产甘露聚糖酶海洋微生物的筛选及酶学性质研究  

Screening of Mannanase-producing Marine Microorganisms and Characterization of the Enzyme

作　　者：李云程[1,2] 林娟[1,2] 梁燕辉[1,2] 叶秀云[1,2] 朱凡[1,2] 

机构地区：[1]福州大学生物科学与工程学院,福州350116 [2]福建省海洋酶工程重点实验室,福州350116

出　　处：《中国食品学报》2015年第12期66-73,共8页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家海洋局海洋公益性行业科研专项(201305015);国家科技部"863计划"课题(2013AA102101)

摘　　要：目的：筛选产甘露聚糖酶的海洋微生物。方法：采用2216E、海水-高氏一号和海水-PDA培养基对采集的微生物进行分离纯化,采用平板透明圈法对菌株进行初筛,并对初筛中HC值较大的菌株通过发酵测酶活复筛;采用16S r DNA序列分析,并结合细胞和菌落形态观察及部分生理生化试验,对菌株进行鉴定;利用硫酸铵盐析法制备粗酶液,研究甘露聚糖酶作用的酶学特性。结果：从台湾海峡采集的海水和海泥样品中,分离得到细菌467株,霉菌145株,放线菌10株,初筛得到64株有透明圈的菌株,复筛得到菌株B555,其酶活力最高为28.18 U/m L,经鉴定为芽孢杆菌（Bacillus sp.）。菌株B555所产甘露聚糖酶的最适反应p H为7.0,且在p H 5.0~10.0时较稳定;最适反应温度为50~55℃,在55℃时半衰期约为150 min;1 mmol/L的金属离子Ni＋、Co2＋、Zn2＋、Mg2＋、Ca2＋、Na＋、K＋、Fe3＋、Ba2对酶活力均有抑制作用。在55℃,p H 7.0条件下,该酶对槐豆胶和魔芋粉有很好的底物特异性,Km值分别为4.7和3.33 mg/m L,Vmax值分别为588.23和476.19μmol/（m L·min）。可应用于魔芋甘露低聚糖的酶法制备。Objective： To screen and identify mannanse-producing marine microorganisms and characterize the enzyme. Method： 2216E, seawater-PDA and seawater-Gause＇s Synthetic Agar Media were used to isolate marine microorganisms. The first screening of mannanase-producing microorganisms was through plate transparent ring method. Furthermore, morphological, physiological, biochemical and molecular biology methods were applied to identify the strains. The crude enzyme was prepared by ammonium sulfate fractionation to analyze theeffects of temperature, pH and metal icons on the enzyme activity. Result： There are 467 bacteria, 145 molds and 10 actinomycetes strains screened from the seawater and mud samples from Taiwan Strait. Strain B555 with the highest enzyme activity of 28.18 U/mL was identified as Bacillus sp. The mannanse produced by strain B555 showed optimal catalytic activity at pH 7.0 and 50-55 %. Its halflife is approximate 150 min under 55 ℃. It was inhibited by Ni＋, Co2＋, Zn2＋, Mg2＋, Ca2＋, Na＋, K＋, Fe3＋, Ba2＋ at the concentration of 1 mmol/L. At 55 ℃ and pH 7.0, the Km and Vmax of the mannanase on locust bean gum were 4.7 mg/ mL and 588.23 pLmol/（mL-min）, and on konjac powder were 3.33 mg/mL and 476.19 μmol/（mL-min）, respectively. Con- clusion： The mannanse will be valuable for Enzymatic preparation of KOGM.

关 键 词：甘露聚糖酶 海洋微生物 筛选 鉴定 酶学性质 

分 类 号：TQ925[轻工技术与工程—发酵工程]