海藻酸钙固定化面包酵母在有机相中催化苯甲醛和苯乙酮的还原
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摘要
近来，有机相中的细胞催化吸引了越来越多的研究者的关注，已成为生物技术领域一个新的研究热点。
本论文以苯甲醛为底物，对面包酵母与其它8种酵母在水相中和有机相中的还原能力进行了比较，探索了活化培养基组成、活化时间、海藻酸钠浓度、pH值、溶剂种类、共底物种类、初始底物浓度、摇床转速和温度对海藻酸钙固定化面包酵母在有机相中的还原能力的影响，也考察了以6小时和24小时为利用周期时海藻酸钙固定化面包酵母的重复利用的情况。
在所考察的9种酵母中，除假丝酵母2#外，面包酵母和其它8种酵母在水相和有机相中都具有还原能力，其中以假丝酵母1#和面包酵母为最强。
活化培养基以既含氮源又含碳源的YPD培养基为最佳，活化时间以1天和4天最为合适。固定化面包酵母在不进行预先活化的情况下在有机相中也具有还原能力。无论是活化酵母还是未活化酵母，产物浓度都随酵母量的增加而增大，并且在酵母量少时增加的幅度较大。
海藻酸钠浓度在1-5％之间都可用于固定化面包酵母，其中浓度为3％时制备的固定化面包酵母催化性能最佳。在不同pH的缓冲液制备的固定化面包酵母中，以pH10为最佳。
溶剂种类对固定化面包酵母的还原能力的影响极大，一般来说细胞活性随LogP值的增大而增大。在所考察的共底物中，大部分醇类物质并不能促进产物浓度的提高，反而有降低产物浓度的趋势。只有高级醇在己烷中才有利于提高产物浓度。
初始底物浓度对固定化面包酵母的催化性能影响较大，当初始底物浓
浙江大学硕士论文 摘要
度从 660 mM增大到 990 mM时，使得凝胶球内底物浓度从 41．2 InM增
大到 84．3 mM，由此造成凝胶球内的产物浓度有较大的下降。
产物浓度随转速增大几乎成线性增大。以已烷为溶剂时，最佳温度为
35℃，以乙酸丁酯为溶剂时，最佳温度为30℃。
以24小时为利用周期时，海藻酸钙固定化面包酵母只能重复利用两
次，以6小时为利用周期时，可重复利用6次以上。
以苯乙酮为底物时，海藻酸钠浓度、溶剂种类、共底物种类和初始底
物浓度对海藻酸钙固定化面包酵母在有机相中还原能力的影响相似。但最
佳 pH与以苯甲醛为底物时稍有不同，为 pH 4和 pH。
Recently, catalysis by the whole cells in organic solvent has attracted attention of more and more researchers and has become a new hotspot in the field of biotechnology.
In this thesis, the reduction ability of benzaldehyde by baker's yeast and another 8 strains of yeast in water and in organic solvent was studied, the effects of medium constitute, cultivation time, sodium alginate concentration, pH, organic solvent, co-substrate, initial concentration of substrate, rev of orbital shake and temperature on the reduction by Calcium-Alginate-immobilized baker's yeast in organic solvent were studied, and the reuse of Calcium-Alginate-immobilized baker's yeast with 6 and 24 hours as a period was tested. The substrate of all the above studies is benzaldehyde.
Except for Cancida 2". cultivated baker's yeast and another 7 strains of yeast all can reduce benzaldehyde to benzyl alcohol in water and in organic solvent.
When baker's yeast cultivated in YPD medium for 1 or 4 days, the ability of reduction of baker's yeast is highest. Baker's yeast without precultivation also has the ability of reduction. Product concentration would be increased with increase of quantity of cells, and product concentration increase more quickly when quantity of yeast is little.
Sodium alginate solution whose concentration is within 1-5% can be used to immobilize baker's yeast, and the immobilized baker's yeast made in alginate concentration of 3% had higher ability of reduction than that in other
concentration. When pH of the buffer used to dissolve yeast is 10, the highest product concentration could be obtained.
Organic solvent would affect on the ability of reduction by immobilized baker's yeast. In general, the higher LogP of solvent is, the higher the ability of reduction the yeast has. Within the so-substrate studied, a majority of alcohols could not increase the concentration of product. Only with higher alcohols as co-substrate in hexane, it could get higher product concentration.
When the initial substrate concentration increase from 660 mM to 990 mM, the substrate concentration within bead varies from 41.2 mM to 84.3 mM. With the increase of substrate concentration, the product concentration in solvent and within bead all decrease greatly.
The product concentration increases almost linearly with rev of orbital shake increasing. The optimum temperature is 35℃ when solvent is hexane and 30℃ when solvent is butylacetate.
The immobilized yeast could be reused for 2 times with 24 hours as a culture period and for more than 6 times with 6 hours as a culture period.
When the substrate is acetophenon, the effect of concentration of alginate. sorts of organic solvent, sorts of co-substrate and initial concentration of substrate on the reduction of Calcium-Alginate-immobilized baker's yeast in organic solvent is similar to the results as the substrate was benzaldehy. But the best pH is pH 4 and pH 10, perhaps because the enzymes participate in the reduction of benzaldehyde and acetophenon are slightly different.
本论文以苯甲醛为底物，对面包酵母与其它8种酵母在水相中和有机相中的还原能力进行了比较，探索了活化培养基组成、活化时间、海藻酸钠浓度、pH值、溶剂种类、共底物种类、初始底物浓度、摇床转速和温度对海藻酸钙固定化面包酵母在有机相中的还原能力的影响，也考察了以6小时和24小时为利用周期时海藻酸钙固定化面包酵母的重复利用的情况。
在所考察的9种酵母中，除假丝酵母2#外，面包酵母和其它8种酵母在水相和有机相中都具有还原能力，其中以假丝酵母1#和面包酵母为最强。
活化培养基以既含氮源又含碳源的YPD培养基为最佳，活化时间以1天和4天最为合适。固定化面包酵母在不进行预先活化的情况下在有机相中也具有还原能力。无论是活化酵母还是未活化酵母，产物浓度都随酵母量的增加而增大，并且在酵母量少时增加的幅度较大。
海藻酸钠浓度在1-5％之间都可用于固定化面包酵母，其中浓度为3％时制备的固定化面包酵母催化性能最佳。在不同pH的缓冲液制备的固定化面包酵母中，以pH10为最佳。
溶剂种类对固定化面包酵母的还原能力的影响极大，一般来说细胞活性随LogP值的增大而增大。在所考察的共底物中，大部分醇类物质并不能促进产物浓度的提高，反而有降低产物浓度的趋势。只有高级醇在己烷中才有利于提高产物浓度。
初始底物浓度对固定化面包酵母的催化性能影响较大，当初始底物浓
浙江大学硕士论文 摘要
度从 660 mM增大到 990 mM时，使得凝胶球内底物浓度从 41．2 InM增
大到 84．3 mM，由此造成凝胶球内的产物浓度有较大的下降。
产物浓度随转速增大几乎成线性增大。以已烷为溶剂时，最佳温度为
35℃，以乙酸丁酯为溶剂时，最佳温度为30℃。
以24小时为利用周期时，海藻酸钙固定化面包酵母只能重复利用两
次，以6小时为利用周期时，可重复利用6次以上。
以苯乙酮为底物时，海藻酸钠浓度、溶剂种类、共底物种类和初始底
物浓度对海藻酸钙固定化面包酵母在有机相中还原能力的影响相似。但最
佳 pH与以苯甲醛为底物时稍有不同，为 pH 4和 pH。
Recently, catalysis by the whole cells in organic solvent has attracted attention of more and more researchers and has become a new hotspot in the field of biotechnology.
In this thesis, the reduction ability of benzaldehyde by baker's yeast and another 8 strains of yeast in water and in organic solvent was studied, the effects of medium constitute, cultivation time, sodium alginate concentration, pH, organic solvent, co-substrate, initial concentration of substrate, rev of orbital shake and temperature on the reduction by Calcium-Alginate-immobilized baker's yeast in organic solvent were studied, and the reuse of Calcium-Alginate-immobilized baker's yeast with 6 and 24 hours as a period was tested. The substrate of all the above studies is benzaldehyde.
Except for Cancida 2". cultivated baker's yeast and another 7 strains of yeast all can reduce benzaldehyde to benzyl alcohol in water and in organic solvent.
When baker's yeast cultivated in YPD medium for 1 or 4 days, the ability of reduction of baker's yeast is highest. Baker's yeast without precultivation also has the ability of reduction. Product concentration would be increased with increase of quantity of cells, and product concentration increase more quickly when quantity of yeast is little.
Sodium alginate solution whose concentration is within 1-5% can be used to immobilize baker's yeast, and the immobilized baker's yeast made in alginate concentration of 3% had higher ability of reduction than that in other
concentration. When pH of the buffer used to dissolve yeast is 10, the highest product concentration could be obtained.
Organic solvent would affect on the ability of reduction by immobilized baker's yeast. In general, the higher LogP of solvent is, the higher the ability of reduction the yeast has. Within the so-substrate studied, a majority of alcohols could not increase the concentration of product. Only with higher alcohols as co-substrate in hexane, it could get higher product concentration.
When the initial substrate concentration increase from 660 mM to 990 mM, the substrate concentration within bead varies from 41.2 mM to 84.3 mM. With the increase of substrate concentration, the product concentration in solvent and within bead all decrease greatly.
The product concentration increases almost linearly with rev of orbital shake increasing. The optimum temperature is 35℃ when solvent is hexane and 30℃ when solvent is butylacetate.
The immobilized yeast could be reused for 2 times with 24 hours as a culture period and for more than 6 times with 6 hours as a culture period.
When the substrate is acetophenon, the effect of concentration of alginate. sorts of organic solvent, sorts of co-substrate and initial concentration of substrate on the reduction of Calcium-Alginate-immobilized baker's yeast in organic solvent is similar to the results as the substrate was benzaldehy. But the best pH is pH 4 and pH 10, perhaps because the enzymes participate in the reduction of benzaldehyde and acetophenon are slightly different.
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