细菌碳酸酐酶催化碳酸钙沉积的动力学和形态学研究
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摘要
自然界中碳酸钙(CaCO_3)的沉积过程极为缓慢,在CaCO_3-H2O-CO2反应体系中CO2 + H2O ? HCO_3- + H+为限速步骤,可利用生物催化剂来加速这一反应的进行。碳酸酐酶(Carbonic anhydrase, CA)是一种以锌为活性中心的金属酶,可以显著催化上述反应的进行。我们前期研究发现微生物CA在方解石沉积中具有显著促进作用。本文采用气体扩散体系,在不同初始pH、酶浓度和Ca~(2+)浓度条件下进一步研究细菌CA催化CaCO_3沉积的动力学,并采用XRD、FTIR和FESEM对沉积过程中CaCO_3晶体的形态学进行分析,获得的主要结果如下:
(1)不同初始pH下细菌CA催化CaCO_3沉积过程中Ca~(2+)沉积量变化符合指数模型。在实验pH范围内(pH 6.0~8.0),CaCO_3沉积速率随初始pH的增加而增大。XRD和FESEM分析结果表明,CA存在时生成的CaCO_3晶体主要为方解石,晶体不太规则,且粒径较大。随时间的增加,晶体逐渐由棱形变为类锥形或聚集成不规则形体。
(2)不同酶浓度下细菌CA催化CaCO_3沉积过程中Ca~(2+)沉积量变化符合指数模型。酶浓度在0.2~2.0 U/mL时有利于CaCO_3的沉积,而当酶浓度为8.0 U/mL时CaCO_3沉积速率受到抑制,说明细菌CA催化CaCO_3沉积时酶浓度并不是越大越有利。XRD、FTIR和FESEM分析结果表明,不同酶浓度下CaCO_3晶体的形态差异较大,且较低浓度CA作用下有球霰石存在,而较高浓度CA作用下有利于方解石的形成。
(3)不同初始Ca~(2+)浓度下细菌CA催化CaCO_3沉积过程中Ca~(2+)沉积量变化不仅符合指数模型也符合多项式模型。在一定范围内CaCO_3沉积速率随初始Ca~(2+)浓度的增加而增大,但过高的初始Ca~(2+)浓度(100 mmol/L)使细菌CA催化沉积CaCO_3的作用效果受到一定程度的影响。XRD、FTIR和FESEM分析结果表明,初始Ca~(2+)浓度对细菌CA作用下产生的CaCO_3晶体的形态有较大影响,而且,较低的初始Ca~(2+)浓度有利于球霰石的形成,而较高的初始Ca~(2+)浓度有利于方解石的形成。
综合上述结果认为,细菌CA催化CaCO_3沉积过程中,初始pH、酶浓度和盐浓度不仅影响CaCO_3沉积速率,而且对CaCO_3的晶型晶貌产生较大影响,可以利用上述不同初始条件来诱导产生不同晶型晶貌的CaCO_3材料。细菌CA对CaCO_3沉积的作用除了催化作用外,还与细菌CA的酶蛋白对Ca~(2+)的静电吸附以及酶蛋白对晶体晶面的选择性吸附有关。本文结果为丰富岩溶动力学理论提供了一定的科学依据,并为CaCO_3生物矿化材料的制备提供了新思路。
The precipitation process of calcium carbonate(CaCO_3)is very slow in nature. In the reactive system of CaCO_3-H2O-CO2, the reaction CO2 + H2O ? H + + HCO_3- is a rate-limiting step. A biological catalyst could be used to accelerate this reaction. Carbonic Anhydrase(CA)is a zinc-containing metalloenzyme that can remarkably catalyze the above reaction, and with a zinc ion as active centre. Our previous studies found that microbial CA had significant promoting effect on calcite precipitation. In this paper, the kinetics of CaCO_3 precipitation catalyzed by bacterial CA at different initial pH, enzyme concentrations and Ca~(2+) concentrations through the gaseous diffusion systems were further investigated, and the morphology of CaCO_3 crystals obtained in the precipitation process was analyzed using XRD, FTIR and FESEM. The main results are as follows.
(1) The change of deposited Ca~(2+) during the process of CaCO_3 precipitation catalyzed by bacterial CA at different initial pH was well fitted by exponential model. In the experimental pH range (pH 6.0~8.0), the precipitation rate of CaCO_3 increased with initial pH. The results of XRD and FESEM analysis indicated that the CaCO_3 crystals were mainly calcite in the presence of bacterial CA. The crystals were not regular and their size was bigger. With increasing contact time, the crystals gradually changed from prism shape to pyramid-like shape or assembled into irregular polyhedral shape.
(2) The change of deposited Ca~(2+) during the process of CaCO_3 precipitation catalyzed by bacterial CA at different enzyme concentrations was well fitted by exponential model. The enzyme concentrations of 0.2~2.0 U/mL were beneficial to CaCO_3 precipitation, however, the CaCO_3 precipitation was inhibited when the enzyme concentration was at 8.0 U/mL, which indicated that overhigh enzyme concentration was not more favorable for CaCO_3 precipitation catalyzed by bacterial CA. The results of XRD, FTIR and FESEM analysis showed that there were significant differences in the morphologies of CaCO_3 crystals among different enzyme concentrations. Vaterite was present at lower concentration of CA, and the higher concentration of CA favored the formation of calcite.
(3) The change of deposited Ca~(2+) during the process of CaCO_3 precipitation catalyzed by bacterial CA at different initial Ca~(2+) concentrations was well fitted by not only exponential model but also polynomial model. The precipitation rate of CaCO_3 increased with the initial concentration of Ca~(2+), but overhigh initial concentration (100 mmol/L) of Ca~(2+) had a certain influence on the effect of CaCO_3 precipitation catalyzed by bacterial CA. The results of XRD, FTIR and FESEM analysis showed that the initial Ca~(2+) concentration had greater effect on the morphology of CaCO_3 crystals formed in the presence of bacterial CA. The lower initial Ca~(2+) concentration favored the formation of vaterite and the higher initial Ca~(2+) concentration favored the formation of calcite.
In summary, the initial pH, enzyme concentration and Ca~(2+) concentration, et al., could not only influence the rate of CaCO_3 precipitation catalyzed by bacterial CA, but also greatly influence the polymorph and morphology of CaCO_3 crystals. Therefore, different crystalline feature of CaCO_3 materials could be induced by bacterial CA under above different initial conditions. The role of bacterial CA in CaCO_3 precipitation was related to the electrostatic adsorption of CA enzyme protein on Ca~(2+) and the selective adsorption of CA enzyme protein on the crystal faces, except for the enzymatic catalysis. The results in this paper provide scientific base for enriching the theory of karst dynamics, and also provide a new idea for preparation of CaCO_3 biomineralization materials.
(1)不同初始pH下细菌CA催化CaCO_3沉积过程中Ca~(2+)沉积量变化符合指数模型。在实验pH范围内(pH 6.0~8.0),CaCO_3沉积速率随初始pH的增加而增大。XRD和FESEM分析结果表明,CA存在时生成的CaCO_3晶体主要为方解石,晶体不太规则,且粒径较大。随时间的增加,晶体逐渐由棱形变为类锥形或聚集成不规则形体。
(2)不同酶浓度下细菌CA催化CaCO_3沉积过程中Ca~(2+)沉积量变化符合指数模型。酶浓度在0.2~2.0 U/mL时有利于CaCO_3的沉积,而当酶浓度为8.0 U/mL时CaCO_3沉积速率受到抑制,说明细菌CA催化CaCO_3沉积时酶浓度并不是越大越有利。XRD、FTIR和FESEM分析结果表明,不同酶浓度下CaCO_3晶体的形态差异较大,且较低浓度CA作用下有球霰石存在,而较高浓度CA作用下有利于方解石的形成。
(3)不同初始Ca~(2+)浓度下细菌CA催化CaCO_3沉积过程中Ca~(2+)沉积量变化不仅符合指数模型也符合多项式模型。在一定范围内CaCO_3沉积速率随初始Ca~(2+)浓度的增加而增大,但过高的初始Ca~(2+)浓度(100 mmol/L)使细菌CA催化沉积CaCO_3的作用效果受到一定程度的影响。XRD、FTIR和FESEM分析结果表明,初始Ca~(2+)浓度对细菌CA作用下产生的CaCO_3晶体的形态有较大影响,而且,较低的初始Ca~(2+)浓度有利于球霰石的形成,而较高的初始Ca~(2+)浓度有利于方解石的形成。
综合上述结果认为,细菌CA催化CaCO_3沉积过程中,初始pH、酶浓度和盐浓度不仅影响CaCO_3沉积速率,而且对CaCO_3的晶型晶貌产生较大影响,可以利用上述不同初始条件来诱导产生不同晶型晶貌的CaCO_3材料。细菌CA对CaCO_3沉积的作用除了催化作用外,还与细菌CA的酶蛋白对Ca~(2+)的静电吸附以及酶蛋白对晶体晶面的选择性吸附有关。本文结果为丰富岩溶动力学理论提供了一定的科学依据,并为CaCO_3生物矿化材料的制备提供了新思路。
The precipitation process of calcium carbonate(CaCO_3)is very slow in nature. In the reactive system of CaCO_3-H2O-CO2, the reaction CO2 + H2O ? H + + HCO_3- is a rate-limiting step. A biological catalyst could be used to accelerate this reaction. Carbonic Anhydrase(CA)is a zinc-containing metalloenzyme that can remarkably catalyze the above reaction, and with a zinc ion as active centre. Our previous studies found that microbial CA had significant promoting effect on calcite precipitation. In this paper, the kinetics of CaCO_3 precipitation catalyzed by bacterial CA at different initial pH, enzyme concentrations and Ca~(2+) concentrations through the gaseous diffusion systems were further investigated, and the morphology of CaCO_3 crystals obtained in the precipitation process was analyzed using XRD, FTIR and FESEM. The main results are as follows.
(1) The change of deposited Ca~(2+) during the process of CaCO_3 precipitation catalyzed by bacterial CA at different initial pH was well fitted by exponential model. In the experimental pH range (pH 6.0~8.0), the precipitation rate of CaCO_3 increased with initial pH. The results of XRD and FESEM analysis indicated that the CaCO_3 crystals were mainly calcite in the presence of bacterial CA. The crystals were not regular and their size was bigger. With increasing contact time, the crystals gradually changed from prism shape to pyramid-like shape or assembled into irregular polyhedral shape.
(2) The change of deposited Ca~(2+) during the process of CaCO_3 precipitation catalyzed by bacterial CA at different enzyme concentrations was well fitted by exponential model. The enzyme concentrations of 0.2~2.0 U/mL were beneficial to CaCO_3 precipitation, however, the CaCO_3 precipitation was inhibited when the enzyme concentration was at 8.0 U/mL, which indicated that overhigh enzyme concentration was not more favorable for CaCO_3 precipitation catalyzed by bacterial CA. The results of XRD, FTIR and FESEM analysis showed that there were significant differences in the morphologies of CaCO_3 crystals among different enzyme concentrations. Vaterite was present at lower concentration of CA, and the higher concentration of CA favored the formation of calcite.
(3) The change of deposited Ca~(2+) during the process of CaCO_3 precipitation catalyzed by bacterial CA at different initial Ca~(2+) concentrations was well fitted by not only exponential model but also polynomial model. The precipitation rate of CaCO_3 increased with the initial concentration of Ca~(2+), but overhigh initial concentration (100 mmol/L) of Ca~(2+) had a certain influence on the effect of CaCO_3 precipitation catalyzed by bacterial CA. The results of XRD, FTIR and FESEM analysis showed that the initial Ca~(2+) concentration had greater effect on the morphology of CaCO_3 crystals formed in the presence of bacterial CA. The lower initial Ca~(2+) concentration favored the formation of vaterite and the higher initial Ca~(2+) concentration favored the formation of calcite.
In summary, the initial pH, enzyme concentration and Ca~(2+) concentration, et al., could not only influence the rate of CaCO_3 precipitation catalyzed by bacterial CA, but also greatly influence the polymorph and morphology of CaCO_3 crystals. Therefore, different crystalline feature of CaCO_3 materials could be induced by bacterial CA under above different initial conditions. The role of bacterial CA in CaCO_3 precipitation was related to the electrostatic adsorption of CA enzyme protein on Ca~(2+) and the selective adsorption of CA enzyme protein on the crystal faces, except for the enzymatic catalysis. The results in this paper provide scientific base for enriching the theory of karst dynamics, and also provide a new idea for preparation of CaCO_3 biomineralization materials.
引文
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