Design and characterisation of an enzyme system for inulin hydrolysis

The optimal conditions for inulin hydrolysis using a commercial inulinase preparation, either free or immobilised in activated Amberlite were established by factorial design and surface response methodology. The immobilised biocatalyst displayed highest activity at pH 5.5 and 50 °C, whereas the optimum pH for the free form was slightly more acidic (4.5), and the optimum temperature was a little higher (55 °C). The model system estimated optimal pH and temperature values of 5.4 and 52 °C for the immobilised system and 4.9 and 56 °C for the free system. Michaelis–Menten type kinetics adequately described both free and immobilised bioconversion systems, which were evaluated under the respective optimal pH and temperature conditions. The use of a non-linear regression method for the determination of the kinetic parameters provided a best fit to the experimental data, as compared to a conventional Lineweaver–Burk linearisation. The K_m for inulin of the free biocatalyst was 153 g l⁻¹ at 55 °C and pH 4.5, whereas the apparent K_m for inulin of the immobilised biocatalyst was 108 g l⁻¹ at pH 5.5 and 50 °C. The reutilisation of the immobilised biocatalyst throughout consecutive batches was evaluated. A significant decrease of enzyme activity was observed in the first two batches, after which the system exhibited significant stability. The low cost of the support, the stability of the immobilised biocatalyst towards pH and temperature and its high affinity for the substrate suggests its potential for inulin hydrolysis.     

Inulinase from Rhodotorula mucilaginosa: immobilization and application in the production of fructooligosaccharides

The crude extract containing inulinase from Rhodotorula mucilaginosa was obtained by submerged fermentation. Inulinase was immobilized on chicken eggshell by physical adsorption and covalent crosslinking, using glutaraldehyde as a crosslinking reagent, and Celite by adsorption. Fructooligosaccharides production was performed using immobilized inulinase (5%, w/v) and inulin substrate solution under experimental conditions evaluated through Doehlert experimental design. The production of inulinase was optimized for concentrations of D-glucose and yeast extract at 12.5 and 0.5 g/L, respectively, resulting in an optimal activity of 0.62 U. The optimal pH and temperature for enzyme activity were 8.0 and 75 °C, respectively, leading to an optimal activity of 3.54 U. The highest immobilization efficiency (46.27%) was obtained upon immobilization on Celite. Immobilization by adsorption to eggshell allowed for specific activity of 4.15 U/g, and adsorption to Celite resulted in specific activity of 3.70 U/g. The highest titer in fructooligosaccharides was obtained with an initial inulin concentration of 250 g/L (25%, w/v), and a reaction time of 16 h. Hence, immobilized inulinase proved to be a promising catalyst for fructooligosaccharides production since the formulation is performed through a simple, low-cost, and large-scale applicable methodology.

     

Immobilization of inulinase for sucrose hydrolysis

A commercial inulinase preparation immobilized on various supports was used for sucrose hydrolysis. Entrapment and encapsulation in Ca-alginate and entrapment in an alginate–silicate sol–gel matrix were evaluated. Best results were obtained with Ca-alginate beads. The influence of sodium-alginate concentration on the immobilization yield was assessed. Inulinase entrapped in Ca-alginate beads displayed high activity in the range 50–60 °C, whereas the optimum for the free enzyme was 60 °C. The optimum pH of the immobilized enzyme was slightly more acidic (4.0) than the one observed for the free form (4.5). The apparent K_M for sucrose of the immobilized inulinase was 184 mM, as compared to 82 mM for free inulinase, as a result of diffusion resistances.     

固定化渗透乳酸克鲁维酵母(Kluyveromyces Lactis)连续水解乳清的研究

研究了一种利用固定化渗透乳酸克鲁维酵母催化水解乳清中乳糖的技术,分析了制备工艺、水解条件对于固定化细胞活性的影响。结果表明,在细胞添加量为60%、交联剂为1%BaCl2,凝胶颗粒大小为1～1.5mm时,固定化细胞的活性可达到78.819 U/g,固定化细胞最适反应温度为36℃,最适pH值7.0,相对于游离细胞,固定化细胞在对温度及pH的适应性上均有所提高,具有较好的应用前景。此外初步探索了一种复合流速连续水解的工艺,即在水解不同阶段,采用了不同的流速进行水解。结果表明,这种复合流速连续水解工艺可以在一定程度上缩短水解时间,提高水解的效率。     

克鲁维酵母突变株UV-G-40-3菊粉酶性质的研究

研究了克鲁维酵母突变株（Ｋｌｕｙｖｅｒｏｍｙｃｅｓ－ＵＶ－Ｇ－４０－３）所产菊粉酶的分布为胞外酶∶胞壁酶∶胞内酶比是５．７∶１．６∶１。该酶Ｓ／Ｉ为５．３,最适温度为５０℃,最适ｐＨ为４．５,在５０℃以下、ｐＨ４．５～８的范围内比较稳定,４℃贮存稳定性好,１４ｄ后仍保持７６％活力,为外切型菊粉酶,酶解粗菊糖（洋姜提取液）活性为纯菊糖的４倍。     

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。     

用于高果糖浆制备的宛氏拟青霉嗜热嗜酸菊粉酶酶学特性分析

为实现酶法水解菊糖制备高果糖浆,从宛氏拟青霉（Paecilomyces variotii）XS27发酵液中分离纯化菊粉酶,并对其酶学特性进行研究。发酵液经过硫酸铵盐析、透析、DEAE-Sepharose Fast Flow层析、Sephacry S-100分子筛过滤层析,得到电泳纯的菊粉酶,比活力327.4 U/mg,纯化倍数37.85。十二烷基硫酸钠-聚丙烯酰氨凝胶电泳测得菊粉酶为单一亚基的酶蛋白,分子质量62.0 kDa。菊粉酶能在较宽的pH值范围（3.5～6.5）内保持高活性,最适作用pH 4.0。在温度40～65 ℃之间,酶活力较高,最适作用温度为60 ℃。薄层色谱分析显示菊粉酶水解菊糖最终产物为果糖。以菊糖为底物,酶的Km和Vmax分别为5.93 μmol/L和75.18 μmol/（L·min）。Mg2＋、Mn2＋、Ca2＋对酶有显著激活作用,Ba2＋、Ni2＋和Hg2＋对酶有一定抑制作用。β-巯基乙醇、二硫苏糖醇和乙二胺四乙酸对酶有抑制作用,表面活性剂（十二烷基硫酸钠、Tween 80和Trition X100）以及乙醇对酶活力没有影响。从宛氏拟青霉XS27发酵液中分离纯化的菊粉酶在强酸高热的环境下具有强活性和稳定性,对表面活性剂乙醇有高耐受性,适合于果葡糖浆的工业化生产。     

Enzymatic synthesis of fructooligosaccharides by inulinases from Aspergillus niger and Kluyveromyces marxianus NRRL Y-7571 in aqueous–organic medium

This work is focused on the synthesis of the fructooligosaccharides (FOS) from sucrose and inulin, using free, immobilized and pre-treated immobilized inulinase from Kluyveromyces marxianus NRRL Y 7571 and Aspergillus niger in an aqueous–organic system. Initially, the influence of pre-treatment using four different gases, propane, n-butane, CO₂ and liquefied petroleum gas (LPG), was investigated towards FOS production and best results were found when both enzymes were previously treated with LPG. The best reaction yields were obtained when the immobilized enzymes were treated with LPG. Considering FOS synthesis using the enzyme from A. niger, yields of 26.62% of GF2 (kestose), 30.62% of GF3 (nystose) and 8.47% of GF4 (fructosyl nystose) were achieved using sucrose as substrate. Using inulinases from K. marxianus NRRL Y 7571, 11.89% of GF2 and 20.83% of GF3 were obtained, using inulin as substrate. However, promising results were achieved using the free form of inulinase from A. niger (77.19% of GF2; 14.03% of GF3 and 0.07% of GF4) using inulin as substrate.     

Production and Properties of Microbial Inulinases: Recent Advances

Microbial inulinases find application in enzymatic hydrolysis of inulin for production of fructose (sweetener) and inulooligosaccharides (prebiotics). Inulin is one of the most abundant nonstructural polysaccharides found widely dispersed in plants and awaits judicious utilization. Demands for an alternative healthy sweetener and multifunctional fructooligosaccharides have prompted investigators to explore microorganisms for inulinase production and to develop bioprocesses for production of high-fructose syrup and oligosaccharides based on enzymatic hydrolysis of raw inulin. Inulinases have been characterized in several molds, yeasts and a few bacteria. Recently, there has been a spurt of interest in finding novel inulinase producers, cloning and expression of inulinase gene in heterologous hosts and use of crude plant inulin and agro-industrial media in both submerged and solid state fermentation for inulinase production. The review discusses current knowledge on production, properties and applications of microbial inulinases and looks into the recent advances in the field.     

对菊粉酶产生菌B-3-3发酵条件、酶的分布及酶学特性的研究

经过对pH、摇瓶装量、底物浓度及碳源种类与酶合成之关系进行研究,发现B-3-3菌株在碱性条件,较大通气量,和较高的碳源浓度下产菊粉酶较高,可达21u/ml。当以蔗糖代替菊粉为碳源时,产酶更高。而其它所试糖类较小或没有诱导菊粉酶合成的作用。改进种子液的制作和种子液培养基的成份,大大缩短了酶合成的时间,并提高了酶的产量。 对B-3-3的菊粉酶在胞内外分布进行了研究,结果为胞内酶:胞外酶约为1∶2;pH和温度对酶的活性和稳定性影响较大,其最适pH为6.5,最适温度55℃。在pH6～8的范围内和55℃以下,酶较稳定。     

菊粉酶产酶菌株的诱变选育及产酶条件的研究

以克鲁维酵母（Ｋｌｕｙｖｅｒｏｍｙｃｅｓ）野生株为出发菌株,经紫外线处理筛选到一株产菊粉酶较高的突变株ＵＶ－Ｇ－４０－３,并对其进行了发酵条件的研究,在菊糖３％、蛋白胨３％、酵母膏１％、ｐＨ５．５、３６℃,装量３０ｍｌ／３００ｍｌ,２４０ｒ／ｍｉｎ摇瓶培养３３ｈ,酶活比野生株提高近一倍,为１３．９Ｕ／ｍｌ。     

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.     

菊粉酶解及其酶解液对实验性糖尿病动物血糖影响的研究

以10％菊粉提取液为原料,在固定化酶与底物体积比为1：5、pH45、温度50℃、搅拌速度148r／min的条件下,用固定化菊粉酶酸解3h,转化率可达87％。酶解产物中,果糖占76％,葡萄糖占13％,低聚果糖占11％。此酶解液用于实验性糖尿病大鼠试验,证明血糖基本保持不变,而对照组大鼠的血糖升高30％左右。且酶解液中所含的低聚果糖又是对健康有益的双歧杆菌生长因子。故此酶解液可望开发为适合糖尿病患者饮用的产品。     

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.     

Raw galacto-oligosaccharide purification by consecutive lactose hydrolysis and selective bioconversion

Galacto-oligosaccharide (GOS) yields obtained by enzymatic transgalactosylation with β-galactosidases are rather low, so purification of the raw GOS is a major issue. In the quest for improving GOS purification, an additional step of lactose hydrolysis is proposed, using immobilised β-galactosidase and permeabilised cells expressing β-galactosidase activity as biocatalysts. To select the best operational conditions for lactose hydrolysis in raw GOS employing both biocatalysts different enzyme:substrate (lactose) mass ratios were evaluated. Bioconversion of the lactose-hydrolysed raw GOS obtained with the two biocatalyst forms were compared, and biocatalyst reuse was evaluated. Lactose hydrolysis of raw GOS allowed the removal of around 70% of the lactose content and increased the relative GOS content by 20%. Incorporation of the lactose hydrolysis step led to a purity and recovery of GOS of 96–97% and 92–96%, respectively, depending on the biocatalyst used.     

海藻酸钠包埋法固定青霉产菊粉酶的研究

利用海藻酸钠包埋法固定青霉(penicillium)产菊粉酶,研究了最佳固定化条件及固定化酶性质,为固定化菊粉酶应用于果糖工业生产提供理论依据。结果表明:海藻酸钠浓度为4%,氯化钙浓度为1%时,固定化酶活力最高且机械强度好;加酶量越少,固定化效率越高;固定化酶与游离酶的最适pH为4.5,但固定化酶在pH4到6.5范围内相对酶活要比游离酶酶活高;固定化酶与游离酶的最适温度均为55℃,在35℃到55℃范围内两者酶活变化不大,在55℃到75℃范围内固定化酶的温度适应性比游离酶的好;在重复利用方面,将固定化酶反复利用7次,酶活仍为原酶活的50.6%。由此说明,固定化酶成本低,利用率高,在工业生产方面具有利用价值。     

Ambient and low temperature winemaking by immobilized cells on brewer’s spent grains: Effect on volatile composition

Repeated batch wine fermentations were conducted using immobilized yeast cells on brewer’s spent grains as well as free cells, at 25, 20, 15 and 10 °C. The operational stability of the biocatalyst was good and no decrease of its activity was observed, even at 10 °C. Ethanol and wine productivities were high, showing the suitability of the biocatalyst for low temperature winemaking. The interaction effect of immobilization and temperature was statistically significant in most cases. Immobilized cells produced wines with a higher content of ethyl and acetate esters as well as volatile fatty acids, at temperatures of 15 and 10 °C, whereas the opposite was observed for free cells at higher temperatures. Same amounts of higher alcohols were produced by both immobilized and free cells. These amounts decreased dramatically with temperature drop. Wines produced by immobilized cells at low fermentation temperatures (10–15 °C) were characterized by a potentially better flavour due to a better ratio of esters to alcohols.     

响应面法优化Aspergillus niger X-6发酵生产菊粉酶工艺的研究

为提高Aspergillus niger X-6 发酵生产菊粉酶的产率,运用Plackett-Burmen 方法对麸皮、菊粉、蛋白胨、酵母膏添加量和发酵时间、温度、pH 值、接种量8 个影响因素进行考察,筛选出麸皮添加量、发酵时间和pH值为3 个显著影响因素,然后采用Box-Behnken 中心组合和响应面法对上述3 个因素进行产酶条件的进一步优化,建立菊粉酶产率的二次多项式数学模型,并分析模型的有效性与因子间的交互作用。结果表明：黑曲霉发酵产菊粉酶的优化工艺参数为：麸皮添加量4.64%、发酵时间81.5h、pH6.0。在此条件下,产酶活力达20.42U/mL,与优化前相比提高了30.81%。     

Sucrose hydrolysis by gelatin-immobilized inulinase from Kluyveromyces marxianus var. bulgaricus

The crude cell-free medium from a culture of Kluyveromyces marxianus var. bulgaricus was immobilized in a gelatin-water support, with an immobilization yield of 82.60% for inulinase activity. The optimum pH for both free and immobilized inulinase was the same (3.5) and the optimum temperatures were 55 °C for the free and 60 °C for the immobilized enzyme. The Arrhenius plots were linear and activation energies were 56.20 (free enzyme) and 20.27 kJ/mol K (immobilized enzyme). The kinetic parameters were calculated by Lineweaver–Burk plots and the V_max and K_m were 37.60 IU/mg protein and 61.83 mM for the free inulinase and 31.45 IU/mg protein and 149.28 mM for the immobilized enzyme, respectively. The operational stability of the immobilized inulinase was studied in a continuous fixed-bed column reactor for 33 days, at the end of which the sucrose conversion was 58.12%.