金针菇漆酶活性测定的最佳反应条件及液体培养胞外酶的研究

研究了ABTS[(2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)]法测定金针菇漆酶活性的最佳反应条件,确定了显色反应的最佳条件为:适宜测定波长为410nm、体系pH4.0、反应温度40℃。对金针菇LP03液体培养过程中的胞外酶活性进行检测,研究了漆酶、纤维素酶、木聚糖酶、过氧化物酶、锰过氧化物酶和淀粉酶的变化规律。在整个培养过程中这6种酶的活性存在很大差异。     

Laccase and Mn-peroxidase production by Coriolus hirsutus strain 075 in a jar fermentor

The white-rot fungus Coriolus hirsutus strain 075 excretes considerable amounts of laccase and Mn-peroxidase into culture broth over a brief production time. The effects of agitation speed, temperature, aeration and inoculum amount on laccase production using a 10-l fermentor were studied. The optimum fermentation conditions were a 15% inoculum, an aeration rate of 0.88 vvm, an agitation speed of 160 rpm, and a temperature of 28 degrees C. By optimizing the fermentation conditions, the laccase activity reached 80+/-3 U/ml in 3 d and the purified enzyme output was 30 mg/l. The laccase and Mn-peroxidase were purified by means of isoelectrofocusing and ion-exchange chromatography. The pIs of the laccase isoenzymes were 4.2 and 4.5. Mn-peroxidase had only one isoenzyme with a pI of 3.2. The optimum pH was 4.5 for laccase with syringaldazine as the substrate and 5.0-5.3 for Mn-peroxidase with Mn(+2) and H2O2 as the substrates. The laccase and Mn-peroxidase retained 50% of their activities at 50 degrees C after 55 h and 12 h of incubation time, respectively.     

Purification and characterization of an l-amino acid oxidase from Pseudomonas sp. AIU 813

An l-amino acid oxidase was found from a newly isolated strain, Pseudomonas sp. AIU 813. This enzyme was remarkably induced by incubation with l-lysine as a nitrogen source, and efficiently purified using an affinity chromatography with l-lysine as ligand. The enzyme oxidized l-lysine, l-ornithine and l-arginine, but not other l-amino acids and d-amino acids. The oxidase activity for l-lysine was detected in a wide pH range, and its optimal was pH 7.0. In contrast, the oxidase activity for l-ornithine and l-arginine was not shown in acidic region from pH 6.5, and optimal pH for both substrates was 9.0. The enzyme was a flavoprotein and composed of two identical subunits with molecular mass of 54.5?kDa. The N-terminal amino acid sequence was similar to that of putative flavin-containing amine oxidase and putative tryptophan 2-monooxygenase, but not to that of l-amino acid oxidases.     

Catalytic activity of laccase hosted in reversed micelles

Nanostructured reversed micelles induce a high laccase activity in organic solvents, because enzymes can maintain their highly dimensional structure in water pools of reversed micelles [RMs]. Laccase attracts considerable attention as a novel industrial enzyme due to its high capability to catalyze the oxidation of aromatic compounds. The catalytic activities of lyophilized laccase and laccase entrapped in RMs were compared using an oxidative reaction. Laccase hosted in an anionic RM effectively catalyzed the oxidative reaction in various organic solvents, while lyophilized laccase exhibited no such catalytic activity. To optimize the preparation and reaction conditions for laccase in RMs, we examined the effects of pH of water pools of RMs, the concentrations of both enzyme and surfactant for the preparation of RMs, the hydration ratio (Wo), and the reaction temperature on laccase catalytic activity in organic media. Laccase entrapped in RMs exhibited the highest catalytic activity in isooctane under the following conditions: bis-2-ethylhexyl sulfosuccinate sodium salt (AOT) of 100 mM, pH 6.0, Wo=40, and reaction temperature of 60 degrees C. Under the optimum conditions, environmental pollutants such as bisphenol A, 2,4-dichlorophenol and 2,4,6-trichlorophenol were effectively degraded in 3 h.     

A SPECTROPHOTOMETRIC ASSAY FOR LACCASE USING o-TOLIDINE

A spectrophotometric method for measuring laccase activity using o-tolidine has been developed. Oxidation of o-tolidine by laccase to a blue colored product corresponded with increases in absorbancies at 366 and 630 nm. This oxidation reaction and increases in absorbance at 366 and 630 nm could also be mimicked using hypochlorite, periodate and UV light in place of laccase. After a lag period, the assay was linear in absorbance with time, although the duration of linear region appeared to be affected by the pH. When assayed from 0.025 to 7 mM tolidine, maximum oxidation of substrate occurred using 3 mM o-tolidine. Oxidation of o-tolidine exhibited a pH dependency and showed an apparent pH optimum at approximately 5.0. The utility of this assay was shown by determining laccase activity in various fungal extracts.     

Cloning, over-expression and characterization of an alkali-tolerant endo-β-1,4-mannanase from Penicillium freii F63

A glycosyl hydrolase family 5 endo-β-mannanase gene (man5F63) was cloned from Penicillium freii F63 and overexpressed in Pichia pastoris. man5F63 contained an open reading frame of 1260 bp that encoded a polypeptide of 419 amino acids including a putative 18-residue signal peptide. The recombinant enzyme (rMan5F63) was secreted into the culture supernatant to near electrophoretic homogeneity with a high yield (1.1 gl(-1) in flask). Its apparent molecular weight was approximately 72.0 kDa, 29.0 kDa higher than the theoretical molecular mass. rMan5F63 was optimal at pH 4.5 and 60 °C and exhibited good stability over a broad pH range from acidic to alkaline (>85.0% activity at pH 4.0-9.0, >70.0% activity at pH 10.0 and 43.7% even at pH 12.0). The activity of rMan5F63 was significantly enhanced in the presence of Co(2+), Cu(2+), Mn(2+) and β-mercaptoethanol and was strongly inhibited by Hg(2+) and SDS. The specific activity, K(m) and V(max) values were 47.5 U mg(-1), 7.8 mg ml(-1) and 70.4 μmol min(-1)mg(-1), respectively, for locust bean gum, and 40.3 U mg(-1), 2.3 mg ml(-1) and 61.7 μmol min(-1)mg(-1), respectively, for konjac flour. All these favorable enzymatic properties make it cost-effective to commercialization and valuable in various industries.     

白腐菌液体菌种培养条件的试验研究

对3种白腐菌的液体菌种培养条件进行了优化研究.结果表明,黄孢原毛平革菌液体菌种培养的较优条件为培养时间4 d、初始pH6.0、装量50 mL、琼脂添加量0.2%,W3液体菌种培养的较优条件为培养时间5 d、初始pH6.5、装量50 mL、琼脂添加量0.3%,变色栓菌液体菌种培养的较优条件为培养时间5 d、初始pH6.5、装量75 mL、琼脂添加量0.1%.     

竹黄菌液态发酵产漆酶培养条件的优化

分离筛选到一株产漆酶的竹黄菌,采用单因子相互比较法,研究了该菌株的最适发酵产酶条件。确定了最适的碳源为2 g/dL可溶性淀粉、最适的氮源为0.8 g/dL的酵母膏,以及最适铜离子浓度为2.4 mmol/L,在自然pH 5.0,装液量为50 mL,30℃、200 r/min摇瓶振荡培养64 h下,产漆酶酶活水平达120 000 U/L,比优化前提高近17倍。该竹黄菌株与已报道的白腐真菌相比,具有发酵周期短且产漆酶酶活较高的优点。     

Isolation of five laccase gene sequences from the white-rot fungus Trametes sanguinea by PCR, and cloning, characterization and expression of the laccase cDNA in yeasts

To obtain laccase-gene-specific sequences from the white-rot fungus Trametes sanguinea M85-2, a PCR screening method was used. Degenerate primers were designed based on highly conserved copper-binding regions I and IV of known laccases and used to amplify laccase sequences from T. sanguinea M85-2 genomic DNA. A single 1.6-kbp DNA band was amplified and cloned into a vector. Partial sequences of 21 clones were classified into five groups (lcc1-5) and the deduced amino acid sequences were all homologous to known laccase sequences. Based on the partial sequence of lcc1, the 5'-end of its cDNA was obtained by a PCR termed 5' rapid amplification of cDNA ends (5'-RACE), and RT-PCR was then carried out using the 5'-primer and the poly-dT primer to obtain the full-length lcc1 cDNA. The obtained cDNA encoded a protein consisting of 518 amino acid residues and its first 21 amino acid residues were predicted to be the signal peptide for secretion. The conserved characteristic structures of laccase, such as copper-binding ligands, N-glycosylation sites, and cysteine residues for disulfide bridges, were observed. The genomic DNA sequence of the lcc1 gene was also cloned by PCR method and the sequence revealed 10 introns. The lcc1 cDNA was inserted into yeast vectors for heterologous expression by Saccharomyces cerevisiae and Pichia pastoris. Phenol-oxidizing activity was detected from transformants of the yeasts, indicating that the obtained cDNA encodes a laccase. Previously, two laccase isozymes were biochemically characterized and purified from T. sanguinea M85-2. Using the sequential PCR method presented here, we have obtained partial sequences of at least five laccase genes and one cDNA clone encoding a protein with laccase activity but without any enzymatic information, suggesting that expressed enzymes under restricted conditions may not represent all the isozymes in target microorganisms. PCR cloning and heterologous expression of the cloned genes can be an alternative method of screening enzymes if these enzymes have conserved sequences.     

金针菇多糖提取新工艺的优化

对金针菇子实体多糖的提取新工艺进行了系统的优化研究。结果表明 ,子实体多糖提取的最优工艺为 :原料经预处理 ,经 0 .15 %的纤维素酶在 40℃、pH4 5条件下水解 3h ,再用 2 0倍样品重量的水在 10 0℃、pH6 5条件下浸提 1h ,过滤 ,滤液经MWCO5 0 0 0的超滤膜在40℃、0 .2MPa下浓缩至原体积的 1/3后用终体积分数为 70 %的乙醇沉淀 ,经脱蛋白干燥 ,得到纯品金针菇多糖 ,产率为 19 9g/g(干重 ) ,说明用超滤浓缩代替热浓缩是可行的。     

凝固型金针菇酸奶的研制

探讨了以金针菇发酵上清液或发酵混合液与鲜牛奶按不同比例混合对凝固型酸奶品质的影响．结果表明：添加10％金针菇发酵上清液或15％发酵混合液到鲜牛奶中，再加入6％的蔗糖和0．2％黄原胶，采用普通酸奶的制作工艺可生产出具有菇味清香，酸甜适口、凝乳均匀、色泽淡黄的凝固型金针菇酸奶。     

Purification and characterization of trypsin from the viscera of sardine (Sardina pilchardus)

Trypsin from the viscera of Sardina pilchardus was purified by fractionation with ammonium sulphate, heat treatment and Sephadex G-100 gel filtration with a ninefold increase in specific activity and 9% recovery. The molecular weight of the enzyme was estimated to be 25,000 Da on SDS–PAGE. This enzyme showed esterase-specific activity on Nα-benzoyl-l-arginine ethyl ester (BAEE). The purified enzyme was inhibited by benzamidine, a synthetic trypsin inhibitor, and phenylmethylsulphonyl fluoride (PMSF) a serine-protease inhibitor, but was not inhibited by the β-mercaptoethanol. The optimum pH and temperature for the enzyme activity were pH 8.0 and 60 °C, respectively. The relative activity at pH 9.0 was 95.5% and the enzyme showed pH stability between 6.0 and 9.0. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGYECQKYSQ. S. pilchardus trypsin, which showed high homology to other fish trypsins, had a charged Lys residue at position 9, where Pro or Ala are common in fish trypsins. The enzyme was strongly inhibited by Zn²⁺ and Cu²⁺.     

阿魏萃取物对阿魏菇发酵培养液中3 种酶酶活力的影响

以阿魏菇NB-1 为液体发酵菌种,在发酵培养液中添加阿魏的三氯甲烷萃取物,27.5℃培养7d,测定发酵液中漆酶、多酚氧化酶(PPO)、蛋白酶以及菌丝体中PPO 酶活力。结果表明：阿魏三氯甲烷萃取物添加量为10、50、100mg/100mL 时,发酵液中漆酶酶活力分别是对照的40、50、50 倍;发酵液PPO 酶活力分别是对照的4.43、30.14、43.14 倍;菌丝体中PPO 酶活力分别是对照的1.6、0.93、1.06 倍,萃取物对菌丝体PPO 酶活力产生了双重作用;发酵液蛋白酶酶活力分别为对照的0.99、0.85、0.67 倍,表现出随阿魏三氯甲烷萃取物剂量增加,酶活力降低的趋势。     

Coriolus versicolor菌丝球用于造纸废水脱色的探讨

对白腐真菌(Coriolus versicolor)产生漆酶进行了研究,探讨了培养因子对漆酶产量的影响,结果发现该菌产漆酶的最适初始PH值为4.5.提高微量元素浓度或添加藜芦醇都可使C.versicolor的产酶能力增加,添加Tween80会有一定的促进作用.采C.versicolor菌丝球进行重复分批产酶试验,结果表明菌丝球的稳定性很好,同一批菌丝球可连续利用10次,平均每批酶活力可保持在9.6IU/ml,产酶能力优于聚氨酯泡沫固定化菌丝.将粗酶液和少量ABTS用于造纸废水脱色试验,在酶活力为5IU/ml、色度浓度186倍条件下,经过24h反应,脱色率达到80%.     

New alkaline trypsin from the intestine of Grey triggerfish (Balistes capriscus) with high activity at low temperature: Purification and characterisation

A highly alkaline trypsin from the intestine of Grey triggerfish (Balistes capriscus), with high activity at low temperature, was purified and characterised. The enzyme was purified to homogeneity using acetone precipitation, Sephadex G-100 gel filtration and Mono Q-Sepharose anion-exchange chromatography, with a 13.9-fold increase in specific activity and 41.3% recovery. The molecular weight of the purified alkaline trypsin was estimated to be 23.2 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and size exclusion chromatography. Purified trypsin appeared as a single band on native–PAGE. Interestingly, the enzyme was highly active over a wide range of pH, from 9.0 to 11.5, with an optimum at pH 10.5, using Nα-benzoyl-DL-arginine-p-nitroanilide (BAPNA) as a substrate. The relative activities at pH 9.0, 11.5 and 12.0 were 86.5%, 92.6% and 52.4%, respectively. The enzyme was extremely stable in the pH range 7.0–12.0. In addition, the enzyme had high activity at low and moderate temperatures with an optimum at around 40 °C and had more than 80% of its maximum activity at 20 °C. The purified enzyme was strongly inhibited by soybean trypsin inhibitor (SBTI) and phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor. The enzyme showed extreme stability towards oxidising agents, retaining about 87% and 80% of its initial activity after 1 h incubation at 40 °C in the presence of 1% sodium perborate and 1% H₂O₂, respectively. In addition, the enzyme showed excellent stability and compatibility with some commercial solid detergents.     

金针菇多糖-Zn~(2+)螯合物对L929肿瘤细胞的增殖抑制作用及其抗氧化活性

比较研究金针菇多糖-Zn~(2+)螯合前后的抗肿瘤作用与其体外抗氧化活性。通过小鼠成纤维肉瘤细胞L929培养实验,观察不同质量浓度金针菇多糖溶液对体外培养L929细胞形态的影响,四甲基偶氮唑蓝(methyl thiazolyl tetrazolium,MTT)实验评价金针菇多糖对L929细胞生长和增殖的影响。结果表明:在质量浓度为5~50μg/mL时,金针菇粗多糖及其纯化组分对L929细胞的抑制作用不强烈,金针菇多糖-Zn~(2+)对L929细胞有较强的抑制作用。相比较金针菇多糖直接作用于L929细胞,金针菇多糖介导的RAW 264.7巨噬细胞上清液对L929细胞的抑制率作用显著增强。同时,以对1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基、超氧阴离子自由基(O_2~-·)、羟自由基(·OH)、H_2O_2的清除率为评价指标,体外抗氧化实验结果显示金针菇多糖-Zn~(2+)抗氧化活性较螯合Zn~(2+)前有所提高。即金针菇多糖中Zn含量的提高有助于提升其抗氧化活性。     

Enzymatic characterization of transglutaminase from Streptomyces mobaraensis DSM 40587 in high salt and effect of enzymatic cross-linking of yak milk proteins on functional properties of stirred yogurt

Streptomyces transglutaminase (TGase) purified from high-salt medium was characterized and applied into yak yogurts. The purified enzyme presented a Michaelis constant of 40.47 mmol and a maximum velocity of 44.44 U/mg of protein for N-carboxybenzoyl-l-glutaminyl-glycine in the hydroxamate procedure. The purified TGase exhibited optimum activity at 55°C and pH 6.0. The enzyme was not stable above 50°C and was stable within a pH range of 5.0 to 10.0 at 4°C for 12h and pH 5.0 to 9.0 at 37°C for 30 min. The TGase activity was not affected by Ca(2+), K(+), Ba(2+), or Na(+), but slightly inhibited by Fe(2+), Mg(2+), and Mn(2+), and strongly by Cu(2+) and Zn(2+). To explore yak milk products, it was used to produce yogurt and TGase was used. It was found that TGase-catalyzed cross-linking was effective in improving functional properties of stirred yak yogurt. Treated yogurt produced a strong acid gel, higher consistency, cohesiveness, index of viscosity, and creamier mouth feel than the untreated product. Furthermore, yak yogurt treated with TGase presented lower wet yak hair or sweat odor, or both. Therefore, TGase can be used to pave the way for exploration of novel yak products to overcome the issues of peculiar wet yak hair or sweat odor, or both.     

漆酶及其在食品工业中的应用

漆酶来源广泛、催化性能优异,在食品工业中具有应用价值。本文介绍了漆酶的来源与一般催化特性,就漆酶在食品工业中的研究与应用进展予以重点综述,并对其应用前景予以展望。     

Loss of rutin and antioxidant activity of asparagus juice caused by a pectolytic enzyme preparation from Aspergillus niger

We found that a commercial pectolytic enzyme preparation from Aspergillus niger (pectinase AN) contained laccase activity that decreased rutin content and antioxidant activity of asparagus juice. This research investigated the effects of pH, temperature, and concentration of pectinase AN on pectinase AN’s laccase activity to decrease rutin content and antioxidant activity of asparagus juice. Asparagus juice was incubated with pectinase AN at different pHs (3.2, 4.5 and 5.8), temperatures (25, 37, and 50 °C) and enzyme concentrations (0.1%, 0.5% and 1%). Rutin content and antioxidant activity of samples was determined by HPLC and 2,2′-diphenyl-1-picrylhydrazyl (DPPH) free radical method, respectively. The rate of loss of rutin and antioxidant activity of asparagus juice was smaller at pH 3.2 than at pH 4.5 and pH 5.8, smaller for 0.1% pectinase AN than 0.5% and 1% pectinase AN. The rate of loss of rutin of asparagus juice was greater at 25 °C than at the other two temperatures. Pectinase AN can decrease rutin content and antioxidant activity of asparagus juice at the selected conditions. But rutin content and antioxidant activity of asparagus juice produced using pectinase AN could be less decreased at pH 3.2 and 0.1% of enzyme with less than 2 h of incubation time. This information was helpful for juice industry to produce juices with high antioxidant activity using pectinase AN.     

亲和层析法分离纯化纳豆激酶

目的：以纳豆激酶的作用底物纤维蛋白为配基制备亲和层析胶,分离纯化纳豆激酶。方法：纳豆杆菌发酵液经硫酸铵分段盐析、透析得粗酶,在4℃条件亲和层析法分离纯化,利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)对酶纯度进行检验。结果：经亲和层析得纳豆激酶为电泳纯,纯化倍数为8.3,回收率43.9%。纯酶的最适pH值和温度分别为pH7.0～9.0、50℃;温度高于60℃纤溶酶活性迅速下降;在pH6.0～10.0范围内稳定性好;Mg2＋对其有微弱激活作用,Cu2+有显著抑制作用。结论：以琼脂糖包埋纤维蛋白制备的层析胶可用于纳豆激酶的快速分离纯化。