α-乙酰乳酸脱羧酶的基因克隆及在大肠杆菌和酿酒酵母中的表达

以ｐＵＣ１９质粒为载体,以Ｅ．ｃｏｌｉ ＪＭ１０９为受体,构建了含α－乙酰乳酸脱羧酶（α－ＡＬＤＣ）基因的地衣芽孢杆菌Ｂａｃｉｌｌｕｓ ｌｉｃｈｅｎｉｆｏｒｍｉｓ ＡＳ１０１０６的基因文库,得到４８００个重组转化子中均含有４～１０ｋｂ的外源插入ＤＮＡ片段,从基因文库中筛选到６个阳性克隆,对其中１个克隆的α－乙酰乳酸脱羧酶基因片段进行亚克隆分析表明,该α－乙酰乳酸脱羧酶基因位于１．６ｋｂ的ＢａｍＨⅠ     

醋酸杆菌α-乙酰乳酸脱羧酶基因克隆、表达及在啤酒发酵中的作用

根据α－乙酰乳酸脱羧酶（α－ａｃｅｔｏｌａｃｔａｔｅｄｅｃａｒｂｏｘｙｌａｓｅ,α－ＡＬＤＣ）基因的碱基序列,用ＰＣＲ方法从醋酸杆菌（Ａｃｅｔｏｂａｃｔｅｒａｃｅｔｉ）中克隆出了０．９９ｋｂ的ＤＮＡ片段,经ＤＮＡ测序后证明该片段是α－乙酰乳酸脱羧酶基因．将该基因重组到质粒ｐＢＶ２２０中,转化大肠杆菌,筛选获得具有α－ＡＬＤＣ活性的重组子菌株．酶活检测表明,重组子细胞表达的α－ＡＬＤＣ活性是供体菌的１１００倍．将得到的α－ＡＬＤＣ粗提后用于啤酒发酵试验,降低了啤酒中双乙酰的含量     

产气肠杆菌α—乙酰乳酸脱羧酶基因的克隆和表达及影响重组酶活性？…

用ＰＣＲ方法从产气肠杆菌（Ｅｎｔｅｒｏｂａｃｔｅｒ ａｅｒｏｇｅｎｅｓ）中克隆出０．９ｋｂ的ＤＮＡ片段,经ＤＮＡ测序证明是α－乙酰乳酸脱羧酶（α－ａｃｅｔｏｌａｃｔａｔｅ ｄｅｃａｒｂｏｘｙｌａｓｅ,α－ＡＬＤＣ）基因。将α－ＡＬＤＣ基因重组到质粒,ｐＢＶ２２０后,转化大肠杆菌,经筛选获得的高效表达的重组子菌株。     

超高温古生菌强烈炽热球菌(Pyrococcus furiosus)的生理代谢

本文述及Ｐｙｒｏｃｏｃｃｕｓ ｆｕｒｉｏｓｕｓ的丙酮酸代谢、麦芽糖发酵（高温糖酵解途径）、由丙酮酸糖原异生途径、还原性末端产物－－Ｌ－丙氨酸的形成和钨对代谢类型的影响等。     

甲壳质脱乙酰基酶的研究概况及进展

甲壳质脱乙酰基酶（ｃｈｉｔｉｎｄｅａｃｅｔｙｌａｓｅ）最初是从真菌毛霉（Ｍｕｃｏｒ．ｒｏｕｘｉ）分离纯化的一种乙酰基转移酶。这种酶可以催化脱去甲壳质分子中Ｎ－乙酰葡糖胺链上的乙酰基,而使之变成壳多糖［１］。除几种真菌外,在昆虫中也发现了这种酶的存在［２］。真菌的甲壳质脱乙酰基酶主要参与真菌细胞壁的形成［３］,还与真菌自溶的过程中的细胞壁裂解有关［４］。最近又发现它参与植物和病原微生物的相互作...     

乳酸菌风味代谢物质的基因调控

乳酸菌的主要风味代谢物质包括丁二酮,乙醛以及各种氨基酸。利用基因工程和代谢工程的相关技术提高乙醛和丁二酮产量,是当前乳酸菌研究的热点之一。乙醛的代谢调控主要是针对丝氨酸羟甲基转移酶的表达进行调控,或是针对丙酮酸脱羧酶和NADH氧化酶的表达采用联合调控策略;而丁二酮的代谢调控则主要集中于乳酸脱氢酶、NADH氧化酶、α-乙酰乳酸合成酶和α-乙酰乳酸脱羧酶中任意两种关键酶基因间的联合调控,并且存在进行乳酸脱氢酶,α-乙酰乳酸合成酶和α-乙酰乳酸脱羧酶3种关键酶基因联合调控的可行性。     

[^3H]Cortisol及[^3H]Dexamethasone在大鼠肝细胞膜上的特异结合位点

本研究应用［３Ｈ］ｃｏｒｔｉｓｏｌ和［３Ｈ］ｄｅｘａｍｅｔｈａｓｏｎｅ（ＤＥＸ）两种配基,观察到大鼠肝细胞膜上存在一类糖皮质激素（ＧＣ）特异结合位点。这些位点与ＧＣ的结合具有饱和性、高亲和力及低容量。其平衡解离常数（Ｋｄ）分别为１２．８４±６．５８ｎｍｏｌ／Ｌ和４０．２７±２３．４４ｎｍｏｌ／Ｌ;最大结合容量（Ｂｍａｘ）分别为２．５７±１．８４ｐｍｏｌ／ｍｇ蛋白质与０．６４±０．１８ｐｍｏｌ／ｍｇ蛋白质（ｃｏｒｔｉｓｏｌ,ｎ＝４;ＤＥＸ,ｎ＝３;±ＳＥ）。动力学实验数据所得的Ｋｄ值与Ｓｃａｔｃｈａｒｄ分析所得的Ｋｄ值结果基本一致。［３Ｈ］ｃｏｒｔｉｓｏｌ和［３Ｈ］ＤＥＸ饱和结合实验数据用Ｓｃａｔｃｈａｒｄ作图分析,均显示为直线。Ｈｉｌｌ系数则分别为０．９８８０和０．９９９０．竞争抑制实验结果表明,ｃｏｒｔｉｓｏｌ对［３Ｈ］ｃｏｒｔｉｓｏｌ的结合位点有高度特异性竞争,比其它几种类固醇（强的松、黄体酮、ＲＵ４８６、ＤＥＸ）的竞争力至少强４０倍以上．用放射自显影技术进行研究,也提供了［３Ｈ］ｃｏｒｔｉｓｏｌ特异结合银粒位于大鼠肝细胞膜上的依据。     

肽基脯氨酰异构酶在细胞核内的功能

有许多酶具有调节转录因子的功能,例如蛋白激酶、磷酸酯酶和乙酰转移酶等。目前Ｌｅｖｅｒｓｏｎ等提出一种新的酶调节机制：肽基脯氨酰异构酶（Ｐｅｐｔｉｄｙｌ－ｐｒｏｌｙｌｉｓｏｍｅｒａｓｅｓ,ＰＰＩａｓｅ）参与调节转录因子的活性［１］。转录因子ｃ－Ｍｙｂ结...     

几株乳酸菌的分离鉴定

通过厌氧分离技术,从鸡肠道和西红柿花面分离得到五株产乳酸细菌,根据《伯杰细菌鉴定手册》（第八版）［１］鉴定ＳＢ１、ＳＢ２４５１、ＳＢ３１５１均为干酪乳杆菌（Ｌ．ｃａｓｅｉ）,Ａ、ＳＡ则可能是乳酸菌的一个新种。五株菌均为同型发酵,乳酸产量均达到９６％以上．对抗生素等药物及低ｐＨ有一定耐受性,是益生素饲料添加剂的优良菌种［２］。     

乳酸乳酸球菌基因表达调控研究进展

乳酸乳酸球菌基因表达调控研究进展还连栋（中国科学院微生物研究所,北京１０００８０）乳酸球菌属（Ｌａｃｔｏｃｏｃｃｕｓ）包括５个种,其中乳酸乳酸球菌（Ｌａｃｔｏｃｏｃｃｕｓｌａｃｔｉｓ）是最典型的一个种。乳酸乳酸球菌以前被称之为乳酸菌或Ｎ群链球菌（Ｓｔ...     

Alternative oxidase from Arum and soybean: Its stabilization during purification

Complete purification of the alternative oxidase from plant mitochondria has not been achieved successfully, because of its instability on solubilization. We report here that the addition of pyruvate to the isolation medium stabilizes the activity of the solubilized enzyme. A procedure is described for the rapid isolation and partial purification of the cyanide-insensitive alternative oxidase from both Arum maculatum and soybean cotyledon ( Glycine max ) mitochondria. The degree of purification was 16- and 74-fold for Arum and soybean enzyme, respectively. The specific activities increased from 1 300 to 20 300 nmol oxygen consumed mg⁻¹ protein min⁻¹ (using duroquinol as substrate) after purification for the Arum erizyme and from 6 to 445 nmol oxygen consumed mg⁻¹ protein min⁻¹ for the soybean enzyme. A turnover for the partially purified Arum enzyme was estimated to be 47 electrons s⁻¹.
The partially purified enzyme from both Arum and soybean cotyledon mitochondria was sensitive to alternative oxidase inhibitors such as salicylhydroxamic acid, n -propyl gallate and octyl gallate, but not to myxottriazol, KCN or antimycin A. The activity of the enzyme could be stimulated by pyruvate, but not by malate and suceinate. The stability of the purified enzyme was also dependent on the continued presence of pyruvate. In the absence of pyruvace, the enzyme activity was lost in a time-dependent manner and the ability of pyruvate to recover the activity was also irreversibly lost.     

Purification, characterisation and mutagenesis of highly expressed recombinant yeast pyruvate kinase.

Recombinant yeast pyruvate kinase has been purified from a strain of Saccharomyces cerevisiae expressing the enzyme to very high levels. Expression was from a multicopy plasmid under the control of the yeast phosphoglycerate kinase promoter. The gene was expressed in the absence of the genomically encoded pyruvate kinase, using a strain of yeast in which the pyruvate kinase gene has been disrupted by the insertion of the yeast Ura3 gene. The purification procedure minimised proteolytic artefacts and enabled the convenient purification of 15-20 mg enzyme from 11 culture. The purified enzyme was characterised by a high specific activity and by a lack of proteolytic degradation. Two active-site mutants of yeast pyruvate kinase have been produced, expressed and characterised in this system and preliminary results are described.     

Oligomeric forms of plant acetolactate synthase depend on flavin adenine dinucleotide

Acetolactate synthase (ALS, EC 4.1.3.18) has been extracted and partially purified from etiolated barley shoots (Hordeum vulgare L.). Multiple forms of this enzyme were separated by gel filtration and/or anion-exchange chromatography using fast protein liquid chromatography. It could be demonstrated that these two species are in equilibrium, which strongly depends on the structural role of flavin adenine dinucleotide and pyruvate. With 50 micromolar of flavin adenine dinucleotide in the medium most of the ALS aggregates as a high molecular weight form (M_r = 440,000), while 50 millimolar pyruvate facilitates dissociation into the smaller form (M_r = 200,000). Data are presented to show that two enzymatically active forms are not isozymes but different oligomeric species or aggregates of the basic 58-kilodalton subunit of ALS. These different ALS species exhibit little difference in feedback inhibition by valine, leucine and isoleucine or in inhibition by the sulfonylurea herbicide chlorsulfuron. Both aggregation forms show a broad pH-optimum between 6.5 and 7. Furthermore, the affinity for pyruvate and the amount of directly-formed acetoin indicate similar properties of these separated ALS forms.     

The crystal structures of Klebsiella pneumoniae acetolactate synthase with enzyme-bound cofactor and with an unusual intermediate

Acetohydroxyacid synthase (AHAS) and acetolactate synthase (ALS) are thiamine diphosphate (ThDP)-dependent enzymes that catalyze the decarboxylation of pyruvate to give a cofactor-bound hydroxyethyl group, which is transferred to a second molecule of pyruvate to give 2-acetolactate. AHAS is found in plants, fungi, and bacteria, is involved in the biosynthesis of the branched-chain amino acids, and contains non-catalytic FAD. ALS is found only in some bacteria, is a catabolic enzyme required for the butanediol fermentation, and does not contain FAD. Here we report the 2.3-A crystal structure of Klebsiella pneumoniae ALS. The overall structure is similar to AHAS except for a groove that accommodates FAD in AHAS, which is filled with amino acid side chains in ALS. The ThDP cofactor has an unusual conformation that is unprecedented among the 26 known three-dimensional structures of nine ThDP-dependent enzymes, including AHAS. This conformation suggests a novel mechanism for ALS. A second structure, at 2.0 A, is described in which the enzyme is trapped halfway through the catalytic cycle so that it contains the hydroxyethyl intermediate bound to ThDP. The cofactor has a tricyclic structure that has not been observed previously in any ThDP-dependent enzyme, although similar structures are well known for free thiamine. This structure is consistent with our proposed mechanism and probably results from an intramolecular proton transfer within a tricyclic carbanion that is the true reaction intermediate. Modeling of the second molecule of pyruvate into the active site of the enzyme with the bound intermediate is consistent with the stereochemistry and specificity of ALS.     

A valine-resistant mutant ofArabidopsis thaliana displays an acetolactate synthase with altered feedback control

A valine-resistant mutant line, VAL-2, ofArabidopsis thaliana (L.) Heynh. was identified by screening M 2 populations of ethylmethane-sulfonate-mutagenized seeds. The resistance was found to be due to a single, dominant, nuclear gene mutation. Assay of acetolactate synthase (ALS) indicated that the valine resistance in this mutant is caused by decreased sensitivity of ALS to the branched-chain amino acids, valine, leucine andisoleucine. A two fold decrease in apparentK _m value for pyruvate of the mutant ALS enzyme was detected compared with that of the wild type. The sensitivity of the ALS enzyme to sulfonylurea, imidazolinone and triazolopyrimidine herbicides was not altered in the mutant. At the plant growth level the mutant was also resistant to valine plus leucine, but was sensitive to leucine orisoleucine alone. The mutant gene,var1, maps, or is very closely linked, toCSR1, the gene encoding acetolactate synthase inArabidopsis.Abbreviations ALS acetolactate synthase - BCAA branched-chain amino acid - CS chlorsulfuron - IM imidazolinone - SU sulfonylurea - TP triazolopyrimidine We thank Dr. George W. Haughn for providing Arabidopsis lines MSU12, MSU15, MSU21, MSU22 and MSU23. This work was supported by a Research Grant from the Natural Sciences and Engineering Research Council of Canada to J.K., K.W. is grateful for a University of Saskatchewan Graduate Scholarship.     

Resolution of brewer's yeast pyruvate decarboxylase into multiple isoforms with similar subunit structure and activity using high-performance liquid chromatography.

A method for the purification of brewer's yeast pyruvate decarboxylase (EC 4.1.1.1) that resolves the enzyme into multiple active isoforms was developed. Seven activity fractions are resolved by DEAE HPLC chromatography. Among these fractions, three distinct subunit composition isoforms are apparent by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: alpha 4, a homotetrameric holoenzyme consisting of the lower mass subunit; alpha 2 beta 2, a heterotetrameric holoenzyme consisting of lower and higher mass subunits; and beta 4, a homotetrameric holoenzyme consisting of the higher mass subunit. Beta 4 is a heretofore unreported form which may represent the unproteolyzed form of the enzyme. The Km and Vmax for the alpha 4 and beta 4 isoforms are identical within the limits of experimental error, as is their behavior vis-à-vis the allosteric regulator pyruvamide. All active isoforms exist as tetramers according to gel filtration analysis under native conditions. The purification has been successfully applied to pyruvate decarboxylase isolated from two different species of yeast and therefore is likely to be of general utility for purification of this enzyme from other yeast sources. Conditions under which all three isoforms demonstrate exceptional stability, making them amenable to prolonged physicochemical studies at 4 degrees C and even at room temperature are reported.     

Formation of β-cyanoalanine and pyruvate by Acacia georginae

Pyruvate is formed on incubation of l-cysteine with acetone powder preparations of Acacia georginae but in the presence of cyanide, β-cyanoalanine is produced and pyruvate production is highly depressed. The pH optimum for pyruvate production is 8·5. In the presence of fluoride (1·5 mM), the pH profile is unchanged and in the presence of cyanide (1·5 mM), minimal pyruvate production occurs at pH 8·5. Although addition of pyridoxal phosphate had no influence on pyruvate or β-Cyanoalanine production, these processes were prevented by sodium borohydride, an inhibitor of pyridoxal enzymes. Neither l-serine nor O-acetyl-l-serine serve as alternative substrates for pyruvate production. β-Fluoroalanine was not detected on incubating fluoride with an enzyme preparation from A. georginae acetone powders.     

Effect of variation of Klebsiella pneumoniae acetolactate synthase expression on metabolic flux redistribution in Escherichia coli

Escherichia coli strains carrying the Bacillus subtilis acetolactate synthase (ALS) gene were previously shown to produce less acetate with higher ATP yields. Metabolic flux analysis was used to show that excess pyruvate was channeled into the less inhibitory product, acetoin. To further understand the role of intrinsic enzymatic properties and the effect of variations in enzyme levels in the alternation of metabolic fluxes, we constructed a chromosomal integrant of the Klebsiella pneumoniae ALS gene. The reported in vitro Michaelis-Menten constants (K(m)) for the Bacillus and the Klebsiella ALS are 13.0 mM and 8.0 mM, respectively. Furthermore, expression of the Klebsiella ALS is under the control of an inducible trp promoter system. Shake-flask experiments showed a linear induction response (the ALS activity changes from about 9 to 223 U/mg of protein when the inducer concentration [IAA] varied from 0 to 40 mg/L). Chemostat experiments showed a similar induction response. Interactions between the branched reactions catalyzed by the PFL, LDH, and the ALS enzymes at the pyruvate node were examined. The results indicate the importance of in vivo enzyme activities in the redistribution of metabolic fluxes.     

N5-(L-1-carboxyethyl)-L-ornithine:NADP+ oxidoreductase from Streptococcus lactis. Purification and partial characterization

N5-(L-1-Carboxyethyl)-L-ornithine:NADP+ oxidoreductase (EC 1.5.1.-) from Streptococcus lactis K1 has been purified 8,000-fold to homogeneity. The NADPH-dependent enzyme mediates the reductive condensation between pyruvic acid and the delta- or epsilon-amino groups of L-ornithine and L-lysine to form N5-(L-1-carboxyethyl)-L-ornithine and N6-(L-1-carboxyethyl)-L-lysine, respectively. The five-step purification procedure involves ion-exchange (DE52 and phosphocellulose P-11), gel filtration (Ultrogel AcA 44), and affinity chromatography (2',5'-ADP-Sepharose 4B). Approximately 100-200 micrograms of purified enzyme of specific activity 40 units/mg were obtained from 60 g of cells, wet weight. Anionic polyacrylamide gel electrophoresis revealed a single enzymatically active protein band, whereas three species (pI 4.8-5.1) were detected by analytical electrofocusing. The purified enzyme is active over a broad pH range of 6.5-9.0 and is stable to heating at 50 degrees C for 10 min. Substrate Km values were determined to be: NADPH, 6.6 microM; pyruvate, 150 microM; ornithine, 3.3 mM; and lysine, 18.2 mM. The oxidoreductase has a relative molecular mass (Mr = 150,000) as estimated by high pressure liquid chromatography exclusion chromatography and by polyacrylamide gradient gel electrophoresis. Conventional gel filtration indicated an Mr = 78,000, and a single protein band of Mr = 38,000 was revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is composed of identical subunits of Mr = 38,000, which may associate to yield both dimeric and tetrameric forms. Polyclonal antibody to the purified protein inhibited enzyme activity. The amino acid composition of the enzyme is reported, and the sequence of the first 37 amino acids from the NH2 terminus has been determined by stepwise Edman degradation.     

Stimulation of glucagon of in vivo phosphorylation of rat hepatic pyruvate kinase

Rat hepatic pyruvate kinase (type L) has been purified to homogeneity by a simple, rapid procedure involving DEAE-cellulose chromatography and elution from a blue Sepharose column. The enzyme was homogeneous by the criteria of sodium dodecyl sulfate disc gel electrophoresis, had a subunit molecular weight of 57,000, and a specific activity of 558 units/mg of protein at 30 degrees. In order to test whether the enzyme is phosphorylated in vivo, rats were injected with radioactive inorganic phosphate. Incorporation into pyruvate kinase was determined after purification of the enzyme to homogeneity as well as after specific immunoprecipitation of the enzyme from partially purified preparations. Sodium dodecyl sulfate disc gel electrophoresis revealed that 32P was incorporated into the enzyme in both cases. Glucagon administration in vivo resulted in a 200 to 300% increase in the incorporation of 32P into the enzyme which was correlated with an inhibition of enzyme activity and an elevation of hepatic levels of cyclic AMP. These results represent the first demonstration of in vivo phosphorylation of a hepatic glycolytic enzyme and strongly support the hypothesis that glucagon regulates pyruvate kinase activity, at least in part, by a phosphorylation mechanism.