Loofa (Luffa cylindrica) sponge: Review of development of the biomatrix as a tool for biotechnological applications

The review discusses the development of loofa sponge (Luffa cylindrica) as a biotechnological tool and the diversity of applications in which it has been successfully used since it was first reported as a matrix for the immobilization of microbiological cells in 1993. The fibro‐vascular reticulated structure, made up of an open network of random lattices of small cross‐sections coupled with very high porosity (79–93%), having very low density (0.02–0.04 g/cm³), and high specific pore volume (21–29 cm³/g), has the characteristics of a carrier/scaffold well‐suited for cell immobilization. This has been confirmed through the immobilization of cells of diverse types, including filamentous and microalgae, fungi, bacteria, yeasts, higher plants, and human and rat hepatocytes. The cells immobilized in loofa sponge have performed well and better than free suspended cells and those immobilized in conventionally used natural and synthetic polymeric materials for the production of ethanol, organic acids, enzymes, and secondary metabolites. The loofa‐immobilized cell systems have been efficiently used for the treatment of wastewaters containing toxic metals, dyes, and chlorinated compounds, and the technology has been used to develop biofilms for the remediation of domestic and industrial wastewaters rich in inorganic and organic matter. In addition, three‐dimensional loofa sponge scaffolds for hepatocyte culture have been suggested to have the potential for development into a bioartificial liver device. Loofa sponge is a cost‐effective, eco‐friendly, and easy to handle matrix that has been used successfully as a biotechnological tool in a variety of systems, purposes, and applications. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:573–600, 2013     

Performance of Luffa cylindrica as an immobilization matrix for the biotransformation of cholesterol by Mycobacterium species

Abstract

The microbial cleavage of the side chain of cholesterol is a slow process due to the low solubility of the substrate in aqueous media (< 1 μM). Cell immobilization has been shown to be an efficient technology for enhancing the yield of cholesterol biotransformation. In these experiments, living cells of Mycobacterium sp. DSM 2966 and Mycobacterium sp. DSM 2967 were immobilized by passive adsorption on different types of solid carriers. As compared to the control and other solid supports, Luffa cylindrica resulted in a 3–4-fold increase of the specific side chain cleavage activity after 7 days of incubation. Luffa cylindrica had no significant negative influence on cell growth. Furthermore, it is a natural, inexpensive, non-toxic and mechanically strong material and therefore suitable for follow-up experiments.     

Cortical Aerenchyma formation in hypocotyl and adventitious roots of Luffa cylindrica subjected to soil flooding

BACKGROUND AND AIMS: Aerenchyma formation is thought to be one of the important morphological adaptations to hypoxic stress. Although sponge gourd is an annual vegetable upland crop, in response to flooding the hypocotyl and newly formed adventitious roots create aerenchyma that is neither schizogenous nor lysigenous, but is produced by radial elongation of cortical cells. The aim of this study is to characterize the morphological changes in flooded tissues and the pattern of cortical aerenchyma formation, and to analyse the relative amount of aerenchyma formed. METHODS: Plants were harvested at 16 d after the flooding treatment was initiated. The root system was observed, and sections of fresh materials (hypocotyl, tap root and adventitious root) were viewed with a light or fluorescence microscope. Distributions of porosity along adventitious roots were estimated by a pycnometer method. KEY RESULTS: Under flooded conditions, a considerable part of the root system consisted of new adventitious roots which soon emerged and grew quickly over the soil surface. The outer cortical cells of these roots and those of the hypocotyl elongated radially and contributed to the development of large intercellular spaces. The elongated cortical cells of adventitious roots were clearly T-shaped, and occurred regularly in mesh-like lacunate structures. In these positions, slits were formed in the epidermis. In the roots, the enlargement of the gas space system began close to the apex in the cortical cell layers immediately beneath the epidermis. The porosity along these roots was 11-45 %. In non-flooded plants, adventitious roots were not formed and no aerenchyma developed in the hypocotyl or tap root. CONCLUSIONS: Sponge gourd aerenchyma is produced by the unique radial elongation of cells that make the expansigeny. These morphological changes seem to enhance flooding tolerance by promoting tissue gas exchange, and sponge gourd might thereby adapt to flooding stress.     

Blueprint for a lipase support: Use of hydrophobic controlled-pore glasses as model systems

For the commercial exploitation of lipase biocatalysis to be successful, it is essential that effective supports are selected for lipase immobilization. In this study hydrophobic controlled-pore glasses have been used as model systems for the immobilization of Rhizomucor miehei lipase. The effect of pore diameter and surface chemistry on enzyme efficiency in a typical esterification reaction under essentially nonaqueous conditions has been examined. It has been found that pore diameters of at least 35 nm are needed for the lipase to be able to utilize the internal volume of the support particles in the immobilization process. Despite the small size of the substrates in the esterification reaction, even larger pores (>100 nm) are required for the lipase efficiency to become independent of pore diameter; below 100 nm lipase activity and efficiency are markedly reduced. It has also been shown that the chemical nature of the hydrophobic surface plays an important part in catalyst design. Although lipase will adsorb readily to a wide range of hydrophobic groups, the highest catalyst activities are obtained when the glass surface is derivatized to give long alkyl chains; the presence of unsaturated derivatives gonerally leads to a reduction in activity. (c) 1994 John Wiley & Sons, Inc.     

普通丝瓜始雌花节位遗传分析

选用始雌花节位有差异的普通丝瓜品种配制‘五叶香丝瓜’ב短圆筒丝瓜’(L1×L2)和‘短圆筒丝瓜’ב蛇形丝瓜’(L2×L3)2套组合,通过调查P1、P2、F1、B1、B2和F2植株的始雌花节位,利用主基因 多基因混合遗传模型联合分离分析了始雌花节位遗传规律。结果显示:L1×L2始雌花节位遗传符合2对加性-显性-上位性主基因 加性-显性多基因遗传模型,L2×L3的遗传符合1对加性主基因 加性-显性多基因遗传模型;L1×L2组合的B1、B2和F2群体遗传率(主基因 多基因)分别为66.13%、51.29%和68.27%,L2×L3组合的B1、B2和F2群体遗传率(主基因 多基因)分别为82.02%、64.87%和65.62%;L1×L2组合B1、B2和F2群体的环境方差占总表型方差的比例分别是23.43%、48.69%和31.73%,L2×L3组合B1、B2和F2群体的环境方差占总表型方差的比例分别是34.27%、55.40%和34.38%。结果表明:普通丝瓜始雌花节位是由主基因和多基因控制的数量性状,早熟性(较低的始雌花节位)不太可能通过杂种优势来实现;始雌花节位遗传不稳定,易受环境因素的影响,但定向选择会有较好的效果。     

Effects of cholinium-based ionic liquids on Aspergillus niger lipase: Stabilizers or inhibitors

Lipases are well-known biocatalysts used in several industrial processes/applications. Thus, as with other enzymes, changes in their surrounding environment and/or their thermodynamic parameters can induce structural changes that can increase, decrease, or even inhibit their catalytic activity. The use of ionic compounds as solvents or additives is a common approach for adjusting reaction conditions and, consequently, for controlling the biocatalytic activity of enzymes. Herein, to elucidate the effects of ionic compounds on the structure of lipase, the stability and enzymatic activity of lipase from Aspergillus niger in aqueous solutions (at 0.05, 0.10, 0.50, and 1.00 M) of six cholinium-based ionic liquids (cholinium chloride [Ch]Cl; cholinium acetate ([Ch][Ac]); cholinium propanoate ([Ch][Prop]); cholinium butanoate ([Ch][But]); cholinium pentanoate ([Ch][Pent]); and cholinium hexanoate ([Ch][Hex])) were evaluated over 24 hr. The enzymatic activity of lipase was maintained or enhanced in the lower concentrations of all the [Ch]⁺-ILs (below 0.1 M). [Ch][Ac] maintained the biocatalytic behavior of lipase, independent of the IL concentration and incubation time. However, above 0.1 M, [Ch][Pent] and [Ch][Hex] caused complete inhibition of the catalytic activity of the enzyme, demonstrating that the increase in the anionic alkyl chain length strongly affected the conformation of the lipase. The hydrophobicity and concentration of the [Ch]⁺-ILs play an important role in the enzyme activity, and these parameters can be controlled by adjusting the anionic alkyl chain length. The inhibitory effects of [Ch][Pent] and [Ch][Hex] may be of great interest to the pharmaceutical industry to induce pharmacological inhibition of gastric and pancreatic lipases.     

黑曲霉(Aspergillus niger)脂肪酶的纯化及性质分析

从黑曲霉发酵液中经硫酸铵分级沉淀,Phenyl-Sepharose疏水柱层析,DEAE-Sepharose 4B阴离子交换柱得到电泳纯的脂肪酶,纯化倍数达10倍,回收率50％。对脂肪酶的性质分析表明：该酶分子质量约为35kDa,最适温度和最适pH分别为37℃和9．5,50℃以下和pH6．0～11．0之间保持稳定,属于碱性脂肪酶。Mg^2＋、Ca^2＋、Cu^2＋、Zn^2＋、Co^2＋、Mn2^＋对该酶有激活作用,而Al^3＋、Fe^2＋、Fe^3＋对酶有严重抑制作用。变性剂盐酸胍和脲对其未见显著影响,而SDS强烈抑制其酶活。用不同氨基酸修饰剂对酶进行修饰,其中NBS和PMSF强烈抑制该酶活性,NBSF和DTT在低浓度下对酶活影响不大,2,3-丁二酮在高浓度下影响其活性。外加稳定剂如NaCl、PEG、甘油、山梨醇、海藻酸钠,均可不同程度的延长脂肪酶的半衰期。在一定质量比条件下,该酶有良好的抗蛋白酶性质。     

固定化脂肪酶性质及其应用研究

利用以四甲氧基硅烷(TMOS)和甲基三甲氧基硅烷(MTMS)为前驱体的溶胶-凝胶法(sol-gel)固定洋葱假单胞菌属脂肪酶,考查了固定化酶和游离酶的酶学性质及催化不同油脂酯交换合成生物柴油的情况。结果表明,80℃以下固定化酶能保持80%以上的酶活,而游离酶在50℃以后活力急剧下降,到80℃残余酶活约为10%;固定化酶在体积分数50%的甲醇中处理48 h能保持85%的酶活,在体积分数90%的乙醇中处理48h能保持31%的酶活,而游离酶残余酶活只有69%和0;在酯交换反应中固定化酶的催化效率比游离酶高10%~20%,且固定化酶重复使用11次后仍能保持60%的酶活。结果显示,酶经过固定化后稳定性和催化活性显著提高。     

Utilization of discard bovine bone as a support for immobilization of recombinant Rhizopus oryzae lipase expressed in Pichia pastoris

In this study the possibility of using discard bovine bone as support for immobilization of Rhizopus oryzae lipase expressed in Pichia pastoris was analyzed. Discard bovine bone were milled and then subjected to a chemical treatment with acetone in order to remove lipids and blood traces. Two types of supports were evaluated: bovine bone and calcined bovine bone for 2 h at 600°C. Supports were characterized by: ICP, SEM, XRD, FTIR, XPS, and N₂ adsorption isotherms. Calcined bovine bone presented appropriate characteristics for the lipase immobilization due to the removal of collagen: high porosity, large surface area and suitable porous structure. Biocatalysts were prepared with different initial enzyme load. For the equilibrium adsorption studies, the Langmuir isotherm was used to fit the data results. The immobilization occurs in monolayer to a value of 35 UA mg^?1. The activities of biocatalysts were tested in transesterification reaction of olive oil. For the enzyme load used in the test, a final yield percentage of 49.6 was achieved after six methanol additions and 180 min of reaction, similar values were obtained using Relizyme as support. Therefore, the bovine bone discard is an economical and appropriate choice for use support immobilization of enzymes. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1246–1253, 2016     

Stability analysis ofBacillus stearothermopilus L1 lipase fused with a cellulose-binding domain

This study was designed to investigate the stability of a lipase fused with a cellulose-binding domain (CBD) to cellulase. The fusion protein was derived from a gene cluster of a CBD fragment of a cellulase gene inTrichoderma hazianum and a lipase gene inBacillus stearothermophilus L1. Due to the CBD, this lipase can be immobilized to a cellulose material. Factors affecting the lipase stability were divided into the reaction-independent factors (RIF), and the reaction-dependent factors (RDF). RIF includes the reaction conditions such as pH and temperature, whereas substrate limitation and product inhibition are examples of RDF. As pH 10 and 50°C were found to be optimum reaction conditions for oil hydrolysis by this lipase, the stability of the free and the immobilized lipase was studied under these conditions. Avicel (microcrystal-line cellulose) was used as a support for lipase immobilization. The effects of both RIF and RDF on the enzyme activity were less for the immobilized lipase than for the free lipase. Due to the irreversible binding of CBD to Avicel and the high stability of the immobilized lipase, the enzyme activity after five times of use was over 70% of the initial activity.     

Study of support materials for sol-gel immobilized lipase

Abstract

A variety of support materials for sol-gel immobilized lipase were considered based on their ability to provide superior sol-gel adhesion, load protein, and synthesize methyl oleate. A standard approach was developed to formulate the supported lipase sol-gels and to allow comparison of the resulting hybrid materials. These supported sol-gels are proposed as an alternative immobilization regime to overcome some challenges associated with enzymatic biodiesel production such as enzyme stability and cost. The support materials considered were 6–12 mesh silica gel, Celite^® R633, Celite^® R632, Celite^® R647, anion-exchange resin AG3-X4, and Quartzel^® felt. Each support material exhibited unique properties that would be beneficial for this application including: Quartzel^® felt had the highest initial sol-gel capacity (62.5 mL/g) and sol-gel adhesion (1100 mg sol-gel/g material); silica gel had the most uniform coating of deposited sol-gel; the anion-exchange resin AG3-X4 supported sol-gel had the highest protein loading (1060 μg lipase/g) and reaction rate [1.25 mM/(min g-material)]; the Celite^® support series were the most thermally stable and had the lowest water content; and the Celite^® R632 supported lipase sol-gels had the highest 6 h biodiesel conversion per gram of supported material (68%) and enzymatic activity [9.4 mmol/(min g-lipase)]. The supported sol-gels with the highest enzymatic properties (conversion, activity, and reaction rate) were those supported on Celite^® R632, anion-exchange resin AG3-X4, and Quartzel^®. These supported sol-gels had superior performance in comparison with the unsupported sol-gels. Based on this study, the lipase sol-gel support material with the most potential for biodiesel production is Celite^® R632.     

Evaluation of cellulose-binding domain fused to a lipase for the lipase immobilization

A cellulose-binding domain (CBD) fragment of a cellulase gene of Trichoderma hazianum was fused to a lipase gene of Bacillus stearothermophilus L1 to make a gene cluster for CBD-BSL lipase. The specific activity of CBD-BSL lipase for oil hydrolysis increased by 33% after being immobilized on Avicel (microcrystalline cellulose), whereas those of CBD-BSL lipase and BSL lipase decreased by 16% and 54%, respectively, after being immobilized on silica gel. Although the loss of activity of an enzyme immobilized by adsorption has been reported previously, the loss of activity of the CBD-BSL lipase immobilized on Avicel was less than 3% after 12 h due to the irreversible binding of CBD to Avicel.     

Synthesis and characterization of cyclodextrin-based polymers as a support for immobilization of Candida rugosa lipase

The objective of this study was to prepare cross-linked β-cyclodextrin polymers for immobilization of Candida rugosa lipase. The structures of synthesized macrocyclic compounds were characterized by Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA) and scanning electron microscope (SEM) techniques. Properties of the immobilized systems were assessed and their performance on hydrolytic reaction were evaluated and compared with the free enzyme. The influence of activation agents (glutaraldehyde (GA) and hexamethylene diisocyanate (HMDI)) and thermal and pH stabilities of the biocatalyst was evaluated. After the optimization of immobilization process, the physical and chemical characterization of immobilized lipase was performed. Obtained data showed that the immobilized enzyme seemed better and offered some advantages in comparison with free enzyme. It can be observed that the free lipase loses its initial activity within around 80 min at 60 °C, while the immobilized lipases retain their initial activities of about 56% by HMDI and 82% by GA after 120 min of heat treatment at 60 °C.Results showed that the specific activity of the immobilized lipase with glutaraldehyde was 62.75 U/mg protein, which is 28.13 times higher than that of the immobilized lipase with HMDI.     

荧光标记脂肪酶的固定化及其稳定性

将标记有荧光探针FITC(异硫氰基荧光素)的脂肪酶固定化,通过测定活性和荧光光谱,探究各种因素对固定化后荧光标记脂肪酶性质的影响,并分析活性、构象和荧光光谱三者之间的联系。研究结果表明:在固定化脂肪酶过程中,聚乙二醇400二丙烯酸酯能形成合理的网格结构,使酶活较高;配体诱导酶的催化构象,使酶活性提高到未诱导酶的2倍以上;配体抽提能使脂肪酶活性中心得到释放从而提高催化活力。固定化脂肪酶的稳定性大大提高,在90℃、强酸强碱下固定化酶仍保有原酶70%、60%以上的活性;用盐酸胍、脲等溶解变性剂浸泡15d后,酶活性仍然可以保持初始活性的70%以上。荧光光谱能较好地反映脂肪酶的活性和构象变化,最适pH和温度下脂肪酶的荧光强度最低,在溶解变性剂中,荧光强度随时间延长而逐渐降低,这表明不同条件下脂肪酶构象经历的去折叠过程不同。     

Isolation of a fluffy mutant of Aspergillus niger from chemostat culture and its potential use as a morphologically stable host for protein production

Chemostat cultivation of Aspergillus niger and other filamentous fungi is often hindered by the spontaneous appearance of morphologic mutants. Using the Variomixing bioreactor and applying different chemostat conditions we tried to optimize morphologic stability in both ammonium- and glucose-limited cultures. In most cultivations mutants with fluffy (aconidial) morphology became dominant. From an ammonium-limited culture, a fluffy mutant was isolated and genetically characterized using the parasexual cycle. The mutant contained a single morphological mutation, causing an increased colony radial growth rate. The fluffy mutant was subjected to transformation and finally conidiospores from a forced heterokaryon were shown to be a proper inoculum for fluffy strain cultivation.     

二氧化硅纳米材料固定中性脂肪酶的条件优化及其特性

以二氧化硅纳米材料为载体,采用吸附法对脂肪酶进行固定化,研究了不同条件对固定化脂肪酶的催化活性的影响,得到最佳的固定化条件:给酶量为28300U/g,固定化温度为45oC,pH值为7.5,时间为10h,此时固定化酶的活力约为3867U/g载体。固定化酶的最适反应温度为45oC,比游离酶的反应温度高5oC,最适pH下降到5.5,低于游离酶的反应pH(pH7)。固定化酶的热稳定性和pH稳定性较游离酶有了很大的提高,其在70oC以下能保持70%以上的酶活力,而游离酶在50oC下残余酶活力仅为30%。在pH5～8的范围内,固定化酶的酶活力能保持50%以上,而游离酶只能保持20%左右。用固定化的中性脂肪酶催化不同的油品,即大豆油、菜籽油及泔水油生产生物柴油,菜籽油的酯化率最高。     

Environment friendly crosslinked chitosan as a matrix for selective adsorption and purification of lipase of Aspergillus niger

Chitosan and its derivatives have been used as affinity matrices for purification of lipase from Aspergillus niger NCIM 1207. Trimellitic anhydride (TMA)-crosslinked deacetylated chitin adsorbed lipase selectively, yielding approximately 5-fold purification of the crude lipase with 70% yield. Further 9-fold purification occurred on eluting through Sephacryl-100. These results suggest that chitosan derivatives can be used as inexpensive biopolymer matrices for the purification of lipases for industrial applications.     

黑曲霉组成型表面展示南极假丝酵母脂肪酶B及其发酵调控

黑曲霉表面展示南极假丝酵母脂肪酶B(CALB)可有效应用于食品、化妆品、医药等行业。以黑曲霉Aspergillus niger SH-1为宿主细胞构建诱导型糖化酶基因启动子表面展示CALB,在较高浓度葡萄糖碳源的发酵中CALB表达会受到抑制,发酵后期菌体容易出现菌丝断裂和展示酶活力下降等问题。采用组成型3-磷酸甘油醛脱氢酶基因启动子替代诱导型糖化酶基因启动子的细胞表面展示CALB黑曲霉菌株可有效解决上述问题,该菌株不但可以利用葡萄糖,而且还能利用木糖为发酵碳源,以木糖为碳源发酵在144 h展示酶水平达到1 100.28 U/g。文中探讨了甘蔗渣水解液发酵生产黑曲霉表面展示CALB,初步达到预期的结果,为甘蔗渣的综合利用提供了新途径。     

Crosslinked aggregates of Rhizopus oryzae lipase as industrial biocatalysts: Preparation, optimization, characterization, and application for enantioselective resolution reactions

Lipase from Rhizopus oryzae (ROL) was immobilized as crosslinked enzyme aggregate (CLEA) via precipitation with ammonium sulfate and simultaneous crosslinking with glutaraldehyde. The optimum conditions of the immobilization process were determined. Lipase CLEAs showed a twofold increase in activity when Tween 80‐pretreated lipase was used for CLEA preparation. CLEAs were shown to have several advantages compared to free lipase. CLEAs were more stable at 50°C and 60°C as well as for a wide range of pH. After incubation at 50°C, CLEA showed 74% of initial activity whereas free enzyme was totally inactivated. Reduction of Schiff bases has been performed for the first time in the CLEA preparation process significantly improving the chemically modified CLEAs' reusability, thus providing an enzyme with high potential for recycling even under aqueous reaction conditions where enzyme leakage is, in general, one of the major problems. The CLEA retained 91% activity after 10 cycles in aqueous medium. The immobilized enzyme was used for kinetic resolution reactions. Results showed that immobilization had an enhancing effect on the conversion (c) as well as on the enantiomeric ratio (E). ROL CLEA displayed five times higher enantioselectivity for the hydrolysis of (R,S)‐1‐phenylethyl acetate and likewise 1.5 times higher enantioselectivity for the transesterification of racemic (R–S)‐1‐phenylethanol with vinylacetate. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 937–945, 2012 This article was published online on June 26, 2012. An edit was subsequently requested. This notice is included in the online and print versions to indicate that both have been corrected [27 June 2012].     

Immobilization of lipase onto a photo-crosslinked polymer network: Characterization and polymerization applications

Abstract

The recovery of activity of lipases immobilized onto a photo-crosslinked polymer network was 76.0% and 41.0% for entrapment and adsorption methods, respectively. Both entrapped and adsorbed immobilized enzymes were very stable, retaining more than 60% of their activity over the range of temperatures studied. Immobilization by either method protected their relative activities nearly 96% at 70°C. The optimum pH was 8.0 for immobilized enzymes and 6.0 for the free enzyme at 40°C, while the relative activities after storage at 0–4°C for 30 days were 98% and 75% using entrapment and adsorption methods, respectively. These results indicated that lipase immobilized by entrapment and adsorption not only had good activity recovery, but also remarkable stability, better reusability and application adaptability than free lipase. Also, it can be safely stated that, photo-crosslinked polymer network can be used as alternative supports for immobilization of lipase for enzymatic polymerization reactions. In the ring-opening polymerization of ?-caprolactone, polymerization rates were clearly affected as monomer conversions were 58% and 49% and the highest molecular weights (M_n) obtained were 7890 and 5600 gmol^{? 1} for entrapment and adsorption methods, respectively.