一株产几丁质脱乙酰酶海洋细菌的筛选、鉴定及发酵优化
|
摘要
利用对硝基-N-乙酰苯胺筛选培养基从海州湾海域海泥中筛选获得产几丁质脱乙酰酶细菌MCDA02。经过形态学、生理生化和16S rDNA序列分析初步鉴定该菌株为解角质素微杆菌。通过单因素优化和正交试验优化,得出最优发酵条件。在单因素优化基础上,利用正交试验优化获得菌株MCDA02最优发酵条件为发酵温度25℃,培养基起始p H7.0,装液量50 m L/250 m L,几丁质3%,发酵时间48 h。在此发酵条件下,菌株MCDA02发酵水平达到158.47 U/m L,是优化前的3.2倍。试验结果为菌株MCDA02几丁质脱乙酰酶的进一步研究奠定基础。
A strain MCDA02 producing chitin deacetylase was screened with the media added nitro-N-acetyl aniline from the marinemud of Haizhou bay. Then,it was identified as Microbacterium keratanolyticum through morphological,biochemical characteristics,and16 S r DNA sequence analysis. With single-factor strategy and orthogonal experiments,the fermentation condition was optimized as followings :fermentation period of 48 h,initial p H of 7.0,culture temperature of 30℃,50 m L of liquid medium in a 250 m L flask,and 3% chitin. Underthe optimized conditions,the highest chitin deacetylase activity of strain MCDA02 reached 158.47 U/m L,which was about 3.2 times of thatbefore optimization. The results laid a foundation for further study of the chitin deacetylase from the strain MCDA02.
A strain MCDA02 producing chitin deacetylase was screened with the media added nitro-N-acetyl aniline from the marinemud of Haizhou bay. Then,it was identified as Microbacterium keratanolyticum through morphological,biochemical characteristics,and16 S r DNA sequence analysis. With single-factor strategy and orthogonal experiments,the fermentation condition was optimized as followings :fermentation period of 48 h,initial p H of 7.0,culture temperature of 30℃,50 m L of liquid medium in a 250 m L flask,and 3% chitin. Underthe optimized conditions,the highest chitin deacetylase activity of strain MCDA02 reached 158.47 U/m L,which was about 3.2 times of thatbefore optimization. The results laid a foundation for further study of the chitin deacetylase from the strain MCDA02.
引文
[1]Khor E,Lim LY.Implantable applications of chitin andchitosan[J].Biomaterials,2003,24(13):2339-2349.
[2]Hou J,Han J,Cai L,et al.Characterization of genes for chitincatabolism in Haloferax mediterranei[J].Appl MicrobiolBiotechnol,2014,98(3):1185-1194.
[3]Win N,Stevens W.Shrimp chitin as substrate for fungal chitindeacetylase[J].Appl Microbiol Biotechnol,2001,57(3):334-341.
[4]Bartnicki-Garcia S,Bracker CE,Reyes E,et al.Isolation ofchitosomes from taxonomically diverse fungi and synthesis of chitinmicrofibrils in vitro[J].Experimental Mycology,1978,2(2):173-192.
[5]张菁菁,董文宾,缑敬轩.几丁质脱乙酰酶菌株的筛选鉴定及酶学性质[J].食品工业科技,2012,33(6):244-246.
[6]Songsiriritthigul C,Lapboonrueng S,Pechsrichuang P,et al.Expression and characterization of Bacillus licheniformis chitinase(Chi A),suitable for bioconversion of chitin waste[J].Bioresource Technology,2010,101(11):4096-4103.
[7]Methacanon P,Prasitsilp M,Pothsree T,et al.HeterogeneousN-deacetylation of squid chitin in alkaline solution[J].Carbohydrate Polymers,2003,52(2):119-123.
[8]王皓,吴丽,朱小花.甲壳素脱乙酰酶的研究概况及展望[J].中国生物工程杂志,2015,35(1):96-103.
[9]Wang SL,Peng JH,Liang TW,et al.Purification and characterizationof a chitosanase from Serratia marcescens TKU011[J].Carbohydrate Research,2008,343(8):1316-1323.
[10]Kuroiwa T,Noguchi Y,Nakajima M,et al.Production of chitosanoligosaccharides using chitosanase immobilized on amylose-coatedmagnetic nanoparticles[J].Process Biochemistry,2008,43(1):62-69.
[11]Zhao Y,Nguyen V,Jo G,et al.Purification and characterizationof chitin deacetylase from Mortierella sp.DY-52[J].Journal ofBiotechnology,2008,136:S304.
[12]Chatterjee S,Chatterjee S,Chatterjee BP,et al.Enhancement ofgrowth and chitosan production by Rhizopus oryzae in whey mediumby plant growth hormones[J].International Journal of BiologicalMacromolecules,2008,42(2):120-126.
[13]Cai J,Yang J,Du Y,et al.Purification and characterization of chitindeacetylase from Scopulariopsis brevicaulis[J].CarbohydratePolymers,2006,65(2):211-217.
[14]Li H,Xu H,Li S,et al.Strain improvement and metabolic fluxmodeling of wild-type and mutant Alcaligenes sp.NX-3 forsynthesis of exopolysaccharide welan gum[J].Biotechnology andBioprocess Engineering,2010,15(5):777-784.
[15]Sun Y,Zhang J,Wu S,et al.Statistical optimization for productionof chitin deacetylase from Rhodococcus erythropolis HG05[J].Carbohydrate Polymers,2014,102:649-652.
[16]Wang Y,Xia G,Wu C,et al.Porous chitosan doped with grapheneoxide as highly effective adsorbent for methyl orange and amidoblack 10B[J].Carbohydrate Polymers,2015,115:686-693.
[17]刘建军,赵祥颖,刘丽萍.几丁质脱乙酰酶(CDA)的研究进展[J].山东食品发酵,2007(4):40-46.
[18]Buchana RE,Gibbons NE.Bergey’s Manual of DeterminativeBacteriology[M].8th Edition.Beijing:Science Press,1984:382-533.
[19]应聪萍,王瑶,李永成.一株产几丁质脱乙酰酶丝状真菌的发酵条件优化研究[J].食品工业科技,2016(1):170-174.
[20]贾志娟.北极深海沉积物宏基因组文库构建及一种新的几丁质脱乙酰酶基因鉴定[D].武汉:华中师范大学,2012.
[21]闫晓平,赵丹,郭巍,等.美国白蛾几丁质脱乙酰酶的克隆,表达及酶学性质[J].中国农业科学,2017,50(5):849-858.
[2]Hou J,Han J,Cai L,et al.Characterization of genes for chitincatabolism in Haloferax mediterranei[J].Appl MicrobiolBiotechnol,2014,98(3):1185-1194.
[3]Win N,Stevens W.Shrimp chitin as substrate for fungal chitindeacetylase[J].Appl Microbiol Biotechnol,2001,57(3):334-341.
[4]Bartnicki-Garcia S,Bracker CE,Reyes E,et al.Isolation ofchitosomes from taxonomically diverse fungi and synthesis of chitinmicrofibrils in vitro[J].Experimental Mycology,1978,2(2):173-192.
[5]张菁菁,董文宾,缑敬轩.几丁质脱乙酰酶菌株的筛选鉴定及酶学性质[J].食品工业科技,2012,33(6):244-246.
[6]Songsiriritthigul C,Lapboonrueng S,Pechsrichuang P,et al.Expression and characterization of Bacillus licheniformis chitinase(Chi A),suitable for bioconversion of chitin waste[J].Bioresource Technology,2010,101(11):4096-4103.
[7]Methacanon P,Prasitsilp M,Pothsree T,et al.HeterogeneousN-deacetylation of squid chitin in alkaline solution[J].Carbohydrate Polymers,2003,52(2):119-123.
[8]王皓,吴丽,朱小花.甲壳素脱乙酰酶的研究概况及展望[J].中国生物工程杂志,2015,35(1):96-103.
[9]Wang SL,Peng JH,Liang TW,et al.Purification and characterizationof a chitosanase from Serratia marcescens TKU011[J].Carbohydrate Research,2008,343(8):1316-1323.
[10]Kuroiwa T,Noguchi Y,Nakajima M,et al.Production of chitosanoligosaccharides using chitosanase immobilized on amylose-coatedmagnetic nanoparticles[J].Process Biochemistry,2008,43(1):62-69.
[11]Zhao Y,Nguyen V,Jo G,et al.Purification and characterizationof chitin deacetylase from Mortierella sp.DY-52[J].Journal ofBiotechnology,2008,136:S304.
[12]Chatterjee S,Chatterjee S,Chatterjee BP,et al.Enhancement ofgrowth and chitosan production by Rhizopus oryzae in whey mediumby plant growth hormones[J].International Journal of BiologicalMacromolecules,2008,42(2):120-126.
[13]Cai J,Yang J,Du Y,et al.Purification and characterization of chitindeacetylase from Scopulariopsis brevicaulis[J].CarbohydratePolymers,2006,65(2):211-217.
[14]Li H,Xu H,Li S,et al.Strain improvement and metabolic fluxmodeling of wild-type and mutant Alcaligenes sp.NX-3 forsynthesis of exopolysaccharide welan gum[J].Biotechnology andBioprocess Engineering,2010,15(5):777-784.
[15]Sun Y,Zhang J,Wu S,et al.Statistical optimization for productionof chitin deacetylase from Rhodococcus erythropolis HG05[J].Carbohydrate Polymers,2014,102:649-652.
[16]Wang Y,Xia G,Wu C,et al.Porous chitosan doped with grapheneoxide as highly effective adsorbent for methyl orange and amidoblack 10B[J].Carbohydrate Polymers,2015,115:686-693.
[17]刘建军,赵祥颖,刘丽萍.几丁质脱乙酰酶(CDA)的研究进展[J].山东食品发酵,2007(4):40-46.
[18]Buchana RE,Gibbons NE.Bergey’s Manual of DeterminativeBacteriology[M].8th Edition.Beijing:Science Press,1984:382-533.
[19]应聪萍,王瑶,李永成.一株产几丁质脱乙酰酶丝状真菌的发酵条件优化研究[J].食品工业科技,2016(1):170-174.
[20]贾志娟.北极深海沉积物宏基因组文库构建及一种新的几丁质脱乙酰酶基因鉴定[D].武汉:华中师范大学,2012.
[21]闫晓平,赵丹,郭巍,等.美国白蛾几丁质脱乙酰酶的克隆,表达及酶学性质[J].中国农业科学,2017,50(5):849-858.