角质酶及其在纺织工业中的应用

详细综述了国内外对角质酶的研究概况,包括角质酶的主要来源,角质酶基因的克隆与表达,以及关于角质酶的发酵研究。着重阐述了目前角质酶在棉纤维的生物精炼,羊毛的防毡缩整理,以及合成纤维的生物改性等方面的应用进展。另外,作为推动纺织工业清洁生产的关键酶制剂,笔者对未来角质酶在纺织工业中的应用前景作了简要展望。     

马拉色菌致病相关因子的研究进展

马拉色菌是人类和温血动物皮肤的常驻菌,亦是一种条件致病性真菌,它可以引起花斑癣、马拉色菌毛囊炎、脂溢性皮炎等多种疾病。脂酶、蛋白酶、磷脂酶和脂氧合酶等为马拉色菌主要侵袭性酶。马拉色菌与角质形成细胞共培养可引起角质形成细胞多种形态学改变、细胞因子含量变化和细胞凋亡。     

角质酶在生物可降解聚酯聚己二酸/对苯二甲酸丁二醇酯降解中的应用

随着废弃塑料带来的环境污染越来越严重,生物可降解聚酯已成为大众关注的焦点。聚己二酸/对苯二甲酸丁二醇酯[poly(butylene adipate-co-terephthalate),PBAT]是脂肪族和芳香族共聚形成的生物可降解聚酯,兼具两者的优异性能。针对PBAT在自然条件下对降解环境要求严格且降解周期长的不足之处,本研究探究了角质酶在PBAT降解中的应用和对苯二甲酸-丁二醇酯(butylene terephthalate,BT)含量对PBAT生物降解性的影响,以实现对PBAT降解速率的提升。选取5种不同来源的聚酯降解酶对PBAT进行降解应用并比较出降解效果最优的酶,并测定了含有不同BT含量的PBAT聚酯的降解效率。结果表明,角质酶ICCG为降解效果最好的酶,且BT含量越高PBAT的降解率越低。此外,还确定了角质酶ICCG对高BT含量的PBAT(H)降解的最适温度、最适缓冲液类型、最适pH、最适E/S(enzyme to substrate)和最适底物浓度比分别为75℃、Tris-HCl、9.0、0.4%和1.0%。本研究结果可为角质酶在PBAT降解中的应用提供一定的理论依据和实验...     

植物角质膜及其渗透性与抗旱性研究进展

角质膜覆盖于陆生植物的地上部分,是植物与外部环境之间的第一道屏障,保护植物免遭各种生物和非生物胁迫。本文就植物角质膜的结构、成分、生物合成的途径及机理、渗透性及温度、湿度、活性剂对角质膜渗透性的影响,角质膜与植物抗旱性关系的研究进展做系统综述,并对植物角质膜研究中存在的问题进行了探讨。     

核盘菌角质酶的克隆、表达及活性分析

角质酶可降解脂肪族或芳香族聚酯,对聚对苯二甲酸乙二醇酯也具有较好的降解作用,但目前市面上角质酶产品非常稀缺,因此寻求一种高效表达的角质酶用于工程酶的开发非常有必要.本研究从核盘菌中克隆得到8个角质酶基因,利用PCR结合RT-PCR技术筛选出主效基因SsCut-52,利用大肠杆菌Escherichia coli BL21...     

产角质酶酵母菌的发现与鉴定及产酶条件的优化

为得到有效水解杜仲角质层的角质酶,通过生物酶法提取杜仲中的活性成分。从病变的杜仲叶中分离出一株能高产角质酶的菌种,鉴定分析证实该菌株是胶红酵母(Rhodotorula mucilaginosa),这是首次从植物病原真菌以外发现产角质酶的酵母菌,并对该菌株发酵产角质酶的条件进行优化。这一发现意义重大,因为酵母菌比植物病原菌安全许多,更利于大规模生产。     

嗜热子囊菌利用短链有机酸生产角质酶

以嗜热子囊菌(Thermobifida fusca WSH03-11)发酵生产角质酶为模型,研究微生物利用市政污泥厌氧酸化所产短链有机酸为碳源发酵生产高附加值产品的可能。发现：(1)以丁酸、丙酸和乙酸为碳源时,有机酸和氮元素浓度分别为8.0 g/L和1.5 g/L有利于角质酶的生产;而以乳酸为碳源时,最适有机酸和氮源浓度分别为3.0 g/L和1.0 g/L;(2)改变诱导物角质的浓度,以丁酸、丙酸、乙酸和乳酸为碳源,分别比优化前提高了31.0%、13.3%、43.8%和73.2%;(3)在四种有机酸中,T. fusca WSH03-11利用乙酸的速率最快,平均比消耗速率是丙酸的1.3倍,丁酸的2.0倍及乳酸的2.2倍;以丁酸为碳源时的酶活(52.4 U/mL)是乳酸的1.7倍、乙酸的2.5倍和丙酸的3.2倍;角质酶对乳酸的得率(12.70 u/mg)分别是丁酸的1.4倍、丙酸的3.0倍和乙酸的3.8倍;(4)以混合酸为碳源生产角质酶,T. fusca WSH03-11优先利用乙酸,而对丁酸的利用受到抑制。进一步研究发现,混合酸中0.5 g/L的乙酸将导致丁酸的消耗量降低66.7%。这是首次利用混合酸作碳源发酵生产角质酶的研究报道。这一研究结果进一步确证了利用市政污泥厌氧酸化所产有机酸为碳源发酵生产高附加值产品的可行性,为以廉价碳源生产角质酶奠定了良好的基础。     

嗜热子囊菌利用短链有机酸生产角质酶

以嗜热子囊菌(Thermobifida fusca WSH03-11)发酵生产角质酶为模型,研究微生物利用市政污泥厌氧酸化所产短链有机酸为碳源发酵生产高附加值产品的可能。发现：(1)以丁酸、丙酸和乙酸为碳源时,有机酸和氮元素浓度分别为8.0 g/L和1.5 g/L有利于角质酶的生产;而以乳酸为碳源时,最适有机酸和氮源浓度分别为3.0 g/L和1.0 g/L;(2)改变诱导物角质的浓度,以丁酸、丙酸、乙酸和乳酸为碳源,分别比优化前提高了31.0%、13.3%、43.8%和73.2%;(3)在四种有机酸中,T. fusca WSH03-11利用乙酸的速率最快,平均比消耗速率是丙酸的1.3倍,丁酸的2.0倍及乳酸的2.2倍;以丁酸为碳源时的酶活(52.4 U/mL)是乳酸的1.7倍、乙酸的2.5倍和丙酸的3.2倍;角质酶对乳酸的得率(12.70 u/mg)分别是丁酸的1.4倍、丙酸的3.0倍和乙酸的3.8倍;(4)以混合酸为碳源生产角质酶,T. fusca WSH03-11优先利用乙酸,而对丁酸的利用受到抑制。进一步研究发现,混合酸中0.5 g/L的乙酸将导致丁酸的消耗量降低66.7%。这是首次利用混合酸作碳源发酵生产角质酶的研究报道。这一研究结果进一步确证了利用市政污泥厌氧酸化所产有机酸为碳源发酵生产高附加值产品的可行性,为以廉价碳源生产角质酶奠定了良好的基础。     

特异腐质霉角质酶-锚定肽融合蛋白的特性及在处理再生纸胶黏物中的应用

随着森林木材资源的减少,废纸回收利用越来越受到人们的重视.然而,废纸回收利用过程中产生的胶黏物会对再生纸的生产造成严重影响.生物法控制胶黏物主要是通过酶断裂胶黏物组分之间的酯键,防止胶黏物的絮凝,具有高效、专一、无污染等优势.角质酶是一种丝氨酸酯酶,可降解胶黏物中的部分成分.相关研究表明,锚定肽tachystatin ...     

重组角质酶的发酵制备及其对涤纶纤维的表面改性

对大肠杆菌表达嗜热子囊菌Thermobifida fusca角质酶的摇瓶诱导条件及3 L发酵罐扩大培养进行了研究,并探讨了角质酶对涤纶纤维的改性作用。结果表明,在摇瓶培养中,采用工业级TB培养基,用2 g/L乳糖诱导,菌体培养至对数生长前期添加0.5％甘氨酸,角质酶产量可达到128 U/mL。在3 L发酵罐扩大培养中,补料培养生物量 (OD600) 最大达到35,角质酶酶活最高达506 U/mL,是迄今国内外报道细菌来源角质酶的最高水平。紫外分光光度法分析初步表明涤纶纤维经角质酶水解产生了对苯二甲酸类物质     

Production of cutinase by Thermomonospora fusca ATCC 27730

Ten strains belonging to various Thermomonospora species were tested for their ability to hydrolyse the insoluble plant polyester cutin. One strain, the thermophile T . fusca ATCC 27730, was found to produce a highly inducible cutinase when grown in broth medium containing purified apple cv. Golden Delicious cutin. Apple pomace, tomato peel, potato suberin and commercial cork were also shown to induce cutinase production. Addition of glucose to the culture medium either at the beginning of fermentation or after 2 days of incubation in the presence of apple cutin led to repression of cutinase production. The cutinase was active against a wide range of cutins, including those isolated from other apple cultivars as well as tomato, cucumber, grapefruit, and green pepper. Cutinase activity in the induced culture supernatant fluids exhibited a half-life of over 60 min at 70 °C and a pH optimum of 11·0. Some potential applications for cutinases are discussed.     

Isolation of a Fusarium solani mutant reduced in cutinase activity and virulence.

Fusarium solani isolate T-8 produces an extracellular enzyme, cutinase, which catalyzes the degradation of cutin in the plant cuticle. Cutinase activity can be measured by the hydrolysis of either the artifical substrate, p-nitrophenylbutyrate (PNB), or radioactive cutin containing [14C]palmitic acid. In the present study, the culture filtrate contained basal levels of cutinase when T-8 was grown on acetate as a sole source of carbon. After mutagenesis, a cutinase-defective mutant (PNB-1) was identified by screening acetate-grown colonies for a loss of PNBase activity. The mutant possessed an 80 to 90% reduction in cutinase activity when grown for 3 to 5 days on acetate- or cutin-containing medium. Induction of cutinase by cutin or hydrolyzed cutin after growth on glucose medium was similarly reduced. Kinetic analysis indicated that cutinase from the mutant possessed a near normal Km for PNB and a 92% reduction in Vmax. Fluorography and Western blotting of 15% sodium dodecyl sulfate-polyacrylamide gels of separated 35S-labeled proteins from cutin induction medium revealed that in the mutant the 22,000-molecular-weight band corresponding to cutinase was reduced approximately 85%. The virulence of the mutant in a pea stem bioassay was decreased by 55% and was restored to nearly the parental level by the addition of purified cutinase. The data suggest that the mutant synthesizes reduced quantities of a functional and immunoreactive cutinase enzyme and that cutinase plays a critical role in infection. The PNB1 mutation may be within a regulatory gene or a promoter for cutinase.     

Isolation and characterization of a cutinase from Fusarium roseum culmorum and its immunological comparison with cutinases from F. solani pisi.

Fusarium roseum culmorum, grown on apple cutin as the sole source of carbon, was shown to produce a cutin depolymerizing enzyme. From the extracellular fluid of these F. roseum cultures, a cutinase and a nonspecific esterase were isolated utilizing Sephadex G-100, QAE-Sephadex, and SP-Sephadex chromatography. The homogeneity of the cutinase was verified by polyacrylamide disc gel electrophoresis. The molecular weight of the cutinase was estimated to be 24,300 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Electrophoretic mobility of this enzyme was between that of Cutinases I and II from Fusarium solani pisi. The F. roseum cutinase hydrolyzed p-nitrophenyl butyrate and cutin, but not p-nitrophenyl palmitate, while the nonspecific esterase hydrolyzed the long-chain esters. Amino acid composition of F. roseum cutinase was found to be similar to that of F. solani pisi Cutinase I except for differences in the number of serine, valine, and cysteine residues. The time-course, protein concentration dependence, substrate concentration dependence, and pH optimum (10.0 for cutin hydrolysis) of the F. roseum cutinase was similar to the cutinases from F. solani pisi. The F. roseum cutinase was inhibited by diisopropylfluorophosphate and paraoxon, and the [³H]diisopropylphosphate group was covalently attached to the enzyme upon treatment with tritiated diisopropylfluorophosphate. Therefore, it is concluded that catalysis by cutinase involves an “active serine.” Immunochemical studies with a rabbit antibody prepared against F. solani pisi Cutinase I demonstrated that Cutinase II from this organism was immunologically very similar to, but not identical to, Cutinase I. On the other hand, the cutinase from F. roseum was immunologically quite different from the cutinases isolated from F. solani pisi in that it did not cross-react with anticutinase I. However, all three cutinases were virtually identical in their sensitivity to inhibition by anticutinase I, and all three enzymes were virtually completely inhibited by the anticutinase I.     

Evidence for a constitutive cytoplasmic cutinase in ungerminated conidia of Botrytis cinerea Pers.: Fr.

Cytoplasmic soluble proteins from ungerminated conidia of Botrytis cinerea exhibited cutinase activity, while cell wall binding proteins lacked this activity. Cutinase activity in proteins extracted from cell walls and cytoplasm of ungerminated conidia of Botrytis cinerea was determined using p-nitrophenyl butyrate (PNB) and TLC analysis of products derived from hydrolysis of [³H]cutin. Treatment of conidia with indoxyl acetate, a substrate indicative of non-specific esterase and cutinase activity, also gave a positive reaction in the cytoplasm of ungerminated conidia. The possible role of a putative constitutive cutinase in the cytoplasm of conidia in the early stages of infection of plants by B. cinerea is discussed.     

Synthesis and characterization of a new cutinase substrate, 4-nitrophenyl (16-methyl sulfone ester) hexadecanoate

Phytopathogenic fungi penetrate plants by breaking down the cuticular barrier with cutinase. Cutinases are extracellular hydrolytic enzymes that degrade cutin, a polyester composed of hydroxy and epoxy fatty acids. Until now, cutinase has been recognized by its ability to release labeled cutin monomers or by a non-specific esterase assay based on the hydrolysis of p-nitrophenyl esters of short fatty acids. In this work, an insoluble p-nitrophenyl derivative was synthesized and purified, and its structure was determined to be 4-nitrophenyl (16-methyl sulfone ester) hexadecanoate (pNMSEH) by nuclear magnetic resonance (H+ NMR) analysis. pNMSEH was tested as a new cutinase substrate with Pseudomonas mandocino cutinase and porcine liver esterase. While a linear release over time of p-nitrophenol (pNP) was recorded in the presence of cutinase, no response was obtained with the esterase. The calculated kinetic parameters of pNMSEH hydrolysis by cutinase revealed a high specificity (Km=1.8mM), albeit a low catalytic rate (Vmax=10.5 micromol min(-l)l(-1)). This new synthetic substrate may be helpful for detecting and assaying cutinase activity in mixed solutions, such as crude fungal extracellular extracts.     

Production,characterization and applications of microbial cutinases

Cutinase is an enzyme that catalyses the degradation of insoluble biopolyester cutin, a structural component of plants. This enzyme has some properties of lipase and esterase. Because of its unique nature, it has potential of being an industrially important enzyme. Some of the useful applications of cutinase include hydrolysis of fats and oils, esterification and transesterification reactions. This enzyme is mainly produced by phytopathogenic fungi, but there are several bacteria which are known to produce cutinase. In this article, the production, purification, characterizations, enhancement of activity and stability, immobilization of the enzyme and its applications in various industries have been discussed.     

Identification and characterization of bacterial cutinase

Cutinase, which exists in both fungi and bacteria, catalyzes the cleavage of the ester bonds of cutin. Fungal cutinases have been extensively studied, however, reports on bacterial cutinases have been limited due to the lack of knowledge concerning the identity of their open reading frames. In the present study, the cutinase from Thermobifida fusca was induced by cutin and purified to homogeneity by following p-nitrophenyl butyrate hydrolyzing activity. Peptide mass fingerprinting analysis of the wild-type enzyme matched two proteins, Tfu_0883 and Tfu_0882, which are 93% identical in sequence. Both proteins were cloned and overexpressed in their mature form. Recombinant Tfu_0883 and Tfu_0882 display very similar enzymatic properties and were confirmed to be cutinases by their capability to hydrolyze the ester bonds of cutin. Comparative characterization of Fusarium solani pisi and T. fusca cutinases indicated that they have similar substrate specificity and catalytic properties except that the T. fusca enzymes are thermally more stable. Homology modeling revealed that T. fusca cutinases adopt an alpha/beta-hydrolase fold that exhibits both similarities and variations from the fungal cutinase structure. A serine hydrolase catalytic mechanism involving a Ser(170)-His(248)-Asp(216) (Tfu_0883 numbering) catalytic triad was supported by active site-directed inhibition studies and mutational analyses. This is the first report of cutinase encoding genes from bacterial sources.     

Purification and characterization of cutinase from a fluorescent Pseudomonas putida bacterial strain isolated from phyllosphere

Cutinase, an extracellular enzyme, was induced by cutin in a fluorescent Pseudomonas putida strain that was found to be cohabiting with an apparently nitrogen-fixing Corynebacterium. This enzyme was purified from the culture fluid by acetone precipitation followed by chromatography on DEAE-cellulose, QAE-Sephadex, Sepharose 6B, and Sephadex G-100. The purified enzyme showed a single band when subjected to polyacrylamide electrophoresis and the enzymatic activity coincided with the protein band. Sodium dodecyl sulfate-polyacrylamide electrophoresis showed a single band at a molecular weight of 30,000 and gel filtration of the native enzyme through a calibrated Sephadex G-100 column indicated a molecular weight of 30,000, showing that the enzyme is a monomer. The amino acid composition of bacterial cutinase is distinctly different from that of fungal or plant cutinases. This bacterial cutinase showed a broad pH optimum between 8.5 and 10.5 with 3H-labeled apple cutin as the substrate. Linear rates of cutin hydrolysis were measured up to 20 min of incubation time and 4 mg/ml of cutin gave the maximum hydrolysis rate. This cutinase catalyzed hydrolysis of p-nitrophenyl esters of C4 to C16 fatty acids with decreasing V and increasing Km for the longer chain esters. It did not hydrolyze tripalmitoyl glycerol or trioleyl glycerol, indicating that this is not a general lipase. Active serine-directed reagents such as organophosphates and organoboronic acids severely inhibited the enzyme, suggesting that bacterial cutinase is an "active serine" enzyme. Neither thiol-directed reagents nor metal ion chelators had any effect on this enzyme. Antibody raised against purified enzyme gave a single precipitin line on Ouchterlony double diffusion analysis. Western blot analysis of the extracellular fluid of induced Ps. putida showed a single band at 30 kDa. No immunological cross-reactivity was detected between the present bacterial enzyme and the fungal enzyme from Fusarium solani pisi when rabbit antibodies against either enzyme was used.     

Fusarium polycaprolactone depolymerase is cutinase.

Polycaprolactone (PCL), a synthetic polyester, is degraded by a variety of microorganisms, including some phytopathogens. Many phytopathogens secrete cutinase, a serine hydrolase that degrades cutin, the structural polymer of the plant cuticle. We compared wild-type strains and a cutinase-negative gene replacement mutant strain of Fusarium solani f. sp. pisi (D. J. Stahl and W. Schäfer, Plant Cell 4:621-629, 1992) and a wild-type strain of Fusarium moniliforme to show that Fusarium cutinase is a PCL depolymerase. The wild-type strains, but not the mutant strain, (i) degraded PCL and used it as a source of carbon and energy, (ii) showed induction of secreted PCL depolymerase and an esterase activity of cutinase when grown in the presence of cutin, and (iii) showed induction of PCL depolymerase and an esterase activity of cutinase when grown in the presence of a hydrolysate of PCL, which contains PCL oligomers that are structurally similar to the natural inducers of cutinase. These results together with other details of regulation and conditions for optimal enzyme activity indicate that the Fusarium PCL depolymerase, required for degradation and utilization of PCL, is cutinase.