组成酶、诱导酶及相关试题分析

本文较详细地介绍了微生物细胞内的组成酶和诱导酶,并通过相关试题分析进一步阐述了这两类酶的生物学特性,旨在帮助学生更好地认识这两类酶的区别。

产碱杆菌DN25降氰酶产酶条件初步优化

以实验室保藏的一株具有高效降氰活性的菌株Alcaligenes sp. DN25为研究对象,通过分析其降解产物,发现终产物中存在甲酸,初步判断此菌株降氰反应为氰水解途径。在培养基中添加KCN来考察底物对菌体产酶的诱导作用,结果发现菌体的活力没有提高,表明菌株DN25所产降氰酶应为组成型酶。在培养基中添加4种金属离子和4种含硫物质,其中微量金属离子对菌体的生长和活性均有抑制;Na2SO4和Na2S2O3对菌体生长和产酶均无明显影响;DL-半胱氨酸对菌体产酶有明显促进作用,DL-甲硫氨酸可同时提高菌体的生长量和产酶水平,从而提高发酵液比活力。研究结果为降氰酶的大量获得以及酶制剂在氰污染治理方面的应用研究奠定了基础。

盾壳霉寄生核盘菌过程中胞壁降解酶的变化及对核盘菌的影响

通过测定盾壳霉寄生核盘菌过程中几丁质酶和β-1,3-葡聚糖酶的变化及对核盘菌菌丝和菌组织的影响,结果表明:盾壳霉在寄生核盘菌初期其几丁质酶和β-1,3-葡聚糖酶的活性均会明显的升高,具有明显的诱导酶特征,同时盾壳霉对照在没有核盘菌存在时,也能检测到几丁质酶和β-1,3-葡聚糖酶活性,说明盾壳霉产生的几丁质酶和β-1,3-葡聚糖酶都有组成酶存在。核盘菌菌丝和菌核组织的大量消解都出现在这两种酶活性达到高峰之后,表明两种酶协同作用更有利于核盘菌菌丝和菌核组织的消解腐烂。

微生物代谢的调节知识点例析

微生物在长期的进化过程中,形成了一整套完善的代谢调节体系,以保证各种代谢活动经济而高效地进行,其生命活动的调节方式是酶合成的调节和酶活性的调节．现浅析如下．

Effect of dietary genistein on growth performance,digestive enzyme activity,and body composition of Nile tilapia Oreochromis niloticus

An 8-week feeding experiment was performed to evaluate the effect of dietary genistein on growth performance,body composition,and digestive enzymes activity of juvenile Nile tilapia(Oreochromis niloticus).Four isonitrogenous and isoenergetic diets were formulated containing four graded supplements of genistein:0,30,300,and 3 000 μg/g.Each diet was randomly assigned in triplicate to tanks stocked with 15 juvenile tilapia(10.47±1.24 g).The results show that 30 and 300 μg/gdietary genistein had no significant effect on growth performance of Nile tilapia,but the higher level of genistein(3 000 μg/g)significantly depressed the final body weight and specific growth rate.There was no significant difference in survival rate,feed intake,feed efficiency ratio or whole body composition among all dietary treatments.An assay of digestive enzymes showed that the diet containing 3 000 μg/ggenistein decreased stomach and hepatopancreas protease activity,and amylase activity in the liver and intestine,while a dietary level of300 μg/g genistein depressed stomach protease and intestine amylase activities.However,no significant difference in stomach amylase activity was found among dietary treatments.Overall,the results of the present study indicate that a high level of dietary genistein(3 000 μg/g,or above) would significantly reduce the growth of Nile tilapia,partly because of its inhibitory effect on the activity of major digestive enzymes.Accordingly,the detrimental effects of genistein,as found in soybean products,should not be ignored when applied as an alternative ingredient source in aquaculture.

Human intestinal acyl-CoA synthetase 5 is sensitive to the inhibitor triacsin C

AIM:To investigate whether human acyl-CoA synthetase 5(ACSL5) is sensitive to the ACSL inhibitor triacsin C.METHODS:The ACSL isoforms ACSL1 and ACSL5 from rat as well as human ACSL5 were cloned and recombinantly expressed as 6xHis-tagged enzymes.Ni 2+-affinity purified recombinant enzymes were assayed at pH 7.5 or pH 9.5 in the presence or absence of triacsin C.In addition,ACSL5 transfected CaCo2 cells and intestinal human mucosa were monitored.ACSL5 expression in cellular systems was verified using Western blot and immunofluorescence.The ACSL assay mix included TrisHCl(pH 7.4),ATP,CoA,EDTA,DTT,MgCl 2,[9,103 H] palmitic acid,and triton X-100.The 200 μL reaction was initiated with the addition of solubilized,purified recombinant proteins or cellular lysates.Reactions were terminated after 10,30 or 60 min of incubation with Doles medium.RESULTS:Expression of soluble recombinant ACSL proteins was found after incubation with isopropyl betaD-1-thiogalactopyranoside and after ultracentrifugation these were further purified to near homogeneity with Ni 2+-affinity chromatography.Triacsin C selectively and strongly inhibited recombinant human ACSL5 protein at pH 7.5 and pH 9.5,as well as recombinant rat ACSL1(sensitive control),but not recombinant rat ACSL5(insensitive control).The IC50 for human ACSL5 was about 10 μmol/L.The inhibitory triacsin C effect was similar for different incubation times(10,30 and 60 min) and was not modified by the N-or C-terminal location of the 6xHis-tag.In order to evaluate ACSL5 sensitivity to triacsin C in a cellular environment,stable human ACSL5 CaCo2 transfectants and mechanically dissected normal human intestinal mucosa with high physiological expression of ACSL5 were analyzed.In both models,ACSL5 peak activity was found at pH 7.5 and pH 9.5,corresponding to the properties of recombinant human ACSL5 protein.In the presence of triacsin C(25 μmol/L),total ACSL activity was dramatically diminished in human ACSL5 transfectants as well as in ACSL5-rich human intestinal mucosa.CONCLUSION:The data strongly indicate that human ACSL5 is sensitive to triacsin C and does not compensate for other triacsin C-sensitive ACSL isoforms.

Recent advances in the molecular genetics of resin biosynthesis and genetic engineering strategies to improve defenses in conifers

Since the first terpenoid synthase cDNA was obtained by the reverse genetic approach from grand fir, great progress in the molecular genetics of terpenoid formation has been made with angiosperms and genes encoding a monoterpene synthase, a sesquiterpene synthase, and a diterpene synthase. Tree killing bark beetles and their vectored fungal pathogens are the most destructive agents of conifer forests worldwide. Conifers defend against attack by the constitutive and inducible production of oleoresin that accumulates at the wound site to kill invaders and both flush and seal the injury. Although toxic to the bark beetle and fungal pathogen, oleoresin also plays a central role in the chemical ecology of these boring insects. Recent advances in the molecular genetics of terpenoid biosynthesis provide evidence for the evolutionary origins of oleoresin and permit consideration of genetic engineering strategies to improve conifer defenses as a component of modern forest biotechnology. This review described enzymes of resin biosynthesis, structural feathers of genes genomic intron and exon organization, pathway organization and evolution, resin production and accumulation, interactions between conifer and bark beetle, and engineering strategies to improve conifer defenses.

Production of Poly（3-hydroxybutyrate-co-3-hydroxyhexanoate） by Recombinant Pseudomonas stutzeri 1317 from Unrelated Carbon Sources

Synthetic biology promises to simplify the construction of metabolic pathways by assembling the de- tached modules of the whole pathway. This gives new approaches for the microbial production of industrial products such as polyhydroxyalkanoates （PHA）. In this study, to produce poly（3-hydroxybutyrate-co-3-hydroxyhexanoate） （PHBHHx） by Pseudomonas stutzeri 1317 from unrelated carbon sources such as glucose, the phaCl-phaZ-phaC2 operon of P. stutzeri 1317 was knocked out to generate the PHA deficient mutant P. stutzeri 1317LF. Then three modules containing phaCahAReBRe, phaCahBReGep and phaCAhPah were introduced into P. stutzeri 1317LF separately The shake flask results indicated that the precursor supply and PHA synthase activity were the vital factors for the PHBHHx accumulation of P. stutzeri 1317LF. Furthermore, the PHBHHx accumulation of the recombinants from different carbon resources were performed. The highest PHBHHx content was 23.7% （by mass） with 58.6% （by mole） 3HB fraction. These results provide basis for further improving the PHBHHx accumulation of P. stutzeri from unrelated carbon sources.

The complete mitochondrial genome of Triuncina daii(Lepidoptera:Bombycidae) and its phylogenetic implications

The bombycid moth, Triuncina daii Xing Wang ＆ Zolotuhin, 2015, plays an important role for analyzing the phylogenetic relationships of the family Bombycidae （Lepidoptera： Bombycoidea）. Here we first describe the complete mitochondrial genome （mitogenome） of T. daii, which includes thirteen protein-coding genes （PCGs）, twenty-two transfer RNA genes, two ribosomal RNA genes and an A＋T-rich region, and we find the mitogenome is 15,482 bp in length （GenBank no. KY091643）. The genes order and orientation in the T. daii mitogenome are similar to other sequenced lepidopteran species. Except for cox1, all of the PCGs started with ATN. Twelve PCGs stopped at TAA except for cox1 which stops at a single T. Thirteen PCGs of available species are used to demonstrate the inner phylogenetic relationships of Bombycoidea. The bombycid species form a monophyletic clade with a bootstrap value of 100% and a posterior probability of 1.00.

The Expression of Ca N and CaMK is Associated with Lipogenesis in the Muscle of Chicken

Intramuscular fat （IMF） content in chickens significantly contributes to meat quality. The main objective of this study was to assess the expression of calcineudn （CAN） and Ca^2＋/calmodutin-dependent protein kinase （CAME） in lipogene- sis in chicken muscle. The chickens were slaughtered and sampled at the ages of 4, 8, and 16 weeks, respectively. IMF content and the expression of CaN subunits and CaMK isoforms were measured in thigh muscle tissue. The results showed that the IMF contents were higher in chickens at the age of 16 weeks compared with those in chickens at the ages of 4 and 8 weeks （P〈0.05）. The expression levels of fatty acid synthase （FAS） and fatty acid translocase CD36 （FAT/CD36） mRNA in 16-week-old chickens were all significantly up-regulated compared with those in 4-week-old chickens （P〈0.05）. The mRNA levels of CaNB and CaMK IV in 16-week-old chickens were significantly lower than those in 4-week-old chickens （P〈0.05）. But the CaMK II mRNA levels in 16-week-old chickens were significantly higher than those in 4-week-old chickens （P〈0.05）. To investigate the roles of CaMK and CaN in adipogenesis, SV cells were incubated in standard adipogenesis medium for 24 h and treated with specific inhibitor of CaMK and CaN. The ex- pressions of CCAAT/enhancer binding protein β（C/EBPJ3）, sterol regulatory element- binding protein 1 （SREBP1） and peroxisome proliferation-activated receptor ）, （PPARy） were dramatically enhanced by CsA and CaN inhibitor （P〈0.05）. KN93, a CaMK Ⅱ inhibitor, dramatically repressed the expression of those lipogenic genes （P〈0.05）. All the results above indicated that CaN and CaMK had different effects on adipogenesis in the muscle of chickens.

Expression, purification and activity assay of recombinant human thymidylate synthase

Thymidylate synthase （TS, E.C.2.1.1.45） catalyzes a critical reaction in the only pathway of de novo synthesis of thymidylate （dTMP） in human cells, and is an important target of chemotherapy. To evaluate the inhibitory activities of novel compounds to TS, a convenient method of activity assay using 6x His-tagged recombinant human TS （rhTS） was established and 49 novel synthetic folate analogues were screened to discover potential TS inhibitors. During the process, 4 novel compounds were found to effectively inhibit TS, while the IC 50 of a positive control raltitrexed was 3.4 μM in this assay.

Discovery of thiazostatin siderphores from Streptomyces sp. HMU0027 with a genomic DNA-based PCR assay targeting nonribosomal peptide synthetase

Nonribosomal peptides （NRPs） represent a large family of natural products with great diversities of chemical structures and biological activities. The peptide backbones of NRPs are synthesized by nonribosomal peptide synthetases （NRPSs） that minimally consist of one adenylation （A） domain, one peptidyl carrier protein （PCP） and one condensation （C） domain. In this study, we carded out a PCR screening and identified 21 ＂positive＂ strains from 100 actinomycete strains with the degenerate primers designed from the conserved sequences of A domains of NRPSs. One of the 21 ＂positive＂ strains, Streptomyces sp. HMU0027, was selected for large-scale fermentation （9 L） based on HPLC analysis, and subsequent isolation and structural elucidation afforded seven NRPS-synthesized thiazostatin siderophore analogues （1-7）. Compound 1 was a new compound containing an unusual linkage between a phenolate siderophore and a sugar moiety. These results laid the foundation for further biosynthetic research of these thiazostatin analogues and highlighted the power of genome mining technologies based on biosynthetic knowledge in NRP discovery.

Dynamic m^6A modification and its emerging regulatory role in m RNA splicing

Recent studies on enzymes regulating dynamic N6-methyl-adenosine （m6A） in RNA together with the findings from m6A-methylated RNA immunoprecipitation followed by high-throughput sequencing （MeRIP-seq/m6A- seq） have revealed a broad biological role of m6A in RNA processing, development, differentiation, metabolism and fertility. RNA m6A methylation is catalyzed by a multi- component methyltransferase complex composed of at least three subunits： METTL3, METTL14 and Wilms tumor 1-associated protein （WTAP）, in which METTL3 and METTL14 serve as catalytic subunits, while WTAP as reg- ulatory subunit. Dioxygenases FTO and ALKBH5, as the first two known m6A demethylases, catalyze m6A removal. Five m6A-binding proteins are classified into cytoplasmic YT521-B homology （YTH） domain-containing family YT- HDF1-3 and nuclear YTHDC1-2. Perturbation of enzy- matic activities catalyzing dynamic m6A results in altered expression of thousands of genes and affects mRNA stability and splicing at the cellular level. Here, we summarize recent discoveries about m6A methyltransferases （writers）,demethylases （erasers） and binding proteins （readers）, and further discuss the potential impacts of m6A on RNA pro- cessing, especially on mRNA splicing.

Antisense expression of Gossypium barbadense UGD6 in Arabidopsis thaliana significantly alters cell wall composition

Uridine diphosphate-glucose dehydrogenase （UGD, EC1.1.1 glucuronate）, a critical precursor of cell wall polysaccharides 22 oxidizes UDP-Glc （UDP-D-glucose） to UDP-GlcA （UDP-D- GbUGD6 from Gossypium barbadense is more highly expressed late in the elongation of cotton fibers （15 d post-anthesis （DPA）） and during the stage of secondary cell wall thickening （30 DPA）. Subcellular localization analysis in onion epidermis revealed that fluorescently labeled GbUGD6 protein was distribut- ed throughout the cell membrane, as well as the nucleus and vacuoles. Examination of UGD function in Arabidopsis revealed that the antisense GbUGD6 lines had shorter roots, deferred blossoming, compared to wild-type plants. Activities of associated enzymes were also affected by UGD reduction, and biochemical analysis of cell wall samples showed an increase in cellulose levels and a decrease in UGP-GlcA contents. The results of the present study as well as previous studies on UGD support the conclusion that UGD plays a major role in synthesizing polysaccharides synthesis in the cell wall.

Soil Enzyme Activities and Organic Matter Composition Affected by 26 Years of Continuous Cropping

The study was to determine the long-term effects of subtropical monoculture and rotational cropping systems and fertilization on soil enzyme activities and soil C, N, and P levels. Cropping systems included continuous sorghum(Sorghum bicolor L.), cotton(Gossypium hirsutum L.), corn(Zea mays L.), and cotton/sorghum rotations after 26 years of treatment imposition. Soil under continuous sorghum and continuous corn had 15% and 11%, respectively, greater C concentrations than soil under continuous cotton.Organic C was 10% higher at 0–7.5 cm than at 7.5–15 cm. Total N followed similar trends with soil depth as organic C. Continuous sorghum had 19% higher total N than other crop species and rotations. With fertilization, continuous cotton had the highest total P at 0–7.5 cm and sorghum had the highest at 7.5–15 cm. Soil total P was 14% higher at 0–7.5 than at 7.5–15 cm, and fertilization increased 15% total P compared to unfertilized soil. Arylsulfatase, alkaline phosphatase, and β-d-glucosidase activity were the highest for sorghum and the lowest for cotton. Rotation increased enzyme activities compared to continuous cotton but not for continuous sorghum. Of all crop species and rotations, continuous cotton generally showed the lowest levels of organic matter and enzyme activities after 26 years. Fertilization significantly increased the yields for all cropping systems, but rotation had no significant effect on either sorghum or cotton lint yield compared to each crop grown in monoculture. Long-term cropping did not increase soil organic matter levels beyond short-term gains, indicating the difficulty in promoting C sequestration in subtropical soils.

Activating transcription factor 5 regulates lipid metabolism in adipocytes

Activating transcription factor 5(ATF5) is a member of the activating transcription factor/cA MP response element binding protein(ATF/CREB) family, and is highly expressed in liver and adipose tissue. Previous reports have shown that ATF5 promoted 3T3-L1 preadipocytes differentiation. In this study, we found that ATF5 was highly expressed in mature adipocytes, suggesting a potential role of ATF5 in mature adipocytes, which has not been reported previously. To understand the function of ATF5 in mature adipocytes, we knocked down the expression of ATF5 in 3T3-L1 mature adipocytes and observed decreased lipid droplets. Consistent with the in vitro experiment, the knockdown of ATF5 in white adipose tissue led to less adipose tissue and smaller adipocytes size. Further research revealed that the inhibition of ATF5 diminished the adipocytes size via the inhibition of fatty acid synthetase, stearyl coenzyme A desaturation enzyme 1, and the induction of carnitine palmitoyl transferase 1, one key enzyme of lipid metabolism. In addition, ATF5 knockdown in inguinal white adipose tissue improved whole body insulin sensitivity.Our work provides a new understanding of ATF5 function in mature adipocytes and a potential therapeutic target of diabetes.

Effect of acupotomy on nitric oxide synthase and beta-endorphin in third lumbar vertebrae transverse process syndrome model rats

OBJECTIVE： To explore the long-term effects and pain relief mechanism of acupotomy by observing changes in nitric oxide synthase （NOS） and beta-en- dorphin （~3-EP） in the hypothalamus, spinal cord, and peripheral blood of rats with third lumbar ver- tebrae （L3） transverse process syndrome. METHODS： Twenty-eight SD rats were randomly as- signed to normal, model, electroacupuncture （EA）, and acupotomy group. The last three groups were put through an operation to emulate L3 transverse process syndrome. Fourteen days after the simulation operation, EA and acupotomy treatments were applied to the respective groups. Fifty-six days afterthe simulation operation, biochemistry tests and enzyme-linked immunosorbent assay were used to measure NOS and 13-EP in the hypothalamus, spinal cord, and peripheral blood. RESULTS： Rats with the simulation operation showed significantly higher levels of NOS and II3-EP in the hypothalamus, spinal cord, and peripheral blood than those in the normal group. The EA and acupotomy groups had significantly lower levels of NOS and β-EP than those in the model group. There was no statistical difference between the EA and acupotomy groups. CONCLUSION： EA and acupotomy treatments significantly lowered NOS and β-EP levels in the hypothalamus, spinal cord, and peripheral blood and alleviated L3 transverse process syndrome.

Effect of nitric oxide synthase inhibitor on proteoglycan metabolism in repaired articular cartilage in rabbits

Objective: To study the effect of nitric oxide synthase inhibitor, S-methyl thiocarbamate (SMT), on proteoglycan metabolism in repaired articular cartilage in rabbits. Methods: Twenty-four male New Zealand white rabbits, aged 8 months and weighing 2.5 kg±0.2 kg, were used in this study. Cartilage defects in full thickness were created on the intercondylar articular surface of bilateral femurs of all the rabbits. Then the rabbits were randomly divided into 3 groups (n=8 in each group). The defects in one group were filled with fibrin glue impregnated with recombinant human bone morphogenetic protein-2 (rhBMP-2, BMP group), in one group with fibrin glue impregnated with rhBMP-2 and hypodermic injection with SMT (SMT group) and in the other group with nothing (control group). All the animals were killed at one year postoperatively. The tissue sections were stained with safranine O-fast green and analyzed by Quantiment 500 system to determine the content of glycosaminoglycan through measuring the percentage of safranine O-stained area, the thickness of cartilages and the mean gray scale (average stain intensity). Radiolabelled sodium sulphate ( Na 2 35SO 4) was used to assess the proteoglycan synthesis. Results: At one year postoperatively, the percentage of safranine O-stained area, the mean gray scale and the cartilage thickness of the repaired tissues in SMT group were significantly higher than those of BMP group (P< 0.01) and the control group (P< 0.05). Result of incorporation of Na 2 35SO 4 showed that the proteoglycan synthesis in SMT group was higher than those of BMP group and the control group (P< 0.01). Conclusions: SMT, a nitric oxide synthase inhibitor, can significantly increase the content of glycosaminoglycan and proteoglycan synthesis, and computer-based image analysis is a reliable method for evaluating proteoglycan metabolism.

A euryarchaeal histone modulates strand displacement synthesis by replicative DNA polymerases

Euryarchaeota and Crenarchaeota, the two main lineages of the domain Archaea, encode different chromatin proteins and differ in the use of replicative DNA polymerases. Crenarchaea possess a single family B DNA polymerase(Pol B), which is capable of strand displacement modulated by the chromatin proteins Cren7 and Sul7 d. Euryarchaea have two distinct replicative DNA polymerases, PolB and PolD, a family D DNA polymerase. Here we characterized the strand displacement activities of Pol B and Pol D from the hyperthermophilic euryarchaeon Pyrococcus furiosus and investigated the influence of HPf A1, a homolog of eukaryotic histones from P. furiosus, on these activities. We showed that both Pol B and Pol D were efficient in strand displacement. HPf A1 inhibited DNA strand displacement by both DNA polymerases but exhibited little effect on the displacement of a RNA strand annealed to single-stranded template DNA. This is consistent with the finding that HPf A1 bound more tightly to double-stranded DNA than to a RNA:DNA hybrid. Our results suggest that, although crenarchaea and euryarchaea differ in chromosomal packaging, they share similar mechanisms in modulating strand displacement by DNA polymerases during lagging strand DNA synthesis.

New insights into the role of DNA synthesis in meiotic recombination

Meiosis comprises two rounds of nuclear division following a single phase of DNA replication, leading to the production of haploid gametes and is essential for sexual reproduction in eukaryotes. Unlike mitosis, meiosis involves homologous chromosome pairing, synapsis, and recombination during prophase I. Meiotic recombination not only ensures the accurate segregation of homologs, but also redistributes alleles among offspring. DNA synthesis is a critical process during meiotic recombination, but our understanding of the proteins that execute and regulate it is limited. This review summarizes the recent advances in defining the role of DNA synthesis in meiotic recombina- tion through analyses of DNA synthesis genes, with specific emphasis on DNA polymerases （e.g., Pole and PolS）, replication processivity factor RFC1 and translesion polymerases （e.g., Pol~）. We also present a new double strand break repair model for meiotic recombination, which includes lagging strand DNA synthesis and leading strand elongation. Finally, we propose that DNA synthesis is one of critical factors for discriminating meiotic recombination pathways and that this differentiation may be conserved among eukaryotes.