红景天苷生物、化学和生物催化合成的分子理论及应用

红景天苷是红景天属植物的主要活性成分之一,研究红景天苷的高效合成具有重要的科研和应用价值。本文全面概述近年来合成红景天苷的研究状况,主要包括生物合成途径、化学合成途径、生物催化合成途径。对生物合成途径中红景天苷的代谢合成途径、关键酶及基因、实践现状进行了分析;总结了以Koenigs-Knorr法为理论基础的化学合成的研究进展;对具有广泛发展前景的体外生物催化合成状况进行了理论与实践的概述。通过对这些合成方法的展望,为人们了解合成红景天苷的研究现状、进一步深入研究提供参考。     

红景天苷生物合成机制及其基因工程研究进展

红景天苷主要来源于红景天,是红景天属植物的重要活性物质,具有抗辐射、抗缺氧、抗肿瘤等多种药用功效。由于红景天野生资源少,且红景天苷在红景天中含量极少、提取率低,因此,研究提高红景天苷产量的生产方法具有重要的意义和价值。在研究红景天苷生物合成机制的基础上,通过基因工程技术来提高红景天苷的产量具有一定的前景和研究价值。综述了红景天苷的生物合成机制、代谢途径中的关键酶和利用代谢工程合成红景天苷的方法,并对其研究前景进行了展望,以期为红景天苷的生物合成相关研究提供参考。     

红景天甙的生物合成及其关键代谢酶研究

红景天甙是红景天属植物的主要药效成分,是一种很有前途的环境适应药物,具有抗疲劳、抗衰老、抗微波辐射、抗病毒及抗肿瘤等特异功效,尤其在军事、航天、运动及保健医学上具重要应用价值,近年来备受关注。本文在介绍了红景天属植物的开发利用价值及资源现状的基础上,重点探索了红景天甙生物合成的可能途径。认为其甙元酪醇是经由莽草酸途径合成,再由UDP葡萄糖基转移酶（Glucosyltransferase）催化葡萄糖和酪醇合成红景天甙,而红景天甙又可能在β-D-葡萄糖苷酶作用下降解为甙元酪醇和葡萄糖。文章阐述了参与红景天甙代谢的上述两个关键酶的研究现状及前景。     

微生物源α-半乳糖苷酶的研究进展

介绍了微生物源α-半乳糖苷酶的生理生化特性、合成调控机制的研究进展情况及其在食品、饲料、医药工业等领域的一些应用。Α-半乳糖苷酶均是糖蛋白,不同来源的α-半乳糖苷酶的作用基质特异性差别较大,作用基质特异性差别是由蛋白质部分N-末端氨基酸序列决定的。不同微生物来源的α-半乳糖苷酶其最佳作用条件、pH稳定性及耐热性差异较大。微生物α-半乳糖苷酶是一种诱导酶,其合成受多个基因的调控,高浓度的葡萄糖能抑制其合成。     

微生物源a—半乳糖苷酶的研究进展

介绍了微生物源a-半乳糖苷酶的生理生化特性、合成调控机制的研究进展情况及其在食品、饲料、医药工业等领域的一些应用。a-半乳糖苷酶均是糖蛋白,不同来源的a-半乳糖苷酶的作用基质特异性差别较大,作用基质特异性差别是由蛋白质部分N-末端氨基酸序列决定的。不同微生物来源的a-半乳糖苷酶其最佳作用条件、pH稳定性及耐热性差异较大。微生物a-半乳糖苷酶是一种诱导酶,其合成受多个基因的调控,高浓度的葡萄糖能抑制其合成。     

微生物糖苷酶的新型突变酶——硫代糖苷酶的产生及应用

微生物糖苷酶的酸碱功能氨基酸突变酶能催化硫代糖苷的合成,这类酶被称为硫代糖苷酶。目前发展的硫代糖苷酶有β-硫代葡糖苷酶、β-硫代甘露糖苷酶、β-硫代半乳糖苷酶、α-硫代木糖苷酶和α-硫代葡糖苷酶,来源于细菌和古细菌,能合成多种硫代糖苷。最近,硫代糖苷酶被应用于糖蛋白的糖基化修饰,首次人工合成硫代糖蛋白。微生物糖苷酶合成功能的新延伸,对糖生物学、生物技术和制药业的发展将有着重要意义。     

微生物糖苷酶的新型突变酶——硫代糖苷酶的产生及应用

微生物糖苷酶的酸碱功能氨基酸突变酶能催化硫代糖苷的合成,这类酶被称为硫代糖苷酶。目前发展的硫代糖苷酶有β-硫代葡糖苷酶、β-硫代甘露糖苷酶、β-硫代半乳糖苷酶、α-硫代木糖苷酶和α-硫代葡糖苷酶,来源于细菌和古细菌,能合成多种硫代糖苷。最近,硫代糖苷酶被应用于糖蛋白的糖基化修饰,首次人工合成硫代糖蛋白。微生物糖苷酶合成功能的新延伸,对糖生物学、生物技术和制药业的发展将有着重要意义。     

四川阿坝产野生大花红景天不同器官中红景天苷含量的比较

大花红景天[Rhodiola crenulata（Hook.f.et Thoms）H．Ohba]为药材红景天的基原植物,是珍稀的药食兼用天然植物资源之一[1-2]。红景天具有益气活血、通脉平喘、扶正固本的作用[1],主要含黄酮类、有机酸类、多糖类、苷类、香豆素类和挥发性成分等,其中包含的重要生物活性成分有红景天苷、红景天素、酪醇、二苯甲基六氢吡啶和超氧化物歧化酶（SOD）等,还含有维生素及人体必需的多种大量和微量元素,营养价值极高[3-5]。红景天具有抗疲劳、抗缺氧、强壮机体、抗心肌缺血、抗肿瘤和保护神经细胞等作用,红景天药材或其提取物已成为多种中成药、食品、饮料和化妆品的重要原料旧[6]。     

非水相介质中β-葡萄糖苷酶催化合成红景天甙的研究

本文探讨了在非水相介质中酶催化逆水解反应合成红景天甙的新方法,研究了非水相反应体系、酪醇浓度、D-葡萄糖浓度、反应时间、pH值对β-葡萄糖苷酶催化酪醇和D-葡萄糖合成红景天甙的影响,优化了酶法合成红景天甙的条件,使其转化率达到了17．7％,即7．39g/L,远远高于米曲霉整体细胞催化的产量（0．7g／L）。该方法可望应用于其它具有生理活性糖苷类化合物的高效酶促合成,具有潜在的应用价值。     

唾液酸苷酶的催化机理及水解与合成寡糖进展

唾液酸苷酶(EC.3.2.1.18)是一类重要的糖苷水解酶,在动物和微生物中广泛存在.该类酶催化寡糖或糖缀合物上非还原末端唾液酸水解,具有重要的生物学功能,如参与溶酶体降解代谢物、癌症发生、微生物致病等多种生理和病理过程.除了水解活性外,有的唾液酸苷酶还具有转糖基活性,能够以唾液酸单糖或糖苷为糖基供体,催化唾液酸转移到受体分子上,一步合成寡糖和糖苷化合物.这种合成活性对于唾液酸相关糖链的大量获得具有重要意义,有利于推动该类寡糖的基础研究及其在食品和医药中的应用.本文综述了唾液酸苷酶的结构和催化机理、生理功能、转糖基作用及其在寡糖合成中的应用.     

High yield production of salidroside in the suspension culture of Rhodiola sachalinensis

Salidroside has been identified as the most potent ingredient of the Chinese medicine herb, Rhodiola sachalinensis. Since the natural supply of this herb is rapidly decreasing, we established a compact callus aggregate (CCA) strain and culturing system for high yield salidroside production. Several callus strains induced from the explants originated from root, stem, leaf and cotyledon of R. sachalinensis were established and screened for rapid growth rate, high salidroside content and easy propagation in suspension culture condition. The CCA strain was established from a callus strain initiated from the cotyledon. The kinetics of dry weight accumulation and cellular salidroside content in various culture conditions for the strain was determined. For high salidroside production, the optimal inoculum amount was 10% and the optimal concentration for 6-benzylaminopurine and indole-3-butyric acid added in the liquid medium was 5 and 2.5 mg l-1, respectively. The acidic culture medium and a faster shaking speed favored the salidroside accumulation. The addition of 2,4-D, in the liquid MS medium and the utilization of L-tyrosol for chemical feeding enhanced salidroside production. Using a proper combination of culture condition and treatment, salidroside accumulation could reach 57.72 mg g-1 dry weight, that was 5-10-fold higher than that detected in field-grown plants. The corresponding salidroside yield was 555.13 mg l-1, a level suitable for cost effective commercial production to compensate the natural resource shortage of R. sachalinensis.     

Strategies for the production of cell wall‐deconstructing enzymes in lignocellulosic biomass and their utilization for biofuel production

Microbial cell wall‐deconstructing enzymes are widely used in the food, wine, pulp and paper, textile, and detergent industries and will be heavily utilized by cellulosic biorefineries in the production of fuels and chemicals. Due to their ability to use freely available solar energy, genetically engineered bioenergy crops provide an attractive alternative to microbial bioreactors for the production of cell wall‐deconstructing enzymes. This review article summarizes the efforts made within the last decade on the production of cell wall‐deconstructing enzymes in planta for use in the deconstruction of lignocellulosic biomass. A number of strategies have been employed to increase enzyme yields and limit negative impacts on plant growth and development including targeting heterologous enzymes into specific subcellular compartments using signal peptides, using tissue‐specific or inducible promoters to limit the expression of enzymes to certain portions of the plant or certain times, and fusion of amplification sequences upstream of the coding region to enhance expression. We also summarize methods that have been used to access and maintain activity of plant‐generated enzymes when used in conjunction with thermochemical pretreatments for the production of lignocellulosic biofuels.     

脂肪酶高产菌株选育和菌种库的建立

从山东省济南市植物油厂、肉联厂、乳品厂、菜市场等处的含油土壤中分离筛选到80余株脂肪酶活性较高的产生菌,包括细菌、霉菌、酵母等各种类型,我们对其中的部分菌株进行了形态学及酶学性质的初步研究。一株酶活较高的菌株Y-11经鉴定为丝孢酵母属（Trichosporon）,用紫外线及亚硝酸对其进行了双重诱变、然后用制霉菌素及琥珀酸钠筛选耐药性突变株,使酶活提高155%,并将筛选到的菌株建立一个能够产生各具特色的脂肪酶的菌种库,为今后进一步开展脂肪酶应用研究打下基础。     

Impact of wild-type and genetically modified Pseudomonas fluorescens on soil enzyme activities and microbial population structure in the rhizosphere of pea

The aim of this study was to determine the impact of wild-type along with functionally and nonfunctionally modified Pseudomonas fluorescens strains in the rhizosphere. The wild-type F113 strain carried a gene encoding the production of the antibiotic 2,4-diacetylphloroglucinol (DAPG) useful in plant disease control, and was marked with a lacZY gene cassette. The first modified strain was a functional modification of strain F113 with repressed production of DAPG, creating the DAPG-negative strain F113 G22. The second paired comparison was a nonfunctional modification of wild-type (unmarked) strain SBW25, constructed to carry marker genes only, creating strain SBW25 EeZY-6KX. Significant perturbations were found in the indigenous bacterial population structure, with the F113 (DAPG+) strain causing a shift towards slower growing colonies (K strategists) compared with the nonantibiotic-producing derivative (F113 G22) and the SBW25 strains. The DAPG+ strain also significantly reduced, in comparison with the other inocula, the total Pseudomonas populations but did not affect the total microbial populations. The survival of F113 and F113 G22 were an order of magnitude lower than the SBW 25 strains. The DAPG+ strain caused a significant decrease in the shoot-to-root ratio in comparison to the control and other inoculants, indicating plant stress. F113 increased soil alkaline phosphatase, phosphodiesterase and aryl sulphatase activities compared to the other inocula, which themselves reduced the same enzyme activities compared to the control. In contrast to this, the β-glucosidase, β-galactosidase and N -acetyl glucosaminidase activities decreased with the inoculation of the DAPG+ strain. These results indicate that soil enzymes are sensitive to the impact of inoculation with genetically modified microorganisms (GMMs).     

Recombinant microbial systems for improved β-galactosidase production and biotechnological applications

β-Galactosidases (EC 3.2.1.23) constitute a large family of proteins that are known to catalyze both hydrolytic and transgalactosylation reactions. The hydrolytic activity has been applied in the food industry for decades for reducing the lactose content in milk, while the transgalactosylation activity has been used to synthesize galacto-oligosaccharides and galactose containing chemicals in recent years. The main focus of this review is on the expression and production of Aspergillus niger, Kluyveromyces lactis and bacterial β-galactosidases in different microbial hosts. Furthermore, emphasis is given on the reported applications of the recombinant enzymes. Current developments on novel β-galactosidases, derived from newly identified microbial sources or by protein engineering means, together with the use of efficient recombinant microbial production systems are converting this enzyme into a relevant synthetic tool. Thermostable β-galactosidases (cold-adapted or thermophilic) in addition to the growing market for functional foods will likely redouble its industrial interest.     

萜烯生物合成中关键酶的研究进展*

萜烯类化合物是一类高度多样化的天然产物,具有抗肿瘤、抗氧化及免疫调节等生理活性,因此被广泛应用于医药健康、食品、化妆品领域。然而,直接从自然资源中获取萜烯类化合物效率低、成本高,且往往对生态环境产生不利影响,不能实现绿色可持续生产。微生物合成萜烯类化合物近年来备受关注,研究人员从合成途径的构建与调控、关键酶的理性及半理性改造、发酵工艺优化等多个方面进行了探究,取得了丰硕的成果。其中,合成途径中关键酶的催化效率是影响微生物生产萜烯类化合物的重要因素。针对关键酶的研究对于提高微生物合成萜烯类化合物的能力,推动该类天然产物微生物生产的大规模应用具有重要意义。对萜烯类化合物合成途径中的3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)、1-脱氧-D-木酮糖-5-磷酸合酶(DXS)、异戊二烯基二磷酸合成酶(IDS)和萜烯合酶(TPS)4种关键酶的研究进行了综述,并总结讨论了如何通过代谢工程和蛋白质工程手段以及合成生物学技术调节关键酶的催化活性,提高微生物合成萜烯类化合物的效率,对未来利用微生物合成萜烯类化合物的发展进行了展望。     

Engineering dicamba selectivity in crops: a search for appropriate degradative enzyme(s)

The biotechnologial approaches to conferring crop selectivity to herbicides have been demonstrated for a number of compounds such as glyphosate, glufosinate, imidazolinones and cyclohexanediones. Imidazolinone-resistant and cyclohexanedione-resistant maize lines are already in the market. There are several other effective and environmentally benign herbicides such as dicamba, for which engineering crop selectivity is desirable, to broaden the product utility in different crops and provide new solutions for weed control. One of the most effective approaches to conferring dicamba selectivity in crops is to incorporate a gene for its rapid metabolism. It is advantageous to have different dicamba-metabolizing enzymes in order to maximize the chances of at least one functioning optimally in a plant environment. Three different metabolizing enzymes are currently available to engineer crop selectivity. The first one is the folate-dependent O-demethylase from Clostridium thermoaceticum, that converts dicamba to herbicidally inactive 3,6-dichlorosalicylate. The second enzyme is the NADH-dependent, multi-component monooxygenase from Pseudomonas maltophilia DI-6 that also converts dicamba to 3,6-dichlorosalicylate. The third enzyme is from corn endosperm cultures that catalyzes the 5-hydroxylation of dicamba. The merits of these three enzymes are discussed with respect to conferring crop selectivity to dicamba. In addition, a rapid microbial screen was conceived for discovery of new dicamba-degrading bacteria, which resulted in identification of Pseudomonas orvilla. This bacteria degraded dicamba by the same pathway, perhaps using a similar enzyme system as Pseudomonas maltophilia DI-6. However, the microbial screen has the potential to identify novel bacteria that degrade dicamba by a different pathway, providing more options for metabolizing enzymes to confer herbicide selectivity in crops. Received 13 February 1997/ Accepted in revised form 26 June 1997     

Application of metagenomic techniques in mining enzymes from microbial communities for biofuel synthesis

Feedstock for biofuel synthesis is transitioning to lignocelluosic biomass to address criticism over competition between first generation biofuels and food production. As microbial catalysis is increasingly applied for the conversion of biomass to biofuels, increased import has been placed on the development of novel enzymes. With revolutionary advances in sequencer technology and metagenomic sequencing, mining enzymes from microbial communities for biofuel synthesis is becoming more and more practical. The present article highlights the latest research progress on the special characteristics of metagenomic sequencing, which has been a powerful tool for new enzyme discovery and gene functional analysis in the biomass energy field. Critical enzymes recently developed for the pretreatment and conversion of lignocellulosic materials are evaluated with respect to their activity and stability, with additional explorations into xylanase, laccase, amylase, chitinase, and lipolytic biocatalysts for other biomass feedstocks.     

Validation of leaf and microbial pectinases: commercial launching of a new platform technology

Almost all current genetically modified plant commercial products are derived from seeds. The first protein product made in leaves for commercial use is reported here. Leaf pectinases are validated here with eight liquid commercial microbial enzyme products for textile or juice industry applications. Leaf pectinases are functional in broad pH/temperature ranges as crude leaf extracts, while most commercial enzyme products showed significant loss at alkaline pH or higher temperature, essential for various textile applications. In contrast to commercial liquid enzymes requiring cold storage/transportation, leaf pectinase powder was stored up to 16 months at ambient temperature without loss of enzyme activity. Commercial pectinase products showed much higher enzyme protein PAGE than crude leaf extracts with comparable enzyme activity without protease inhibitors. Natural cotton fibre does not absorb water due to hydrophobic nature of waxes and pectins. After bioscouring with pectinase, measurement of contact‐angle water droplet absorption by the FAMAS videos showed 33 or 63 (leaf pectinase), 61 or 64 (commercial pectinase) milliseconds , well below the 10‐second industry requirements. First marker‐free lettuce plants expressing pectinases were also created by removal of the antibiotic resistance aadA gene. Leaf pectinase powder efficiently clarified orange juice pulp similar to several microbial enzyme products. Commercial pilot scale biomass production of tobacco leaves expressing different pectinases showed that hydroponic growth at Fraunhofer yielded 10 times lower leaf biomass per plant than soil‐grown plants in the greenhouse. Pectinase enzyme yield from the greenhouse plants was double that of Fraunhofer. Thus, this leaf‐production platform offers a novel, low‐cost approach for enzyme production by elimination of fermentation, purification, concentration, formulation and cold chain.     

Fungal biotechnology

Fungi are used in many industrial processes, such as the production of enzymes, vitamins, polysaccharides, polyhydric alcohols, pigments, lipids, and glycolipids. Some of these products are produced commercially while others are potentially valuable in biotechnology. Fungal secondary metabolites are extremely important to our health and nutrition and have tremendous economic impact. In addition to the multiple reaction sequences of fermentations, fungi are extremely useful in carrying out biotransformation processes. These are becoming essential to the fine-chemical industry in the production of single-isomer intermediates. Recombinant DNA technology, which includes yeasts and other fungi as hosts, has markedly increased markets for microbial enzymes. Molecular manipulations have been added to mutational techniques as a means of increasing titers and yields of microbial processes and in the discovery of new drugs. Today, fungal biology is a major participant in global industry. Moreover, the best is yet to come as genomes of additional species are sequenced at some level (cDNA, complete genomes, expressed sequence tags) and gene and protein arrays become available.