黑曲霉乳糖酶固定化

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学生实验报告-黑曲霉-2014

实验一黑曲霉孢子固定化及其对淀粉糖化的效果分析一、目的要求1. 学习、掌握用包埋法固定微生物细胞的技术及固定化细胞增殖技术。

2. 了解固定化细胞的应用技术。

3. 掌握评价固定化细胞应用效果的方法。

二、试剂①马铃薯斜面培养基：马铃薯200g，葡萄糖20g，琼脂15~20g，定容至1000ml，pH值自然。

②生长培养基：同上（不加琼脂），200ml，灭菌。

③发酵培养基：配制1%、2%、3%、4%的可溶性淀粉溶液各200ml，pH=5.0，灭菌。

④3g海藻酸钠溶于80ml蒸馏水中，灭菌。

⑤0.1M CaCl2溶液250ml，灭菌。

⑥无菌水，500ml，灭菌。

三、实验步骤1. 制备黑曲霉孢子悬液将斜面黑曲霉孢子刮下，用无菌水制成5×108个/ ml孢子的悬液10 ml。

2. 制备海藻酸钠菌悬液将10 ml孢子悬液加入到80ml海藻酸钠溶液中，加入10ml无菌水定容至100ml，配成海藻酸钠浓度为3%、黑曲霉孢子浓度为5×107个/ ml的海藻酸钠菌悬液。

3. 制粒和固定用制粒装置将海藻酸钠菌悬液滴入0.1M CaCl2溶液中使之成粒。

固定化球直径为3mm，室温下固定5小时。

4. 固定化细胞预培养固定化球用无菌水冲洗三次，称重，每瓶15-20g装入生长培养基，25~28℃，220r/min震荡预培养36小时。

5. 摇床发酵培养：固定化球过滤，无菌水冲洗后转入发酵培养基。

对照：0.5%、1%、2%可溶性淀粉，28℃，220r/min震荡培养。

处理：含有15-20g固定化凝胶球的相同浓度的可溶性淀粉，50~60℃，220r/min，24小时6. 还原糖测定用3,5-二硝基水杨酸比色法测还原糖生成量。

取3支25ml刻度试管，编号（对照1管，处理每个浓度2管），按下表精确加入待测液和试剂。

单位：ml对照处理① 处理② 待测液（？%淀粉）1 1 1 蒸馏水1 1 1 3,5-二硝基水杨酸 1.5 1.5 1.5摇匀，沸水浴中，5分钟，取出后立即放入盛有冷水的烧杯中冷却至室温，蒸馏水定容至25ml 。

固定化酶生产低乳糖牛乳的研究

ｃｏｓｎｉ．ｅｃｎｉｏｓｒｄｃｏｏｄｔｎｆｌｌｔｓｍｉｎｅｕａｅｓｂｌｆｅｉｂｌｅｃｓｅｆｌ－ｅｒｓｌｋｎＴｏｔｕｕｏｕｔｎｃｎｉｏｓｏｗ— ｃｅｌａｄｔｓｇｔｉｔｏｔｉｇｈｎｐｉｉｏａｏｋｈａｉｙｈｍｍｏｉｄｌｔｅｉｔｌｄｂｄｉａａｎｈｉｅｚ
（．１中国环境管理干部学院生态学系，河北秦皇岛０６０）６０４（．２河北农业大学食品科技学院，河北保定０１０）７０１，
摘要：以离子交换树脂Ｄ５为载体，１１采用吸附交联法固定化黑曲霉来源乳糖酶，并将固定化酶装填于填பைடு நூலகம்床反应器中处理牛乳，
研究固定化酶连续生产低乳糖乳的条件和使用稳定性。试验结果表明：在５０℃下，牛乳以０５／ｎ的流速通．ｍＬｍｉ３过反应器生产低乳糖乳效果最好，可获得７．％的乳糖水解率，达到低乳糖乳的要求。固定化酶在最适条件下连续水解牛乳，每隔２ｐ６缓冲９７０ｈ用Ｈ５．
（．ｐｒｎｆｏｄＳｉｎｅａｄＴｃｎｌｇ，ｒｕｔｒｉｅｓｙｏＨｅｅ，ｏｉｇ０１０，ｉａ２ＤｅａｔｔＦｏｃｅｃｅｈｏｏｙＡｇｉｌｅｍｅｏｎｃｕＵｎｖｒｉｆｂｉＢａｄｎ７０１Ｃｈｎ）ｔ
ｒａｔｒ、ｅｅｒｓａｃｅ．ｓｌｓｏｄａ，ｆｒｃｎｉｕｕｒｄｃｏｆｌｗ— ｃｏｅｍｉｅｆｅ－ｅｅｃｏ，ｅｂｓｖｌｃｔｄｅｃｏ Ⅳ ｒｅｅｒｈｄＲｅｕｔｈｗｅｔｔｏｏｔｏｓｐｏｕｔｎｏｈｎｉｏｌｔｓｌｉｔｌｄｂｄｒａｔｒｔｅｔｅｏｉａａｋｎｈｉｈｙｎｒａｔｎｔｍｐｒｔｒｅｃｏｅｅａｕｅｗｅｅ０５ｉｒ．３ｍＬｍｎａｄ５ｌｉｎ０℃ ，ｅｐｃｖｌ，ｎｅｗｈｃｈｅｈｄｏｙｉｒｔｆａｔｓｅｃｅ９７，ｅｉｇｔｅｒｑｅｔｒｓｅｔｅｙｕｄｒｉｈｔｉｙｒｌｓａｅｏｌｃｏｅｒａｈｄ７．％ｍｅｔｈｅｕｓｓｎ

乳糖酶的生产技术及其在食品工业应用研究进展.介绍

乳糖酶的生产技术及其在食品工业应用研究进展摘要：乳糖酶亦称β-半乳糖苷酶，在工业生产中有广泛的应用，本文通过来源及其性质、基础研究与应用等方面对乳糖酶进行综述。

关键词：乳糖酶；固定化；应用乳是各种哺乳动物哺育其幼仔最理想的天然食物。

它富含优质蛋白质、乳脂、乳糖等营养成分和钙、磷、钾等矿物质以及多种维生素，还含有多种免疫物质、酶、激素等具有生理活性调节功能的生物活性物质。

乳糖是哺乳动物乳汁中特有的糖类，它是由一分子葡萄糖和一分子半乳糖组成的双糖，其合成步骤为：以葡萄糖为前体物质，一部分葡萄糖先转化为半乳糖，然后经乳糖合成酶催化。

半乳糖与葡萄糖结合，形成乳糖。

人体摄入乳糖后，在消化过程中，经乳糖酶催化，分解为葡萄糖和半乳糖。

乳糖是矿物质的载体，能促进钙、磷吸收及整理肠道，其分解产物半乳糖是婴儿脑发育的必需物质，参与脑组织及其神经系统的构成。

但是，机体却不能直接利用乳糖，乳糖必须经乳糖酶分解为单糖后才能被吸收和利用（杨卉新等，2014）。

若乳糖酶缺乏者一次摄入较多乳糖，乳糖未能及时被消化吸收，进入结肠后被肠道细菌分解，产生大量乳酸、甲酸等短链脂肪酸和氢气，造成渗透压升高，使肠腔中的水分增多，引起腹涨、肠鸣、肠绞痛直至发生水泻等症状，总称为乳糖不耐受症。

乳糖不耐受症状，在中国人群中发生率很高，因此限制了很大一部分国人对牛奶的摄入，而牛奶又是人类良好的优质蛋白、矿物质及维生素的天然来源，故乳糖酶缺乏问题显得尤为突出（张玉英，2014）。

1889年荷兰生物学家，Beijerineek首次报道了乳糖酶可水解乳糖以来，人们对于乳糖酶的研究日趋完整（蒋世琼，2000）。

目前，解决乳糖不耐受的最佳方法是用乳糖酶水解乳糖来生产低乳糖或无乳糖乳制品。

而现在商业乳糖酶中乳糖酶的最适温度在37℃左右或者更高（P Nicholas，2002）。

国外学者经多年研究，已成功地找到产乳糖酶的微生物，并研制了一系列乳糖酶商品，现已投入市场。

固定化乳糖酶制备低乳糖牛奶的研究进展

因此，就中国乳品业来说，一方面牛乳是人类最佳的饮食，需要大力普及全民营养知识，让广大消费者自觉饮用牛乳，以更好地增加国民体质；另一方面，由于乳糖不耐症，有很多人无法
充分利用牛乳，这对牛乳的生产及消费的增长具有较大的阻碍作用。降低牛乳中的乳糖含量，对于满足乳糖不耐症患者的乳品消费需求，以及促进我国乳品工业的发展具有重要的意义。
卜建斌，云战友［23］以酰肼颗粒为载体、戊二醛为交联剂，通过共价结合制得固定化乳糖酶，对它的特性进行了研究。结果表明，固定化乳糖酶的最适反应温度为 38 41℃ ，最适反应 pH 值为 6. 5 7. 0，动力学常数 Km = 304. 87mmol / L， Vmax = 0. 10mmol / （ g · min），固定化酶在较低温度下的使用稳定性好，在 10℃ 下反应 65 个批次后活性仅降低 15. 8% ，而在室温下（ 24 26℃ ）经过 65 批的反应活性下降将近 50% 。 2. 4 多种方法共用
196
2. 2 交联法（ cross － linking）使酶与带两个以上的多官能团试剂进行交联
反应，生成不溶于水的二维交联聚集体。作为交联剂的有形成希夫碱的戊二醛，形成肤键的异氰酸酯，发生重氮偶合反应的双重氮联苯胺等，其中戊二醛最为常用［13］。但是交联法一般不单独使用，而是与其他方法结合一块使用。
酶会使牛奶掺入外来蛋白以及使用游离乳酶酶提高了生产成本，从而使乳糖酶的应用受到限制。将乳糖酶固
定化后既可以重复使用，又能连续操作，且缩短了处理时间，从而明显降低了使用成本，因此对于乳糖酶的
固定化受到了酶学专家的关注。本文简要介绍了乳糖和乳糖酶及其分类、乳糖酶的固定化方法及其应用，包

乳糖酶的工业生产原理

乳糖酶的工业生产原理
乳糖酶是一种酶，它能够将乳糖分解成葡萄糖和半乳糖。

乳糖酶的工业生产原理主要包括以下几个步骤：
1. 菌种培养：首先，培养乳糖酶产生菌株。

常用的菌株有放线菌、大肠杆菌等。

这些菌株经过筛选和改良，具有较高的乳糖酶产量。

2. 酶的提取与纯化：将培养得到的菌株进行破碎、离心等操作，获取到含有乳糖酶的菌液。

接下来，通过过滤、酶解和纯化等步骤将乳糖酶从菌液中分离出来，获得较纯的乳糖酶。

3. 乳糖酶的固定化：为了提高乳糖酶的稳定性和重复使用性，常常将乳糖酶固定在固体载体上。

常用的固体载体有树脂、硅胶、海藻酸钙等。

将乳糖酶与固体载体相混合，通过吸附、交联等方法将酶固定在载体上，形成固定化乳糖酶。

4. 生产过程的优化和控制：在乳糖酶的工业生产过程中，需要优化和控制多种因素，包括合适的菌种培养条件、培养基的配方和浓度、酶的提取条件、固定化乳糖酶的反应条件等。

5. 产品的提取和纯化：最后，将固定化乳糖酶的反应体系进行分离和纯化，以获得高纯度的乳糖酶产品。

乳糖酶的工业生产原理可以根据具体的生产需求和工艺技术进行调整和改进。

乳糖固定化实验报告

一、实验目的1. 了解乳糖酶的性质和作用。

2. 掌握乳糖酶固定化的基本原理和方法。

3. 研究不同固定化方法对乳糖酶活性的影响。

4. 探讨固定化乳糖酶在乳糖水解中的应用。

二、实验原理乳糖酶（β-半乳糖苷酶）是一种能够催化乳糖水解为葡萄糖和半乳糖的酶。

乳糖不耐受症患者由于体内缺乏乳糖酶，无法消化乳糖，导致食用乳制品后出现腹胀、腹泻等症状。

固定化乳糖酶可以克服传统酶制剂的缺点，如酶活性不稳定、易失活等，从而提高乳糖水解效率。

本实验采用化学结合法将乳糖酶固定化在载体上，通过比较不同固定化方法对酶活性的影响，筛选出最佳的固定化方法。

三、实验材料与仪器材料：1. 乳糖酶2. 载体：壳聚糖、明胶、海藻酸钠3. 乳糖4. 磷酸盐缓冲液5. pH计6. 离心机7. 酶标仪仪器：1. 烧杯2. 移液器3. 恒温水浴锅4. 电子天平5. 显微镜四、实验步骤1. 乳糖酶溶液的制备：将乳糖酶用磷酸盐缓冲液溶解，配制成一定浓度的酶溶液。

2. 载体的制备：a. 壳聚糖：将壳聚糖用磷酸盐缓冲液溶解，配制成一定浓度的溶液。

b. 明胶：将明胶用磷酸盐缓冲液溶解，配制成一定浓度的溶液。

c. 海藻酸钠：将海藻酸钠用磷酸盐缓冲液溶解，配制成一定浓度的溶液。

3. 乳糖酶固定化：a. 壳聚糖固定化：将壳聚糖溶液与乳糖酶溶液混合，搅拌均匀，加入一定量的交联剂戊二醛，反应一定时间后，用离心机分离固定化酶。

b. 明胶固定化：将明胶溶液与乳糖酶溶液混合，搅拌均匀，加入一定量的交联剂戊二醛，反应一定时间后，用离心机分离固定化酶。

c. 海藻酸钠固定化：将海藻酸钠溶液与乳糖酶溶液混合，搅拌均匀，加入一定量的交联剂戊二醛，反应一定时间后，用离心机分离固定化酶。

4. 固定化酶的活性测定：将固定化酶分别与乳糖溶液混合，在一定条件下进行水解反应，通过测定水解产物的浓度来计算酶的活性。

5. 结果分析：比较不同固定化方法对乳糖酶活性的影响，筛选出最佳的固定化方法。

五、实验结果与分析1. 固定化酶的活性：通过实验发现，壳聚糖固定化酶的活性最高，明胶固定化酶的活性次之，海藻酸钠固定化酶的活性最低。

黑曲霉的固定化

Removal of heavy metals from industrial wastewater by free and immobilized cells of Aspergillus nigerK.Tsekova a ,*,D.Todorova a ,S.Ganeva ba Stephan Angeloff Institute of Microbiology e Bulgarian Academy of Sciences,Acad.G.Bonchev,Str.,bl.26,1113So ﬁa,Bulgaria bFaculty of Chemistry e So ﬁa University,1164So ﬁa,Bulgariaa r t i c l e i n f oArticle history:Received 24March 2010Received in revised form 5May 2010Accepted 9May 2010Available online 7June 2010Keywords:Biosorption Heavy metals Wastewater Immobilization Aspergillus nigera b s t r a c tAspergillus niger ,strain B 77,was immobilized by inclusion in two different polymers:polyvinyl e alcohol hydrogel (PVA)and Ca e alginate.The biomass/polymer matrices were formed into equal size unites of the cubes and spheres,and the resulting biomass/polymer matrices were used to remove heavy metals (Cu 2þ,Mn 2þ,Zn 2þ,Ni 2þ,Fe 3þ,Pb 2þ,Cd 2þ)from wastewater in shake ﬂask experiments.Total biosorption capacities of the biosorbents were in the following order:free cells (33.3mg/g)<PVA e biomass (39.8mg/g)<Ca alginate e biomass (44.6mg/g).The metal removal ef ﬁciencies of the beads Ca alginate e biomass were 96.2%for Cd 2þ;90.0%for Pb 2þ;80.0%for Fe 3þ;72.8%for Cu 2þ;55.4%for Zn 2þ;54.4%for Ni 2þand 52.3%for Mn 2þ,while the removal ef ﬁciencies of cubes PVA e biomass for the same heavy metals ions were:95.0%;88.0%;80.0%;67.1%;58.5%;48.9%and 44.6%,respectively.The results obtained from these experiments,were compared with those using dispersed biomass as a sorbent.Promising results were obtained in the laboratory,as effective metal removals were observed.Ó2010Elsevier Ltd.All rights reserved.1.IntroductionThe increase use of heavy metals in difference industrial activ-ities causes their existence in wastewater.The discharge of heavy metal ions in industrial ef ﬂuent is of great concern because of their toxic effect on living species,even at very low concentrations.The detoxi ﬁcation of metal ions from industrial ef ﬂuent using biosorption processes is an area of extensive research during the last years (Volesky and Holan,1995;Tripathi et al.,2007).Biosorption is an innovative and low cost effective method for the removal of toxic substances from wastewaters (Zouboulis et al.,2003;Silva et al.,2009;Chatterjee et al.,2010).Fungi are recognized for their superior ability to produce a large variety of extra cellular proteins,organic acids,enzymes and other metabolites,and their waste biomass may be used as effective biosorbent for removal,reduction and detoxi ﬁcation of industrial ef ﬂuents ingredients (Gupta and Mukerji,2001;Christian et al.,2005).Various fungal species under the genus Aspergillus ,Penicil-lium and Rhizopus have been shown to be effective in biosorption of heavy metals from polluted ef ﬂuents both as immobilized cells and in the mobilized state (Kapoor and Viraraghvan,1995;Leitão,2009;Tsekova et al.,2010a,b ).During the last decade many research works have been focused on the development of immobilized systems of microorganisms into polymeric matrices suitable for metal ions uptake applications (Zouboulis et al.,2003;Tsekova et al.,2008;Mata et al.,2009).Biosorption has been considered as a promising technology for the removal of low levels of toxic metals from industrials ef ﬂuents and natural waters.In view of potential applications in remediation of heavy metals from aqueous solutions the immobilization of the biomass is generally necessary.Immobilized cells are usually easier to handle,require less complex separation systems,allow a high biomass density to be maintained and provide a greater opportu-nity for reuse and recovery.Despite the current interest in microbial detoxi ﬁcation of ef ﬂuents,relatively little work has been concerned with charac-terization of metal uptake by ﬁlamentous fungi,particularly when the heavy metals present at different and low concentrations.The purpose of this study was to determine the ability of Aspergillus niger (free and immobilized biomass)to remove toxic metals from an industrial wastewater by batch system.2.Materials and methods 2.1.Sample collection siteWastewater samples were from copper production factory in Pirdop,Bulgaria.The samples were collected for a relative long*Corresponding author.Tel.:þ35929793167;fax:þ35928700109.E-mail addresses:ktsekova@microbio.bas.bg (K.Tsekova),sganeva@chem.uni-so ﬁa.bg (S.Ganeva).Contents lists available at ScienceDirectInternational Biodeterioration &Biodegradationjou rn al homepage:/locate/ibiod0964-8305/$e see front matter Ó2010Elsevier Ltd.All rights reserved.doi:10.1016/j.ibiod.2010.05.003International Biodeterioration &Biodegradation 64(2010)447e 451period of time(April e November2009)in high e grade plastic bottles of1.5l capacity and stored in a refrigerator at4 C.From all taken wastes a representative sample was prepared mixing150ml from each separate bottle.2.2.Determination of heavy metal concentrationsThe initial concentration of the metals in the wastewater and samples after biosorption treatment were determined using an Atomic Absorption Spectrophotometer Perkin Elmer Analyst400, air-acetyleneﬂame.Cadmium and lead after biosorption were determined using electrothermal atomic absorption spectrom-eter Zeeman Perkin Elmer3030,pyrolytically coated graphite tubes and optimized temperature program for modiﬁer freeETAAS.2.3.Preparation of biosorbents2.3.1.Microorganisms,medium and cultivationThe mutant strain A.niger B77(A.niger),an industrial producer of glucoamylase(N 65/1980,National Bank of Industrial Micro-organisms and Cell Cultures,Bulgaria)was used in this study. Spores from established culture(6e7days old)incubated on potato glucose agar slants at30 C were used for the preparation of inocula.The liquid growth medium consisted of(g/L):glucose30 and corn steep liqueur45;pH adjusted to4.8.Cultivation of A.niger was carried out in500e ml Erlenmeyer ﬂasks with75ml growth medium on a rotary shaker at30 C.After 48h of cultivation,the mycelium was harvested byﬁltration from the medium,washed twice with distilled water and stored at4 C until use.The dry weight of the free fungal biomass(FC)was determined after drying at85 C for48h.2.3.2.Immobilizing materials and techniquesPoly(vinyl)alcohol(PVA)hydrogel was obtained as describe earlier(Tsekova et al.,2008).The PVA e hydrogel used for immo-bilization was cut into small pieces(4Â4Â4mm in size).They were washed with distilled water and than submerged in500-ml Erlenmeyerﬂasks containing75ml growth medium(previously autoclaved for20min at120 C).The carrier pieces were inoculated with10ml A.niger spores suspension(1Â106/ml)were cultivated as described above.After48h of incubation,the PVA e immobi-lized biomass of A.niger was harvested from the medium,washed twice with distilled water and stored at4 C until used as a bio-sorbent.The dry weight of PVA e biomass was determined as described for FC.Method described by El e Naggar et al.(2006)was used to prepare Ca alginate beads the spore suspension(10ml,1Â106/ml) was mixed with Na e alginate prior to its stabilization.Beads of approximately4mm diameter were obtained by selecting an appropriate oriﬁce size through which the polymer/spores mixed passed.The cultivation was carried out as described above.After 48h of incubation,the Ca alginate e immobilized biomass of A.niger was harvested from the medium,washed twice with distilled water and stored at4 C until used as a biosorbent.The dry weight of Ca alginate e biomass was determined as described for FC.2.4.Batch biosorption studiesBatch biosorption experiments were carried out in500ml Erlenmeyerﬂasks,as follows:preweighed biosorbent samples (wet or immobilized biomass)with concentration varying from0.1 to0.5g/l(dry weight)were examined.Each sample was added to 100ml of real wastewater,containing heavy metal ions.Biosorption studies were performed at different initial pH from5.5to7.5using 0.1N NaOH.The mixtures were then agitated at120rpm on a rotary shaker up to30min at25 C.Then the content of theﬂasks was separated byﬁltration using a Whatman N 1ﬁlter paper.2.5.Metal uptake(q)Uptake of metal ions was calculated from a metal mass balance yielding:q¼VðC iÀC fÞ(1) where:q is mg metal ions per g dry biosorbent;V is the reaction volume(l),C i and C f are the initial and residual metal concen-trations(mg/l),respectively,and m is the amount of dry bio-sorbent(g).The concentration of the metal ions in theﬁltrates was deter-mined using atomic absorption spectrophotometer with an air/ acetyleneﬂame(model2380;Perkin Elmer,Uberlingen,Germany).Aliquots of wet biomass as well as of immobilized biomass, followed by drying for48h at85 C,were considered as dry biosorbent to calculate the uptake.The efﬁciency of heavy metal removal was calculated from the amount of metal ions adsorbed on the biosorbent and the amount of metal ions available in the wastewater,as the following equation:%removal¼mg heavy metal ions removedmg heavy metal ions availableÂ100(2)2.6.ReproducibilityAll the experiments were run in triplicates and controls were also run on same pattern without addition of biosorbent.The data shown are average from three separate experiments.Table1Characteristics of the biosorption process for removal of heavy metals from waste-water using free biomass as a sorbent.Performance of biosorbentof A.niger biomassHeavy metals in mixed solution,mg/lCu Zn Ni Fe Pb Cd Mn Initial concentrations mg/l7 1.30.90.20.050.0813 Equilibrium concentrations mg/l 2.8 1.10.460.060.0060.0048.1 Equilibrium time min20151555520 Heavy metal uptake mg/g140.66 1.50.460.150.2516.3 Removal efﬁciency%6015.448.970889537.7 Free biomass(m)0.3g/l;pH e6.5;number of parallels:(n)3;relative standard deviation(RSD)3e8%.Table2Characteristics of the biosorption process for removal of heavy metals from waste-water:PVA e biomass m¼0.3g/l;pH e6.5.Performance of biosorbent Heavy metals in mixed solution,mg/lCu Zn Ni Fe Pb Cd Mn Initial concentrations mg/l7 1.30.90.20.050.0813 Equilibrium concentrations mg/l 2.30.540.460.040.0060.0047.2 Equilibrium time min1510555515 Heavy metal uptake mg/g15.6 2.5 1.50.530.140.2519.3 Removal efﬁciency%67.158.548.980889544.6 Number of parallels:(n)3;relative standard deviation(RSD)3e8%.K.Tsekova et al./International Biodeterioration&Biodegradation64(2010)447e451 4483.Results and discussion3.1.Sorption of heavy metals from wastewater by freeand immobilized cells of A.niger3.1.1.Sorption of heavy metals from industrial wastewater by free biomass of A.niger (FC)A.niger biomass absorbed Fe 3þ,Pb 2þand Cd 2þions from industrial wastewater more rapidly than other ions (Table 1)within 15e 20min.Experiments indicated that sorption equilibrium reached much faster in case of industrial wastewater sample (up to 20min)in comparison to single ions solution (up to 30min)using same biosorbent (Tsekova et al.,2010a ).These results are impor-tant,as equilibrium time is one of the important parameters for selecting a wastewater treatment system.This may be due to the presence of co e metal ions in the industrial ef ﬂuents as well as to the differences in the heavy metal ions concentrations (Muhammad et al.,2009;Chatterjee et al.,2010).The removal percentages order at equilibrium was:Cd 2þ(95%)>Pb 2þ(88%)>Fe 3þ(70%)>Cu 2þ(60%)>Ni 2þ(48.9%)>Mn 2þ(37.7%)>Zn 2þ(15.4%)3.1.2.Sorption of heavy metals by immobilized biomass of A.nigerThe effect of immobilization of A.niger on PVA e hydrogel as well as on Ca alginate on the removal of heavy metal ions by adsorption was investigated.The metal removal study,illustrated in Tables 2,3showed that their removals were affected by immobilization of A.niger in comparison to the removal ef ﬁciency by free biomass (Table 1).In general,the both immobilized biosorbents displayed higher bio-sorption capacities for Mn 2þand Cu 2þ,presented in the ef ﬂuent at higher initial concentrations.There was however considerabledifference in total biosorption capability of the test fungal bio-sorbents.Immobilized on Ca alginate cells of A.niger showed highest total biosorption capacity of 71.6mg/g for all heavy metal ions (Table 3),followed by PVA immobilized cells(68.9mg/g,Table 2)and free cells(59.3mg/g j ,Table 1),respectively.Meanwhile,the superior biosorption potential of Ca alginate e immobilized cells (22.6mg/g and 17mg/g)over PVA e immobilized ones (19.3mg/g and 15.6mg/g)was observed in the case of Mn 2þand Cu 2þions,respectively.In general,removal ef ﬁciency of the test biosorbents,for available metal ions,was observed to follow the sequence in following mode:FC (59.3%)<PVA e biomass (68.9%)<Ca alginate e biomass (71.6%).The both immobilized biosorbents displayed high removal potential for Cd 2,Pb 2þ,Fe 3þ,in comparison to other heavy metal ions from the industrial ef ﬂuent.At the same time the immobilized biomass in Ca alginate exhibited the highest biosorption potency toward Mn 2þand Cu 2þions,that ’s why it was chosen for the following investigations.3.1.3.Effect of pHFig.1shows the effect of pH on the biosorption of different metals by A.niger immobilized biomass.Removal ef ﬁciency was analyzed over a pH range 5.5e 7.5.The results show that the metal sorption was a function of pH,as the pH increased from 5.5to 6.5,adsorption capacity increased at ﬁrst for all metals.Maximum adsorption occurs at pH 6.5for Fe 3þ,Cu 2þ,Pb 2þand Cd 2þ,and at pH 7.5for Zn 2þ,Ni2þand Mn 2þ.The maximum removal ef ﬁciencies (%)for the different metals by Ca alginate e biomass were 96.3%for Cd 2þ(at pH 7.5)>90%for Pb 2þ(at pH 7.5)>80%for Fe 3þ(at pH 6.5)>72.8%for Cu 2þ(at pH 6.5)>61.5%for Mn 2þ(at pH 7.5)>59.7%for Zn 2þ(at pH 7.5)>58.9%for Ni 2þ(at pH 7.5).After pH 7.5the ef ﬁciency of the metal removal process increases drastically due to the formation of metal hydroxides with their respective metal ions (Zouboulis et al.,2003).This is mostly due to the metal precipitation as hydrox-ides which depend on the pH and ion concentration,but not due to the biosorption (Al e Qodah,2006;AjayKumar et al.,2009).pH value is one of the main factors in biosorption ef ﬁciency of different biosorbents.The different pH binding pro ﬁles for different metal ions are due to the nature of the chemical interactions of metal ions with the biosorbent.Solution pH in ﬂuences surface metal binding sites of the biosorbents and the chemistry of the cell walls,as well as physicochemistry and hydrolysis of the metals.Table 3Characteristics of the biosorption process for removal of heavy metals from waste-water:Ca alginate e biomass m ¼0.3g/l;pH e 6.5.Performance of biosorbentHeavy metals in mixed solution,mg/l CuZnNiFePbCdMn Initial concentrationsmg/l 7 1.30.90.20.050.0813Equilibrium concentrations mg/l 1.90.580.410.040.0050.003 6.2Equilibrium time min 1510555515Heavy metal uptake mg/g 17 2.4 1.60.530.150.3022.6Removal ef ﬁciency%72.855.454.4809096.252.3Number of parallels:(n)3;relative standard deviation (RSD)3e 8%.102030405060708090100Cu Zn Ni Fe Pb Cd Mnr e m o v a l , %pH - 6.5pH - 7.5Fig.1.Effect of pH on metal ions removal from wastewater using Ca alginate e immobilized cells of Aspergillus niger as a biosorbent.(m 0.3g/l dry weight).Vertical bars show standard error of means of three replicates.K.Tsekova et al./International Biodeterioration &Biodegradation 64(2010)447e 4514493.1.4.Effect of adsorbent weight (g/l)The effect of adsorbent weight (g/l)on the adsorption ef ﬁciency of the best fungal biosorbent (Ca alginate e immobilized cells)is shown on Fig.2.Adsorption experiments were carried out at different biosorbent doses ranging from 0.1to 0.5g/l in mixed ions solution.It was observed as a general trend that there is an increase of the removal percentage with increase in adsorbent weight from 0.1to 0.3g/l.The maximum removal of the most heavy metal ions was attained at an adsorbent dose of 0.3g/l with no further signi ﬁcant increase in the removal percentage at higher biosorbent concentration tested was observed.In the case of Fe 3þ,Mn 2þand Ni 2þmaximum removal was attained at 0.5g/l of adsorbent weight.Removal ef ﬁciency increases for Fe 3þfrom 80%till 90%,Mn 2þfrom 52.3%till 61.5%,and for Ni 2þfrom 58.9%till 79%when the sorbent mass increases from 0.3g/l to 0.5g/l.These results are in agreement with previously studies on many other adsorbents (Yu et al.,2001;Dakiky et al.,2002).As the biosorbent mass increases the number of available binding sites or surface area for the heavy metal ions also increased.However,the removal ef ﬁciency of Pb 2þand Cd 2þretained constant when the biosorbent weight increased.Accord-ing to the previous works,higher biosorbent dose could produce a “screening ”effect on the binding sites,thus resulting in lower heavy metal uptake (Yahaya et al.,2009;Tsekova et al.,2010a,b ).3.1.5.Secondary chemical treatment of the ﬁltrate after biosorptionTo the ﬁltrate obtained after biosorption 5ml 2%sodium diethyldithiocarbamate (NaDDTC)solution and 4g activated char-coal were added,mixed for 10min and ﬁltered through Whatman N 1ﬁlter paper.In this second ﬁltrate the concentration of all the examined heavy metals was below the detection limit of the measurement method.Such successful procedure for completely removing of heavy metals from ef ﬂuents has been not reported in the literature yet.4.ConclusionThe ability of A.niger biomass to bind and remove heavy metals,i.e.Cu 2þ,Zn 2þ,Ni 2þ,Pb 2þ,Cd 2þ,Fe 3þ,Mn 2þfrom real wastewater was investigated.To overcome the separation problems of using freely suspended biomass form,as well as,mass loss after regen-eration of the biosorbent,the biomass was immobilized in the polymer matrixes (PVA and Ca alginate gels).Biosorption studies of Ca alginate e A.niger beads have been found to be effective in removing of relatively low concentrations of these seven heavy metals from wastewater.The process was mainly in ﬂuenced by pH and biosorbent dose.At pH 6.5and biosorbent dose of 0.5g/l dry weight the removal ef ﬁciencies obtained for Ca alginate e biomass beads were:for Cd 2þe 96.2%;for Pb 2þe 90%;for Fe 3þe 90%;for Cu 2þe 73.5%;for Ni 2þe 70.9%for Zn 2þe 60.9%and Mn 2þe 61.5%.The results obtained showed that immobilized biomass of A.niger ,appears as a possible biosorbent to be used for treatment of metal e polluted industrial wastewaters.The secondary chemical treatment with NaDDTC-activated charcoal showed complete removal of all the studied heavy metals.AcknowledgementsThis work was supported by the National Science Fund at the Ministry of Education and Science of Republic of Bulgaria (Grant DOO2-185/2008)and Operative Program Human Resources (Grant BG051PO001e 3.3.04/32).ReferencesAjayKumar,A.V.,Darwish,N.A.,Hilal,N.,2009.Study of various parameters in thebiosorption on heavy metals on activated sludge (Special Issue for Environ-ment).World Applied Sciences Journal 5,32e 40.Al e Qodah,Z.,2006.Biosorption of heavy metal ions from aqueous solutions byactivated sludge.Desalination 196,164e 176.Chatterjee,S.K.,Bhattacharjee,I.,Chandra,G.,2010.Biosorption of heavy metalsfrom industrial waste water by Geobacillus thermodenitri ﬁcans .Journal of Hazardous Materials 175,117e 125.Christian,V.,Shrivastava,R.,Shukla,D.,Modi,H.A.,Vyas,B.R.M.,2005.Degradationof xenobiotic compounds by lignin e degrading white e rote fungi:enzy-mology and mechanism involved.Indian Journal of Experimental Biology 43,301e 312.Dakiky,M.,Khamis,M.,Manassra,A.,Mer ’eb,M.,2002.Selective adsorption ofchromium (VI)in industrial wastewater using low e cost abundantly available adsorbents.Advance Environmental Research 6(4),533e 540.El e Naggar,M.Y.,El e Assar,S.A.,Youseff,A.Y.,El e Sersy,N.A.,Beltagy,E.A.,2006.Extracellular b e mannanase production by the immobilization of the locally isolated Aspergillus niger .International Journal of Agriculture &Biology 8(1),57e 62.Gupta,R.,Mukerji,K.G.,2001.Bioremediation:Past,present and future.In:Tewari,R.,Mukerji,K.G.,Gupta,J.K.,Gupta,L.K.(Eds.),Role of microbes in the management of environmental pollution.A.P.H.Publishing Corp,New Delhi,pp.73e 81.Kapoor, A.,Viraraghvan,T.,1995.Fungal biosorption:an alternative treatmentoption for heavy metal bearing wastewater:a review.Bioresource Technology 53,195e 206.Cu Zn Ni Fe Pb Cd Mnr e m o v a l , %m - 0.1g/l m - 0.3g/l m - 0.5g/lFig.2.Effect of sorbent dose on metal ions removal from wastewater using Ca alginate e immobilized cells of Aspergillus niger as a biosorbent.(pH 6.5).Vertical bars show standard error of means of three replicates.K.Tsekova et al./International Biodeterioration &Biodegradation 64(2010)447e 451450Leitão, A.L.,2009.Potential of Penicillium species in the bioremediationﬁeld.International Journal of Environmental Research and Public Health6, 1393e1417.Mata,Y.N.,Blázquez,M.L.,Ballester,A.,González,F.,Munoz,J.A.,2009.Biosorption of cadmium,lead and copper with calcium alginate xerogels and immobilized Fucus vesiculosus.Journal of Hazardous Materials163,555e562. Muhammad,R.,Nadeem,R.,Hanif,M.A.,Ansari,T.M.,Rehman,K.U.,2009.Pb(II) biosorption from hazardous aqueous streams using Gossypium hirsutum (Cotton)waste biomass.Journal of Hazardous Materials161,88e94.Silva,R.M.P.,Rodriguez,A.Á.,De Oca,J.M.G.M.,Moreno,D.C.,2009.Biosorption of chromium,copper,manganese and zinc by Pseudomonas aeruginosa AT18 isolated from a site contaminated with petroleum.Bioresource Technology100, 1533e1538.Tripathi,A.K.,Harsh,N.S.K.,Gupta,N.,2007.Fungal treatment of industrial efﬂu-ents:a mini e review.Life Science Journal4(2),78e81.Tsekova,K.,Christova,D.,Todorova,D.,Ivanova,S.,2008.Biosorption of ternary mixture of heavy metals by entrapped in PVA e hydrogel biomass of Penicillium cyclopium.Comptes rendus de l`Academie bulgare des Sciences61(9),1175e1180.Tsekova,K.,Todorova,D.,Dencheva,V.,Ganeva,S.,2010a.Biosorption of copper(II)and cadmium(II)from aqueous solutions by free and immobilized biomass ofAspergillus niger.Bioresource Technology101,1727e1731.Tsekova,K.,Christova,D.,Dencheva,V.,Ganeva,S.,2010b.Biosorption of binarymixture of copper and cobalt by free and immobilized biomass of Penicilliumptes rendus de l`Academie bulgare des Sciences63(1),85e90.Volesky,B.,Holan,Z.R.,1995.Biosorption of heavy metals.Biotechnology Progress11(3),235e250.Yahaya,Y.A.,Don,M.M.,Bhatia,S.,2009.Biosorption of copper(II)onto immobilizedcells of Pycnoporus sanguineus from aqueous solution:equilibrium and kineticstudies.Journal of Hazardous Materials161,189e195.Yu,B.,Zhang,Y.,Shukla,S.S.,Dorris,K.L.,2001.The removal of heavy metals fromaqueous solutions by sawdust adsorption:removal of lead and comparison ofits adsorption with copper.Journal of Hazardous Materials84,83e94.Zouboulis,A.I.,Matis,K.A.,Loukidou,M., Sebesta,F.,2003.Metal biosorption by PAN e immobilized fungal biomass in simulated wastewaters.Colloids andSurfaces212,185e195.K.Tsekova et al./International Biodeterioration&Biodegradation64(2010)447e451451。

酶的固定化技术及其应用综述

酶的固定化技术及其应用曾鸿雁(西南科技大学,四川,绵阳)摘要:随着工业生物技术和酶工程的不断发展，酶在各个领域的广泛应用，对酶的要求也越来越严格。

本文针对目前酶工程技术之一酶的固定化，对酶的固定化技术及其展望做一综述。

关键词：酶，固定化，技术Immobilization of Enzyme And its Applications Abstract：with the continuous development of biotechnology industrial and enzyme engineering , enzyme are widely used in various fields and the requirements to enzymes also become more and more stringent . This article is to review the enzyme immobilization, which is one of the current enzyme engineering technologiesKey words: enzyme, immobilization, technology一、引言酶是一类具有生物催化性质的高分子物质，其催化性具有专一性强、催化效率高和作用脚尖温和等特点。

但是在实际工业生产中，由于实际环境因素，应用酶的过程出现了一些不足之处：①酶的催化效率不高。

人们在使用酶的过程中，往往要求酶的催化效率要足够高，以加快反应速度，提高劳动生产率，然而实际上很多酶的催化效率不够高而难于满足人们的使用要求。

②酶的稳定性较差。

大多数酶稳定性较差，在高温、强酸、强碱和重金属离子等外界因素的影响下，都容易变形失活。

③酶的一次性使用。

酶一般是在溶液中与底物反应，这样酶在反应系统中，与底物和产物混合在一起，反应结束后，即使酶仍有很高的活力，也难于回收利用。

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黑曲乳糖酶的固定化
方法：吸附交联法
载体：以阳离子交换树脂D151
交联剂：戊二醛
与游离酶性质相比：
最适作用温度：降低
热稳定性：降低。

最适pH值：乳糖酶经固定化处理后,其最适pH值较游离酶稍向碱性方向移动
酸碱稳定性：固定化酶酸碱稳定pH值范围在2.5 ～ 4.5;而游离酶酸碱
稳定pH值范围在5.0 ～7.0。

总之，应用吸附交联法制得的固定化乳糖酶, 较游离酶在牛奶的天然pH值下使用
更为适宜, 且操作稳定性好。

脂环酸芽孢杆菌α-葡萄糖苷酶固定化
方法：物理交联法
载体：壳聚糖
交联剂：戊二醛
最适pH：固定化α-葡萄糖苷酶的最适pH 4.5，与游离酶的pH 5.2 相比，其最适pH 值向酸性方向偏移，具有更强的耐酸性，但对碱的耐受性有所降低。

最适温度：固定化酶的最适温度较游离酶有所提高，从游离酶的52 ℃提高到固定化酶的55 ℃，说明固定化后α-葡萄糖苷酶的热稳定性有所提高。

酸碱稳定性：游离酶的耐碱性较固定化酶弱，而pH 大于9.5 时固定化酶活性降低，小于游离酶。

热稳定性：经固定化处理的α-葡萄糖苷酶的热稳定性有了极大的提高。

当温度65 ℃时，固定化酶保持近100%酶活力，而游离酶完全失去活性。

总之，相对于游离酶，经壳聚糖固定化后酶的活性中心构象更稳固，削弱了不利因素对酶活性的影响，增加了固定化α-葡萄糖苷酶的稳定性，有利于工业化应用。

漆酶固定化
方法：物理吸附法
载体：纳米多孔金
最适pH：固定化后漆酶具有与游离酶相似的活力-pH 轮廓图, 最佳反应pH 都是
4.5。

与游离酶相比固定化不但使漆酶可稳定的pH 范围变宽了(pH 3.5～6.0), 也明显提高了漆酶的耐酸耐碱性
最适温度：固定化酶的最佳反应温度为60 ℃, 较游离酶的40 ℃上升了20 ℃
热稳定性：固定化后漆酶的热稳定性明显提高
重复利用性：固定化酶具有很好的重复利用性
总之，相对于游离态, 固定化漆酶的最适pH 没有改变, 但最适温度却从游离态的40 ℃上
升至60 ℃. 尽管固定化漆酶的催化效率要低于游离漆酶, 然而固定化后漆酶的pH 和热稳定性都明显提高了;重复使用8 次仍能保持初始活力的65%, 且在4 ℃下保存1 个月几乎观察不到明显的活力下降.。