生物酶在造纸工业绿色制造中的应用

近年来随着生物技术的发展,生物酶制剂的生产水平不断提高,促进了酶制剂在生物制浆、生物漂白、废纸生物脱墨、酶法纸浆改善性能及树脂生物控制等方面的应用,体现了酶技术在减轻制浆造纸工业环境污染、改善纸浆抄造性能等方面的潜力。文中重点介绍在不同制浆造纸原料及工艺中酶的选用、复配和应用技术及原理,以及酶制剂的应用效率及其对制浆造纸中节能减排和绿色环保的意义。     

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, techniques for studying the regulatory mechanism of this system are essential to elucidating the cellular and molecular processes of the aforementioned diseases. The use of hemagglutinin derived ubiquitin probes outlined in this paper serves as a valuable tool for the study of this system. This paper details a method that enables the user to perform assays that give a direct visualization of deubiquitinating enzyme activity. Deubiquitinating enzymes control proteasomal degradation and share functional homology at their active sites, which allows the user to investigate the activity of multiple enzymes in one assay. Lysates are obtained through gentle mechanical cell disruption and incubated with active site directed probes. Functional enzymes are tagged with the probes while inactive enzymes remain unbound. By running this assay, the user obtains information on both the activity and potential expression of multiple deubiquitinating enzymes in a fast and easy manner. The current method is significantly more efficient than using individual antibodies for the predicted one hundred deubiquitinating enzymes in the human cell.     

Application of enzymes in the pulp and paper industry

The pulp and paper industry processes huge quantities of lignocellulosic biomass every year. The technology for pulp manufacture is highly diverse, and numerous opportunities exist for the application of microbial enzymes. Historically, enzymes have found some uses in the paper industry, but these have been mainly confined to areas such as modifications of raw starch. However, a wide range of applications in the pulp and paper industry have now been identified. The use of enzymes in the pulp and paper industry has grown rapidly since the mid 1980s. While many applications of enzymes in the pulp and paper industry are still in the research and development stage, several applications have found their way into the mills in an unprecedented short period of time. Currently the most important application of enzymes is in the prebleaching of kraft pulp. Xylanase enzymes have been found to be most effective for that purpose. Xylanase prebleaching technology is now in use at several mills worldwide. This technology has been successfully transferred to full industrial scale in just a few years. The enzymatic pitch control method using lipase was put into practice in a large-scale paper-making process as a routine operation in the early 1990s and was the first case in the world in which an enzyme was successfully applied in the actual paper-making process. Improvement of pulp drainage with enzymes is practiced routinely at mill scale. Enzymatic deinking has also been successfully applied during mill trials and can be expected to expand in application as increasing amounts of newsprint must be deinked and recycled. The University of Georgia has recently opened a pilot plant for deinking of recycled paper. Pulp bleaching with a laccase mediator system has reached pilot plant stage and is expected to be commercialized soon. Enzymatic debarking, enzymatic beating, and reduction of vessel picking with enzymes are still in the R&D stage but hold great promise for reducing energy. Other enzymatic applications, i.e., removal of shives and slime, retting of flax fibers, and selective removal of xylan, are also expected to have a profound impact on the future technology of the pulp and paper-making process.     

低温酶及其冷适应性机制研究进展

生活在极地、深海等常年低温环境中的低温微生物和部分变温生物,能够通过产生低温酶来适应在低温环境中的生长、繁殖和代谢。这些酶在低温或常温条件下具有很高的催化活性,对热却很敏感,在高温时能够快速失活,因此在实际的生产和生活中具有广泛的应用前景。基于这些特性,低温酶的冷适应性机制研究成为了当前的一个热点。较系统地综述了低温酶的来源、特点、生产状况以及具有代表性的几种低温酶的冷适应性机制。     

温度对嗜冷酵母糖代谢途径某些关键酶的活性效应

对嗜冷酵母Y18和酿酒酵母细胞中EMP途径和TCA循环中一些关键酶的温度特性进行了比较研究。Y18细胞中,1,6二磷酸果糖醛缩酶、琥珀酸脱氢酶和己糖激酶对温度很敏感,符合Feller提出的冷活性的概念属于冷活性酶类。柠檬酸合成酶的温度特性类似于中温酶。Α酮戊二酸脱氢酶存在不同温度特性的同功酶。通过对嗜冷酵母和中温酶母细胞中琥珀酸脱氢酶的Km值进行比较,结果显示嗜冷酵母琥珀酸脱氢酶在20℃具有较低的Km值。另外还对嗜冷菌细胞中代谢酶类的一些特点进行了讨论。     

Identification of protein phosphatases dephosphorylating mRNP proteins from cryptobiotic gastrulae of the brine shrimp A. salina

In the cytosol of A. salina cryptobiotic gastrulae at least five protein phosphatases active on phosphorylase a have been detected by ion exchange chromatography on DEAE-cellulose. Only two of these enzymes (PP-X and PP-Y) are active in mRNP dephosphorylation. Both enzymes are insensitive to inhibitor-1 and -2 and stimulation of enzymatic activity (2.5-fold with PP-X and 6.5-fold with PP-Y) can be accomplished by ethanol treatment of the native enzymes, or freeze-thawing in the presence of 1.7% (v/v) 2-mercaptoethanol. These properties allow PP-X and PP-Y to be classified as type-2A enzymes according to the nomenclature of Cohen. This paper is the first report of protein phosphatases capable of dephosphorylating mRNP proteins.     

Production of xylanases by mangrove fungi from the Philippines and their application in enzymatic pretreatment of recycled paper pulps

Mangrove fungi are vastly unexplored for enzymes with industrial application. This study aimed to assess the biocatalytic activity of mangrove fungal xylanases on recycled paper pulp. Forty-four mangrove fungal (MF) isolates were initially screened for xylanolytic activity in minimal medium with corn cob xylan as the sole carbon source. Eight MF were further cultivated under submerged fermentation for the production of crude xylanases. These crude enzymes were then characterized and tested for the pretreatment of recycled paper pulps. Results showed that 93 % of the tested MF isolates exhibited xylanolytic activity in solid medium. In submerged fermentation, salinity improved the growth of the fungal isolates but did not influence xylanase production. The crude xylanases were mostly optimally active at 50 °C and pH 7. Changes in pH had a greater effect on xylanase stability than temperature. More than half of the activity was lost at pH 9 for majority of the crude enzymes. However, two thermophilic xylanases from Fusarium sp. KAWIT-A and Aureobasidium sp. 2LIPA-M and one alkaliphilic xylanase from Phomopsis sp. MACA-J were also produced. All crude enzymes exhibited cellulase activities ranging from 4 to 21 U/ml. Enzymatic pretreatment of recycled paper pulps with 5 % consistency produced 70–650 mg of reducing sugars per gram of pulp at 50 °C after 60 min. The release of high amounts of reducing sugars showed the potential of mangrove fungal crude xylanases in the local paper and pulp industry. The diverse properties shown by the tested crude enzymes also indicate its potential applications to other enzyme-requiring industries.     

alpha-1 Antitrypsin phenotypes determined by isoelectric focusing of the cysteine-antitrypsin mixed disulfide in serum.

The methods available for analysis of sugar kinase activity are usually either complicated or time consuming. Coupled assays, aside from the added cost of coupling enzymes and substrates, present problems due to the pH optima, activators, and inhibitors of the coupling enzymes. Direct separation of the product requires either ion exchange (1) or paper chromatography (2,3). The former requires constant attention and the latter usually takes either overnight for the completion of a chromatogram or a great deal of elution solvent (200 ml) for DEAE paper discs (3).Those enzymes which form phosphorylated products from nonionic substrates (hexokinases, glycerol kinase, phosphoribosyl-transferases, etc.) may be conveniently assayed by chromatograhic separation of a radioactive phosphorylated product from the radioactive nonionic substrate, where the product remains at the origin. In such assays, no interfering coupling enzymes are used and the product can be directly and sensitively measured. The only current limitation with such methods is the time required for the separation of the phosphorylated product. It would be advantageous to obtain the enzyme's activity in as short a time as possible.We present here a method of paper chromatographic separation of phosphorylated product from nonionic substrate which requires only approximately two hours, uses a large petri dish, very little chromatographic grade paper, and almost no attention.     

Accessory enzymes influence cellulase hydrolysis of the model substrate and the realistic lignocellulosic biomass

The potential of cellulase enzymes in the developing and ongoing “biorefinery” industry has provided a great motivation to develop an efficient cellulase mixture. Recent work has shown how important the role that the so-called accessory enzymes can play in an effective enzymatic hydrolysis. In this study, three newest Novozymes Cellic CTec cellulase preparations (CTec 1/2/3) were compared to hydrolyze steam pretreated lignocellulosic substrates and model substances at an identical FPA loading. These cellulase preparations were found to display significantly different hydrolytic performances irrelevant with the FPA. And this difference was even observed on the filter paper itself when the FPA based assay was revisited. The analysis of specific enzyme activity in cellulase preparations demonstrated that different accessory enzymes were mainly responsible for the discrepancy of enzymatic hydrolysis between diversified substrates and various cellulases. Such the active role of accessory enzymes present in cellulase preparations was finally verified by supplementation with β-glucosidase, xylanase and lytic polysaccharide monooxygenases AA9. This paper provides new insights into the role of accessory enzymes, which can further provide a useful reference for the rational customization of cellulase cocktails in order to realize an efficient conversion of natural lignocellulosic substrates.     

Application of immobilized enzyme technologies for the textile industry: a review

Abstract

A number of enzymes have applications in the textile sector, as several natural fibres (e.g. cotton, wool and flax) can be subjected to processing using such natural biocatalysts. In the latest two decades, demand for industrial enzymes has increased considerably; however, the textile industry requires highly stable enzymes, and good performance at extreme values of pH and temperature. New strategies are continuously emerging for the immobilization of enzymes with superior efficiency and usage. Enzymatic immobilization can stabilize enzymes and extend their useful life. Additionally, reduction in effluent treatment costs and improvement of efficiency is possible. This paper briefly reviews the most recent research efforts pertaining to immobilization of enzymes with potential application in the textile industry.     

Endoplasmic reticulum marker enzymes in Golgi fractions--what does this mean?

NADPH cytochrome c (cyt c) reductase and glucose-6-phosphatase, two enzymes thought to be restricted to the endoplasmic reticulum (ER) and widely used as ER markers, are present in isolated Golgi fractions assayed immediately after their isolation. Both enzymes are rapidly inactivated in fractions stored at 0 degrees C in 0.25 M sucrose, conditions which do not affect the activity of other enzymes in the same preparation. The inactivation process was shown to be dependent on time and protein concentration and could be prevented by EDTA and catalase. Morphological evidence shows that extensive membrane damage occurs parallel with the inactivation. Taken together with the immunological data in the companion paper, the findings indicate that the enzymes NADPH cyt c reductase and probably glucose-6-phosphate are indigenous components of Golgi membranes.     

嗜热β-甘露聚糖酶的研究进展

β-甘露聚糖酶在生物能源、饲料、食品和纺织等工业中均有着重要的应用前景。其属于半纤维素酶类,广泛存在于动植物和微生物中,微生物来源尤为广泛。随着极端微生物和极端酶的广泛研究,嗜热甘露聚糖酶因其在高温环境中具有较高酶活性和稳定性而倍受关注,并取得了较大的研究进展。本文综述了β-甘露聚糖酶的来源、分类和水解催化方式,以及嗜热甘露聚糖酶的优势和其在基因资源挖掘、重组表达以及分子改良方面的研究进展,展望了嗜热β-甘露聚糖酶未来可能的研究方向和发展前景。     

Fungal enzyme sets for plant polysaccharide degradation

Enzymatic degradation of plant polysaccharides has many industrial applications, such as within the paper, food, and feed industry and for sustainable production of fuels and chemicals. Cellulose, hemicelluloses, and pectins are the main components of plant cell wall polysaccharides. These polysaccharides are often tightly packed, contain many different sugar residues, and are branched with a diversity of structures. To enable efficient degradation of these polysaccharides, fungi produce an extensive set of carbohydrate-active enzymes. The variety of the enzyme set differs between fungi and often corresponds to the requirements of its habitat. Carbohydrate-active enzymes can be organized in different families based on the amino acid sequence of the structurally related catalytic modules. Fungal enzymes involved in plant polysaccharide degradation are assigned to at least 35 glycoside hydrolase families, three carbohydrate esterase families and six polysaccharide lyase families. This mini-review will discuss the enzymes needed for complete degradation of plant polysaccharides and will give an overview of the latest developments concerning fungal carbohydrate-active enzymes and their corresponding families.     

Complete dechlorination of tetrachlorohydroquinone by cell extracts of pentachlorophenol-induced Rhodococcus chlorophenolicus.

In this paper we describe the sequence of reactions leading from tetrachloro-para-hydroquinone to 1,2,4-trihydroxybenzene by inducible enzymes of Rhodococcus chlorophenolicus. Tetrachlorohydroquinone was first converted to a dichlorotrihydroxybenzene in a reaction involving both hydrolytic and reductive dechlorination; no trichlorinated intermediate was detected. Dichlorotrihydroxybenzene was subsequently reductively dechlorinated to a monochlorotrihydroxybenzene and finally to 1,2,4-trihydroxybenzene. The cell extract also catalyzed, at a lower rate, reductive dechlorination of trichlorohydroquinone, mainly to 2,3-dichlorohydroquinone. To our knowledge this is the first demonstration of reductive aromatic dechlorination by bacterial enzymes.     

Characterisation and application of glycanases secreted by Aspergillus terreus CCMI 498 and Trichoderma viride CCMI 84 for enzymatic deinking of mixed office wastepaper

Two enzymatic extracts obtained from xylan-grown Aspergillus terreus CCMI 498 and cellulose-grown Trichoderma viride CCMI 84 were characterised for different glycanase activities. Both strains produce extracellular endoxylanase and endoglucanase enzymes. The enzymes optimal activity was found in the temperature range of 45–60 °C. Endoglucanase systems show identical activity profiles towards temperature, regardless of the strain and inducing substrate. Conversely, the endoxylanases produced by both strains showed maximal activity at different pH values (from 4.5 to 5.5), being the more acidic xylanase produced by T. viride grown on cellulose. The endoglucanase activities have an optimum pH at 4.5–5.0. The endoxylanase and endoglucanase activities exhibited high stability at 50 °C and pH 5.0. Mannanase, β-xylosidase, and amylase activities were also found, being the first two activities only present for T. viride extract. These two enzymatic extracts were used for mixed office wastepaper (MOW) deinking. When the enzymatic extract from T. viride was used, a further increase of 24% in ink removal was obtained by comparison with the control. Both enzymes contributed to the improvement of the paper strength properties and the obtained results clearly indicate that the effective use of enzymes for deinking can also contribute to the pulp and paper properties improvement.     

High-temperature enzymatic breakdown of cellulose

Cellulose is an abundant and renewable biopolymer that can be used for biofuel generation; however, structural entrapment with other cell wall components hinders enzyme-substrate interactions, a key bottleneck for ethanol production. Biomass is routinely subjected to treatments that facilitate cellulase-cellulose contacts. Cellulases and glucosidases act by hydrolyzing glycosidic bonds of linear glucose β-1,4-linked polymers, producing glucose. Here we describe eight high-temperature-operating cellulases (TCel enzymes) identified from a survey of thermobacterial and archaeal genomes. Three TCel enzymes preferentially hydrolyzed soluble cellulose, while two preferred insoluble cellulose such as cotton linters and filter paper. TCel enzymes had temperature optima ranging from 85°C to 102°C. TCel enzymes were stable, retaining 80% of initial activity after 120 h at 85°C. Two modes of cellulose breakdown, i.e., with endo- and exo-acting glucanases, were detected, and with two-enzyme combinations at 85°C, synergistic cellulase activity was observed for some enzyme combinations.     

Expression of lignocellulolytic enzymes in Pichia pastoris

ABSTRACT: BACKGROUND: Sustainable utilization of plant biomass as renewable source for fuels and chemical building blocks requires a complex mixture of diverse enzymes, including hydrolases which comprise the largest class of lignocellulolytic enzymes. These enzymes need to be available in large amounts at a low price to allow sustainable and economic biotechnological processes. Over the past years Pichia pastoris has become an attractive host for the cost-efficient production and engineering of heterologous (eukaryotic) proteins due to several advantages. RESULTS: In this paper codon optimized genes and synthetic alcohol oxidase 1 promoter variants were used to generate Pichia pastoris strains which individually expressed cellobiohydrolase 1, cellobiohydrolase 2 and beta-mannanase from Trichoderma reesei and xylanase A from Thermomyces lanuginosus. For three of these enzymes even gram quantities of enzyme per liter were obtained by fed-batch cultivation. Additionally, we compared our achieved yields of secreted enzymes and the corresponding activities to literature data. CONCLUSION: In our experiments we could clearly see the importance of gene optimization and strain characterization for successfully improving secretion levels. We also give a basic guideline for understanding the interplay of promoter strength and gene dosage for a successful improvement of the secretory production of lignocellulolytic enzymes in Pichia pastoris.     

Extremophilic proteases as novel and efficient tools in short peptide synthesis

The objective of this review is to outline the crucial role that peptides play in various sectors, including medicine. Different ways of producing these compounds are discussed with an emphasis on the benefits offered by industrial enzyme biotechnology. This paper describes mechanisms of peptide bond formation using a range of proteases with different active site structures. Importantly, these enzymes may be further improved chemically and/or genetically to make them better suited for their various applications and process conditions. The focus is on extremophilic proteases, whose potential does not seem to have been fully appreciated to date. The structure of these proteins is somewhat different from that of the common commercially available enzymes, making them effective at high salinity and high or low temperatures, which are often favorable to peptide synthesis. Examples of such enzymes include halophilic, thermophilic, and psychrophilic proteases; this paper also mentions some promising catalytic proteins which require further study in this respect.     

Structural Basis of Redox Signaling in Photosynthesis: Structure and Function of Ferredoxin:thioredoxin Reductase and Target Enzymes

The role of the ferredoxin:thioredoxin system in the reversible light activation of chloroplast enzymes by thiol-disulfide interchange with thioredoxins is now well established. Recent fruitful collaboration between biochemists and structural biologists, reflected by the shared authorship of the paper, allowed to solve the structures of all of the components of the system, including several target enzymes, thus providing a structural basis for the elucidation of the activation mechanism at a molecular level. In the present Review, these structural data are analyzed in conjunction with the information that was obtained previously through biochemical and site-directed mutagenesis approaches. The unique 4Fe-4S cluster enzyme ferredoxin:thioredoxin reductase (FTR) uses photosynthetically reduced ferredoxin as an electron donor to reduce the disulfide bridge of different thioredoxin isoforms. Thioredoxins in turn reduce regulatory disulfides of various target enzymes. This process triggers conformational changes on these enzymes, allowing them to reach optimal activity. No common activation mechanism can be put forward for these enzymes, as every thioredoxin-regulated protein undergoes specific structural modifications. It is thus important to solve the structures of the individual target enzymes in order to fully understand the molecular mechanism of the redox regulation of each of them.     

Optimization of extracellular endoxylanase, endoglucanase and peroxidase production by Streptomyces sp. F2621 isolated in Turkey

AIMS: To determine the effect of environmental conditions on the production of extracellular lignocellulose-degrading enzymes by Streptomyces sp. F2621 and to assess the potential use of these enzymes in the hydrolysis of lignocellulose material. METHODS AND RESULTS: The production of extracellular lignocellulose-degrading enzymes, endoxylanase, endoglucanase and peroxidase during the growth of Streptomyces sp. F2621 in basal salts-yeast extract medium containing different carbon sources and the effect of a number of environmental parameters (e.g. carbon sources and concentrations, pH and temperature) were investigated. The highest endoxylanase (22.41 U ml(-1)) and peroxidase (0.58 U ml(-1)) activities were obtained after 2-4 days of incubation at 30 degrees C in a basal salts medium containing 0.4% (w/v) oat spelt xylan and 0.6% (w/v) yeast extract, corresponding to C : N ratio of 6 : 1. Cell-free extracellular enzyme preparations from the strain were capable of releasing both sugar and aromatic compounds during incubation with eucalyptus paper pulp, straw and xylan. Overall, 9.3% hydrolysis of xylan occurred after 24-h incubation. However the rates of hydrolysis of paper pulp and straw were approximately twofold less than xylan hydrolysis, although the total percentage hydrolysis of available substrate (24.5% and 16.3%, respectively) was greater than xylan hydrolysis. CONCLUSIONS: The high levels of enzyme production achieved under batch cultivation conditions, coupled with no significant production of endoglucanase during the growth phase of organism and the release of both sugar and aromatic compounds from paper pulp and straw signify the suitability for these enzymes for industrial applications such as pulp and paper production. SIGNIFICANCE AND IMPACT OF THE STUDY: The results highlight the environmental conditions for the production of extracellular lignocellulose-degrading enzymes by Streptomyces sp. F2621 and suggest the use of streptomycetes and/or their enzymes in industrial processes.