【作者】 李兴峰；

【导师】 贾英民；

【作者基本信息】 河北农业大学 ， 农产品加工及贮藏工程， 2004， 硕士

【摘要】 本论文系统研究了产乳糖酶微生物的分离筛选、发酵工艺和酶学性质，发现： （1）通过产酶菌株的分离筛选，建立了一套快速、准确的乳糖酶高产菌株筛选方法：以ONPG作为产酶指示物，采用0.2ml的离心管为初筛工具；确定了合理、客观的综合考察酶活和实际应用中的主要酶学性质两个方面，以OHA；LHA、LHA／OHA；作用温度及热稳定性、作用pH及pH稳定性多个指标组成的三级复筛方法。提出了一种快速、客观的乳糖酶活力测定程序：引入修正系数OHA／LHA，以ONPG为表观指标，除以修正系数，来代替LHA。这样，既能提高测定速度，又能真实反映乳糖水解能力。最终，采用ONPC作为产酶指示物，从保定新天香乳品公司生产车间附近的土壤中筛得一高产菌株，发酵液中OHA=2.24 U／ml、LHA=1.71 U／ml、OHA／LHA=1.31、最适温度70℃、最适pH4.0、热稳定性好（70℃，10min）。经菌落形态观察、分生孢子头显微观察，初步鉴定为黑曲霉，是生产食品酶制剂的安全菌株。将D_2-26菌株编号为Aspergillus niger D_2-26。 （2）发酵工艺优化表明，黑曲霉D_2-26菌株产酶优化的培养基为：果胶0.3％、玉米浆2％、豆粕粉1.5％、硝酸铵0.2％、K₂HPO₄ 1％、NP-5 0.3％。优化发酵条件为：接种量为孢子浓度10⁴／ml，发酵培养基的起始pH为5.5，装液量为30／250mL，摇床30℃，140rpm，培养7天。经过发酵工艺优化，黑曲霉D_2-26菌株产酶活力可达6.53 U／ml。 （3）酶学性质研究发现，D_2-26菌株乳糖酶的基本酶学性质为：最适作用温度为70℃，作用温度较宽，70℃处理10min，酶活力基本无损失，属于高温乳糖酶；最适作用pH为4，作用pH较宽（2.5—6.5），酶的pH稳定范围在2.5—7之间；K⁺、Ca²⁺、Zn²⁺、Mg²⁺、Pb²⁺、EDTA对乳糖酶的水解无抑制或激活作用；Fe²⁺、Cu²⁺、Mn²⁺对水解有不同程度的的激活，其中，Fe²⁺在低浓度下有较高激活作用，Cu²⁺、Mn²⁺在高浓度下激活较明显。更多还原

【Abstract】 Systematic study on the screening of lactase-yielding strains, liquid fermentation of lactase high producing strain D2-26 and enzyme properties were carried out in this paper. The main results were as following:1. We improved tool of primary screening and found a reasonable method of repeated screening: using 0.2ml epondolf tube as new tool of primary screening; method of the second screening has three standards, including OHA, LHA and primary enzyme properties. The feasible assay procedure of lactase activity was put forward, namely using OHA and OHA/LHA represent LHA .The high-yielding strain D2-26 was screened by using ONPG as indicator from soil samples around production plant of Baoding Tensun Dairy CO.LTD, D2-26 showed relative high enzymatic activity in liquid fermentation, the OHA, LHA, OHA/LHA was 2.24U/ml, 1.71U/ml, 1.31 respectively. Optimal temperature was 70 ℃, optimal pH was 4.0. The enzyme had a good thermostability (70℃,10min). After primary identification the lactase-producing organism was identified as Aspergillus niger and safety strain.2. Optimized culture medium in liquid fermentation were: pectin 0.5%, corn liquor 2%, soybean powder 1.5%, N^NOs 0.2%, K2HPO4 1%, NP-5 0.3%. Optimized culture condition were:104 spore per mil inoculum quantity, pH5.5, 30癈,140rpm, 30/250mL. After optimum of liquid fermentation, the maximum enzyme activity of D2.26 was 6.53 U/ml.3.The test of crude enzyme indicated that optimal temperature of D2.26 lactase was 70 ℃, reaction temperature was relatively extensive. The lactase had good thermostability up to 70℃ and was higher temperature lactase. Optimal pH was 4.0 and it was stable at pH 2.5-7 at room temperature. The metal irons K+, Ca2+, Zn2+, Mg2+, Pb2+, EDTA have no inhibition on lactase .Fe2+, Cu2+, Mn2+ had different activation,Fe2+ has high activation at low density while Cu2+, Mn2+ have high activation at high density.更多还原