María Elena Flores

β‐Xylosidase and xylanase characterization and production by Streptomyces sp. CH‐M‐1035

Extracellular xylanase activity and cell‐bound β‐xylosidase production by a selected strain of Streptomyces sp. CH‐M‐1035 was characterized during growth on three xylans, sugar cane bagasse pith and lemon peel as sole carbon source. The cell‐bound β‐xylosidase and extracellular endoxylanase had pH optima of 6·0 and 5·0, and temperature optima of 50°C and 60°C, respectively. The highest level of β‐xylosidase activity was obtained when Streptomyces sp. CH‐M‐1035 was grown on larchwood xylan, whereas the maximal endoxylanase production was found on lemon peel. Reducing sugars accumulated in the culture media when Streptomyces sp. CH‐M‐1035 was grown on xylans, but not on agroindustrial residues.

Role of vitamin B12 on methylmalonyl-CoA mutase activity

Vitamin B12 is an organometallic compound with important metabolic derivatives that act as cofactors of certain enzymes, which have been grouped into three subfamilies depending on their cofactors. Among them, methylmalonyl-CoA mutase (MCM) has been extensively studied. This enzyme catalyzes the reversible isomerization of L-methylmalonyl-CoA to succinyl-CoA using adenosylcobalamin (AdoCbl) as a cofactor participating in the generation of radicals that allow isomerization of the substrate. The crystal structure of MCM determined in Propionibacterium freudenreichii var. shermanii has helped to elucidate the role of this cofactor AdoCbl in the reaction to specify the mechanism by which radicals are generated from the coenzyme and to clarify the interactions between the enzyme, coenzyme, and substrate. The existence of human methylmalonic acidemia (MMA) due to the presence of mutations in MCM shows the importance of its role in metabolism. The recent crystallization of the human MCM has shown that despite being similar to the bacterial protein, there are significant differences in the structural organization of the two proteins. Recent studies have identified the involvement of an accessory protein called MMAA, which interacts with MCM to prevent MCM’s inactivation or acts as a chaperone to promote regeneration of inactivated enzyme. The interdisciplinary studies using this protein as a model in different organisms have helped to elucidate the mechanism of action of this isomerase, the impact of mutations at a functional level and their repercussion in the development and progression of MMA in humans. It is still necessary to study the mechanisms involved in more detail using new methods.

Physiological studies on induction and catabolite repression of β-xylosidase and endoxylanase in Streptomyces sp. CH-M-1035

Abstract The specificity of induction of β-xylosidase and endoxylanase from Streptomyces sp. CH-M-1035 was investigated using mono-, di- and poly-saccharides, among other compounds. This microorganism was able to grow on all carbon sources except cellulose microcrystalline and methyl-β- D -xylopyranoside. β-Xylosidase was induced by larchwood xylan, birchwood xylan, oat spelts xylan and D -xylose. Basal levels of activity were obtained in sucrose, lactose, maltose and pectin. Endoxylanase activity was induced in presence of xylans, D -xylose, sucrose and D -arabinose. Both enzymes, β-xylosidase and endoxylanase, were repressed by the addition of glucose, glycerol and succinic acid to the culture medium containing 1% birchwood xylan. On the other hand, addition of methyl-β- D -xylopyranoside in the same conditions induced β-xylosidase but not endoxylanase synthesis. An unexpected stimulatory effect was observed on β-xylosidase biosynthesis when pyruvic acid was added to the culture medium independently of the xylan used.

Isolation of endopolygalacturonase hyperproducing mutants ofAspergillus sp. CH-Y-1043

SummaryWith the aim of obtaining hyperproducing strains of pectinases,Aspergillus sp. CH-Y-1043 was mutated with NTG and mutants resistant to glycerol catabolic repression were selected. Among the mutants obtained, CH-SS/ M63 produced an endo-PG activity 400% higher than the wild type, using lemon peel as the sole carbon source.

Opposite effect of Hsp90α and Hsp90β on eNOS ability to produce nitric oxide or superoxide anion in human embryonic kidney cells.

Heat shock protein 90 subfamily is composed by two cytosolic isoforms known as Hsp90α and Hsp90β. Endothelial nitric oxide synthase (eNOS) is regulated by Hsp90, however the specific role of each Hsp90 isoform on NO production has not been established. This study was designed to evaluate the effect of Hsp90α and Hsp90β over-expression on eNOS/NO pathway. Rat Hsp90α and Hsp90β were cloned into pcDNA3.1(+) and transfected in human embryonic kidney cells (HEK-293). Hsp90α and Hsp90β transfection was corroborated by Western blot analysis and their effect on NO production (NO2/NO3), eNOS protein and its phosphorylation at Ser1177 and Thr495, as well as Akt/PKB Ser473 phosphorylation was determined. The interaction of Hsp90α and Hsp90β with eNOS and the dimer/monomer ratio of Hsp90, as well as O2- generation were also assessed. After transfection, Hsp90α and Hsp90β levels were significantly increased in HEK-293 cells. The Hsp90α over-expression induced a significant increase in NO2/NO3 levels, an effect that was associated with increased phosphorylation of eNOS Ser 1177 and Akt/PKB Ser473, as well as with a greater Hsp90α dimerization. Noteworthy, pcHsp90β transfection reduced significantly NO2/NO3 and increased O2- generation. These effects were associated with a reduction of eNOS dimeric conformation, increased eNOS Thr495 phosphorylation, reduced Akt/PKB phosphorylation, and by a greater amount of monomeric Hsp90β conformation. These data show for first time that Hsp90α and Hsp90β differentially modulate NO and O2- generation by eNOS through promoting changes in eNOS conformation and phosphorylation state.

Improvement of pectinase production by interspecific hybrids of Aspergillus strains

Protoplast fusion induced by polyethylene glycol and Ca2+, was performed between auxotrophic mutants of pectinolytic fungi Aspergillus sp. CH‐Y‐1043 (A13) ade− and Aspergillus flavipes ATCC‐16795 (F7) lys−. Prototrophic colonies were developed on minimal medium with a fusion frequency of 1·0×10−2. The reversion frequency of the mutation in spores and protoplasts was low and ranged from 2·0 to 4·0×10−7. Four prototrophic hybrids (HH, HE, HF and HJ) exhibited enhanced production of endo‐pectinase and pectin‐lyase. The highest production was observed in HJ ; maximum activities were 150 and 160% respectively, whereas the exo‐pectinase production was similar to the wild‐type strain Aspergillus sp. CH‐Y‐1043. Hybrid HJ showed the greatest growth ; nevertheless, specific endo‐pectinase and pectin‐lyase activities were higher in all hybrids than those produced by the wild‐type strains.

Glucose and glycerol repression of α-amylase in Streptomyces kanamyceticus and isolation of deregulated mutants

SummaryGlucose and glycerol at concentrations of 2 % negatively affected amylase synthesis in plate and submerged Streptomyces kanamyceticus cultures. This microorganism was insensitive to growth inhibition by glucose analogs and deregulated mutants were identified by a clearing zone around colonies grown on starch and glycerol or glucose, and selected. Three kinds of mutants were obtained: one insensitive to glucose (Mutant 41), another insensitive to glycerol repression (Mutant E) and the last (Mutant 29) an amylase-hyperproducing mutant, albeit regulated by glucose or glycerol like the wild type. The levels of glucokinase, an enzyme involved in catabolite regulation of Enterobacteria, were determined and results showed no differences between the parental strain and the mutants.

Protoplasts from pectinolytic fungi: isolation, regeneration and pectinolytic enzyme production

S. Solís, M.E. FLORES AND C. HUITRON. 1996. Protoplast release in pectinolytic strain mutants of Aspergillus sp. CH‐Y‐1043 (A13) and Aspergillus flavipes ATCC‐16795 (F7) is described. Optimum yield of protoplasts A13 was obtained in a lapse of 1 h when commercially lytic enzymes of Trichoderma harzanium (2 mg ml−1) were added in 0.05 mol 1−1 citrate‐phosphate buffer pH 5.0 containing 0.7 mol 1−1 KCl and 10 mg ml−1 BSA. Best results in F7 were obtained when the protoplasting system of A13 was supplemented with 10 mg ml−1Aureobasidium sp. lytic enzymes. Isolated protoplasts in A13 and F7 were capable of a high regeneration frequency of 87% and 53% when 0.7 mol 1−1 KCl and sorbitol were used as osmotic stabilizers. Endo‐P, Exo‐P and pectin lyase production were not modified during the process of regeneration.

Heat shock protein 72 (Hsp72) specific induction and temporal stability in urine samples as a reliable biomarker of acute kidney injury (AKI).

Abstract We demonstrated that urinary heat shock protein of 72 KDa (Hsp72) is a sensitive biomarker for the early detection of acute kidney injury (AKI). However, whether Hsp72 induction during an AKI episode is kidney-specific is unknown, as well as, the degree of Hsp72 stability in urine samples. In rats that underwent bilateral renal ischemia and reperfusion (I/R), Hsp72 levels were evaluated in several tissues and in collected urines under different storage and temperature conditions, as well as in variable numbers of freeze-thaw cycles. The effect of room temperature and five freeze-thaw cycles on urinary Hsp72 levels was also evaluated in urine samples from AKI patients. We found that Hsp72 increased exclusively in the renal cortex of I/R group, emphasizing its performance as an AKI biomarker. Urinary-Hsp72 remained constant at room temperature (48 h), during 9 months of storage and was not affected by five freeze/thaw cycles.

Function, kinetic properties, crystallization, and regulation of microbial malate dehydrogenase

Malate dehydrogenase (MDH) is an enzyme widely distributed among living organisms and is a key protein in the central oxidative pathway. It catalyzes the interconversion between malate and oxaloacetate using NAD+ or NADP+ as a cofactor. Surprisingly, this enzyme has been extensively studied in eukaryotes but there are few reportsabout this enzyme in prokaryotes. It is necessary to review the relevant information to gain a better understanding of the function of this enzyme. Our review of the data generated from studies in bacteria shows much diversity in their molecular properties, including weight, oligomeric states, cofactor and substrate binding affinities, as well as differences in the direction of the enzymatic reaction. Furthermore, due to the importance of its function, the transcription and activity of this enzyme are rigorously regulated. Crystal structures of MDH from different bacterial sources led to the identification of the regions involved in substrate and cofactor binding and the residues important for the dimer-dimer interface. This structural information allows one to make direct modifications to improve the enzyme catalysis by increasing its activity, cofactor binding capacity, substrate specificity, and thermostability. A comparative analysis of the phylogenetic reconstruction of MDH reveals interesting facts about its evolutionary history, dividing this superfamily of proteins into two principle clades and establishing relationships between MDHs from different cellular compartments from archaea, bacteria, and eukaryotes.概 要苹果酸脱氢酶 (MDH) 广泛存在于动物、 植物以及微生物体内, 是生物体进行糖代谢的关键酶之一。 在辅酶 I (NAD+) 或辅酶 II (NADP+) 的作用下, 能够催化草酰乙酸和苹果酸之间相互转化。 虽然目前真核微生物中 MDH 已被广泛研究, 但是对原核生物中的这种酶却鲜有报道。 因此, 有必要对 MDH 的相关研究信息进行综述, 以期更好地了解这种酶的功能。 本文综述了细菌相关研究的各种数据信息, 进一步挖掘 MDH 的分子多样性, 包括分子量、 低聚态、 辅因子与底物的结合力, 以及酶反应方向的差异等。 通过对不同细菌来源的 MDH 的晶体结构的分析, 可鉴别底物与辅因子结合的部位以及形成二聚体的重要残基。 对这些结构信息的了解将有利于指导研究人员对酶的结构进行修饰从而提高其催化能力, 比如增加酶的活性、 辅助因子的结合能力、 底物特异性和热稳定性等。 另外, 本文通过分析比较 MDH 系统发生树的重建, 将其蛋白超家族分成两个主分支, 同时在古生菌、 细菌和真核微生物等不同细胞的 MDH 之间建立联系。