Stability evaluation of an immobilized enzyme system for inulin hydrolysis

The stability of free and Amberlite-immobilized inulinase, aiming at inulin hydrolysis was evaluated. The apparent activation energy of the biotransformation decreased when the immobilized biocatalyst was used, suggesting diffusional limitations, despite a decrease in the optimal temperature for catalytic activity for the immobilized biocatalyst. Thermal deactivation, of both forms of the biocatalyst, was evaluated by the linear inverted model. Inulinase immobilization consistently enhanced half-life of the enzyme, which increased up to 6-fold, as compared to the free form. Mean enzymatic activity was computed for both forms of the biocatalyst, and evidenced a decrease of optimal temperature with increased incubation periods. The deactivation energies estimated by an Arrhenius plot, evidenced a decrease of roughly 20% when free inulinase was used. The immobilized biocatalyst was effectively reused in successive batch runs for the hydrolysis of a 5% inulin solution.     

STABILIZATION OF THE ACTIVITY OF β-GALACTOSIDASE IN PERMEABILIZED IMMOBILIZED CELLS FOR HYDROLYSIS OF LACTOSE IN MILK

A potentially low cost β‐galactosidase was prepared as a crude permeabil‐ized cell mass of the yeast Kluyveromyces lactis that had been grown in ultrafiltered cheese whey. The enzyme preparation was immobilized in alginate beads. Milk lactose hydrolysis rates were 25% higher in manganese alginate beads than in calcium alginate beads. The immobilized biocatalyst lost activity when stored in calcium chloride solution, however, storage in 5 mM DTT or 50% glycerol allowed the biocatalyst to be recycled at least 5 times without any loss of activity.     

Hydrolysis of sucrose by invertase immobilized on nylon-6 microbeads

A commercial extracellular invertase (EC 3.2.1.26) from Saccharomyces cerevisiae has been inmobilized by covalent bonding on novel microbeads of nylon-6 using glutaraldehyde. The enzyme was strongly bound on the support, immobilized with an efficiency factor of 0.93. The biocatalyst showed a maximum enzyme activity when immobilized at pH 5.0, but optimum pH activity for both immobilized and free invertases was 5.5. The optimum temperatures for immobilized and free enzymes were 60 and 65 °C, respectively. Kinetic parameters were determined for immobilized and free invertases: V_max values were 1.37 and 1.06 mmol min⁻¹ mg⁻¹, respectively. The K_m and K_i values were 0.029 and 0.71 M for immobilized invertase and 0.024 and 0.69 M for free invertase. It was found that the thermal stability of the immobilized invertase with regard to the free one increased by 25% at 50 °C, 38% at 60 °C and 750% at 70 °C. The immobilized biocatalyst was tested in a tubular fixed-bed reactor to investigate its possible application for continuous sucrose hydrolysis. The effects of two different sugar concentrations and three flow rates on the productivity of the reactor and on the specific productivity of the biocatalyst were studied. The system demonstrated a very good productivity up to 2.0 M sugar concentration, with conversion factors of 0.95 and 0.97, depending on sucrose concentration in the feeding. This approach may serve as a simple technique and can be a feasible alternative to continuous sucrose hydrolysis in a fixed bed reactor for the preparation of fructose-rich syrup.     

脂肪酶固定化及其在食品领域中应用的研究进展

脂肪酶作为一种绿色的生物催化剂广泛应用在食品等工业领域,但是其结构极易被破坏且在环境中稳定性较差,固定化脂肪酶是提高其稳定性和重复利用率的关键技术。近年来新型材料的不断出现,为脂肪酶的固定化带来了契机。由于脂肪酶是作用于油水界面的特殊酶,固定化材料的物理和化学性能会直接改变固定化酶的催化特性,提高酶活力、选择性和稳定性等。本文综述了近年来固定化脂肪酶的材料和方法,及其在食品加工领域中的应用,并展望了固定化脂肪酶的发展前景。     

Preparation of immobilized Trametes pubescens laccase on a cryogel-type polymeric carrier and application of the biocatalyst to apple juice phenolic compounds oxidation

A new biocatalyst was prepared by the immobilization of a Trametes pubescens laccase, into a wide-pore poly(vinyl alcohol) cryogel. The known enzyme was produced and purified by the previously described procedure. The resulted laccase (yield 40%) has an activity of 46.4 U mg⁻¹ and 12.51 mg mL⁻¹ protein content. The enzyme was subsequently immobilized in a functionalized macroporous cryogel beads by a covalent immobilization technique. The time dependence of the immobilization process and the enzyme loading of the carrier material (5.2 mg g⁻¹ cryogel) were determined by measuring the decrease of protein amount in the enzyme solution. In conversion experiments, a higher stability of the immobilized biocatalyst compared to the free enzyme was evidenced. Steady-state kinetic characterization of four phenols (catechol, caffeic and chlorogenic acids, and catechin) has been performed with free and immobilized laccase, the catalytic parameters being determined and compared. The effect of both laccases (free and immobilized) on the phenol content of retailed apple juice samples, having the same initial composition, was also investigated by working in batch conversion. The variation in phenolic compound content has been compared with that of an untreated apple juice sample having initially the same content of phenolic compounds. A number of advantages resulted in using the immobilized laccase for the apple juice treatment (conservation to some extent of enzyme activity, higher content of phenols preserved, easy separation of the enzyme from the apple juice, therefore avoiding the possible unhealthy effects due to the remaining protein, etc.).     

粥化酶在果蔬加工中的应用

研究了黑曲霉所产粥化酶在不同果蔬加工中的应用以及在苹果及南瓜汁加工中的应用条件．结果表明，使用粥化酶可以使果蔬的出汁率及果蔬汁澄清度有不同程度的提高．通过试验确定了苹果和南瓜汁加工工艺中酶解及澄清的最佳条件．     

固定化脂酶催化合成生物柴油的研究

探讨了酶法制备生物柴油的过程,通过脂肪酶酯化和醇解两种工艺路线合成生物柴油的试验研究,考察了反应条件如醇油比、催化剂用量、反应温度、反应时间等对酯化率和产品纯度的影响.试验表明,采用分批加入甲醇的酯化工艺,酯化率可以达到95%以上;采用醇解工艺菜籽油酯化率达95%以上,产品经GC分析其纯度可达98%以上,固定化酶的半衰期至少达100 d.     

Configuration of a bioreactor for milk lactose hydrolysis

Permeabilized microbial cells can be used as a crude enzyme preparation for industrial applications. Immobilization and process recycling can compensate for the low specific activity of this preparation. For biomass immobilization, the common support is alginate beads; however, its low surface area and the low biomass concentration limit the activity. We here describe a biocatalyst consisting of a paste of permeabilized Kluyveromyces lactis cells gelled with manganese alginate over a semicircular stainless steel screen. A ratio of wet permeabilized biomass to alginate of 50:4 (wt/wt) resulted in a paste with maximum immobilized beta-galactosidase activity and maximum gel biomass retention. The biocatalysts retained activity better when stored in milk at 4 degrees C than in 50% glycerol. The unused biocatalysts stored in milk did not lose activity after 50 d. However, repeated use of the same biocatalyst 40 times resulted in almost 50% loss of activity. A bioreactor design with two different conditions of operation were tested for milk lactose hydrolysis using this biocatalyst. The bioreactor was operated at 40 degrees C as packed bed or with recirculation, similar to a continuous stirred tank reactor. The continuous system with recirculation resulted in 82.9% lactose hydrolysis at a residence time of 285.5 min (flow of 2.0 ml/min), indicating the potential of this system for processing low lactose milk, or even in processing other substrates, using an appropriate biocatalyst.     

聚乙烯醇环氧化载体制备及在植酸酶固定化中的应用

该文以聚乙烯醇为原料,利用戊二醛为交联剂,聚合得到交联球形聚乙烯醇,并将其与环氧氯丙烷反应用以固载环氧基团,得到可用于酶固定化的载体。讨论了交联聚乙烯醇球环氧基的固载条件和酶的固定化条件。植酸酶经过固定化后,机械性能和化学稳定性都得到提高,可以重复多次对植酸钠进行水解反应。同时对该自由酶和固定化酶进行酶学特性研究。发现自由酶和固定化酶的最适pH值分别为5.5和7.0,最适反应温度分别为55℃和70℃。植酸酶经固定化,提高了酶的操作、温度、pH值和贮藏稳定性。     

聚乙烯醇复合凝胶固定化黑曲霉细胞研究

以聚乙烯醇(PVA)复合凝胶为载体,利用冻融法固定产α葡萄糖转苷酶的黑曲霉M1菌丝。由于PVA冻胶的多孔性,高分子底物可以穿透载体与细胞酶直接反应。细胞经过固定化后,机械性能和化学稳定性都得到提高,可以重复多次对甲基αD葡萄糖苷进行水解反应。同时对该自由细胞和固定化细胞进行酶学特性研究。发现2者的最适pH值同为pH6.0,最适反应温度分别为55℃、60℃。固定化细胞的Vm值为2.84μmol/(L·min·g),大于自由细胞的Vm1.71μmol/[L·(min·g)],固定化细胞的Km(11.88mmol/L)小于自由细胞的Km(20.50mmol/L)。细胞经固定化,提高了细胞酶的操作、温度和贮藏稳定性。     

Microbial production of inulo-oligosaccharides by an endoinulinase from Pseudomonas sp. expressed in Escherichia coli

In an attempt to utilize the whole cell as a biocatalyst for inulo-oligosaccharide (IOS) production from inulin, the endoinulinase gene (inu1) of Pseudomonas sp. was cloned into the plasmid pBR322 using EcoRI restriction endonuclease and Escherichia coli HB101 as the host strain. The endoinulinase from E. coli HB101/pKMG50 was constitutively expressed, producing a high yield of IOS (78%). In a batchwise reaction, the initial enzyme concentration determined the total oligosaccharide yield, and excess enzyme decreased the total oligosaccharide yield due to the formation of high amounts of free sugars such as glucose and fructose. The recombinant E. coli expressing endoinulinase activity were immobilized on a polystyrene carrier material, resulting in a dramatically enhanced thermal stability of the enzyme. Continuous production of IOS from inulin was also carried out at 50 degrees C using a bioreactor packed with the immobilized cells. Under the optimal operation conditions, continuous production of IOS was achieved with a productivity of 150 g/l.h for 17 d at 50 degrees C without significant loss of initial activity.     

交联青霉素G酰化酶聚集体制备及其催化特性研究

目的考察交联青霉素G酰化酶聚集体催化特性,探讨其应用潜力。方法以硫酸铵为沉淀剂获得青霉素G酰化酶蛋白沉淀,戊二醛交联沉淀的蛋白质制备交联酶聚集体,在此基础上研究交联酶聚集体的催化特性。结果交联粪产碱杆菌来源青霉素G酰化酶聚集体的最适反应温度55℃,最适pH9．0,其酸碱和热稳定性均优于游离酶,重复使用13批次几乎无酶活性丢失,反应20批次后仍有50．9％的活性。结论交联酶聚集体的稳定性较游离酶好,具有一定的工业应用前景。     

The effect of a commercial pectolytic enzyme on grape skin cell wall degradation and colour evolution during the maceration process

The effect of a macerating enzyme and maceration time on the structure of grape skin cell walls and on wine quality was studied by making wines with different maceration times with and without the addition of enzyme. The results show that the addition of a macerating enzyme accelerates the extraction of phenolic compounds, reducing the maceration time needed for high quality winemaking. The degradation of grape skin cell walls was influenced by the enzyme, which mainly affected the pectin fraction of the cell wall.     

交联青霉素G酰化酶聚集体制备及其催化特性研究

目的考察交联青霉素G酰化酶聚集体催化特性,探讨其应用潜力。方法以硫酸铵为沉淀剂获得青霉素G酰化酶蛋白沉淀,戊二醛交联沉淀的蛋白质制备交联酶聚集体,在此基础上研究交联酶聚集体的催化特性。结果交联粪产碱杆菌来源青霉素G酰化酶聚集体的最适反应温度55℃,最适pH 9.0,其酸碱和热稳定性均优于游离酶,重复使用13批次几乎无酶活性丢失,反应20批次后仍有50.9%的活性。结论交联酶聚集体的稳定性较游离酶好,具有一定的工业应用前景。     

Kefir Immobilized on Corn Grains as Biocatalyst for Lactic Acid Fermentation and Sourdough Bread Making

Abstract: The natural mixed culture kefir was immobilized on boiled corn grains to produce an efficient biocatalyst for lactic acid fermentation with direct applications in food production, such as sourdough bread making. The immobilized biocatalyst was initially evaluated for its efficiency for lactic acid production by fermentation of cheese whey at various temperatures. The immobilized cells increased the fermentation rate and enhanced lactic acid production compared to free kefir cells. Maximum lactic acid yield (68.8 g/100 g) and lactic acid productivity (12.6 g/L per day) were obtained during fermentation by immobilized cells at 37 °C. The immobilized biocatalyst was then assessed as culture for sourdough bread making. The produced sourdough breads had satisfactory specific loaf volumes and good sensory characteristics. Specifically, bread made by addition of 60% w/w sourdough containing kefir immobilized on corn was more resistant regarding mould spoilage (appearance during the 11^th day), probably due to higher lactic acid produced (2.86 g/Kg of bread) compared to the control samples. The sourdough breads made with the immobilized biocatalyst had aroma profiles similar to that of the control samples as shown by headspace SPME GC‐MS analysis.     

Freeze-dried wheat supported biocatalyst for low temperature wine making

A new biocatalyst was prepared by immobilization of Saccharomyces cerevisiae AXAZ-1 yeast cells on whole wheat grains. This biocatalyst was subjected to freeze-drying and the effects of several protective agents and storage at 5 °C on viability and fermentative activity of yeasts cells were studied. Glycerol was the best protective agent that preserved the viability of immobilized yeast cells even after 9 month storage. After freeze-drying the biocatalyst was used for repeated batch wine making at extremely low temperatures until 5 °C. The produced wines were analyzed for volatile byproducts by GC and GC/MS and the results showed that the freeze-dried immobilized biocatalysts, with glycerol as protectant, produced wines with higher formation of esters than free cells, having at least similar aromatic profile to those produced by wet biocatalysts.     

粥化酶及其在果蔬加工中的应用

综述了我国果蔬加工的现状,根据目前加工过程中单一使用果胶酶还存在不足的情况,提出了一种新型的果蔬加工用酶——粥化酶。它是一种复合酶,主要包括果胶酶、纤维素酶、半纤维素酶、蛋白酶、淀粉酶等,可以提高果蔬汁的出汁率,增加澄清度,在果蔬加工中具有广阔的应用前景。     

Continuous Hydrolysis of Fructans in Jerusalem Artichoke Extracts using Immobilized Nonviable Cells of Kluyveromyces marxianus

Non-viable immobilized cells of Kluyveromyces marxianus in alginate beads having inulase (ß-2,1-fructan fructano-hydrolyase E.C.3.2.1.7) acitivity were used as biocatalyst in a packed bed reactor. Extracts of Jerusalem artichoke tubers were contacted with the biocatalyst for continuous conversion of the fructan component to fructose. In a bed reactor packed with 100 mL of beads, a volumetric productivity of 136 g/L/hr total reducing sugars was obtained with 98% substrate conversion. When operated continuously for 30 days, a 55% loss in the original activity was observed, giving a half life for the biocatalyst of 28 days.     

无溶剂体系糖酯的酶法合成

以固定化脂肪酶作为生物催化剂在无溶剂体系中初步合成了蔗糖月桂酸酯。探讨了底物摩尔比、pH、初始水活度、水合盐等影响酯化反应的因素，并研究了酯化反应的选择性。     

介孔分子筛MCM-48固定化木瓜蛋白酶性质的研究

以介孔分子筛MCM48为载体、戊二醛作交联剂对木瓜蛋白酶进行了固定化。实验结果表明,与游离酶相比,固定化酶的热稳定性有了显著的提高,其半衰期达到了2500min以上,是游离酶(80min)的30倍以上。固定化酶的pH稳定性和储藏稳定性也有了明显改善。此外,固定化酶还具有很好的操作稳定性,经过12d的连续酶解,酶活力仍保持在初始酶活力的70%以上。