我国自行构建的L-苯丙氨酸工程菌株发酵产酸率达国际先进水平

福建省发展和改革委员会于2009年1月17日对福建省麦丹生物集团有限公司和福建沙县侨丹实业有限公司共同承担的"L-苯丙氨酸高产基因工程菌的构建和应用"项目进行科技成果鉴定,由中国工程院院士、浙江工业大学原校长沈寅初教授和清华大学曹竹安教授等专家组成的鉴定委员会,对该项目给予很高的评价,认为所构建的工程菌株ES-4573-2其产酸率达国际先进水平,并足以形成具有自主知识产权的科技成果,使我国L-苯丙氨酸生产进入国际先进行列.L-苯丙氨酸是人体和动物不能在体内自行生物合成的必需氨基酸之一,广泛应用于食品、饲料、医药和日用化工等领域.尤其是低热量、高甜度的二肽甜昧剂-阿可斯巴甜的主要原料,市场需求量大.为进一步提高L-苯丙氨酸产酶产酸水平,增强我国L-苯丙氨酸在国际市场中的竞争力,该项目在两位国家级专家施巧琴教授和吴松刚教授的直接领导下,该课题组在国内外首次应用分子定向协同共进化技术对L-苯丙氨酸代谢途径关键酶基因进行整体改造,构建成突变库,筛选出具有高产酸水平的突变株,取得了显著成效.采用上述技术,工程菌株ES-4573-2经小试、中试进入试产,在采间歇式补料及发酵优化控制的基础上,产酸率较酪氨酸缺陷型宿主菌株提高了292％,取得了重大突破.目前,该工程菌株已在大生产中使用,生产性能稳定,对提高L-苯丙氨酸的产量和质量起了重要的作用,为我国发酵工业的发展作出了贡献.     

酶法生产L-高苯丙氨酸的研究进展

L-高苯丙氨酸(L-homophenylalanine,L-HPA)作为一种重要的非天然氨基酸,是合成治疗高血压的普利类药物等的关键中间体,具有广阔的市场前景。目前L-高苯丙氨酸的合成主要依赖于化学法,但化学合成L-高苯丙氨酸具有原料昂贵、步骤繁琐和污染严重等缺点,限制了广泛应用。因此,国内外研究者对L-高苯丙氨酸的酶法生产进行了深入的研究。本文就目前酶法合成L-高苯丙氨酸的工艺,包括脱氢酶法、转氨酶法、海因酶法和脱羧酶法的研究进展进行了综述,为酶法合成L-高苯丙氨酸提供一定的借鉴,为最终实现L-高苯丙氨酸的酶法工业化生产奠定基础。     

L-苯丙氨酸制备的研究进展

笔者对化学和生物合成L-苯丙氨酸的研究进展进行了综述。首先简述L-苯丙氨酸化学合成和酶促合成方法;然后综述微生物发酵法制备L-苯丙氨酸的研究进展,简单介绍大肠杆菌和谷氨酸棒杆菌发酵法生成L-苯丙氨酸的代谢机制,同时,对发酵法合成L-苯丙氨酸的各项应用研究展开重点介绍;最后,对L-苯丙氨酸在生物领域的发展进行了展望。     

L-苯丙氨酸生产的代谢工程研究

L-苯丙氨酸是一种重要的食品和医药中间体。工业上一般采用酶法和发酵法来生产L-苯丙氨酸。代谢工程的兴起,使得更加理性的改造菌株成为可能,这更加促进了发酵法的广泛应用。主要介绍了代谢工程在L-苯丙氨酸生产菌的改造中的应用情况,其中涉及苯丙氨酸生物合成途径中相关基因及其酶的调控、中央代谢途径的改造和芳香族氨基酸生物合成支路的修饰。并探讨了将来的发展前景。     

固定化酶法拆分制备L-苯丙氨酸的工艺研究

<正> 苯丙氨酸是人体必需的八种氨基酸之一,在人体内有生理作用的是L-型的,故合成制得D L-苯丙氨酸必须进行拆分。六十年代末,日、美等国用固定化氨基酰化酶拆分制备L-氨基酸已应用于工业生产,由于固定化酶法拆分具有高度专一性,可反复使用,反应条件温和,酶不残留在产物中,产物易纯化、收率较高、无三废等优点,这也引起我国许多科研单     

我国小品种氨基酸研究与生产取得重大突破

<正>在我国生物发酵产业中,氨基酸生产占重要地位,L-谷氨酸、L-赖氨酸及L-精氨酸等大宗氨基酸产品的生产规模及生产水平已达到世界先进水平,而小品种氨基酸诸如:L-苯丙氨酸、L-色氨酸、L-缬氨酸及L-异亮氨酸等无论在生产规模还是在生产水平上,均与国外有较大的差距。近年来,福建省麦丹生物集团有限公司及其福州研究院在中国生物发酵产业协会石维忱理事长的关心和指导下,在国家     

酪氨酸流加方式对重组大肠杆菌发酵生产L-苯丙氨酸的影响

研究了流加浓度对酪氨酸重组大肠杆菌Escherichia coli BR-165(pAP-B03)发酵生产L-苯丙氨酸的影响.结果表明,诱导后L-酪氨酸流加加速了菌体的生长,提高了生产强度,缩短了发酵周期.在流加浓度为75 mg/h时,最大菌体干重达到了40.13 g/L(对照11.48 g/L),生产周期缩短到30 h(对照48 h),生产强度达到1.409 g/h/L(对照0.876 g/h/L).但是L-酪氨酸的流加对L-苯丙氨酸的最终产量没有明显的影响,因此可认为流加酪氨酸是减少发酵时间并提高生产强度的有效方法.本研究获得的酪氨酸流加方式对L-苯丙氨酸的工业化生产具有一定的指导意义.     

D-氨基酰化酶拆分D,L-苯丙氨酸制备D-苯丙氨酸

进行了以D,L-苯丙氨酸为原料经D-氨基酰化酶制备D-苯丙氨酸的研究。乙酰-D,L-苯丙氨酸浓度为0.5mol.L-1,给酶量为3×104U.L-1时,24 h拆分率可达到97%。采用阳离子交换树脂进行了拆分液中的D-苯丙氨酸的分离,D-苯丙氨酸的收率为95.4%。采用醋酸酐作为催化剂,在145℃的条件下,乙酰-L-苯丙氨酸可以消旋成乙酰-D,L-苯丙氨酸继续拆分。     

用生物反应器生产L-氨基酸

现已有几种L-氨基酸是用生物反应器中的酶或全细胞生产的。最早采用这种技术的例子之一是日本L-天冬氨酸的生产。最近放大了由肉桂酸和氨生产L-苯丙氨酸的生物反应器法,产物效价达60克/升以上,原料转化率约为90％。     

由D、L-苯丙乳酸盐消旋混合物酶法生产L-苯丙氨酸

本文研究了在酶膜反应器中由D.L苯丙乳酸盐消旋混合物生产L-苯丙氨酸。反应的第一步消旋混合物在羟基异己酸脱氢酶作用下,脱氢生成本丙酮酸;第二步苯丙酮酸在苯丙氨酸脱氨酶作用下,还原胺化成L-苯丙氨酸。从化学计量来看,上述两步反应均需要辅酶参加。第一步需要NAD,第二步需要NADH,在此反应中,辅酶可以直接再生而起到催化作用。由于铺酶被共价结合到聚乙二醇—2000上,所以铺酶就类似于其它三种酶一样保留在反应器中。为了由D.L-本丙乳酸盐能不断连续地生产L-苯丙氨酸,我们设计了反应动力学及反应器体系模型。按照上述模型,我们可以计算出反应器中三种酶的最适比率,辅酶的最适浓度,以及投料中苯丙酮酸的最适浓度。采用这一程序,我们用底物浓度为50mM D.L-苯芮乳酸盐,可获得28克/升L-苯丙氨酸。     

Monitoring of phenylketonuria: a colorimetric method for the determination of plasma phenylalanine using L-phenylalanine dehydrogenase

A simple, rapid, accurate, and precise colorimetric assay for the determination of L-phenylalanine in plasma samples using L-phenylalanine dehydrogenase [L-phenylalanine:NAD+-oxidoreductase (deaminating)] from Rhodococcus sp. M 4 is described. The enzyme catalyzes the NAD-dependent oxidative deamination of L-phenylalanine. However, the equilibrium of reaction favors L-phenylalanine formation. By stoichiometric coupling of this reaction with diaphorase/iodonitro tetrazolium chloride (INT) the formed NADH converts INT to a formazan whereby the reaction is displaced in favor of phenylpyruvate. Using a kinetic approach the increase in absorbance at 492 nm shows linearity over more than 30 min. Deproteinized standard solutions of L-phenylalanine in the range from 30 to 1200 mumol/liter show a linearity between the dAformazan/30 min and the substrate concentration. In phenylketonuria (PKU) plasma samples no interferences caused by L-tyrosine or phenylpyruvic acid are seen. Applicability is demonstrated by comparative determination of plasma L-phenylalanine of treated PKU patients by the colorimetric method and automated amino acid analysis.     

Enzymatic determination of L-phenylalanine and phenylpyruvate with L-phenylalanine dehydrogenase

An enzymatic method is described for the determination of L-phenylalanine or phenylpyruvate using L-phenylalanine dehydrogenase. The enzyme catalyzes the NAD-dependent oxidative deamination of L-phenylalanine or the reductive amination of the 2-oxoacid, respectively. The stoichiometric coupling of the coenzyme allows a direct spectrophotometric assay of the substrate concentration. The equilibrium of the reaction favors L-phenylalanine formation; however, by measuring initial reaction velocities, the enzyme can be used for L-phenylalanine determination, too. Standard solutions of L-phenylalanine in the range of 10-300 microM and of phenylpyruvate (5-100 microM) show a linearity between the value for dENADH/min and the substrate concentration. Besides phenylalanine, the enzyme can convert tyrosine and methionine, and their oxoacids, respectively. The Km values of these substrates are higher. The influence of tyrosine on the determination of phenylalanine was studied and appeared tolerable for certain applications.     

A liquid emulsion membrane process for the separation of amino acids

The method of using liquid emulsion membranes featuring the cation carrier D2EHPA [di-(2-ethylhexyl) phosphoric acid] for the separation of L-phenylalanine is examined. Results from experiments performed under various conditions are discussed and an optimal condition for separation is determined. The selectivity of the liquid emulsion membrane system is discussed. The effects of impurities such as sodium chloride, glucose, lactic acid, and L-tryptophan on the transport of L-phenylalanine are evaluated. It is shown that the liquid emulsion membrane system is a potential operation not only to separate L-phenylalanine but also concentrate it with great efficiency.     

Phenylalanine uptake in isolated renal brush border vesicles.

The uptake of L-phenylalanine into brush border microvilli vesicles and basolateral plasma membrane vesicles isolated from rat kidney cortex by differential centrifugation and free flow electrophoresis was investigated using filtration techniques. Brush border microvilli but not basolateral plasma membrane vesicles take up L-phenylalanine by an Na+-dependent, saturable transport system. The apparent affinity of the transport system for L-phenylalanine is 6.1 mM at 100 mM Na+ and for Na+ 13mM at 1 mM L-phenylalanine. Reduction of the Na+ concentration reduces the apparent affinity of the transport system for L-phenylalanine but does not alter the maximum velocity. In the presence of an electrochemical potential difference of Na+ across the membrane (etaNao greater than etaNai) the brush border microvilli accumulate transiently L-phenylalanine over the concentration in the incubation medium (overshoot pheomenon). This overshoot and the initial rate of uptake are markedly increased when the intravesicular space is rendered electrically more negative by membrane diffusion potentials induced by the use of highly permeant anions, of valinomycin in the presence of an outwardly directed K+ gradient and of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence of an outward-directed proton gradient. These results indicate that the entry of L-phenylalanine across the brush border membrane into the proximal tubular epithelial cells involves cotransport with Na+ and is dependent on the concentration difference of the amino acid, on the concentration difference of Na+ and on the electrical potential difference. The exit of L-phenylalanine across the basolateral plasma membranes is Na+-independent and probably involves facilitated diffusion.     

Regulation of the aromatic pathway in the cyanobacterium Synechococcus sp. strain Pcc6301 (Anacystis nidulans).

A pattern of allosteric control for aromatic biosynthesis in cyanobacteria relies upon early-pathway regulation as the major control point for the entire branched pathway. In Synechococcus sp. strain PCC6301 (Anacystis nidulans), two enzymes which form precursors for L-phenylalanine biosynthesis are subject to control by feedback inhibition. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (first pathway enzyme) is feedback inhibited by L-tyrosine, whereas prephenate dehydratase (enzyme step 9) is feedback inhibited by L-phenylalanine and allosterically activated by L-tyrosine. Mutants lacking feedback inhibition of prephenate dehydratase excreted relatively modest quantities of L-phenylalanine. In contrast, mutants deregulated in allosteric control of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase excreted large quantities of L-phenylalanine (in addition to even greater quantities of L-tyrosine). Clearly, in the latter mutants, the elevated levels of prephenate must overwhelm the inhibition of prephenate dehydratase by L-phenylalanine, an effect assisted by increased intracellular L-tyrosine, an allosteric activator. The results show that early-pathway flow regulated in vivo by 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase is the dominating influence upon metabolite flow-through to L-phenylalanine. L-Tyrosine biosynthesis exemplifies such early-pathway control even more simply, since 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase is the sole regulatory enzyme subject to end-product control by L-tyrosine.     

Shifting the biotransformation pathways of L-phenylalanine into benzaldehyde by Trametes suaveolens CBS 334.85 using HP20 resin

AIMS: The biotransformation of L-phenylalanine into benzaldehyde (bitter almond aroma) was studied in the strain Trametes suaveolens CBS 334.85. METHODS AND RESULTS: Cultures of this fungus were carried out in the absence or in the presence of HP20 resin, a highly selective adsorbent for aromatic compounds. For the identification of the main catabolic pathways of L-phenylalanine, a control medium (without L-phenylalanine) was supplemented with each of the aromatic compounds, previously detected in the culture broth, as precursors. Trametes suaveolens CBS 334.85 was shown to biosynthesize benzyl and p-hydroxybenzyl derivatives, particularly benzaldehyde, and large amounts of 3-phenyl-1-propanol, benzyl and p-hydroxybenzyl alcohols as the products of both cinnamate and phenylpyruvate pathways. CONCLUSION: The addition of HP20 resin, made it possible to direct the catabolism of L- phenylalanine to benzaldehyde, the desired target compound, and to trap it before its transformation into benzyl alcohol. In these conditions, benzaldehyde production was increased 21-fold, from 33 to 710 mg l-1 corresponding to a molar yield of 31%. SIGNIFICANCE AND IMPACT OF THE STUDY: These results showed the good potential of Trametes suaveolens as a biotechnological agent to synthesize natural benzaldehyde which is one of the most important aromatic aldehydes used in the flavour industry.     

Inhibition of intestinal absorption of phenylalanine by phenylalaninol.

Plasma phenylalanine and tyrosine levels in rats which had been orally administered L-phenylalaninol and L-phenylalanine were determined. Since these amino acid levels in rats administered L-phenylalanine solution containing L-phenylalaninol were significantly lower than those in rats administered L-phenylalanine alone. L-phenylalaninol appears to inhibit the intestinal absorption of L-phenylalanine. This effect was more potent than that of cycloleucine. L-phenylalaninol inhibited the phenylalanine transport of everted sacs. The Km value of L-phenylalanine was 3.44 X 10(-3) M and the Ki value of L-phenylalaninol was 7.69 M 10(-3) M from Lineweaver-Burk plots. From these two curves, it appeared that L-phenylalaninol may competitively inhibit the intestinal transport of L-phenylalanine. The effects of L-phenylalanine, L-phenylalaninol and cycloleucine on the urinary excretions of Na+ and K+ in rats were also examined. Potassium excretion which increased on oral administration of L-phenylalanine, was suppressed by the administration of L-phenylalaninol but not administration of cycloleucine. L-phenylalaninol alone enhanced Na+ excretion in urine. These results confirmed that L-phenylalaninol shows inhibitory effects as potent as those of cycloleucine on the intestinal absorption of L-phenylalanine.     

Purification, characterization and induction of L-phenylalanine ammonia-lyase in Phaseolus vulgaris

The enzyme L-phenylalanine ammonia-lyase was purified from leaves of Phaseolus vulgaris by Sephacryl S-200 gel filtration and Sepharose-4-B--succinyl-aminoethyl-L-phenylalanine affinity chromatography. L-Phenylalanine ammonia-lyase was specifically eluted from the affinity matrix with its substrate L-phenylalanine at 20-25 degrees C. The purified enzyme was shown to be homogeneous by gel electrophoresis both in presence and absence of SDS. Its Mr, determined by gel filtration and non-denaturing gel electrophoresis, was 320,000 +/- 9000 and 330,000 +/- 4000 respectively. After SDS electrophoresis only one band of Mr 83,000 +/- 4000 was detected, indicating that the enzyme is an oligomer containing four subunits. The pH optimum of enzyme activity was 8.8-9.2. Ampholyte isoelectrofocusing in polyacrylamide demonstrated the presence of a single charged species at pH 4.2. The homogeneous enzyme catalyzed the deamination of L-phenylalanine to trans-cinnamate but did not catalyze the transamination of L-phenylalanine to L-phenylpyruvate. The enzyme showed Km 1.25 mM for L-phenylalanine. Antibodies to homogeneous L-phenylalanine ammonia-lyase recognised specific epitopes on L-phenylalanine aminotransferase as demonstrated by immunoaffinity purification and immunoblotting. The induction of L-phenylalanine ammonia-lyase activity during phaseollin biosynthesis in the Phaseolus vulgaris--Colletotrichum lindemuthianum interaction was regulated by an increase in enzyme concentration resulting from an increase in de novo synthesis of L-phenylalanine ammonia-lyase protein.     

Structural-based screening of L-phenylglycine aminotransferase using L-phenylalanine as the optimal amino donor: Recycling of L-phenylalanine to produce L-phenylglycine

The aproteinogenic amino acid, L-phenylglycine, is an important side chain building block for some drugs. It would be of great commercial and environmental value to biocatalyse L-phenylalanine to L-phenylglycine, and thus replace the organic synthesis method. To produce L-phenylglycine from L-phenylalanine, an L-phenylglycine aminotransferase was screened and characterized. HpgT_AO showed high homology to α-aminoadipate aminotransferase. The L-phenylalanine binding site was near the residues S26, R401, N201, and G46 in HpgT_AO, and L-phenylalanine formed a hydrogen bond with Asn20, which was similar to the substrate binding mechanism of α-aminoadipate aminotransferase. HpgT_AO showed increased activity in alkalescent environment below 40°C. The kinetic analysis showed that L-phenylalanine had the highest affinity to HpgT_AO, which ensured the recycle biosynthesis of Lphenylglycine from L-phenylalanine. To date, it was the only aminotransferase using L-phenylalanine as an optimal amino donor. The L-phenylglycine biocatalysis operon was also constructed by co-expressing the hmaS, hmo and hpgT by a single plasmid. The first in vitro conversion of L-phenylalanine to L-phenylglycine was achieved by directly using the L-phenylalanine fermentation broth as the raw material.