黑曲霉产单宁酶的研究和有机相中固定化黑曲霉细胞生物合成没食子酸丙酯的初探
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摘要
本研究以产单宁酶黑曲霉菌株为出发菌，通过诱变选育和产酶发酵工艺优化来提高菌种产酶能力，然后对其进行细胞固定化，应用于有机相生物合成抗氧化剂没食子酸丙酯(propyl gallate，PG)的研究。固定化细胞有机相生物合成PG的研究在国内外尚属首次，故本研究在学术上和实际应用中均具有重要意义。
在前人研究的基础上，构建出单宁酶酶活力测定方法：以PG为底物，紫外光区270nm为测定波长，通过没食子酸丙酯的浓度与吸光值之间的线性关系计算出底物的减少量，从而测得单宁酶酶活力。该方法快速简便、灵敏可靠，重复性好、可信度高，适用于胞内单宁酶酶活力的测定。
通过微波诱变与化学诱变相结合的复合选育，最终获得高产单宁酶的黑曲霉L_(21)菌株，产酶可达130.6U/ml。在摇瓶发酵条件下对该菌株的产酶工艺参数包括单宁酸浓度、碳源、氮源、碳氮比、初始pH值、转速和培养温度等因素进行研究，而后采用L_(27)(3~(13))正交试验对上述7个主要因素进行优化。在最优发酵条件下，黑曲霉L_(21)菌株可获得单宁酶酶活力225.8U/ml。
通过6种黑曲霉细胞固定化方法的综合对比，最终选用海藻酸钙包埋法。鉴于固定化细胞在菌丝生长和产酶方面均表现出滞后效应，因此对黑曲霉L_(21)固定化细胞产酶发酵条件中海藻酸钠浓度、CaCl_2浓度、凝胶中孢子浓度、胶珠接种量、单宁酸浓度和装液量这6个因素进行研究，并采用L_(18)(3~7)正交试验进行优化。优化后，黑曲霉L_(21)固定化细胞不仅具有与游离细胞相似的前期产酶过程和产酶峰值，而且还表现出比游离细胞更长的平衡期。
最后，将固定化黑曲霉L_(21)细胞应用于有机相生物催化合成PG。通过对反应体系的选择以及合成工艺参数的初步优化，可获得20％左右的PG产率。
This research thesis reported that a producing tannase strain, Aspergillus niger TA was improved by mutation screening and optimization of its fermentation conditions; Then after cell immobilization, Aspergillus niger cells were applied to biosynthesize antioxidant propyl gallate (PG) in organic media. Since study on PG biosynthesis by immobilized cell in organic media was first reported home and abroad, this research work was meaningful both in science and application.
A new method to determine tannase activity was established, in which PG was selected as substrate and 270nm as measure wavelength. According to the linear relationship between the concentration of PG and the corresponding absorption values, tannase activity was determined from the decreasing amount of substrate. The method exhibited not only simplicity and sensitivity, but also repeatability and accuracy. It was only used to determine the activity of intracellular tannase.
A high-yielding tannase mutant strain, Aspergillus niger L2i was screened from its parent strain by combining microwave irradiation with chemical inducing, and its final tannase activity was 130.6 U/ml. Its fermentation parameters in shaking flasks were studied, including concentration of tannic acid, carbon source and nitrogen source with their proportion, initial pH value, agitation speed and incubation temperature, etc. Above seven factors were optimized with L2?(313) orthogonal experiment. Under the optimum conditions, the activity of tannase from Aspergillus niger L2i was 225.8 U/ml.
After compare with five kinds of cell immobilization methods, calcium alginate entrapment was adopted to immobilize Aspergillus niger L2i cells. For improving hysteresis effect in mycelium growth and tannase producing, the fermentation parameters about Aspergillus niger L2i immobilization cells were studied, including concentration of sodium alginate, CaCl2 and spores, inoculation amount of gel beads, concentration of tannic acid and medium volume in shaking flasks, etc. Above six factors were further optimized by LIg(37) orthogonal experiment. Under these optimum conditions, immobilized Aspergillus niger L2i cells exhibited not only enzyme-producing earlier and enzyme peak value similar to that of free cells, but also stationary phase longer than that of free cells.
At last, immobilized Aspergillus niger L21 cells were applied to biosynthesize PG in organic phase. After reaction system was selected and synthesis parameters preliminary was optimized, about 20% production percent of PG was obtained in the organic media.
在前人研究的基础上，构建出单宁酶酶活力测定方法：以PG为底物，紫外光区270nm为测定波长，通过没食子酸丙酯的浓度与吸光值之间的线性关系计算出底物的减少量，从而测得单宁酶酶活力。该方法快速简便、灵敏可靠，重复性好、可信度高，适用于胞内单宁酶酶活力的测定。
通过微波诱变与化学诱变相结合的复合选育，最终获得高产单宁酶的黑曲霉L_(21)菌株，产酶可达130.6U/ml。在摇瓶发酵条件下对该菌株的产酶工艺参数包括单宁酸浓度、碳源、氮源、碳氮比、初始pH值、转速和培养温度等因素进行研究，而后采用L_(27)(3~(13))正交试验对上述7个主要因素进行优化。在最优发酵条件下，黑曲霉L_(21)菌株可获得单宁酶酶活力225.8U/ml。
通过6种黑曲霉细胞固定化方法的综合对比，最终选用海藻酸钙包埋法。鉴于固定化细胞在菌丝生长和产酶方面均表现出滞后效应，因此对黑曲霉L_(21)固定化细胞产酶发酵条件中海藻酸钠浓度、CaCl_2浓度、凝胶中孢子浓度、胶珠接种量、单宁酸浓度和装液量这6个因素进行研究，并采用L_(18)(3~7)正交试验进行优化。优化后，黑曲霉L_(21)固定化细胞不仅具有与游离细胞相似的前期产酶过程和产酶峰值，而且还表现出比游离细胞更长的平衡期。
最后，将固定化黑曲霉L_(21)细胞应用于有机相生物催化合成PG。通过对反应体系的选择以及合成工艺参数的初步优化，可获得20％左右的PG产率。
This research thesis reported that a producing tannase strain, Aspergillus niger TA was improved by mutation screening and optimization of its fermentation conditions; Then after cell immobilization, Aspergillus niger cells were applied to biosynthesize antioxidant propyl gallate (PG) in organic media. Since study on PG biosynthesis by immobilized cell in organic media was first reported home and abroad, this research work was meaningful both in science and application.
A new method to determine tannase activity was established, in which PG was selected as substrate and 270nm as measure wavelength. According to the linear relationship between the concentration of PG and the corresponding absorption values, tannase activity was determined from the decreasing amount of substrate. The method exhibited not only simplicity and sensitivity, but also repeatability and accuracy. It was only used to determine the activity of intracellular tannase.
A high-yielding tannase mutant strain, Aspergillus niger L2i was screened from its parent strain by combining microwave irradiation with chemical inducing, and its final tannase activity was 130.6 U/ml. Its fermentation parameters in shaking flasks were studied, including concentration of tannic acid, carbon source and nitrogen source with their proportion, initial pH value, agitation speed and incubation temperature, etc. Above seven factors were optimized with L2?(313) orthogonal experiment. Under the optimum conditions, the activity of tannase from Aspergillus niger L2i was 225.8 U/ml.
After compare with five kinds of cell immobilization methods, calcium alginate entrapment was adopted to immobilize Aspergillus niger L2i cells. For improving hysteresis effect in mycelium growth and tannase producing, the fermentation parameters about Aspergillus niger L2i immobilization cells were studied, including concentration of sodium alginate, CaCl2 and spores, inoculation amount of gel beads, concentration of tannic acid and medium volume in shaking flasks, etc. Above six factors were further optimized by LIg(37) orthogonal experiment. Under these optimum conditions, immobilized Aspergillus niger L2i cells exhibited not only enzyme-producing earlier and enzyme peak value similar to that of free cells, but also stationary phase longer than that of free cells.
At last, immobilized Aspergillus niger L21 cells were applied to biosynthesize PG in organic phase. After reaction system was selected and synthesis parameters preliminary was optimized, about 20% production percent of PG was obtained in the organic media.
引文
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