Research Progress and Industrial Application of Leucine Dehydrogenase

微生物脱氢酶催化羰基不对称还原制备光学纯氨基酸及其衍生物具有非常大的优势.亮氨酸脱氢酶能选择性地催化α-酮酸,氨化还原得到α-氨基酸及其衍生物.本文综述了亮氨酸脱氢酶的来源,理化性质,底物特异性,酶基因工程菌构建等方面的内容及研究进展.从辅酶再生策略,酶膜反应器两方面讨论了其工业化应用,并展望了今后的发展前景.     

蜡样芽孢杆菌亮氨酸脱氢酶基因在大肠杆菌中的表达及重组酶性质

本研究采用PCR技术从蜡样芽孢杆菌Bacillus cereus基因组DNA中克隆出亮氨酸脱氢酶基因,构建重组表达质粒p ET28α(+)-ldh,实现在大肠杆菌中的高效表达,并分析重组亮氨酸脱氢酶的酶学性质。结果表明,从Bacillus cereus成功克隆的亮氨酸脱氢酶编码基因约为1 000 bp,表达的重组亮氨酸脱氢酶相对分子质量约为40 k Da。酶学研究结果表明:该酶的最适反应温度为37℃,其热稳定性好,30℃的半衰期长达330 h;最适反应p H为9.5;在p H 7.0~8.0的缓冲液中保存24 h后仍保持原有酶活力的80%以上;金属离子Fe2+对该酶具有明显的促进作用,而EDTA强烈抑制亮氨酸脱氢酶的活性。动力学分析结果表明该酶对底物NADH催化的Km和Vmax分别为0.635 mmol/L和1.54μmol/(L·min)。亮氨酸脱氢酶基因在大肠杆菌中的成功表达为手性氨基酸的生物合成提供了可能。     

亮氨酸脱氢酶与葡萄糖脱氢酶高效共表达制备L-叔亮氨酸北大核心CSCD

【目的】通过不同双基因共表达策略对亮氨酸脱氢酶和葡萄糖脱氢酶基因在大肠杆菌中表达影响的研究,获得具有高辅酶再生效率的双酶共表达重组生物催化剂,实现L-叔亮氨酸"一锅法"高效不对称合成。【方法】以来自于蜡状芽孢杆菌(Bacillus cereus)的亮氨酸脱氢酶(LDH)和来自芽孢菌属(Bacillus sp.)的葡萄糖脱氢酶(GDH)为模板,考察单质粒共表达,双质粒共表达和融合表达等3种共表达策略对重组细胞中亮氨酸脱氢酶和葡萄糖脱氢酶活的影响,比较不同酶活比例和不同催化剂形式对三甲基丙酮酸不对称还原制备L-叔亮氨酸效率的影响。【结果】研究发现不同共表达策略对亮氨酸脱氢酶和葡萄糖脱氢酶的影响存在明显差异。亮氨酸脱氢酶在不同策略下均能够正常表达,而葡萄糖脱氢酶在融合表达时没有活力,当C端含有组氨酸标签时,表达蛋白活性低。通过表达优化,获得3株亮氨酸脱氢酶和葡萄糖脱氢酶高效表达且具有不同酶活比例的重组菌。比较粗酶液和全细胞形式下的催化效率,发现酶活比例及催化剂形式对不对称还原反应效率具有重要影响。确定单质粒串联表达C端不含His标签重组菌E.coli BL21/p ET28a-L-SD-AS-G为最佳催化剂,以粗酶液进行转化时,完全转化0.5 mol/L底物所需菌体量为15 g/L,辅酶量为0.1 mmol/L。【结论】采用单质粒共表达策略,成功构建出1株具有较高亮氨酸脱氢酶和葡萄糖脱氢酶活性的重组菌,实现高效催化TMP合成L-Tle。     

内消旋-二氨基庚二酸脱氢酶不对称合成非天然手性D-氨基酸的研究进展

内消旋-二氨基庚二酸脱氢酶不对称合成非天然的手性D-氨基酸是目前生物催化领域的研究热点。内消旋-二氨基庚二酸脱氢酶具有优良的立体选择性,利用其进行酶催化不对称合成光学纯的手性D-氨基酸,被广泛用于医药、食品、化妆品、精细化学品等领域。为了促进生物催化法在合成手性D-氨基酸方向的进一步发展,本文对内消旋-二氨基庚二酸脱氢酶催化合成D-氨基酸的现状进行了综述。重点介绍了Corynebacterium glutamicum、Ureibacillus thermosphaericus、Symbiobacterium thermophilum来源的内消旋-二氨基庚二酸脱氢酶在新酶的挖掘、催化性能、晶体结构解析、分子改造、功能与催化机制、合成D-氨基酸新途径等方面的研究进展,并对内消旋-二氨基庚二酸脱氢酶的未来研究方向及策略进行了展望。本综述将进一步加深人们对内消旋-二氨基庚二酸脱氢酶的认识,也为具有挑战性的生物合成任务提供信息借鉴。     

利用巨大芽孢杆菌酶系合成L-丙氨酸和L-缬氨酸

本文研究了利用巨大芽孢杆菌ATCC_(39118)酶系合成氨基酸,同时也研究了丙氨酸脱氢酶、缬氨酸脱氢酶及葡萄糖脱氢酶的提纯工艺。所获得的AlaDH、ValDHc和GlcDH的比活性分别为11.2u/mg,7.8u/mg和23.0u/mg。为了进一步探讨由α-酮酸酶法转化成氨基酸的最适条件,我们对以上三种酶的主要性质,包括稳定性,最适pH、动力学常数、底物专一性及底物和产物对酶的抑制作用等进行了测定。同时用粗酶提取液和纯酶进行了由丙酮酸合成L-丙氨酸,由α-酮异戍酸合成L-缬氨酸的批量实验,在转化中葡萄糖脱氢酶作为NADH的再生酶。结果粗酶提取液催化L-丙氨酸产量的克分子转化率为80％,而纯酶催化的克分子转化率增加到92％。L-缬氨酸产量的克分子转化率也类似(93％)。     

用固定化的多酶系统和葡聚糖-NAD~+的合成单元制备必需的L-支链氨基酸

我们制备的合成单元,每种都含有亮氨酸脱氢酶,尿素酶,可溶性的葡聚糖和下列辅酶再生脱氢酶:葡萄糖脱氢酶,酵母酒精脱氢酶、苹果酸脱氢酶或乳酸脱氢酶。合成细胞被填充在一个小柱里,随着葡聚糖-NADH的产生,L亮氨酸,L缬氨酸和L异亮氨酸相继产生。得率的最大值依赖于所用的辅酶产生系统。对于D葡萄糖和葡萄糖脱氢酶系统,得率为83～93％;对于乙醇和酵母酒精脱氢酶系统,得率为90％;对于L-苹果酸和苹果酸脱氢酶系统,得率为45～55％;对于L乳酸和乳酸脱氢酶系统,得率为64～78％。本文还进行了动力学实验,表观Km值如下:α-酮异己酸(KIC),0.33mM;α-酮异戍酸(KIV),0.51mM;DL α-酮-β-甲基-η-戊酮(KMV),0.58mM;尿素,3.52mM;D-葡萄糖,27.82mM;乙醇,3.89mM;L苹果酸3.02mM;L乳酸16.67mM。动力学分析表明,在亮氨酸脱氢酶催化的反应中,KIC,KIV和KMV都是竞争性抑制物,它们的抑制系数是和Km值相关的。     

亮氨酸脱氢酶C端Loop区域的理性设计及多酶级联高效合成l-2-氨基丁酸

亮氨酸脱氢酶 (Leucine dehydrogenase,LDH) 是制备l-2-氨基丁酸的关键限速酶,针对该酶的Loop区域进行改造以提高关键酶的酶活及稳定性从而高效合成l-2-氨基丁酸。通过亮氨酸脱氢酶的分子动力学模拟分析均方根涨落 (Root mean square fluctuation,RMSF) 值,对其波动非常明显的Loop区域合理设计以得到比酶活提高的截短突变体EsLDHD2,其比酶活为野生型的123.2%;此外,由于l-2-氨基丁酸制备过程中苏氨酸脱氨酶催化l-苏氨酸制备2-酮丁酸的速率过快导致多酶催化不平衡,因此双拷贝亮氨酸脱氢酶及甲酸脱氢酶以平衡多酶催化速率,构建多酶级联催化的单细胞E. coli BL21/pACYCDuet-RM,其摩尔转化率相较于E. coli BL21/pACYCDuet-RO提高74.6%;对菌株E. coli BL21/pACYCDuet-RM的全细胞转化条件进行优化,其最适pH、温度、底物浓度分别为7.5、35 ℃和80 g/L,此时摩尔转化率大于99%;在1 L转化体系和最适转化条件下分批加入l-苏氨酸80 g和40 g,l-2-氨基丁酸的产量达97.2 g。总之,该策略为l-2-氨基丁酸的制备提供了绿色、高效的合成方法,具有工业化制备药物前体的巨大潜力。     

α-酮戊二酸与动脉粥样硬化的关系研究进展

α-酮戊二酸(α-ketoglutarate,α-KG)是戊二酸带酮基的衍生物中的一种,是三羧酸循环中重要的代谢中间产物,通过异柠檬酸脱氢酶(IDH)催化异柠檬酸氧化脱羧和谷氨酸脱氢酶催化谷氨酸氧化脱氨产生,是连接细胞内碳-氮代谢的关键节点。动脉粥样硬化(atherosclerosis,As)是一种慢性进行性疾病,病因复杂,且容易引发多种心脑血管疾病。本文从血管内皮细胞功能、自噬、DNA甲基化修饰、能量代谢、血管衰老等方面探讨α-KG与As之间的关系及其调控机制。     

定点突变改变近平滑假丝酵母SCRⅡ催化苯乙酮衍生物底物谱

【目的】通过定点突变技术,改变近平滑假丝酵母短链羰基还原酶Ⅱ(SCRⅡ)催化苯乙酮衍生物的功能,为数种手性芳香醇的生产提供一种高效、安全的新型制备方法。【方法】通过氨基酸序列和蛋白结构比对的方法,选择SCRⅡ的底物结合域中关键氨基酸位点E228实施突变,构建相应的突变株Escherichia coliBL21/pET28a-E228S;以苯乙酮衍生物为底物,对突变株的酶活和生物转化功能进行了分析。【结果】酶活测定结果表明:突变株E.coli BL21/pET28a-E228S催化原始底物2-羟基苯乙酮的酶活仅为原始酶活的25%左右;而催化苯乙酮、4’-甲基苯乙酮、4’-氯苯乙酮的酶活是突变前的7-20倍。突变株E.coli BL21/pET28a-E228S生物转化2-羟基苯乙酮,获得产物(S)-苯基乙二醇的得率不超过10%,而以苯乙酮、4’-甲基苯乙酮、4’-氯苯乙酮为底物时,生物转化产物光学纯度维持在99%,得率高达80%以上。【结论】对底物结合域中的关键氨基酸实施突变,提高了SCRⅡ催化苯乙酮衍生物的底物广谱性,拓展了该酶的生物功能,为理性改造短链羰基还原酶的不对称还原催化功能和手性芳香醇的制备提供了新型途径。     

氨基酸脱氢酶的催化机理、分子改造及合成应用

氨基酸脱氢酶催化可逆的氨基酸氧化脱氨和酮酸的不对称还原胺化反应,热力学上反应平衡倾向于生成氨基酸方向,从原子经济学和对环境影响的角度来看,是具有极大优势的氨基酸合成方法之一。本文将主要阐述近年来在?-氨基酸脱氢酶催化机理、分子改造和合成应用方面的研究进展。     

黑斑蛙早期胚胎发育过程中乳酸脱氨酶同工酶的初步研究

本文采用聚丙烯酰胺凝胶电泳方法,对河南新乡地区黑班蛙（Rananigromaculata）早期胚胎发育过程中（受精后0－324h）乳酸脱氢酶（LDH）同工酶进行了研究。结果表明：LDH1自始至终存在,且活性一直占绝对优势;LDH5比于心跳期出现,该期以后其活性仅次于LDH1;LDH2于襄胚早期开始出现,其活性一直较弱;LDH3和LDH4均于开口期少量出现,以后前者活性一直极弱,后者则呈现一定的活性。与哺乳类及鱼类资料比较,提示两栖类黑斑蛙LDH同工酶有其独特的表达谱式。     

Lactate dehydrogenase activity and its isoenzyme spectrum in the human pancreas in the prenatal period

Lactate dehydrogenase (LDH) activity and the character of its isoenzyme distribution in pancreas of the human embryos and feti of the 5th-13th-week development were studied. It is shown that LDH activity was rather high already in early periods of the embryonic development, peaks of the enzymic activity were observed after 7-8 and 12-13 weeks. The isoenzymic LDH spectrum was characterized by the presence of four isoenzymes: LDH1, LDH2, LDH3, LDH5. Isoenzyme LDH4 was absent in the human pancreas in all the studied periods of embryonic development. The data obtained evidence for intensity of the glycolysis processes at LDH reaction level in the prenatal ontogenesis period and they reflect most probably the processes of development and differentiation ox cellular populations in the given organ.     

The development of lactate and malate dehydrogenases in the C57BL-6 mouse kidney

The development of lactate dehydrogenase (LDH; EC 1.1.1.27) and malate dehydrogenase (MDH; EC 1.1.1.37) was measured in the kidney of male and female $\text{C57BL}\text{6}$ mice from ages prenatal 16 days to 80 days. Maximum reactions rates of the enzymes were measured in vitro by following the reduction of the nicotinamide-adenine dinucleotide spectrophotometrically.Analysis of variance showed no significant sex difference for LDH and MDH. There was a significant sex difference for the ratio LDH:MDH and a significant age difference for LDH, MDH, and the ratio LDH:MDH. In the male and female, LDH activity increased from prenatal 16 days to 30 days. Malate dehydrogenase activity reached adult values at 22 days in the male and at 30 days in the female. The ratio LDH:MDH in the male decreased from prenatal 16 days to 3 days, after which the ratio continued to decline to 20 days at a less rapid rate. This general pattern was also found in the female followed by a further decline in the ratio at 50 days.The development of LDH and MDH in the $\text{C57BL}\text{6}$ mouse is tissue specific and probably parallels the development of the tissue's function. In the case of the kidney, LDH and MDH development may reflect maturation of mitochondrial function and the kidney's ability to concentrate urine.     

中国林蛙早期胚胎发育过程同工酶的研究

本文采用聚丙烯酰胺凝胶电泳方法,对中国林蛙（Rana chensinensis)早期胚胎发育过程中（受精后0－512h)乳酸脱氢酶（LDH）同工酶和酯酶（EST）同工酶进行了研究,结果表明：1．LDH同工酶在受精卵及早期胚胎发育过程中一直存在,并且在胚胎发育的不同时期差异显著,LDH1同工酶在胚胎发育的各个阶段都占绝对优势,而DH5活性从尾芽期开始增强,LDH2,LDH3,LDH4 3种同工酶的活性较;低同时也发现LDH1,LDH3,LDH4同工酶各亚带也发育阶段的特异性,2．EST同工酶在开口期才开始出现。     

Reversibility of the lactate dehydrogenase isozyme shift induced by low oxygen tension

The shift of the lactate dehydrogenase (LDH) isoenzymes in experimental animals under hypoxia from B (aerobic) to A (anaerobic) is confirmed and its reversibility after removal of the animals to room air is reported. These experiments are interpreted as further supportive evidence of a physiological adaptive role for the lactate dehydrogenase enzyme system. The slow adaptive process observed in this experimental situation presumably requires de novo enzyme synthesis, however, in the emergency of a sudden deprivation of oxygen the liberation of preformed A LDH could be a link in the rapid chain of events resulting in depressed cardiac muscle contractility which follows myocardial infarction. A recently proposed comprehensive explanation of the role of LDH isozymes is thus extended.     

Lactate dehydrogenase ontogeny,paternal gene activation,and tetramer assembly in embryos of brook trout,lake trout,and their hybrids

Measurement of lactate dehydrogenase in reciprocal hybrids of trout during development revealed that a maternal effect was involved in the regulation of enzyme levels until resorption of the yolk sac was completed. Malate dehydrogenase specific activities were the same in these embryos and larvae. The more negatively charged B subunits of LDH predominated during early stages of embryogenesis in lake trout and brook trout with an increase in synthesis of A subunits evident as development progressed. Activation of the paternal A gene in reciprocal hybrids occurred at a relatively late stage with the LDH subunit specific to the retina appearing after hatching. Analysis of brook trout progeny from a cross of parental types with a variant and wild-type B subunit suggested nonrandom LDH tetramer assembly which may be genetically controlled.This study was supported by National Science Foundation Grant GB-7271.     

The quantitation of bovine embryo viability using a bioluminescent assay for lactate dehydrogenase

Forty-seven bovine embryos, ranging from the four-cell to expanded blastocyst stage, with grades ranging from excellent to poor, were collected non-surgically from superovulated Holstein heifers. A viability assay based on the measurement of bioluminescent emission from the media surrounding an embryo was tested. This assay measured the activity of the enzyme lactate dehydrogenase (LDH) released into the media by the embryos. Lactate dehydrogenase has been reported to be released into media by nonviable embryos. The assay used is simple, rapid and nonsubjective, requiring approximately 5 min to complete. The LDH assay proved to be a practical method for distinguishing between nonviable and viable embryos. Viability was determined by the observation of embryo development in culture following the LDH assay. The activity of LDH in the media of nonviable embryos was consistently higher than for viable embryos (P<0.001), with no overlap between the two groups. Thus, the LDH assay was shown to be a reliable test of embryo viability.     

X chromosome expression during oogenesis in the mouse.

The activities of glucose-6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase (LDH) have been assayed in mouse oocytes at several stages of follicle development isolated from XX and XO female mice. Throughout the entire growth period the activity of G6PD was proportional to the number of X chromosomes present in the oocyte, whereas no difference in LDH activity was detected between XX and XO oocytes. It is concluded, therefore, that both X chromosomes are functional throughout oogenesis.     

体外培养骨髓巨核细胞脱氢酶活性的细胞化学观察

本文采用液体培养体系结合酶细胞化学方法,对体外培养不同发育阶段的小鼠肾髓巨核细胞乳酸脱氢酶、苹果酸脱氢酶、谷氨酸脱氢酶的活性变化进行了动态观察。在9天培养期间,巨核细胞的增殖数在5—7天达到高峰,并随时间有不同程度分化。对培养3、5、7、9天的巨核细胞进行酶细胞化学研究,结果表明,巨核细胞在发育成熟前,三种酶活性均有增高。提示巨核细胞在分化过程中,糖酵解及三羧酸循环代谢均有增强。     

Refolding of lactate dehydrogenase by zeolite beta

We used zeolite beta as an adsorbing matrix to refold recombinant lactate dehydrogenase (LDH) protein collected as an insoluble aggregate from a bacterial expression system. The adsorption isotherm revealed that 1 g of zeolite adsorbed 200 mg of denatured LDH solubilized with a buffer containing 6 M of guanidine hydrochloride. The pH of the buffer had little effect on the adsorption, but this property was abolished by preincubation of the zeolite with polyethylene glycol (PEG) in a weight ratio of 1:10. These data suggest that the adsorption of LDH depends on the hydrophobicity of the zeolite surface, and that the adsorption of PEG to zeolite is sufficient to release LDH from its surface. LDH was thus released by refolding buffer containing PEG and arginine, and soluble LDH was obtained in its active enzymatic form. The addition of arginine dramatically increased the yield of LDH in a dose‐dependent manner. The overall refolding efficiency was optimized to 35%. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009