [J]. Appl. Environ. Microbiol., 1991, (57): 1669-1674.
Studier,F.W.,Moffatt,B.A.Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes[J].J.Mol.Biol.,1986,(189):113-130.
Suzuki,K.,Ojima,T.,Nishita,K.Purification and cDNA cloning of a cellulase from abalone Haliotis discus hannai[J].Eur.J.Biochem.,2003,(270):771-778.
Tabernero,C.,Coll,P.M.,Fernandez-Abalos,J.M.,Perez,P.,Santamaria,R.I.Cloning and DNA sequencing of bgaA,a gene encoding an endo-beta-1,3-1,4-glucanase,from an alkalophilic Bacillus strain(N137)[J].Appl.Environ.Microbiol.,1994,(60):1213-1220.
Teather,R.M.,Erfle,J.D.DNA sequence of a Fibrobacter succinogenes mixed-linkage beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase) gene[J].J.Bacteriol.,1994,(172):3837-3841.
Teng,D.,Wang,J.H.,Fan,Y.,Yang,Y.L.,Tian,Z.G.,Luo,J.,Yang,G.P.,Zhang,F.Cloning of beta-1,3-1,4-glucanase gene from Bacillus licheniformis EGW039(CGMCC 0635) and its expression in Escherichia coli BL21(DE3)[J].Appl.Microbiol.Biotechnol.,2006,(72):705-712.
Vehmaanpera,J.,Steinbora,G.,Hofemeister,J.Genetic manipulation of Bacillus amyloliquefaciens[J].J.Biotechnol.,1991,(19):221-240.
Wen,T.N.,Chen,J.L.,Lee,S.H.,Yang,N.S.,Shyur,L.F.A truncated Fibrobacter succinogenes 1,3-1,4-beta-d-glucanase with improved enzymatic activity and thermotolerance[J].Biochemistry,2001(44):9197-9205.
Woolley,L.C.,James,D.J.,Manning,K.Purification and properties of an endo-beta-1,4-glucanase from strawberry and down-regulation of the corresponding gene,cell[J].Planta,2001,(214):11-21.
Xu,B.,Hellman,U.,Ersson,B.,Janson,J.C.Purification,characterization and amino-acid sequence analysis of a thermostable,low molecular mass endo-beta-1,4-glucanase from blue mussel,Mytilus edulis[J].Eur.J.Biochem,2000,(267):4970-4977.
Yang,S.,Qiaojuan,Y.,Jiang,Z.,Fan,G.,Wang,L.Biochemical characterization of a novel thermostable beta-1,3-1,4-glucanase(lichenase) from Paecilomyces thermophila[J].J.Agric.Food Chem.,2008,(56):5345-5351.
Yang,X.M.,Xu,L.,Eppstein,L.Production of recombinant human interferon-alpha 1 by Escherichia coli using a computer-controlled cultivation process[J].J.Biotechnol.,1992,(23):291-301.
Yee,L.,Blanch,H.W.Defined media optimization for growth of recombinant Escherichia coli X90[J].Biotechnol.Bioeng.,1993a,(41):221-230.
Yee,L.,Blanch,H.W.Recombinant trypsin production in high cell density fed-batch cultures in Escherichia coli[J].Biotechnol.Bioeng.,1993b,(41):781-790.
Yoon,S.K.,Kang,W.K.,Park,T.H.Fed-batch operation of recombinant Escherichia coli containing trp promoter with controlled specific growth rate[J].Biotechnol.Bioeng.,1994,(43):995-999.
Yu,P.,McKinnon,J.J.,Christensen,D.A.Improving the nutritional value of oat hulls for ruminant animals with pretreatment of a multienzyme cocktail:in vitro studies[J].J.Anim.Sci.,2005,(83):1133-1141.
陈耀祖.有机分析[M].北京高等教育出版社,1984.
费笛波,冯观泉,李孝辉.β-葡聚糖酶高产菌株BS9418F的选育及其发酵条件的研究[J].微生物学杂志,2002,22(1):29-31.
宫春波,谢丽源.β-葡聚糖酶及在啤酒工业中的应用[J].广州食品工业科技,2002,(1):58-59.
顾赛红,孙建义,李卫芬.里氏木霉(Trichoderma reesei)产β-葡聚糖酶和木聚糖酶的条件研究[J].浙江大学学报,2003,29(5):545-549.
贺小贤,姜绪林.黑曲霉β-葡聚糖酶产酶培养基的研究[J].食品工业科技,2003,24(12):21-23.
黄兴奇,宋大新,郑伟军.枯草芽孢杆菌β-1,3-1,4-葡聚糖酶基因(bglS)在酵母菌中的克隆与表达[J].微生物学报,1992,32(3):74-76.
李洪利,黄捷,周丽.亲和层析技术在现代蛋白质研究中的应用及新发展[J].海洋湖沼通报,2005,(3):87-89.
李卫芬,孙建义,顾赛红.β-葡聚糖酶的分离纯化和特性研究[J].菌物研究,2001,20(2):178-183.
林宇野.丝状真菌产生β-葡聚糖酶的研究I.GCN平板的建立及产酶菌株的选育[J].福建师范大学学报,1995,11(3):94-101.
刘妙莲,林宇野,王洁,王薇青.β-葡聚糖酶(EC.3.2.1.73)的研究Ⅱ:β-葡聚糖酶的性质及应用[J].食品与发酵工业,1989,(4):1-5.
刘雨田,郭中文.β-葡聚糖与β-葡聚糖酶[J].畜禽业,2000,(7):42-43.
彭萱.β-葡聚糖酶高产菌Bacillus subtilis 9126-6的发酵性能研究[J].南京农业大学学报,1994,17(1):59-64.
任健,杨艳,谢明杰,曹文伟.细菌的β-1,4-内切葡聚糖酶基因在酿酒酵母(Saccharomyces cerevisiae)中的表达[J].微生物学杂志,1997,17(3):30-36.
石家骥,崔福绵.产β-葡聚糖酶菌种T199的选育及发酵条件[J].微生物学报,2001,41(6):750-752.
孙彦等.生物分离工程[M].化学工业出版社,2005.
孙迎庆,曹淑桂,韩四平.β-葡聚糖苷酶的分离纯化和性质研究[J].中国生物化学与分子生物学报,1998,14(1):82-86.
孙玉英,王瑞明,关凤梅.局限曲霉产β-葡聚糖酶发酵特性的研究[J].酿酒科技,2003,118(4):36-37.
孙玉英,王瑞明,刘庆军.β-葡聚糖酶高产菌株的分离筛选及其新菌种初步鉴定[J].酿酒科技,2004,(2):25-27.
谭会泽,冯定远.饲料中的β-葡聚糖和β-葡聚糖酶的应用[J].畜禽业,2005,(3):18-21.
汤兴俊,何国庆.热稳定性β-葡聚糖酶菌种选育及产酶特性[J].农业生物技术学报,2002,10(4):385-389.
王骥,赵辅昆,陈清西.福寿螺(Ampullaria Crossean)外切-β-1,4-葡聚糖酶/外切-β-1,4-木聚糖酶的分离纯化及性质[C].中国生物化学与分子生物学会第八届会员代表大会暨全国学术会议,上海,2001.
王圆,张朝晖,王蓓,朱海东,芦国营.细菌产β-1,3-1,4-葡聚糖酶的结构、功能及应用进展[J].饲料工业,2005,(26):18-20.
郑毅,陈接锋,马石金.黑曲霉FS25产β-葡聚糖酶发酵特性的研究[J].微生物学通报,2001a,28(4):47-50.
郑毅,陈接锋,吴松刚.黑曲霉(Aspergillus niger)产β-葡聚糖酶固态发酵优化的研究[J].食品与发酵工业,2001b,27(12):7-10.
郑毅,段慧明.快速筛选产β-葡聚糖酶曲霉及抗阻遏育种[J].生物技术,2001c,11(1):21-24.