辣根过氧化物酶同工酶在不同介质中的动力学

本文研究了辣根过氧化物酶［ＥＣ１．１１．１．７］同工酶的联苯胺动力学。结果表明：其酸性酶和碱性酶的最适ｐＨ均为５．８左右。二者最适有机溶剂浓度略有差异：酸性酶最适乙醇浓度为５０％,最适二氧六环浓度为４０％;而碱性酶则分别为６０％和５０％。在水溶剂中,酸性酶为米氏酶,碱性酶为正协同的别构酶;在有机溶剂（如：乙醇、二氧六环）中,酸性酶为正协同的别构酶,碱性酶则仍为正协同的别构酶。即有机溶剂可能使酶构象     

有机溶剂对蜡蚧菌几丁质酶的影响

以蜡蚧菌(Ll)发酵液为材料,经硫酸铵沉淀、离子交换和凝胶过滤,获得部分纯化的Ll几丁质酶(EC3. 2. 1. 14)制剂.研究了不同有机溶剂对Ll几丁质酶的影响.结果表明,丙三醇、甲醛和戊二醛对几丁质酶有抑制作用;丙酮对酶有激活作用;甲醇、乙醇、正丁醇和乙二醇在低浓度时对酶有激活作用,随着浓度的升高表现出抑制作用;二氧六环的浓度低于6 %时对酶的影响不明显,而高于6 %时对酶则有激活作用.     

旋扭山绿豆根瘤菌MXDI6菌株氢酶诱导表达

在自生异养条件下,旋扭山绿豆根瘤菌ＭＸＤＩ６菌株的氢酶诱导表达受气相、ｐＨ值、镍等因子影响：氢酶表达的最适氧浓度为４％,最适氢浓度为１５％,二氧化碳没有明显影响;氢酶表达的ｐＨ值以５．０—６．０为宜;０．５μｍｏｌ／ＬＮｉＣｌ２明显促进吸氢活性,但镍浓度大于１μｍｏｌ／Ｌ则抑制吸氢活性．     

木霉T6木聚糖酶制剂研究

本文研究了木霉Ｔ６（Ｔｒｉｃｈｏｄｅｒｍａ ｓｐ．）产木聚糖酶固态发酵过程和木聚糖酶制剂制备,结果显示,固体曲培养４ｄ时酶活力最高,固体曲最适液固浸提比为７：１。木聚糖酶在６０～６５％硫酸铵饱和度下盐析效果最好。冷冻干燥和４０℃烘干酶粉得率分别为６８．３％和４５．７％。酶最适反应ＰＨ为４．５,最适反应温度５０℃,在不同温度下１小时后的半失活温度为４７．７℃。     

放线菌果胶酶产酶条件及酶促反应条件的研究

从埋麻土壤中分离到放线菌２９８株,经初筛和复筛得到产酶活性较高的一株放线菌５－７１。最适产酶条件是：果胶０．５％,葡萄糖０．５％,蛋白胨０．５％,酵母粉０．１％,Ｋ２ＨＰＯ４ ０．１％,ＫＨ２ＰＯ４ ０．１％,ＮａＣｌ０．１％,ＭｇＳＯ４·７Ｈ２Ｏ０．０５％,ｐＨ８．０。２５℃培养３天达产酶高峰。通气量对产酶影响不大。酶促反应最适条件是：ｐＨ９．６,４５℃,底物果胶浓度０．７５％,作用时间９０ｍｉ     

合成己酸乙酯脂肪酶产生菌的筛选及产酶条件

从２７株脂肪酶产生菌中筛选到能由乙醇和己酸合成己酸乙酯的菌株８株。其中Ｒｈｉｚｏｐｕｓｓｐ．Ｈ－３菌株脂肪酶活力为５０－６０ｕ／ｍｌ,全细胞在有机溶剂中的酯化率可达己酸的９１％。Ｈ３产酶的最适碳源为淀粉或葡萄糖。６％黄豆饼粉加４％蛋白陈复合氮源有利于酶活力的增加。     

谷胱甘肽硫转移酶(GST)的固定化及酶学特性研究

对谷胱甘肽硫转移酶的固定化、游离酶和固定化酶的酶学特性进行了研究,通过试验,确定谷胱甘肽硫转移酶的最佳固定化条件为先用2％壳聚糖吸附酶,然后再加戊二醛交联,交联用戊二醛浓度为1．2％,交联时间6h;游离酶的最适温度为45—55℃,最适pH值为6．5-7．0：固定化酶的最适温度为45-50℃,最适pH值为7．0;游离酶和固定化酶的最适酶促反应时间为30min。     

洗涤剂用碱性纤维素酶的研究（Ⅱ）——酶的提取及性质

产碱性纤维素酶的嗜碱芽孢杆菌ＳＨＹ８－５７２５发酵液经絮凝预处理后,采用混合盐析方法沉淀酶,盐析收率可达９５％以上,所制得酶粉ＣＭＣａｓｅ可达５００ｕ／ｇ以上。酶的最适ｐＨ为８．０和１０．５,稳定ｐＨ范围为４～１２;最适温度为４５℃（ｐＨ９．０,１０ｍｉｎ）稳定温度范围为５０℃（ｐＨ９．０,３０ｍｉｎ）以下;洗衣粉中各种表面活性剂和助剂对ＣＭＣａｓｅ基本无影响,酶的底物特异性表明,该碱性纤维素酶主     

无花果蛋白酶在阴离子交换树脂上的固定化

无花果蛋白酶通过８％戊二醛活化载体,共价结合到聚苯乙烯阴离子交换树脂ＧＭ２０１上,固定化作用在ｐＨ７．７,酶浓度０．８ｍｇ／ｇ树脂,４℃下进行６ｈ。得到的固定化酶表观Ｋ_ｍ值（酪蛋白,１．１１×１０~（－４）ｍｏｌ／Ｌ）小于溶液酶Ｋ_ｍ值（１．９６×１０~（－４）ｍｏｌ／Ｌ）;固定化酶活性在ｐＨ６～８保持稳定,溶液酶最适ｐＨ为７．２;固定化酶最适温度由溶液酶的５０～６０℃移至３７℃;固定化酶２５℃保持７ｄ,重复水解酪蛋白７次后,保留８３．３％活性。固定化酶对酪蛋白水解度达４７．５％,对大豆球蛋白达１１．６％。     

枯草杆菌（Bacillus subtilis）产果胶酶的研究：Ⅰ.菌种选育,鉴定 …

分离到１株高产果胶酶菌株，经形态，生理以及生化指标鉴定，确认为枯草杆菌（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）并命名为枯草杆菌１８。发酵液用９０％硫酸铵沉淀，透析后的粗酶经ＣＭ－５２柱层析，收集酶峰，再过ＳｈｅｐｈａｄｅｘＧ－１００得到部分纯化酶。该酶最适ＰＨ９．０，在ＰＨ９－１１稳定，最适反应温度６０℃，５０℃加热５０ｍｉｎ保存６０％酶活，６０℃加热５０ｍｉｎ酶活则保存１０％，酶的等电点（ｐＩ）４     

Effect of support material and enzyme pretreatment on enantioselectivity of immobilized subtilisin in organic solvents

Subtilisin Carlsberg was covalently attached to five macroporous acrylic supports of varying aquaphilicity (a measure of hydrophilicity). Kinetic parameters of the transesterification of S and R enantiomers of secphenethyl alcohol with vinyl butyrate, catalyzed by various immobilized subtilisins, were determined in anhydrous dioxane and acetonitrile. Enzyme enantioselectivity in acetonitrile, but not in dioxane, correlated with the aquaphilicity of the support; a mechanistic rationale for this phenomenon was proposed. Although the catalytic activity of immobilized subtilisin in anhydrous solvents strongly depended on enzyme pretreatment, the enantioselectivity was essential conserved. (c) 1994 John Wiley & Sons, Inc.     

Schedules for Sectioning Maize Kernels in Paraffin

For paraffin sectioning of maize kernels, the following technic is recommended: Use fresh, turgid kernels and utmost care in removing kernels from the cob and in subdividing into properly oriented slices. Kill and harden in a chrome-acetic-formalin formula. Rinse in water and dehydrate in four grades of dioxane to anhydrous; evacuate with an aspirator and infiltrate with paraffin. If anhydrous dioxane is excessively costly, dehydrate as above to the commercial grade and transfer by intermediate steps to one of the following two-solvent mixtures, using anhydrous ingredients, (A) dioxane and normal butyl alcohol, (B) dioxane and tertiary butyl alcohol, (C) normal butyl alcohol and chloroform, (D) tertiary butyl alcohol and chloroform. Evacuate in the final solvent. (Melted parowax floats on any of the above mixtures, affording gradual, progressive infiltration to pure parowax by periodic decantation and addition of wax.) Finally, transfer to compounded casting wax and cast in paper boats. To prepare a kernel segment for sectioning, fasten to a plastic block, shave the wax from the cutting plane and soak for 12-24 hours, at 35° C, in water containing a trace of safranin or other dye. Mordant starch grains in 1% tannic acid + 1/2% potassium metabisulphite. A wide choice of simple or multiple stains can be used. These methods are also applicable to tough old stems of corn and hemp, and possibly to many caryopses and seeds.     

Simple strategy of reactivation of a partially inactivated penicillin g acylase biocatalyst in organic solvent and its impact on the synthesis of β‐lactam antibiotics

Some reactions of organic synthesis require to be performed in rather aggressive media, like organic solvents, that frequently impair enzyme operational stability to a considerable extent. We have studied the option of developing a reactivation strategy to increase biocatalyst lifespan under such conditions, under the hypothesis that organic solvent enzyme inactivation is a reversible process. Glyoxyl agarose immobilized penicillin G acylase and cross‐linked enzyme aggregates of the enzyme were considered as biocatalysts performing in dioxane medium. Reactivation strategy consisted in re‐incubation in aqueous medium of the partly inactivated biocatalysts in organic medium, best conditions of reactivation being studied with respect to dioxane concentration and level of enzyme inactivation attained prior to reactivation. Best results were obtained with glyoxyl agarose immobilized penicillin G acylase at all levels of residual activity studied, with reactivations up to 50%; for the case of a biocatalyst inactivated down to 75% of its initial activity, full recovery of enzyme activity was obtained after reactivation. The potential of this strategy was evaluated in the thermodynamically controlled synthesis of deacetoxycephalosporin G in a sequential batch reactor operation, where a 20% increase in the cumulative productivity was obtained by including an intermediate stage of reactivation after 50% inactivation. Biotechnol. Bioeng. 2009;103: 472–479. © 2009 Wiley Periodicals, Inc.     

Immobilization and properties of carminomycin 4-O-methyltranferase, the enzyme which catalyzes the final step in the biosynthesis of daunorubicin in Streptomyces peucetius

The carminomycin 4-O-methyltransferase enzyme from Streptomyces peucetius was covalently immobilized on 3M Emphaze ABI-activated beads. Optimal conditions of time, temperature, pH, ionic strength, enzyme, substrate (carminomycin), and cosubstrate (S-adenosyl-L-methionine) concentrations were defined for the immobilization reaction. Protein immobilization yield ranged from 52% to 60%. Including carminomycin during immobilization had a positive effect on the activity of the immobilized enzyme but a strongly negative effect on the coupling efficiency. The immobilized enzyme retained at least 57% of its maximum activity after storage at 4 degrees C for more than 4 months. The properties of the free and immobilized enzyme were compared to determine whether immobilization could alter enzyme activity. Both soluble and bound enzyme exhibited the same pH profile with an optimum near 8.0. Immobilization caused an approximately 50% decrease in the apparent K(m) (K'(m)) for carminomycin while the K'(m) for S-adenosyl-L-methionine was approximately doubled. A 57% decrease in the V(max) value occurred upon immobilization. These changes are discussed in terms of active site modifications as a consequence of the enzyme immobilization. This system has a potential use in bioreactors for improving the conversion of carminomycin to daunorubicin. (c) 1995 John Wiley & Sons, Inc.     

Steady-state and transient-state kinetic studies on the oxidation of 3,4-dimethoxybenzyl alcohol catalyzed by the ligninase of Phanerocheate chrysosporium Burds

Catalysis of the H2O2-dependent oxidation of 3,4-dimethoxybenzyl (veratryl) alcohol by the hemoprotein ligninase isolated from wood-decaying fungus, Phanerochaete chrysosporium Burds, is characterized. The reaction yields veratraldehyde and exhibits a stoichiometry of one H2O2 consumed per aldehyde formed. Ping-pong steady-state kinetics are observed for H2O2 (KM = 29 microM) and veratryl alcohol (KM = 72 microM) at pH 3.5. The magnitude of the turnover number varies from 2 to 3 s-1 at this pH, depending on the preparation of the enzyme. Each preparation of enzyme consists of a mixture of active and inactive enzyme. Extensive steady-state kinetic studies of several different preparations of enzyme, suggest a mechanism in which H2O2 reacts with enzyme to form an intermediate that subsequently reacts with the alcohol to return the enzyme to the resting state. The pH dependence of the overall reaction indicates that an ionization occurs having an apparent pK alpha approximately 3.1. The activity is, thus, nearly zero at pH 5 and increases to a maximum near pH approximately 2. However, the enzyme is unstable at this low pH. Transient-state kinetic studies reveal that, upon reaction of ligninase with H2O2, spectral changes occur in the Soret region, which, by analogy to previous studies of horseradish peroxidase, are consistent with formation of Compounds I and II. The active form of the enzyme appears to react rapidly with H2O2; we observed a positive correlation between the turnover number of the enzyme preparation and the extent of a rapid reaction between H2O2 and ligninase to form Compound I. Free radical cations derived from veratryl alcohol do not appear to be released from the enzyme during catalysis; however, other substrates are known to be converted to cation radicals (Kersten, P., Tien, M., Kalyanaraman, B., and Kirk, T.K. (1985) J. Biol. Chem. 260, 2609-2612). Our results are generally consistent with a classical peroxidase mechanism for the action of ligninase on lignin-like substrates.     

Complete amino acid sequence and characterization of the reaction mechanism of a glucosamine-induced novel alcohol dehydrogenase from Agrobacterium radiobacter (tumefaciens)

A glucosamine-induced novel alcohol dehydrogenase has been isolated from Agrobacterium radiobacter (tumefaciens) and its fundamental properties have been characterized. The enzyme catalyzes NAD-dependent dehydrogenation of aliphatic alcohols and amino alcohols. In this work, the complete amino acid sequence of the alcohol dehydrogenase was determined by PCR method using genomic DNA of A. radiobacter as template. The enzyme comprises 336 amino acids and has a molecular mass of 36 kDa. The primary structure of the enzyme demonstrates a high homology to structures of alcohol dehydrogenases from Shinorhizobium meliloti (83% identity, 90% positive) and Pseudomonas aeruginosa (65% identity, 76% positive). The two Zn(2+) ion binding sites, both the active site and another site that contributed to stabilization of the enzyme, are conserved in those enzymes. Sequences analysis of the NAD-dependent dehydrogenase family using a hypothetical phylogenetic tree indicates that these three enzymes form a new group distinct from other members of the Zn-containing long-chain alcohol dehydrogenase family. The physicochemical properties of alcohol dehydrogenase from A. radiobacter were characterized as follows. (1) Stereospecificity of the hydride transfer from ethanol to NADH was categorized as pro-R type by NMR spectra of NADH formed in the enzymatic reaction using ethanol-D(6) was used as substrate. (2) Optimal pH for all alcohols with no amino group examined was pH 8.5 (of the C(2)-C(6) alcohols, n-amyl alcohol demonstrated the highest activity). Conversely, glucosaminitol was optimally dehydrogenated at pH 10.0. (3) The rate-determining step of the dehydrogenase for ethanol is deprotonation of the enzyme-NAD-Zn-OHCH(2)CH(3) complex to enzyme-NAD-Zn-O(-)CH(2)CH(3) complex and that for glucosaminitol is H(2)O addition to enzyme-Zn-NADH complex.     

Structural and catalytic response to temperature and cosolvents of carboxylesterase EST1 from the extremely thermoacidophilic archaeon Sulfolobus solfataricus P1

The interactive effects of temperature and cosolvents on the kinetic and structural features of a carboxylesterase from the extremely thermoacidophilic archaeon Sulfolobus solfataricus P1 (Sso EST1) were examined. While dimethylformamide, acetonitrile, and dioxane were all found to be deleterious to enzyme function, dimethyl sulfoxide (DMSO) activated Sso EST1 to various extents. This was particularly true at 3.5% (v/v) DMSO, where k(cat) was 20-30% higher than at 1.2% DMSO, over the temperature range of 50-85 degrees C. DMSO compensated for thermal activation in some cases; for example, k(cat) at 60 degrees C in 3.5% DMSO was comparable to k(cat) at 85 degrees C in 1.2% DMSO. The relationship between DMSO activation and enzyme structural characteristics was also investigated. Nuclear magnetic resonance spectroscopy and circular dichroism showed no gross change in enzyme conformation with 3.5% DMSO between 50 and 80 degrees C. However, low levels of DMSO were shown to have a small yet significant change in enzyme conformation. This was evident through the reduction of Sso EST1's melting temperature and changes in the microenvironment of the enzyme's tyrosine and tryptophan residues at 3.5% versus 1.2% (v/v) solvent. Finally, activation parameter analysis based on kinetic data, at 1.2% and 3.5% DMSO, implied an increase in conformational flexibility with additional cosolvent. These results suggest the activating effect of DMSO was related to small changes in the enzyme's structure resulting in an increase in its conformational flexibility. Thus, in addition to their use for solubilizing hydrophobic substrates in water, cosolvents may also serve as activators in applications involving thermostable biocatalysts at sub-optimal temperatures.     

Alcohol and temperature induced conformational transitions in ervatamin B: sequential unfolding of domains

The structural aspects of ervatamin B have been studied in different types of alcohol. This alcohol did not affect the structure or activity of ervatamin B under neutral conditions. At a low pH (3.0), different kinds of alcohol have different effects. Interestingly, at a certain concentration of non-fluorinated, aliphatic, monohydric alcohol, a conformational switch from the predominantly alpha-helical to beta-sheeted state is observed with a complete loss of tertiary structure and proteolytic activity. This is contrary to the observation that alcohol induces mostly the alpha-helical structure in proteins. The O-state of ervatamin B in 50% methanol at pH 3.0 has enhanced the stability towards GuHCl denaturation and shows a biphasic transition. This suggests the presence of two structural parts with different stabilities that unfold in steps. The thermal unfolding of ervatamin B in the O-state is also biphasic, which confirms the presence of two domains in the enzyme structure that unfold sequentially. The differential stabilization of the structural parts may also be a reflection of the differential stabilization of local conformations in methanol. Thermal unfolding of ervatamin B in the absence of alcohol is cooperative, both at neutral and low pH, and can be fitted to a two state model. However, at pH 2.0 the calorimetric profiles show two peaks, which indicates the presence of two structural domains in the enzyme with different thermal stabilities that are denatured more or less independently. With an increase in pH to 3.0 and 4.0, the shape of the DSC profiles change, and the two peaks converge to a predominant single peak. However, the ratio of van't Hoff enthalpy to calorimetric enthalpy is approximated to 2.0, indicating non-cooperativity in thermal unfolding.     

Purification and characterization of a novel thermostable lipase from Pseudomonas cepacia.

A thermostable lipase from Pseudomonas cepacia has been purified to homogeneity as judged by SDS-PAGE and isoelectric focusing. The purification included treatment of the culture supernatant with acrinol, hydrophobic interaction chromatography, and gel filtration. The enzyme was a monomeric protein with M(r) of 36,500 and pI of 5.1. The optimal pH at 50 degrees C and optimal temperature at pH 6.5 were 5.5-6.5 and 55-60 degrees C, respectively, when olive oil was used as the substrate. Simple triglycerides of short and middle chain fatty acids (C < or = 12) were the preferred substrates over those of long chain fatty acids. The enzyme cleaved all the ester bonds of triolein, with some preference for the 1,3-ester bonds. The enzyme retained all its activity even after incubation at 75 degrees C (pH 6.5) for 30 min. Further, the activity was not impaired during 21 h storage at pH 6.5 in 40% water-miscible solvents including methanol, ethanol, acetone, acetonitrile, dimethylformamide, dimethylsulfoxide, and dioxane. The addition of dimethylsulfoxide or acetone to the assay mixture in the range of 0-35% stimulated the enzyme, whereas benzene or n-hexane had an inhibitory effect. These properties together with the N-terminal amino acid sequence confirmed that the enzyme differs from the known Pseudomonas sp. lipases.     

Cooperativity of covalent attachment and ion exchange on alcalase immobilization using glutaraldehyde chemistry: Enzyme stabilization and improved proteolytic activity

Alcalase was scarcely immobilized on monoaminoethyl-N-aminoethyl (MANAE)-agarose beads at different pH values (<20% at pH 7). The enzyme did not immobilize on MANAE-agarose activated with glutaraldehyde at high ionic strength, suggesting a low reactivity of the enzyme with the support functionalized in this manner. However, the immobilization is relatively rapid when using low ionic strength and glutaraldehyde activated support. Using these conditions, the enzyme was immobilized at pH 5, 7, and 9, and in all cases, the activity vs. Boc-Ala-ONp decreased to around 50%. However, the activity vs. casein greatly depends on the immobilization pH, while at pH 5 it is also 50%, at pH 7 it is around 200%, and at pH 9 it is around 140%. All immobilized enzymes were significantly stabilized compared to the free enzyme when inactivated at pH 5, 7, or 9. The highest stability was always observed when the enzyme was immobilized at pH 9, and the worst stability occurred when the enzyme was immobilized at pH 5, in agreement with the reactivity of the amino groups of the enzyme. Stabilization was lower for the three preparations when the inactivation was performed at pH 5. Thus, this is a practical example on how the cooperative effect of ion exchange and covalent immobilization may be used to immobilize an enzyme when only one independent cause of immobilization is unable to immobilize the enzyme, while adjusting the immobilization pH leads to very different properties of the final immobilized enzyme preparation. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2768, 2019.