摘要
本研究利用扩展青霉脂肪酶(Penicillium Expansum Lipase)对豆油
进行水解反应研究。以开发制备一定组分的甘油酯为目的，制备了具有一
定比例含量的甘油三酯、甘油二酯和甘油单酯。在全面研究脂肪酶的基础
上，重点研究了脂肪酶的测活方法、固定化、水解豆油的反应体系和反应
条件以及反应产物中各甘油酯成分的定量，为豆油的生物利用提供理论依
据。
在脂肪酶活力测定中，三丁酸甘油酯和三油酸甘油酯为国外进口的商
品，脂肪酶活力检测的结果稳定性好于国产的橄榄油。同时也发现利用三
丁酸甘油酯作为底物，检测得到的酶活力是用橄榄油和三油酸甘油酯作为
反应底物的 2～3 倍。一般认为是由于前者空间结构简单，酶作用容易发
生，而另外两个底物由于结构比较复杂，而且受到乳化条件的影响，因此
活力较低；另外用三丁酸甘油酯作为底物，最大的优点在于反应只需振荡
或搅拌，不需加乳化剂就能分散在水中，因此在筛选和连续自动滴定以及
动力学研究中特别适用，但因为丁酸为短链酸，检测出的酶活还需用三油
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吉林大学硕士学位毕业论文 高 贵
酸甘油酯或橄榄油来核实。当利用橄榄油作为底物的时候，由于橄榄油生
产条件的不同、生产批次的差异，容易导致橄榄油的结构组成和杂质发生
一定程度的改变，也造成脂肪酶活力检测的不稳定，因此在实验过程中要
注意尽量使用同一厂家、同一生产批次的橄榄油，这样检测出的脂肪酶活
力才具有可比性和参考价值。利用橄榄油作为脂肪酶活力检测的底物时，
通过选择适当的乳化方法和检测方法可以很大程度上提高酶活检测结果
的重复性和可靠性。
在实验的基础上，对几种常用的脂肪酶活力检测方法进行了比较和研
究，认为采用橄榄油作为底物反应底物，利用正确的乳化方法和检测手段，
不但可以获得较为稳定的脂肪酶活力数据，而且条件简单，容易操作，可
以推广使用。
在脂肪酶的固定化研究上，采用硅藻土作为载体，对脂肪酶进行固定
化，研究了固定化条件对固定化脂肪酶的催化活性以及硅藻土吸附脂肪酶
量的影响。得到最适的固定化反应条件：温度为30～35℃； pH值为7.7；
缓冲液离子浓度在0.01 ～ 0.03mol/L之间；载体与脂肪酶的质量比为8：
1。在以上最适固定化的条件下，每克载体上的蛋白载量为 52mg，酶活为
1301μmol·min-1·g-1。以此固定化酶水解豆油，使用三次后酶活仍达到
1137μmol·min-1·g-1。可以认为硅藻土固定化脂肪酶在催化豆油水解反
应中非常可行。
在产物稳定性分析上，反应产物中甘油二酯、甘油单酯、甘油和脂肪
酸是混合在一起的，其中甘油二酯、甘油单酯和脂肪酸是溶到油相的，而
甘油是溶在水相中，借助一定的物理、化学手段可分离得到目的产物。脂
肪酶在反应体系中溶于水中，且酶中有一定含量的培养基成分，若反应产
物分离不彻底，在产物保存中极易造成酯类的进一步水解和酸败，改变产
物的组成，固定化酶由于固定化时非酶成分进行了减除，且酶吸附在载体
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吉林大学硕士学位毕业论文
高 贵
上，比重增强，反应结束后主要沉积在水相中，虽有少量脱落，但经过离
心、灭活处理，产物稳定性非常明显。
在水解产物成分的动态分析上，利用柱层析法、薄层层析法和高效液
相色谱联用质谱对脂肪酶水解豆油产物中甘油酯的成分进行了分析，共鉴
定出9种组分，根据质谱确定了它们的分子式，均为脂肪酸甘油酯。测定
了豆油水解样中各甘油酯的相对含量，采用薄层层析定性分析判断产物中
各甘油酯和脂肪酸的多少，硅胶柱分离计算出各种甘油酯和脂肪酸的含
量；借助液—质联用分析手段确定豆油水解物中的各组分及相对含量，并
对应酸值测定，根据酸值的大小与水解产物中各种成分的相对含量关系建
立了判定脂肪酶水解豆油反应进程的方法。实现了用测酸值的方法估算反
应产物中各物质的相对含量，作为定量指标，手段可行，为酶法催化有机
相反应的时时监控提供依据，它同脂肪酶活力检测方法一样具有理论与实
际意义。
The hydrolysis of soybean oil catalyzed by Penicillium Expansum Lipase
was studied for preparation of natural anti-bubbing agent in this work. The assay
of lipase activity was improved and the immobilization method of lipase was
confirmed. Furthermore, the assay of quantitive determination the component of
soybean oil was studied. Based on the systemic research above, the reaction
conditions of the hydrolysis of soybean oil were screened out. All the work will
provide the theory foundation for the exploration of soybean oil.
In assays of determination lipase activity, the stability of the data with
tributyrin and triolein (obtained from Sigma) as substrates was better than that of
the data with olive oil (obtained from China) as substrate. It could be found that
the lipase activity determined with triolein as substrate was about 2-3 times as
much as the results with olive oil and tributyrin as substrates.
Compared several frequently-used assays of determination lipase activity,
it’s found that pre-emulsification of the substrate and selection of a correct
determination method could give a stable lipase activity data. The method of
determination lipase activity improved in this work could be applied widely
because the assay conditions were simple and the operation was easy.
In immobilization research of lipase, diatomaceous earth was used as
protein carrier. The results of the optimal conditions for lipase immobilization
were that pH is 7.7; temperature range is from 30℃ to 35℃; ionic strength
range is from 0.01mol/Lto 0.03mol/L; the optimal ratio of diatomite and lipase is
8:1. Under the optimal conditions, the protein amount absorbed on the
diatomaceous earth was 52mg/g and the lipase activity was 1301 μ
mol·min-1·g-1.The immobilized lipase activity in hydrolysis of soybean oil was
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吉林大学硕士学位毕业论文 高 贵
remained 1137μmol·min-1·g-1 even after 3 times reused.
In hydrolysis of soybean oil, the optimal conditions were screened out. The
reaction system was 1:2(oil/buffer=v/v); the lipase was 0.25g/20mL（160u/mL）
in buffer，the reaction time was 2.5h and the stir speed was 160rpm.
In this work, the analysis method of hydrolysis product component has also
been improved. TLC was used to segregate the different glyceride and fatty acid
in hydrolysis product components and to analyze the different glyceride and fatty
acid qualitatively. The silica gel column was used to obtain different components.
The different components in hydrolysis product were distinguished by
HPLC-MS. The acid number of hydrolysis product has also been determined.
Through comparison and analysis, it could be found that the relative content of
different components in hydrolysis product possessed some certain relativity with
the acid number. So the acid number could be used as a indicator to estimate the
relative content of different components in hydrolysis product.
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