D-海因酶产生菌的筛选、性质及固定化研究
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摘要
本文以DL-对羟基苯海因(DL-p-HPH)为唯一氮源,从自然资源中进行D-海因酶产生菌株的筛选。利用双层平板法和微孔快速筛选法,共筛得三株有活力的菌株,对其中活力较好的一株进行了深入研究。
对上述菌株进行培养后,加入底物进行生物转化,转化液用TLC法和HPLC法液进行了初步鉴定。结果显示转化液中在与产物对羟基苯甘氨酸(D-p-HPG)标样相同的Rf值处,有对应的斑点出现。后将转化产物用NANO2进行处理后进一步提纯分离,使用了732型阳离子树脂,上柱的流速为2ml／min,而后用0.2mol／L的NaOH溶液以0.4mL／min的速度进行洗脱。将洗脱得到的溶液浓缩结晶,得到纯品后运用HPLC、旋光度和红外进行了测定,确定了产物为D-p-HPG。
在上述研究的基础上,对该菌种进行了16S rRNA鉴定,鉴定结果为革兰氏阴性菌,产碱杆菌属(Alcaligenes sp.)。
通过对该菌的培养条件进行优化,确定其最适的培养条件为：最适碳源为甘油,氮源为酵母膏,最适生长温度为28-30℃,最佳起始pH值为pH7.0,最佳装样体积为50mL／250mL的三角瓶。产酶细胞的生物量在培养了48小时时达到了最高,但酶活在培养了大约24小时时为最高。
此后又对该菌进行了固定化,最终选择PVA-硼酸法,并确定0.1 g／mL的PVA浓度为固定化该细胞的最适浓度,固定化过程中,在饱和的硼酸溶液中加入0.01 g／mL的Mg2+具有较好的效果。固定化后的细胞对pH的耐受范围增大；连续反应7批,保持相对酶活80%以上；储存稳定性较好,4℃下储存一个月,相对酶活仍然在80%左右；固定化细胞的最适反应温度从固定化前的37℃上升到47-50℃,在最适温度下,相对酶活为游离细胞的3倍。采用本文的固定化方法不但提高了细胞的稳定性,同时能较大幅度地提高酶的活力,所以这种方法为以后工业化过程的研究奠定了良好的基础。
The new strains with D-hydantoinase activity have been screeded by use of DL-p-hydroxy-phenylhydantoin (DL-p-HPH) as a single nitrogen source from nature. Totally, three strains which had higher enzyme activity were obtained by double-plate and rapid micro-bacteria methods. The one showed highest enzyme activity was selected to do further study.
Next, the strain screened above was fermented, and the preliminary fermentation products were identified by TLC and HPLC. Compared with the standard samples of D-p-hydroxyphenyIgIycine (D-p-HPG), a conversion product had the same Rf value as the standard sample of D-p-HPG. Then transformation products were purified after treated with NaNO2 by ion-exchange resin 732 in the following conditions:the flow rate of sample solution is about 2ml/min, and the eluted solution was 0.2 mol/L NaOH at a rate of 0.4 ml/min. The effluent was concentrated and crystallized. After that the specific rotation and IR spectrum of the pure product was measured. Then we could confirm the product is D-p-HPG
On the basis above, the bacteria strain was identified as Alcaligenes sp. based on 16S rRNA result.
Culture conditions of the strain were optimized and the optimal cultivation conditions:the optimum carbon source was glycerol, the nitrogen source was the yeast extract, optimum growth temperature was 28-30℃, the best primary pH was pH 7.0. The best culture volume was 50 ml/250ml flask. The cell biomass reached to the highest when cultured for 48 hours, but the enzyme activity had reached to the highest after 24 hours.
Afterwards, bacteria were immobilized by PVA-boric acid method. The optimal concentration of PVA for the immobilization was 0.1 g/ml. During the immobilization process, adding Mg2+ in boric acid solution had better result than adding other cations (Na+、Zn2+、Ca2+). The optimal pH was 8.0 for both of immobilized and free cells. The variation of pH (from 6.5 to 9.5) has little effect on activity of immobilized cells while had dramatic effect on free cells. The immobilized cells can tolerate higher temperature than free cells. The optimal temperature of the immobilized cells was 47℃compared with 37℃of the free cells. Under such conditions, the activity of the immobilized cells can reach about 3 times of the free cells. After 7 batches of reaction, the remained activity of the immobilized cells was still more than 80% of initial activity. After 30 days storage at 4℃the immobilized cells kept more than 80% of its initial activity. For free cells, it kept only 30% of initial activity. In short, the immobilized cells of Alcaligene sp. prepared by PVA entrapment has better pH and temperature tolerant, higher activity and stability compared to free cell and has potential application in the future.
对上述菌株进行培养后,加入底物进行生物转化,转化液用TLC法和HPLC法液进行了初步鉴定。结果显示转化液中在与产物对羟基苯甘氨酸(D-p-HPG)标样相同的Rf值处,有对应的斑点出现。后将转化产物用NANO2进行处理后进一步提纯分离,使用了732型阳离子树脂,上柱的流速为2ml／min,而后用0.2mol／L的NaOH溶液以0.4mL／min的速度进行洗脱。将洗脱得到的溶液浓缩结晶,得到纯品后运用HPLC、旋光度和红外进行了测定,确定了产物为D-p-HPG。
在上述研究的基础上,对该菌种进行了16S rRNA鉴定,鉴定结果为革兰氏阴性菌,产碱杆菌属(Alcaligenes sp.)。
通过对该菌的培养条件进行优化,确定其最适的培养条件为：最适碳源为甘油,氮源为酵母膏,最适生长温度为28-30℃,最佳起始pH值为pH7.0,最佳装样体积为50mL／250mL的三角瓶。产酶细胞的生物量在培养了48小时时达到了最高,但酶活在培养了大约24小时时为最高。
此后又对该菌进行了固定化,最终选择PVA-硼酸法,并确定0.1 g／mL的PVA浓度为固定化该细胞的最适浓度,固定化过程中,在饱和的硼酸溶液中加入0.01 g／mL的Mg2+具有较好的效果。固定化后的细胞对pH的耐受范围增大；连续反应7批,保持相对酶活80%以上；储存稳定性较好,4℃下储存一个月,相对酶活仍然在80%左右；固定化细胞的最适反应温度从固定化前的37℃上升到47-50℃,在最适温度下,相对酶活为游离细胞的3倍。采用本文的固定化方法不但提高了细胞的稳定性,同时能较大幅度地提高酶的活力,所以这种方法为以后工业化过程的研究奠定了良好的基础。
The new strains with D-hydantoinase activity have been screeded by use of DL-p-hydroxy-phenylhydantoin (DL-p-HPH) as a single nitrogen source from nature. Totally, three strains which had higher enzyme activity were obtained by double-plate and rapid micro-bacteria methods. The one showed highest enzyme activity was selected to do further study.
Next, the strain screened above was fermented, and the preliminary fermentation products were identified by TLC and HPLC. Compared with the standard samples of D-p-hydroxyphenyIgIycine (D-p-HPG), a conversion product had the same Rf value as the standard sample of D-p-HPG. Then transformation products were purified after treated with NaNO2 by ion-exchange resin 732 in the following conditions:the flow rate of sample solution is about 2ml/min, and the eluted solution was 0.2 mol/L NaOH at a rate of 0.4 ml/min. The effluent was concentrated and crystallized. After that the specific rotation and IR spectrum of the pure product was measured. Then we could confirm the product is D-p-HPG
On the basis above, the bacteria strain was identified as Alcaligenes sp. based on 16S rRNA result.
Culture conditions of the strain were optimized and the optimal cultivation conditions:the optimum carbon source was glycerol, the nitrogen source was the yeast extract, optimum growth temperature was 28-30℃, the best primary pH was pH 7.0. The best culture volume was 50 ml/250ml flask. The cell biomass reached to the highest when cultured for 48 hours, but the enzyme activity had reached to the highest after 24 hours.
Afterwards, bacteria were immobilized by PVA-boric acid method. The optimal concentration of PVA for the immobilization was 0.1 g/ml. During the immobilization process, adding Mg2+ in boric acid solution had better result than adding other cations (Na+、Zn2+、Ca2+). The optimal pH was 8.0 for both of immobilized and free cells. The variation of pH (from 6.5 to 9.5) has little effect on activity of immobilized cells while had dramatic effect on free cells. The immobilized cells can tolerate higher temperature than free cells. The optimal temperature of the immobilized cells was 47℃compared with 37℃of the free cells. Under such conditions, the activity of the immobilized cells can reach about 3 times of the free cells. After 7 batches of reaction, the remained activity of the immobilized cells was still more than 80% of initial activity. After 30 days storage at 4℃the immobilized cells kept more than 80% of its initial activity. For free cells, it kept only 30% of initial activity. In short, the immobilized cells of Alcaligene sp. prepared by PVA entrapment has better pH and temperature tolerant, higher activity and stability compared to free cell and has potential application in the future.
引文
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[2]Yang H B, Zheng G, Peng X Z, et al. Amino acids and-Tyr-tRNA Tyr deacylase: stereospecificity of the translation machine revisited [J]. FEBS Letters,2003,552:95～ 981.
[3]WakayamaM, Yoshimune K, Hirose Y, et al. Production of D-amino acids by N-acyl-d-amino acid amidohydrolase and its structure and function [J]. J Molecular Catalysis(B),Enzymatic,2003,23:71～78.
[4]Yasuhisa A and Tina L L1 Enzymes acting on peptides containing D-amino acid [J].J Biosc and Bioeng,2000,89:295-306.
[5]Schulze B, Wubbolts GM. Biocatalysis for industrial production of fine chemicals [J].Current Opinion in Biotech,1999,10:609～615.
[6]陈建波,徐毅.生物酶法制备D-对羟基苯甘氨酸的研究[J].生物学杂志,2007,24(5)：8-11.
[7]杨柳，孙万儒.海因酶的研究进展[J].生物工程进展.2001,21(3)：13-16.
[8]A.Louwrier, C.J.Knowles, Enzyme Micobe. Technol.19(1996)562-571.
[9]于平.海因酶研究新进展[J].生物学杂志。2005,22(2)：1-4.
[10]许激扬,吴梧桐,公剑,等.化学酶法合成D_对羟基苯甘氨酸[J].中国药科大学学报.1998,29(5)：394-396.
[11]都立辉,刘芳.16S rRNA基因在细菌菌种鉴定中的应用[J].乳品科学与技术2006,5：207-209.
[12]R.Diirr et al. Distribution of hydantoinase activity in bacterial isolates from geographically distinct environmental sources[J]. Journal of Molecular Catalysis B:Enzymatic 39(2006):160-165.
[13]罗雪,冯瑞山,胡卓逸.海因酶产生菌的微孔快速筛选法[J].华西医科大学学报,2001,32(3)：462-463.
[14]戴连科,胡重浩,许重阳,尹明星.酶法生产D-对羟基苯甘氨酸提取方法综述[J].浙江化工,2008,39(6)：17-18.
[15]戴连科,胡重浩,许重阳,骆红英,徐海斌.纳滤膜在D-对羟基苯甘氨酸提取中的应用[J].
中国医药工业杂志,2009,40(3)：200-202.
[16]Krieg N, et al eds. Bergey's Mannul of Systematic Bacteriology. Vol.1-4. Baltimore: William & Wilkins,1984-1989.
[17]沈萍主编.微生物学[M].高等教育出版社,2000.
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