Biochemical and structural characterization of the intracellular mannanase AaManA of Alicyclobacillus acidocaldarius reveals a novel glycoside hydrolase family belonging to clan GH-A

An intracellular mannanase was identified from the thermoacidophile Alicyclobacillus acidocaldarius Tc-12-31. This enzyme is particularly interesting, because it shows no significant sequence similarity to any known glycoside hydrolase. Gene cloning, biochemical characterization, and structural studies of this novel mannanase are reported in this paper. The gene consists of 963 bp and encodes a 320-amino acid protein, AaManA. Based on its substrate specificity and product profile, AaManA is classified as an endo-beta-1,4-mannanase that is capable of transglycosylation. Kinetic analysis studies revealed that the enzyme required at least five subsites for efficient hydrolysis. The crystal structure at 1.9 angstroms resolution showed that AaManA adopted a (beta/alpha)8-barrel fold. Two catalytic residues were identified: Glu151 at the C terminus of beta-stand beta4 and Glu231 at the C terminus of beta7. Based on the structure of the enzyme and evidence of its transglycosylation activity, AaManA is placed in clan GH-A. Superpositioning of its structure with that of other clan GH-A enzymes revealed that six of the eight GH-A key residues were functionally conserved in AaManA, with the exceptions being residues Thr95 and Cys150. We propose a model of substrate binding in AaManA in which Glu282 interacts with the axial OH-C(2) in-2 subsites. Based on sequence comparisons, the enzyme was assigned to a new glycoside hydrolase family (GH113) that belongs to clan GH-A.     

Genome survey sequencing of red swamp crayfish <Emphasis Type="Italic">Procambarus clarkii</Emphasis>

Red swamp crayfish, Procambarus clarkii, presently is an important aquatic commercial species in China. The crayfish is a hot area of research focus, and its genetic improvement is quite urgent for the crayfish aquaculture in China. However, the knowledge of its genomic landscape is limited. In this study, a survey of P. clarkii genome was investigated based on Illumina’s Solexa sequencing platform. Meanwhile, its genome size was estimated using flow cytometry. Interestingly, the genome size estimated is about 8.50 Gb by flow cytometry and 1.86 Gb with genome survey sequencing. Based on the assembled genome sequences, total of 136,962 genes and 152,268 exons were predicted, and the predicted genes ranged from 150 to 12,807 bp in length. The survey sequences could help accelerate the progress of gene discovery involved in genetic diversity and evolutionary analysis, even though it could not successfully applied for estimation of P. clarkii genome size.     

生物耐铜的分子机理及铜污染环境的生物联合修复

铜是动植物和人类必需的微量元素,缺乏或过多都将产生不良影响。随着社会经济的发展,人类活动对环境的干扰日益加剧,工业和农业生产活动常可导致土壤铜污染,铜已成为土壤重金属污染的主要元素之一。总结了铜在植物体内的自发内稳态调节机制,在细菌和真菌体内的吸收、分布、解毒和调节因子,同时以蚯蚓为例简要阐述了土壤动物对铜的解毒机理;从分子生物学角度对重金属铜在生物体内的代谢机理及生物对环境中过量铜的联合修复研究进展进行了综述,以期为铜污染环境的植物、微生物和动物联合修复的分子机理研究提供借鉴。     

Identification and characterization of a novel salt‐tolerant esterase from a Tibetan glacier metagenomic library

A salt‐tolerant esterase, designated H9Est, was identified from a metagenomic library of the Karuola glacier. H9Est gene comprised 1071 bp and encoded a polypeptide of 357 amino acids with a molecular mass of 40 kDa. Sequence analysis revealed that H9Est belonged to the family IV of bacterial lypolitic enzyme. H9Est was overexpressed in Escherichia coli and the purified enzyme showed hydrolytic activity towards p‐nitrophenyl esters with carbon chain from 2 to 8. The optimal esterase activity was at 40°C and pH 8.0 and the enzyme retained its activity towards some miscible organic solvents such as polyethylene glycol. A three‐dimensional model of H9Est revealed that S200, D294, and H324 formed the H9Est catalytic triad. Circular Dichroism spectra and molecular dynamic simulation indicated that the esterase had a wide denaturation temperature range and flexible loops that would be beneficial for H9Est performance at low temperatures while retaining heat‐resistant features. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:890–899, 2015     

Rational design to improve thermostability and specific activity of the truncated Fibrobacter succinogenes 1,3-1,4-β-d-glucanase

1,3-1,4-β-D-Glucanase has been widely used as a feed additive to help non-ruminant animals digest plant fibers, with potential in increasing nutrition turnover rate and reducing sanitary problems. Engineering of enzymes for better thermostability is of great importance because it not only can broaden their industrial applications, but also facilitate exploring the mechanism of enzyme stability from structural point of view. To obtain enzyme with higher thermostability and specific activity, structure-based rational design was carried out in this study. Eleven mutants of Fibrobacter succinogenes 1,3-1,4-β-D-glucanase were constructed in attempt to improve the enzyme properties. In particular, the crude proteins expressed in Pichia pastoris were examined firstly to ensure that the protein productions meet the need for industrial fermentation. The crude protein of V18Y mutant showed a 2 °C increment of Tm and W203Y showed ～30% increment of the specific activity. To further investigate the structure-function relationship, some mutants were expressed and purified from P. pastoris and Escherichia coli. Notably, the specific activity of purified W203Y which was expressed in E. coli was 63% higher than the wild-type protein. The double mutant V18Y/W203Y showed the same increments of Tm and specific activity as the single mutants did. When expressed and purified from E. coli, V18Y/W203Y showed similar pattern of thermostability increment and 75% higher specific activity. Furthermore, the apo-form and substrate complex structures of V18Y/W203Y were solved by X-ray crystallography. Analyzing protein structure of V18Y/W203Y helps elucidate how the mutations could enhance the protein stability and enzyme activity.     

琥珀酸脱氢酶或琥珀酰辅酶A合成酶缺失促进大肠杆菌积累5-氨基乙酰丙酸

5-氨基乙酰丙酸 (ALA) 是生物体内四吡咯类化合物的合成前体,在农业及医药领域应用广泛,是极具开发价值的高附加值生物基化学品。目前利用外源C4途径的重组大肠杆菌发酵生产ALA的研究主要利用LB培养基并添加葡萄糖和琥珀酸、甘氨酸等合成前体,成本较高。琥珀酸在C4途径中以琥珀酰辅酶A的形式直接参与ALA的合成。文中在以葡萄糖为主要碳源的无机盐培养基中研究了琥珀酰辅酶A下游代谢途径琥珀酸脱氢酶编码基因sdhAB和琥珀酰辅酶A合成酶编码基因sucCD缺失对ALA积累的影响。与仅表达异源ALA合成酶的对照菌株相比,sdhAB和sucCD缺失菌株ALA的产量分别提高了25.59%和12.40%,且ALA的积累不依赖于琥珀酸的添加和LB培养基的使用,从而大幅降低了生产成本,显示出良好的工业应用前景。     

Mouse model of male germ cell apoptosis in response to a lack of hormonal stimulation

As a prerequisite for studies using mutant mice, we established a mouse model for induction of male germ cell apoptosis after deprivation of gonadotropins and intratesticular testosterone (T). We employed a potent long acting gonadotropin-releasing hormone antagonist (GnRH-A), acyline, alone or in combination with an antiandrogen, flutamide for effective induction of germ cell apoptosis in mice. Combined treatment with continuous release of acyline (3 mg/kg BW/day) with flutamide (in the form of sc pellets of 25 mg) resulted in almost the same level of suppression of spermatogenesis, as judged by testis weight and by germ cell apoptotic index, in 2 weeks as that reported for rats after treatment with 1.25 mg/kg BW Nal-Glu GnRH-A for the same time period. Within the study paradigm, the maximum suppression of spermatogenesis occurred after a single sc injection of high (20 mg/kg BW) dose of acyline with flutamide. The combined treatment resulted in complete absence of elongated spermatids. Germ cell counts at stages VII-VIII showed a significant (P < 0.05) reduction in the number of preleptotene (27.1%) and pachytene spermatocytes (81.9%), and round spermatids (96.6%) in acyline + flutamide group in comparison with controls. In fact, treatment with a single high (20 mg/kg BW) dose of acyline combined with flutamide in mice achieved same or greater level of suppression, measured by germ cell counts at stages VII-VIII, in two weeks when compared with those reported after daily treatment with Nal-Glu GnRH-A for 4 weeks in rats. Both plasma and testicular T levels were markedly suppressed after administration of acyline alone either by miniosmotic pump or by a single sc injection. Addition of flutamide to acyline had no discernible effect on plasma or intratesticular T levels when compared with acyline alone. These results demonstrate that optimum suppression of spermatogenesis through increased germ cell death is only possible in mice if total abolition of androgen action is achieved and further emphasize the usefulness of acyline + flutamide treated mice as a suitable model system to study hormonal regulation of testicular germ cell apoptosis.