固定化黑曲霉增殖细胞生产低聚果糖的研究

黑曲霉孢子经海藻酸钙包埋３３ｈ后得到的固定化增殖细胞其酶活性达到最高，机械强度仍保持较高水平（为起始时８０％），最适ｐＨ为５．０，与游离酶相同，最适温度为５５℃，比游离酶高５℃；有害离子对其影响较小，其Ｋｍ（以蔗糖为底物）为８２ｍｍｏｌ．将固定化增殖细胞装入填充式反应柱，产品中低聚果糖含量在５０％～５５％，操作一个月活性保持不变。     

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。     

高纯度低聚果糖的研制

低聚果糖具有优越的生理功能,但是普通级低聚果糖产品含有近50％葡萄糖、蔗糖和果糖,它们没有特殊的功能,并且可能会损害低聚果糖的生理功能．为了制备高纯度低聚果糖,已开发了一些新的技术,例如,采用固定化黑曲霉细胞和葡萄糖异构酶,或采用一种由葡萄糖氧化酶和过氧化物酶组成的双酶体系的酶处理方法和采用模拟移动床的离子交换法．本文简单地介绍这些工艺．     

双酶法生产高纯度低聚果糖的研究

以果糖转移酶作用于蔗糖制得的低聚果糖为原料，将葡萄糖氧化酶及过氧化氢酶协同作用于３０％的总糖，在３０℃，ｐＨ５．０条件下，通气反应１６ｈ，得到纯度为８６．９２％（总低聚果糖占总糖）的低聚果糖。     

高纯度低聚糖的研制

低聚果糖具有优越的生理功能，但是普通级低聚果糖产品含有近50％葡萄糖，蔗糖和果糖，它们没有特殊的功能，并且可能会损害低聚果糖的生理功能。为了制备高纯低聚果糖，已开发了一些新的技术，例如，采用固定化黑曲霉细胞和葡萄糖异构酶，或采用一种由葡萄糖氧化酶和过氧化酶组成的双酶体系的酶处理方法和采用模拟移动床的离子交换法。本简单地介绍了这些工艺。     

制备高含量低聚果糖联产D-阿洛酮糖的工艺研究

制备蔗糖源高含量低聚果糖主要利用色谱分离技术除去普通级低聚果糖产品中的葡萄糖和蔗糖，得到高含量低聚果糖组分和副产物组分。为进一步高值化利用副产物，赋予其功能性及扩大应用范围，在制备低聚果糖工艺技术基础上利用蔗糖转化酶、葡萄糖异构酶、D-阿洛酮糖3-差向异构酶、色谱分离依次处理副产物，并通过单因素试验优化D-阿洛酮糖3-差向异构酶异构反应及色谱分离纯化参数。结果得到：在反应温度45℃、p H5.75、每克底物55 U酶量、反应时间12 h的条件下，底物中果糖质量浓度在90%以上；色谱分离关键参数：洗脱液流量15 L/min，外加压力50 kPa。D-阿洛酮糖最高转化率为27.1%，成品糖浆中D-阿洛酮糖含量>91%，整体工艺总产率为91%。研究结果表明联产工艺技术可行性高，要进行工业化生产还需进一步优化。     

利用酵母提高低聚果糖纯度

将黑曲霉AS0023生产的果糖转移酶作用于底物蔗糖,可制备含量为55%的低聚果糖,但该产品中亦含有30%的副产物葡萄糖.利用酵母消化低聚果糖产品中的葡萄糖,可生产高纯度低聚果糖.将筛选得到具有较弱转化酶活性的酵母SK2.003,经培养后添加于总糖浓度为20%的低聚果糖中,经30℃、250r/min反应24h,制得纯度为80.24%的低聚果糖.     

利用重组葡萄糖脱氢酶提高低聚果糖纯度

用IPTG（诱导基因工程菌）M15／pQE31-gdh表达葡萄糖脱氢酶，纯化，该重组葡萄糖脱氢酶用于高纯度低聚果糖生产研究。以含量为51．11％（低聚果糖／总糖）低聚果糖的产品为底物，葡萄糖脱氢酶（5U／g总糖）作用2h，将低聚果糖含量提高到61，37％，消除了48，81％的葡萄糖。以30％（w／v）蔗糖为底物，利用有果糖基转移酶活性的米曲霉GX0015（15U／g总糖）作用6h后，加入葡萄糖脱氢酶（5U／g总糖）协同作用4h，得到成分为：低聚果糖含量76．87％、葡萄糖11．05％、蔗糖含量5.21％的产品。由果糖基转移酶和葡萄糖脱氢酶组成的系统能够有效降低葡萄糖含量，提高低聚果糖含量。     

低聚果糖研究进展

介绍国内外以蔗糖为基质用活菌体或酶，采用间歇式或连续式生产低聚果糖的方法，以及生产中采用酶母菌、葡萄糖氧化酶、葡萄糖异构酶、糖苷酶消除葡萄糖，提高低聚果糖产率的几种途径。     

粉状高浓低聚果糖和葡萄糖酸钙的生产

粉状高浓低聚果糖和葡萄糖酸钙的生产刘耀东等（台湾糖业公司糖业研究所）由果糖转移酶从反应液蔗糖中生成低聚果糖的最高转化率仅为５０～６０％（Ｗ／Ｖ）。由于有一定量的葡萄糖和蔗糖存在于所产低聚果糖中，特殊用途之低聚果糖仍未可获。采用果糖转移酶和葡萄糖氧化酶...     

固定化酶化学发光型葡萄糖传感器法测定食品中的葡萄糖

制成了固定化酶化学发光型葡萄糖传感器,此传感器可用于对葡萄糖的在线快速检测。用MCM-41介孔分子筛固定葡萄糖氧化酶(GOD),制成GOD酶柱,并用于流动注射化学发光分析。酶柱的使用条件为:柱温40℃,葡萄糖溶液pH=6.0,流速3 r/min。测定的线性范围为1～200 mg/L,检出限为0.2 mg/L,10次测定的RSD为1.8%,回收率在97.81%～102.0%。测定结果与国标方法的测定结果无显著性差异。     

An optical flow injection analysis system for measurement of glucose in tomato

An optical flow injection analysis (FIA) system for glucose measurement in tomato was constructed by using a glucose oxidase (GOD) immobilized reactor and a photodiode sensor, a peristaltic pump and a personal computer. The GOD reactor was crammed with strip-shaped of a GOD immobilized membrane to an acrylic pipe. The GOD was immobilized on polytetrafluoroethylene membrane by crosslinking with glutaraldehyde. Hydrogen peroxide generated by the GOD reaction oxidized luminol to produce chemiluminescence in the presence of horseradish peroxide. The chemiluminescence was detected by using the photodiode. The glucose measurement system was calibrated with standard glucose solutions. The calibration range of the system for glucose was 2.0–100.0 mmol L⁻¹. Measurement time for every sample was within 3 min. Glucose concentrations of a fresh and processed tomato samples measured with this glucose measurement system could be evaluated rapidly and non-laboriously.     

黑曲霉H1-9b 葡萄糖氧化酶的分离纯化及部分性质研究

采用研磨法破碎黑曲霉H1-9b 菌丝体,经pH5.7 磷酸缓冲液抽提获得粗酶液,粗酶液经DEAE-Sepharose离子交换层析和Superdex-200 凝胶过滤层析,得到葡萄糖氧化酶纯品。该酶的比活力为30569.7U/mg,回收率为30.2%,提纯倍数为41.4 倍,分子质量为94.1kD,为单亚基酶。该酶最适温度为37℃,最适pH 值为5.7,在30～40℃温度范围内稳定性较好,在pH4.0～8.0 范围内活性稳定。以不同浓度葡萄糖为底物在最适条件下测得该酶K_m 值为30.69mmol/L,V_max 为21.88μmol/L。Ag^+、Cu²⁺、Zn²⁺ 对该酶活性有较大抑制作用。结果表明,该酶稳定性较好,有一定的应用价值。     

Activity and stability of glucose oxidase in molecular films assembled alternately with polyions

Anionic glucose oxidase (GOD) was assembled alternately with polycations, namely, poly(ethylenimine) (PEI) and poly(dimethyldiallyl-ammonium chloride) (PDDA), in the preparation of molecular films. Enzymatic activity of the films was investigated by sequential redox reaction with glucose, peroxidase (POD) and DA67 dye. The apparent activity was not influenced by substrate diffusion at up to 5 microg of immobilized GOD (at the area of 5 x 5 mm(2) x 2 faces). This is ascribed to the less dense packing of the alternate molecular film compared with Langmuir-Blodgett (LB) films. Immobilized GOD could be released into solution, and its activity was about 80% of native GOD, indicating that the immobilization did not cause significant denaturation. The enzyme activity of the GOD film was maintained for 14 weeks when stored in buffer and in air at 4 degrees C. Activity measurement after incubation at elevated temperatures showed that significant deactivation was not observed up to 50 degrees C. This shows that GOD in the film has higher thermostability than native GOD. The pH profile of the GOD activity in the film became broad and shifted towards higher pH than that of native GOD. The GOD film was also prepared by the premixing method, in which a GOD-polyion complex was assembled alternately with another oppositely-charged polyion. The enzyme activity of the alternate film obtained by premixing was much higher (maximal enhancement, 67-fold) than that of the conventionally assembled films. Better dispersion of GOD in the premixed film appears to enhance the enzyme activity.     

PEI对固定化增殖细胞性质的影响

以海藻酸钙为载体包埋黑曲霉AS0 0 2 3孢子 ,培养后得到的固定化增殖细胞用于生产低聚果糖 .利用材料试验仪研究了聚乙烯亚胺 (PEI)对固定化增殖细胞强度的影响 ,并研究了PEI对固定化增殖细胞酶活及最适 pH值的影响 .结果表明 ,经PEI处理后 ,尽管固定化增殖细胞酶活略有降低 ,最适 pH值由 5.5降至 4 .0 ,但凝胶强度有明显改善 .起始机械强度由 0 .0 6N/粒增加到 0 .2 79N/粒 ,而机械强度衰减率仅为未处理组的 9.2 7% .     

壳聚糖凝胶固定葡萄糖氧化酶制备酶电极的工艺

以壳聚糖作为固定GOD载体 ,研究了壳聚糖溶液的粘度 ,交联剂戊二醛的浓度、用量以及铂丝在酶膜母液中浸涂时间等对葡萄糖传感性能的影响。在优化固定化条件的基础上 ,建立了通过调整固定化条件来适应载体溶液粘度变化的工艺 ,制备出性能基本一致的酶电极     

固定化酶法制备葡萄糖酸钙的研究

目的用固定化葡萄糖氧化酶制备葡萄糖酸钙。方法将葡萄糖氧化酶和过氧化氢酶共价固定到经戊二醛预处理的壳聚糖上,用所得固定化酶催化葡萄糖转化为葡萄糖酸钙。结果固定化葡萄糖氧化酶和过氧化氢酶的回收率分别为86.9%和54.3%;反复用该固定化酶制备葡萄糖酸钙4次后,其酶活性还分别剩余26.5%和21%。结论用固定化酶法制备葡萄糖酸钙,可提高产品纯度,降低成本。     

Inhibition of food-related pathogenic bacteria by god-transformed Penicillium nalgiovense strains.

Two strains of Penicillium nalgiovense, which carried the god gene of Aspergillus niger and had increased glucose oxidase (GOD) activity compared with the wild-type strain, were tested for their ability to suppress the growth of certain food-related pathogenic bacteria. In contrast to the wild type, which showed no antibacterial effect when grown in mixed culture with different bacteria, the two transformed strains were highly antagonistic. The strain that expressed higher amounts of GOD in general had higher inhibitory activity. Both strains showed antibacterial activity against Listeria monocytogenes, Salmonella Enteritidis, and Staphylococcus aureus. The inhibitory activity was dependent on the glucose concentration in the medium. S. aureus was completely inhibited at 1% glucose in the presence of the higher GOD-producing transformant. In contrast, if arabinose was used as a carbon source, no inhibition occurred. If catalase was added to the medium, the inhibitory activity of the transformants was completely inactivated, indicating that the hydrogen peroxide produced was responsible for the antibacterial activity of the transformants.     

产葡萄糖氧化酶菌株的诱变筛选及遗传稳定性研究

采用平板显色初筛、Underbofler滴定法复筛及紫外-硫酸二乙酯复合诱变的方法,从临沂果园土样中得到一株产葡萄糖氧化酶（GOD）活性较好的菌株,命名为Bla-6,其产GOD酶活为（10.50±0.25） U/mL,与原始菌株LPA-4产GOD酶活（6.75 U/mL）相比提高了55.57%。结合5.8S rDNA-ITS区序列系统进化、菌落形态、生长特性、菌体形态等确定其分类地位。结果表明,所得菌株Bla-6被鉴定为黑曲霉（Aspergillus niger）,经8代传代培养后,GOD酶活维持在10.5 U/mL左右,该菌株遗传稳定性较好。     

以黑曲霉生产面包品质改良复合酶的研究

从粮食样品中分离并筛选出 1株可同时产生α 淀粉酶 (FAA)和葡萄糖氧化酶(GOD)的黑曲霉菌株。经试验表明 ,该菌株在适宜的条件下可稳定产生FAA 2 0 0u/mL以上 ,GOD 1 5u/ g以上。产生的 2种复合酶在pH 6～ 7下可表现最高酶活力 ,酶作用的适宜温度为 3 0～ 40℃ ,40℃下保温 3 0min酶活降低 5 0 %左右 ,在 60℃以上 2种酶均基本无活性 ,适宜用作面包制作的品质改良剂