纳米材料固定化植酸酶的制备及其催化效率与影响因素综述

doi:10.16258/j.cnki.1674-5906.2023.10.017

生态环境学报 ›› 2023, Vol. 32 ›› Issue (10): 1889-1900.DOI: 10.16258/j.cnki.1674-5906.2023.10.017

• 综述 • 上一篇

纳米材料固定化植酸酶的制备及其催化效率与影响因素综述

杨晓莉¹^,²(), 毛佳璇¹, 马露冉¹, 徐其静¹^,², 刘雪¹^,²^,^*()

1.西南林业大学生态与环境学院，云南昆明 650224
2.西南林业大学环境修复与健康研究院，云南昆明 650224

收稿日期:2023-07-30 出版日期:2023-10-18 发布日期:2024-01-16
通讯作者: *刘雪。E-mail: liuxue20088002@126.com
*刘雪。E-mail: liuxue20088002@126.com
作者简介:杨晓莉（1998年生），女（彝族），硕士研究生，研究方向为植物营养与环境污染。E-mail: yangxiaoli1102013@126.com
基金资助:
云南省“兴滇英才支持计划”青年人才专项(YNQR-QNRC-2019-027);云南省农业基础研究联合专项(202101BD070001-043);云南省农业基础研究联合专项(202301BD070001-154);国家自然科学基金项目(41867066);国家自然科学基金项目(41907129);大学生创新创业训练计划项目(20210752043);大学生创新创业训练计划项目(20210752014)

Nanomaterial-immobilized Phytase: Preparation, Catalytic Efficiency and Influencing Factors

YANG Xiaoli¹^,²(), MAO Jiaxuan¹, MA Luran¹, XU Qijing¹^,², LIU Xue¹^,²^,^*()

1. Institute of Ecology and Environment, Southwest Forestry University, Kunming 650224, P. R. China
2. Institute of Environment Remediation and Health, Southwest Forestry University, Kunming 650224, P. R. China

Received:2023-07-30 Online:2023-10-18 Published:2024-01-16

摘要/Abstract

摘要：

植酸是土壤磷的重要组分部分，但难以被植物吸收利用，需在专一性酶——植酸酶作用下，经脱磷酸化过程矿化水解释放磷酸（盐）。然而，植酸酶活性极易受环境因素影响，导致酶活性降低甚至失活。如何保持或提高土壤中植酸酶的活性，进而提高土壤内源植酸磷的利用率已引起广泛关注。植酸酶经纳米材料固定化可提高其环境稳定性和活性。总结了不同纳米载体的制备技术、产品特征及优缺点，深入分析了不同纳米载体固定化植酸酶的制备及表征方法、负载酶的酶活性及作用机理。分析发现：1）功能载体固定化植酸酶的环境稳定性（高温、强酸、强碱和金属离子等）和催化水解效率显著优于游离植酸酶，是提高土壤内源植酸磷利用效率的有效途径；2）纳米材料固定化植酸酶的稳定性受载体种类、植酸酶来源与特性及制备方法的影响，应根据植酸酶来源与特性选择适宜的载体种类和固定化方法。针对固定化植酸酶实际应用中的局限性，今后应关注如下几方面：1）稳定性好、活性高的植酸酶生产菌；2）稳定性好、高负载率载体，制备纳米级材料；3）负载率高、成本低的固定化方法，在生产、运输、使用和储存过程中保持酶活；4）不同种类固定化植酸酶在不同种类土壤中的适用性。该综述可为理解纳米材料固定化植酸酶制备技术特征和实际应用影响因素提供理论依据，为开发高负载率、高稳定性固定化植酸酶制剂，进而提高土壤内源植酸磷的生物利用率提供科学依据，对降低外源磷肥施用、降低磷流失污染风险和保障农业生产具有一定的现实意义。

关键词: 纳米材料, 植酸酶, 固定化, 影响因素, 应用效率

Abstract:

Phytate is the pdominant form of soil phosphorus (P), which cannot be directly taken up by plants, but can be hydrolyzed by the specific enzyme phytase via dephosphorylation. However, phytase activity is affected by environmental factors, resulting in reduced activity or deactivation. Consequently, there is growing interest in finding ways to maintain or improve phytase activity in soils to improve the utilization of soil endogenous phytate-P has attracted increasing attentions. Nanomaterial-immobilization can improve the stability and activity of phytase in the environment. This paper provides a review on the preparation technology, product characteristics, advantages and disadvantages of chitosan, hydroxyapatite, graphene oxide and mesoporous silicon dioxide nanocarriers, and analyzes the preparation methods (adsorption, covalent binding, embedding, cross-linking, loading, etc.), characterization methods, loading efficiency, loaded-enzyme activity, and reaction mechanisms of different nanocarriers immobilized phytase. It was found that 1) nanomaterial-immobilized phytase is more stable than free phytase under high temperature, acid, alkali and in the presence of metal ions, which is an effective way to improve the utilization of soil endogenous phytase-P; 2) the activity of nanomaterial-immobilized phytase depends on carrier species, phytase source and characteristics, and preparation methods. Phytase source and characteristics should be considered when selecting suitable carrier and immobilization methods. Given that the practical application of nanomaterial-immobilized phytase is limited, attention can be paid to 1) microbes that produce phytase with high stability and activity; 2) carriers with high stability and loading rate, which can be used to develop nanomaterials; 3) immobilization methods with high loading rate and low cost to maintain enzyme activity during production, transportation, and storage; and 4) the applicability of different immobilized phytases in different soils. The information enhances our understanding of the characteristics of nanomaterial-immobilization technology and limiting factors during practical applications. It also provides technical support for the development of high loading ratio and stability to improve the utilization of soil endogenous phytate-P, which is important for reducing exogenous P fertilizer application, minimizing the risk of P loss, and ensuring sustainable agricultural production.

Key words: nanomaterial, phytase, immobilization, influencing factor, application efficiency

中图分类号:

杨晓莉, 毛佳璇, 马露冉, 徐其静, 刘雪. 纳米材料固定化植酸酶的制备及其催化效率与影响因素综述[J]. 生态环境学报, 2023, 32(10): 1889-1900.

YANG Xiaoli, MAO Jiaxuan, MA Luran, XU Qijing, LIU Xue. Nanomaterial-immobilized Phytase: Preparation, Catalytic Efficiency and Influencing Factors[J]. Ecology and Environment, 2023, 32(10): 1889-1900.

图/表 4

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纳米材料	制备方法	外观特征	优点	缺点	参考文献
壳聚糖	喷雾干燥法	多空微球	产量高, 包封率高, 稳定性好, 释放时间长	易氧化, 微囊致密性较差	Cota‐Arriola et al., 2013
	离子交联法	圆整度不高, 分散性好	反应条件温和、操作简单、药物有效活性高	活性成分易从颗粒表面脱落	Yeon et al., 2019; 吴益栋等2018
	乳化交联法	外观圆整, 表面光滑, 结构致密	合成速度快, 重现性好, 产率高	交联剂对机体有害, 与表面活性剂反应性差	Mao et al., 2016; Riegger et al., 2018
	沉淀析出法	圆整度不高, 分散性好	可有效控制颗粒大小和药物释放	粒子的机械强度差	Chandra et al., 2016
	冷冻干燥法	表面粗糙, 多孔微球, 粒径均匀	操作简单, 多孔微球具有良好的球形度、孔壁较薄和孔径较小	纯壳聚糖基多孔微球使用较少	Song et al., 2021
	反向胶束法	微粒分布均匀	粒子粒径较小, 分布范围小	操作复杂, 需使用大量有机溶剂且纳米粒子难分离	Cota‐Arriola et al., 2013
羟基磷灰石	水热法	表面光滑	结晶度好, 重复性好、水热温度范围广	需在高压高温处理, 步骤繁琐	Jin et al., 2015; An et al., 2016
	沉淀法	块状、针状结构	操作简单、成本低, 适用于工业生产	颗粒质量低, 尺寸大, 粒度分布广, 团聚物多	Cengiz et al., 2008
	固相反应法	轮廓清晰, 形状规则	粒径大小均匀, 晶型完善, 分散性好, 产率高、工艺简单	产物纯度较低, 生产过程能耗较大	Kim et al., 2004; 黄龙霄等, 2015
	溶胶-凝胶法	结晶为六方结构	工艺简单, 产品纯度、均匀度高	凝胶颗粒烧结性差, 干燥收缩大	黄龙霄等, 2015
氧化石墨烯	原位聚合法	纳米片层状	氧化石墨烯与聚合物基体间界面作用增强	基体黏度增大, 聚合物的相对分子质量和单分散性发生改变	张雷等, 2020
	溶液共混法	层状纳米	机械性能和耐热性能好, 工艺简单, 成本低	需使用大量有机溶剂, 易造成环境污染	Yang et al., 2011; 张雷等, 2020
	熔融共混法	不规则片状	分散均匀, 成本低, 安全环保, 利于工业生产	黏度较大, 氧化石墨烯与聚合物界面作用差, 复合材料性能降低	Che et al., 2013; 张雷等, 2020
	乳液共混法	微粒分散	工艺简单、安全环保、复合材料性能改善	pH值影响氧化石墨烯边缘羧基电离程度, 影响亲水性、分散性	张雷等, 2020
	Pickering乳液聚合法	片状、微球	操作简单, 绿色环保, 耐水性能较好, 分散均匀	微球粒径分布较宽	Che et al., 2013; 张雷等, 2020
介孔二氧化硅	模板法	中空微球	操作简单, 介孔体微球外壳的外层厚度达到40 nm, 存储容量大	分散性和介孔分子的有序性较难控制	Fang et al., 2011; 李雨露等, 2022
	选择性刻蚀法	大小均一、单分散的双壳空心微球	操作简单、有效、成本低	刻蚀剂会有残留, 影响其在生物领域中的应用	Tan et al., 2013
	喷雾干燥法	中空颗粒	制备方法简单, 样品的分散性、稳定性较好	干燥产物含有挥发性物质, 对干燥设备和产品工艺技术要求高	李雨露等, 2022

纳米材料	制备方法	外观特征	优点	缺点	参考文献
壳聚糖	喷雾干燥法	多空微球	产量高, 包封率高, 稳定性好, 释放时间长	易氧化, 微囊致密性较差	Cota‐Arriola et al., 2013
	离子交联法	圆整度不高, 分散性好	反应条件温和、操作简单、药物有效活性高	活性成分易从颗粒表面脱落	Yeon et al., 2019; 吴益栋等2018
	乳化交联法	外观圆整, 表面光滑, 结构致密	合成速度快, 重现性好, 产率高	交联剂对机体有害, 与表面活性剂反应性差	Mao et al., 2016; Riegger et al., 2018
	沉淀析出法	圆整度不高, 分散性好	可有效控制颗粒大小和药物释放	粒子的机械强度差	Chandra et al., 2016
	冷冻干燥法	表面粗糙, 多孔微球, 粒径均匀	操作简单, 多孔微球具有良好的球形度、孔壁较薄和孔径较小	纯壳聚糖基多孔微球使用较少	Song et al., 2021
	反向胶束法	微粒分布均匀	粒子粒径较小, 分布范围小	操作复杂, 需使用大量有机溶剂且纳米粒子难分离	Cota‐Arriola et al., 2013
羟基磷灰石	水热法	表面光滑	结晶度好, 重复性好、水热温度范围广	需在高压高温处理, 步骤繁琐	Jin et al., 2015; An et al., 2016
	沉淀法	块状、针状结构	操作简单、成本低, 适用于工业生产	颗粒质量低, 尺寸大, 粒度分布广, 团聚物多	Cengiz et al., 2008
	固相反应法	轮廓清晰, 形状规则	粒径大小均匀, 晶型完善, 分散性好, 产率高、工艺简单	产物纯度较低, 生产过程能耗较大	Kim et al., 2004; 黄龙霄等, 2015
	溶胶-凝胶法	结晶为六方结构	工艺简单, 产品纯度、均匀度高	凝胶颗粒烧结性差, 干燥收缩大	黄龙霄等, 2015
氧化石墨烯	原位聚合法	纳米片层状	氧化石墨烯与聚合物基体间界面作用增强	基体黏度增大, 聚合物的相对分子质量和单分散性发生改变	张雷等, 2020
	溶液共混法	层状纳米	机械性能和耐热性能好, 工艺简单, 成本低	需使用大量有机溶剂, 易造成环境污染	Yang et al., 2011; 张雷等, 2020
	熔融共混法	不规则片状	分散均匀, 成本低, 安全环保, 利于工业生产	黏度较大, 氧化石墨烯与聚合物界面作用差, 复合材料性能降低	Che et al., 2013; 张雷等, 2020
	乳液共混法	微粒分散	工艺简单、安全环保、复合材料性能改善	pH值影响氧化石墨烯边缘羧基电离程度, 影响亲水性、分散性	张雷等, 2020
	Pickering乳液聚合法	片状、微球	操作简单, 绿色环保, 耐水性能较好, 分散均匀	微球粒径分布较宽	Che et al., 2013; 张雷等, 2020
介孔二氧化硅	模板法	中空微球	操作简单, 介孔体微球外壳的外层厚度达到40 nm, 存储容量大	分散性和介孔分子的有序性较难控制	Fang et al., 2011; 李雨露等, 2022
	选择性刻蚀法	大小均一、单分散的双壳空心微球	操作简单、有效、成本低	刻蚀剂会有残留, 影响其在生物领域中的应用	Tan et al., 2013
	喷雾干燥法	中空颗粒	制备方法简单, 样品的分散性、稳定性较好	干燥产物含有挥发性物质, 对干燥设备和产品工艺技术要求高	李雨露等, 2022

载体	植酸酶来源	固定化技术	表征技术	负载率/ %	负载量/ (mg∙g⁻¹)	固定化酶活性/ (U∙g⁻¹)	固定化机理	参考文献
壳聚糖	商业植酸酶	离子凝胶	傅里叶红外光谱、差示扫描量热法	69.2	18	－	壳聚糖阳离子与带负电荷的小分子或聚合物进行静电相互作用 (分子间和分子内交联)	钱浩, 2021
	地芽孢杆菌 Geobacillus sp. TF16	共价结合	－	38	－	3.4	酶利用其蛋白质或碳水化合物部分与壳聚糖结合	Sirin et al., 2017
	赤小豆 Vigna umbellata Thunb	交联	扫描电子显微镜、傅里叶红外光谱	77.5	－	－	壳聚糖的D-葡糖胺-NH₂与戊二醛的-CHO交联反应, 将酶其固定到壳聚糖载体上	Belho et al., 2021
	辣乳菇 Lactarius piperatus	共价结合	X射线衍射、傅里叶红外光谱	87	－	1.42	植酸酶以共价形式与载体表面弱键和游离键结合	Onem et al., 2016
羟基磷灰石	黑曲霉 A. niger	共沉淀	X射线衍射、扫描电子显微镜	70	6	118	羟基磷灰石在氨基功能化后产生共价相互作用, 与酶氨基酸中羧基螯合反应	Coutinho et al., 2020c
羟基磷灰石	黑曲霉 A. niger	吸附	傅里叶红外光谱	100	5	－	羟基磷灰石纳米颗粒上Ca²⁺和植酸酶氨基酸残基在羟基磷灰石和COO⁻上形成离子和配位键	Coutinho et al., 2020d
氧化石墨烯	枯草芽孢杆菌 Bacillus subtilis	吸附- 交联	紫外-可见光谱	74.2	－	3.3	氧化石墨烯的含氧基团, 对酶进行氨氧化	Sun et al., 2014; Dutta et al., 2017
介孔二氧化硅	商业植酸酶	吸附	扫描电子显微镜、透射电子显微镜和 N₂吸附-解吸	－	237	9.53	酶吸附在介孔二氧化硅载体微孔通道内	Xin et al., 2020
介孔二氧化硅	黑曲霉 A. niger	吸附	N₂吸附-解吸	－	6.3	8000	酶吸附在介孔二氧化硅载体微孔通道内	Trouillefou et al., 2015
藻酸钙	酿酒酵母 Saccharomyces cerevisiae CY	包埋	紫外-可见光谱	43	62	0.28	酶分子在溶液中是游离的, 将酶包封在不溶性藻酸钙凝胶中	In et al., 2007
	成团泛菌 Pantoea agglomerans	包埋	紫外-可见光谱	82.3	－	0.022		Greiner, 2008
	地芽孢杆菌 Geobacillus sp.TF16	包埋	－	42	－	5.01		Tan et al., 2013
环氧树脂	鳄梨 Persea americana Mill	吸附、共价结合	化学表征	70.8	0.3	0.1	酶以高的离子强度疏水吸附在疏水载体上, 再与载体之间进行共价反应	Çelem et al., 2009a
功能化多壁碳纳米管	商业植酸酶	共价结合	扫描电子显微镜、圆二色性光谱、zeta电位测量和傅里叶红外光谱	62	110	－	纳米管侧壁的羧酸基团与酶表面的游离胺基结合	Naghshbandi et al., 2018

载体	植酸酶来源	固定化技术	表征技术	负载率/ %	负载量/ (mg∙g⁻¹)	固定化酶活性/ (U∙g⁻¹)	固定化机理	参考文献
壳聚糖	商业植酸酶	离子凝胶	傅里叶红外光谱、差示扫描量热法	69.2	18	－	壳聚糖阳离子与带负电荷的小分子或聚合物进行静电相互作用 (分子间和分子内交联)	钱浩, 2021
	地芽孢杆菌 Geobacillus sp. TF16	共价结合	－	38	－	3.4	酶利用其蛋白质或碳水化合物部分与壳聚糖结合	Sirin et al., 2017
	赤小豆 Vigna umbellata Thunb	交联	扫描电子显微镜、傅里叶红外光谱	77.5	－	－	壳聚糖的D-葡糖胺-NH₂与戊二醛的-CHO交联反应, 将酶其固定到壳聚糖载体上	Belho et al., 2021
	辣乳菇 Lactarius piperatus	共价结合	X射线衍射、傅里叶红外光谱	87	－	1.42	植酸酶以共价形式与载体表面弱键和游离键结合	Onem et al., 2016
羟基磷灰石	黑曲霉 A. niger	共沉淀	X射线衍射、扫描电子显微镜	70	6	118	羟基磷灰石在氨基功能化后产生共价相互作用, 与酶氨基酸中羧基螯合反应	Coutinho et al., 2020c
羟基磷灰石	黑曲霉 A. niger	吸附	傅里叶红外光谱	100	5	－	羟基磷灰石纳米颗粒上Ca²⁺和植酸酶氨基酸残基在羟基磷灰石和COO⁻上形成离子和配位键	Coutinho et al., 2020d
氧化石墨烯	枯草芽孢杆菌 Bacillus subtilis	吸附- 交联	紫外-可见光谱	74.2	－	3.3	氧化石墨烯的含氧基团, 对酶进行氨氧化	Sun et al., 2014; Dutta et al., 2017
介孔二氧化硅	商业植酸酶	吸附	扫描电子显微镜、透射电子显微镜和 N₂吸附-解吸	－	237	9.53	酶吸附在介孔二氧化硅载体微孔通道内	Xin et al., 2020
介孔二氧化硅	黑曲霉 A. niger	吸附	N₂吸附-解吸	－	6.3	8000	酶吸附在介孔二氧化硅载体微孔通道内	Trouillefou et al., 2015
藻酸钙	酿酒酵母 Saccharomyces cerevisiae CY	包埋	紫外-可见光谱	43	62	0.28	酶分子在溶液中是游离的, 将酶包封在不溶性藻酸钙凝胶中	In et al., 2007
	成团泛菌 Pantoea agglomerans	包埋	紫外-可见光谱	82.3	－	0.022		Greiner, 2008
	地芽孢杆菌 Geobacillus sp.TF16	包埋	－	42	－	5.01		Tan et al., 2013
环氧树脂	鳄梨 Persea americana Mill	吸附、共价结合	化学表征	70.8	0.3	0.1	酶以高的离子强度疏水吸附在疏水载体上, 再与载体之间进行共价反应	Çelem et al., 2009a
功能化多壁碳纳米管	商业植酸酶	共价结合	扫描电子显微镜、圆二色性光谱、zeta电位测量和傅里叶红外光谱	62	110	－	纳米管侧壁的羧酸基团与酶表面的游离胺基结合	Naghshbandi et al., 2018

纳米载体	植酸酶来源	水解底物/ 作物	游离酶				固定化酶				应用领域	参考文献
纳米载体	植酸酶来源	水解底物/ 作物	K_m/ μM	水解效率/ 释磷量	pH	温度/ ℃	K_m/ μM	水解效率/ 释磷量	pH	温度/ ℃	应用领域	参考文献
聚乙烯醇壳聚糖	豇豆 V. unguiculata	植酸钠	460	－	5.0	45	13430	－	6.0	65	动物饲料、农业和食品工业	Kamaci et al., 2020
聚乙烯醇	豇豆 V. unguiculata	植酸钠	460	－	5.0	45	1170	－	5.0	65	农业、医疗和食品工业	Duru et al., 2022
壳聚糖	商业植酸酶	植酸钠	446	1.03 mg	5.0	40	1375	1.86 mg	5.0	50	动物饲料	钱浩, 2021
磁铁矿- 壳聚糖	辣乳菇 L. piperatus	植酸钠	－	－	5.0	50	－	－	4.0	50	食品和动物饲料行业	Onem et al., 2016
		绿色小扁豆		36.5%				48.2%
		红色小扁豆		35.9%				49.1%
		豌豆		56.8%				69.0%
		青豆		53.0%				65.8%
		黄豆		62.7%				68.5%
		芸苔属植物		32.7%				43.5%
		玉米		47.1%				53.3%
		干玉米		26.3%				35.5%
		燕麦		26.4%				36.4%
		黑麦		64.6%				68.7%
		小麦		65.7%				75.0%
		蚕豆		45.5%				58.4%
		鹰嘴豆		51.6%				63.0%
		花生		67.5%				75.4%
羟基磷灰石	黑曲霉 A. niger	植酸	－	7.35 μmol∙mL⁻¹	5.0	60	－	7.92 μmol∙mL⁻¹	5.0	60	动物饲料	Coutinho et al., 2020d
介孔二氧化硅	黑曲霉 A. niger	植酸钠	－	0.68 μmol	5.0－5.5	55	－	219 μmol	5.0－5.5	55	植物肥料	Trouillefou et al., 2015
介孔二氧化硅	黑曲霉 A. niger	蒺藜苜蓿	－	4.7 μmol	5.0－5.5	55	－	154 μmol	5.0－5.5	55	植物肥料	Trouillefou et al., 2015
环氧树脂	鳄梨 P. americana Mill	植酸钠	5000	－	4.0	55	12500	－	5.5	55	豆类产品水解酶	Çelem et al., 2009a;
环氧树脂	鳄梨 P. americana Mill	豆浆植酸	5000	56%	4.0	55	12500	65%	5.5	55	豆类产品水解酶	Çelem et al., 2009b
藻酸盐	克雷伯氏菌 Klebsiella	植酸	－	－	5.0	37	－	－	6.0	70	动物饲料	Hidayatullah et al., 2020
藻酸钙	米曲霉 Aspergillus oryzae	植酸钙	200	－	5.0	50	660	－	5.0	50	食品添加剂	Sharma et al., 2023
藻酸盐	紫原青霉 P. purpurogenu	植酸钠	－	－	5.5	37	－	－	4.0	45	环境修复	Awad et al., 2015
藻酸钠	豇豆种子 V. unguiculata	植酸钠	460	－	5.0	45	4660	－	6.0	55	生物技术、生物医学	Duru et al., 2021
Fe₃O₄磁性纳米颗粒	黑麦 Secale cereale	植酸	307	－	6.0	45	455	－	6.0	60	生产肌醇磷酸	Greiner et al., 2013
	黑曲霉 A. niger		54	－	5.0	55	95	－	5.0	65
	艾伯特埃希菌 E. albertii		133	－	4.5	65	220	－	4.5	70
羧化多壁碳纳米管	大肠杆菌 E. coli	植酸钠	－	－	5.5	55	－	－	6.5	55	动物饲料	Naghshbandi et al., 2018
氨基多壁碳纳米管	耶尔森氏菌 Yersinia intermedia	植酸	0.13	－	5.0	50	0.33	－	5.7	70	饲料研制	Lahiji et al., 2021

纳米材料固定化植酸酶的制备及其催化效率与影响因素综述

Nanomaterial-immobilized Phytase: Preparation, Catalytic Efficiency and Influencing Factors

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