菊粉酶酶解菊芋提取液的试验

报道黑曲霉ＡＬ－１５４菊粉酶产生和酶解菊芋提取液的适宜条件：５％菊芋提取液,２％玉米浆、０．３％酵母膏的基本培养基,该酶活力达１４６．４ｕ／ｍｌ,是适培养条件为：ＰＨ４．５－５．０,３０℃,时间４８ｈ,酶解菊芋提取液的最适工艺条件为：ＰＨ５．０,６０℃,底物总糖浓度为１０－２０％,酶用量３．０ｕ／ｇ菊糖,酶解时间１０ｈ,底物降解率达９７．２％,酶解产物中果糖占总糖的８６．１％。     

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。     

固定化菊粉酶的研究

报道了克鲁维酵母突变株（ｋｌｕｙｖｅｒｏｍｙｃｅｓ－ＵＶ－Ｇ－４０－３）所产菊粉酶的固定化及固定化酶的性质，并用固定化菊粉酶酶解洋姜提取液生产果糖，在分批式反应器中，当底物和固定化酶体积比为３．５∶１时，２．５ｈ水解率达到９２．４％，产率为２８．７ｇ／Ｌｈ，在连续填充床反应器中，在稀释率为０．５ｈ－１，转化率达９３．６％，产率为１５．７ｇ／Ｌｈ，半衰期达１１０ｄ以上。     

菊粉酶产菌株的诱变选育及产酶条件的研究

以克鲁维酵母野生为出发菌株,经紫外筛选选到一株产菊粉酶较突变株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。     

黑曲霉固态培养生产纤维素酶的研究

黑曲霉突变株ＤＭ－１是一株产纤维素酶菌株，其中β葡萄糖苷酶活性特别高。采用粗纤维原料固体培养，发酵９６小时（培养温度３１℃），其滤纸酶活和β葡萄糖苷酶活分别为９５和１２００ｍｇ葡萄糖／ｇＤＭｈ。本试验系统研究了各种营养成份和培养条件对ＤＭ－１菌株产纤维素酶的影响。最适发酵培养基为：稻草杆（或麦杆）∶麦麸为６０∶４０、硫酸铵３．０、硫酸镁０．３、玉米浆３．０，加水２００％，自然ｐＨ；环境湿度８５％－９０％。酶反应最适温度和ｐＨ分别为５５℃－６０℃和ｐＨ５．０。酶ｐＨ稳定性较好，在ｐＨ３．０－８．０范围内处理１小时，残余酶活力在８５％以上，该酶经５５℃处理３０ｍｉｎ，剩余酶活力为８６．０％。     

稻米中普鲁兰酶的纯化与性质的研究

稻米的普鲁兰酶（ＰｕｌｌｕｌａｎａｓｅＥ．Ｃ．３．２．１．４１）用ＤＥＡＥ－纤维素吸附，ＳｅｐｈａｄｅｘＧ－１００凝胶过滤，聚丙烯酰胺盘状凝胶电泳纯化，得到了聚丙烯酰胺凝胶电泳均一的纯酶。纯酶作用的最适温度为５０℃，最适ｐＨ为５．５。以普鲁兰（Ｐｕｌｌｕｌａｎ）为底物酶的米氏常数Ｋｍ为０．０１２５％，以支链淀粉为底物，酶的米氏常数Ｋｍ为２．５％，用ＳｅｐｈａｄｅｘＧ－１００凝胶层析法测得酶的分子量为７００００。     

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。     

茶多酚对菠萝蛋白酶的分离及特性的影响研究

本研究用从茶叶中提取的多酚类物质分离菠萝蛋白酶、井研究了菠萝蛋白酶与茶多酚结合后的性质、结果表明,用０．５％的茶多酚提取物对菠萝蛋白酶的沉淀回收率达７８％,茶多酚与菠萝蛋白酶的结合比为１∶６４３（ｍｇ∶ｕ）。等电聚焦电泳图谱表明菠萝皮汁中有６种同工酶组份．这６种酶组份均可与茶多酚结合形成沉淀而得以分离,结合后酶的等电点（ｐＨ３．６０,ｐＨ４．９０,ｐＨ５．３５,ｐＨ６．５０,ｐＨ７．８０,ｐＨ９．２５）、最适ｐＨ值（ｐＨ６．５～８．５）以及最适温度（６０℃）不改变,但酶对底物酪蛋白的亲和力下降（游离酶Ｋｍ＝８．１×１０￣６ｍｏｌ／Ｌ,结合酶Ｋｍ＝１．３９×１０￣５ｍｏｌ／Ｌ）。酶的稳定性有明显的提高,常温下的半衰期由游离酶的５天延长至２７天。酶稳定性的提高与茶多酚的抗氧化特性有关。     

低聚果糖合成酶──果糖转移酶的部分酶学性质研究

研究从土壤中筛选得到的黑曲霉ＡＳ００２３菌株所产的β－果糖转移酶的一些酶学性质。该酶的最适ｐＨ为５．０，最适温度为５０℃，有较好的热稳定性和较宽的ｐＨ（３．５～１０）适宜范围；并研究了１２种化学物质对酶活的影响和底物的特异性。结果显示，钴离子对该酶有一定的激活作用；底物分子越大其反应速度越慢。该酶的米氏常数Ｋ＿ｍ＝４７ｍｍｏｌ，葡萄糖是酶反应的竞争性抑制剂，其Ｋ＿ｉ＝３３０ｍｍｏｌ．当５０％（Ｗ／ｖ）蔗糖与酶在ｐＨ５．０和５０℃作用１６ｈ时，得到５２％（产物／总糖）低聚果糖。     

菊芋块茎制高果糖浆的研究

通过菊芋干片与菊粉提取液的制备、菊粉酶解、精制生产高果糖浆、菊粉酶解的适宜条件为：底物糖浓度１２％，加酶量２６ｕ／ｇ糖，ｐＨ５．０￣５．５，最适温度为５０℃，酶解６ｈ，底物降解率可达９８．５％，菊粉酶解液经活性炭脱色，离子交换树脂处理，减压浓缩等步骤，制得糖浆的固形物含量为７３．８％，果糖含量（占固形物）为８３．６％，同时对糖浆的ＤＥ值、色度、灰分、微生物含量等指标进行了测定。     

Purification and properties of an extracellular inulinase from Rhizopus sp. strain TN-96

A filamentous fungus, Rhizopus sp. strain TN-96, was isolated from rhizosphere soil samples. An extracellular inulinase was purified from the culture filtrate of strain TN-96 grown on inulin by DEAE-Cellulofine A-500 and Sephacryl S-200 HP chromatographies. The enzyme was homogeneous as judged by SDS-polyacrylamide gel electrophoresis, with an apparent M(r) of 83 kDa. The purified enzyme had specific activities of 17 U/mg toward inulin (I) and 0.32 U/mg toward sucrose (S) (I/S ratio, 53). Inulinase activity was optimal at pH 5.5 and 40 degrees C. The inulinase exhibited an apparent K(m) value of 9.0 mM for inulin. The enzyme also hydrolyzed raffinose, but not bacterial levan.     

黑曲霉Aspergillus niger No．26菊粉酶的研究

通过筛选菌种，分离出３株菊粉酶活力较高的菌株，对其中Ｎｏ．２６菌株经过培养基组成的研究，酶活力达３７．１ｕ／ｍｌ。比原活性６．７ｕ／ｍｌ提高５．５倍。经纸上层析鉴定，在菊粉水解过程中，果糖逐步增加，未见中间产物生成，并有很强的水解蔗糖为果糖和葡萄糖，以及水解棉子糖为果糖和蜜二糖的能力。因此该酶主要为外切型菊粉水解酶。该酶在５５℃以下较稳定，最适ｐＨ为３～５，其低而宽的ｐＨ范围有防止微生物污染的能力，具备工业应用的良好性能。     

Purification and immobilisation of inulinase from Aspergillus candidus for producing fructose

A high‐inulinase‐producing strain of Aspergillus candidus (10 units cm⁻³ of medium) for producing fructose from inulin has been identified. The extracellular inulinase from this fungi was purified 56‐fold by ammonium sulphate fractionation, DEAE cellulose and Sephadex G‐150 column chromatography. Invertase to inulinase ratio of 1.8 in culture filtrate was reduced to 0.14 in the purified preparation. The pH and temperature optima were 5.5 and 45°C, respectively. The molecular weight of inulinase was determined as 54±4 kDa. K_m of inulinase with inulin as a substrate was 3.8 mmole dm ⁻³. The purified preparation produced only fructose as the product of inulin, indicating that inulinase has primarily exo‐inulinase activity. Inulinase was immobilised on chitin and casein using glutaraldehyde as a linking agent and on cellulose using FeCl₃‐HCl as a metal chelation agent. Maximum immobilisation of 45.8% was achieved on cellulose. All three immobilised preparations had a higher temperature optima of 55°C. The inulinases immobilised on cellulose and casein were stable at pH 5–7. The cellulose‐immobilised preparation was more stable than the other two preparations after heating for 1 h at 55°C. © 1999 Society of Chemical Industry     

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。     

Purification and Properties of a Thermostable Inulinase (β-D-Fructan Fructohydrolase) from Bacillus stearothermophilus KP1289

A thermophilic soil isolate, Bacillus stearothermophilus KP1289, that grew from 41 °C to 69 °C, produced extracellular inulinases in the presence of inulin. One (inulinase II) of these enzymes was purified to homogeneity. The molecular weight (M_r) and the isoelectric point of the enzyme were estimated as 54,000 and 5.0, respectively. The enzyme was active between 30 and 75 °C and at pH 4.5—8.6 with an optimum at 60 °C and pH 6.1. At 69 °C and pH 7.0 the half-life of the enzyme was 10 min. The enzyme released fructose exo-wise from the non-reducing end of inulin (M_r = 4,5000). The Michaelis constant, catalytic center activity, and specificity constant for inulin at 60 °C and pH 5.0 were 80 mM (360 mg/mL), 460 s^—1, and 5.8 s^—1 mM^—1, respectively. The ratio of specificity constants for inulin, sucrose, and raffinose was 1:0.50:0.16. The enzyme was classified as a thermophilic thermostable β-D -fructan fructohydrolase (EC 3.2.1.80).     

青霉菌B01 产菊粉酶特性的研究及菊粉酶系分析

研究了pH 值、温度、底物浓度、金属离子对青霉菌(Penicillium sp.)B01 所产胞外菊粉酶活性的影响以及酶活随反应时间的变化情况。结果表明,pH4.6 时菊粉酶活力最大,最适反应温度是55℃。在pH3.6～5.4 的范围内,60℃以下酶活较稳定。Ca2+ 对菊粉酶有激活作用,而Cu2+ 对其活性强烈抑制。采用活性聚丙烯酰胺凝胶电泳法(PAGE)对该酶粗液进行蛋白质分析,获得较清晰的6 条蛋白谱带;2,3,5- 氯化三苯基四氮唑(TTC)活性染色结果显示,只有3 条谱带表现为具有菊粉酶活性。对酶解产物用薄层层析(TLC)和高效液相色谱(HPLC)法的分析结果,进一步证实这3 条谱带具有菊粉酶活性,并确定其主要为外切型菊粉酶,产物主要为果糖。     

菊粉酶特性的初步研究

以菊粉为底物研究了pH值、温度、底物浓度、金属离子对菊粉酶活性的影响及酶活随反应时间的变化情况，并测定该菊粉酶的米氏常数和酶活。实验结果表明，该菊粉酶反应的最适pH值为4.7，最适温度为60℃，Ca~(2+)和Zn~(2+)对菊粉酶有激活作用。该菊粉酶的米氏常数Km为0.35mol／L，V_(max)为0.52mg／min。     

雅致放射毛霉YYS-15菌株产菊粉酶发酵条件的研究

在摇瓶条件下对雅致放射毛霉YYS-15产菊粉酶的条件(不同的碳源、氮源、无机盐、初始pH值以及发酵量)进行了研究.经正交试验对其发酵条件优化后,确立了最适宜的发酵条件为菊粉20g/L、蛋白胨25g/L、KNO36g/L、KCl 5g/L、FeSO4·7H2O 0.1g/L、pH值为8.0,在28℃、振荡培养96h后酶活性达到27.36U/mL.     

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.