产壳聚糖酶土壤微生物的筛选鉴定及发酵条件优化
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摘要
对浙江舟山虾蟹养殖场附近的土壤进行针对性地采样,通过壳聚糖平板初筛、摇瓶复筛获得一株具有产壳聚糖酶活性的菌株,编号为ZJOU-AC1。通过形态观察、ITS全序列分析及系统发育进化树的构建,确定ZJOU-AC1为鹿皮色曲霉(Aspergillus cervinus)。对其产酶条件进行初步优化,经优化后的培养基成分为胶体壳聚糖1.5%,(NH_4)_2SO_4 0.4%,KH_2PO_4 0.2%,MgSO_4·7H_2O 0.05%。最适发酵周期为96 h。ZJOU-AC1产酶活力由2.05 U/m L提高到4.85 U/m L,与优化前相比提高了2.36倍。
In order to screen desirable strains, we take sample of soil from areas adjacent to manufacturing locations of those plants which culture shrimps and crabs in Zhoushan. In this study,we had isolated a high Chitosanase-producing fungus ZJOU-AC1 from the soil by primary screening of plate specula and shaking cultivation. Based on standard morphological,physiological properties,ITS r DNA sequence and the construction of phylogenetic tree,the fungus ZJOU-AC1 was identified as Aspergillus cervinus. We also optimized the fermentation conditions to increase its productivity of chitosanase. The optimum medium components for chitosanase production by fungus ZJOU-AC1 were determined and listed as follows :colloid chitosan1.5%,(NH_4)_2SO_4 0.4%,KH_2PO_4 0.2%,MgSO_4·7H_2O 0.05%. The optimum fermentation period is 96 h. The maximal chitosanase activity could reach 4.85 U/m L when ZJOU-AC1 was incubated in flask under the optimum cultural conditions. The activity increased by 2.36 times in comparison with initial activity of 2.05 U/m L.
In order to screen desirable strains, we take sample of soil from areas adjacent to manufacturing locations of those plants which culture shrimps and crabs in Zhoushan. In this study,we had isolated a high Chitosanase-producing fungus ZJOU-AC1 from the soil by primary screening of plate specula and shaking cultivation. Based on standard morphological,physiological properties,ITS r DNA sequence and the construction of phylogenetic tree,the fungus ZJOU-AC1 was identified as Aspergillus cervinus. We also optimized the fermentation conditions to increase its productivity of chitosanase. The optimum medium components for chitosanase production by fungus ZJOU-AC1 were determined and listed as follows :colloid chitosan1.5%,(NH_4)_2SO_4 0.4%,KH_2PO_4 0.2%,MgSO_4·7H_2O 0.05%. The optimum fermentation period is 96 h. The maximal chitosanase activity could reach 4.85 U/m L when ZJOU-AC1 was incubated in flask under the optimum cultural conditions. The activity increased by 2.36 times in comparison with initial activity of 2.05 U/m L.
引文
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[21]ZHU X F,ZHOU Y,FENG J L.Analysis of both chitinase and chitosanase produced by Sphingomonas sp.CJ-5[J].Journal of Zhejiang University,2007,8(11):831-838.
[22]石会会,高艳艳,王秀英,等.产壳聚糖酶菌株的筛选、鉴定及发酵条件优化[J].食品工业,2014(2):54-57.
[23]黄惠莉,苏顺发,许送彬,等.海洋青霉菌壳聚糖酶的分离纯化与性能[J].化学工程,2011,39(10):1-5.
[24]蔡婀娜,黄惠莉,黃双燕.海洋烟曲霉菌产壳聚糖酶的酶学性质初探[J].暨南大学学报(自然科学与医学版),2011,32(5):509-512.
[25]阎贺静,周念波,涂邵勇,等.壳聚糖酶生产菌筛选、鉴定及其酶学性质[J].广东农业科学,2012(24):161-164.
[2]尹爱国,郭春科,龙帅.产壳聚糖酶菌株的筛选及其产酶条件研究[J].甘肃科学学报,2012(4):45-48.
[3]刘杰,许晶,王能飞.一株高产壳聚糖酶细菌的筛选及发酵条件优化[J].湖北农业科学,2014,53(3):517-521.
[4]狄文伟.壳寡糖在蔬菜生产上的应用[J].北方园艺,2016(8):54-55.
[5]罗泉清,高海琪,陈媛,等.壳寡糖修饰的纳米银作为抗菌材料的合成与制备[J].高分子材料科学与工程,2016,32(2):13-18.
[6]陈小娥.曲霉产壳聚糖酶酶学性质及作用机理研究[D].江苏无锡:江南大学,2004.
[7]戴秋萍,方和娣,陶诗,等.二株产壳聚糖酶海洋微生物的筛选和鉴定[J].海洋环境科学,2012,31(3):385-390.
[8]戴芸,朱旭芬.微生物壳聚糖酶的研究概况[J].浙江大学学报(农业与生命科学版),2004,30(2):114-121.
[9]孙玉英,张继泉,王淑军.壳聚糖酶高产菌株的筛选、鉴定及发酵条件的研究[J].食品科学,2009,30(15):164-168.
[10]聂明,魏新林,万佳蓉,等.土壤中选育产壳聚糖酶菌株的研究[J].生物技术,2005,15(5):16-18.
[11]魏景超.真菌鉴定手册[M].上海:上海科学技术出版社,1979.
[12]蔡婉玲,田宝玉,郭菁,等.蛋白酶产生菌的筛选和紫外诱变育种[J].生物技术,2011,21(1):73-76.
[13]曹秀明.壳寡糖及衍生物抗肿瘤作用、免疫调节作用及其机制的研究[D].山东青岛:中国海洋大学,2010.
[14]陈红霞.壳寡糖增强免疫及抗肿瘤活性的研究[D].武汉:华中农业大学,2013.
[15]SUYG,DUYZ,YUAN H,et al.Redox-sensitive polymer-drug conjugate of doxorubicin conjugated stearic acid-g-chitosan oligosaccharide polymeric micelles for cancer therapy[A].Chinese Pharmaceutical Association(CPA)[C].Beijing:Chinese Pharmaceutical Association(CPA),2013.
[16]HUANG G Q,SIN J P,WANG D F,et al.Chitosan oligosaccharide-Ca complex accelerates the depuration of cadmium from chlamys ferrari[J].Journal of Ocean University of China,2012,11(2):219-226.
[17]康立新,周玉玲,马立新.壳聚糖酶的克隆表达与壳寡糖的制备分析[J].生物技术,2012,22(2):20-23.
[18]冯志彬,薛钰,陈国忠,等.1株产壳聚糖酶细菌的分离、鉴定和发酵条件优化[J].食品科学,2016,37(19):171-176.
[19]ANDO A,SAITO A,ARAIS,et al.Molecular characterization of a novel family-46 chitosanase from Pseudomonas sp.A-01[J].Biosci Biotechnol Biochem,2008,72(8):2074-2081.
[20]戴秋萍.产壳聚糖酶海洋微生物菌株的筛选鉴定、发酵产酶条件和酶解产物性能研究[D].杭州:浙江大学,2012.
[21]ZHU X F,ZHOU Y,FENG J L.Analysis of both chitinase and chitosanase produced by Sphingomonas sp.CJ-5[J].Journal of Zhejiang University,2007,8(11):831-838.
[22]石会会,高艳艳,王秀英,等.产壳聚糖酶菌株的筛选、鉴定及发酵条件优化[J].食品工业,2014(2):54-57.
[23]黄惠莉,苏顺发,许送彬,等.海洋青霉菌壳聚糖酶的分离纯化与性能[J].化学工程,2011,39(10):1-5.
[24]蔡婀娜,黄惠莉,黃双燕.海洋烟曲霉菌产壳聚糖酶的酶学性质初探[J].暨南大学学报(自然科学与医学版),2011,32(5):509-512.
[25]阎贺静,周念波,涂邵勇,等.壳聚糖酶生产菌筛选、鉴定及其酶学性质[J].广东农业科学,2012(24):161-164.