添加胰蛋白酶促进Streptomyces hygroscopicus CCTCC M203062产谷氨酰胺转胺酶

研究了添加胰蛋白酶对Streptomyces hygroscopicus CCTCC M203062合成谷氨酰胺转胺酶的影响。结果表明,添加胰蛋白酶可以提高发酵过程中谷氨酰胺转胺酶的酶活。摇瓶培养中,在发酵起始时添加200U/ml的胰蛋白酶,谷氨酰胺转胺酶的酶活最高达到了6.61U/ml,比对照提高了27.1%。初步研究表明,添加胰蛋白酶可以直接切割发酵过程中产生的酶原,使其被快速地转化为成熟酶,因此推测胰蛋白酶提高谷氨酰胺转胺酶酶活的原因是解除了酶原的产物抑制作用,产生更多的酶原,从而促进了产酶。     

添加CTAB促进吸水链霉菌产谷氨酰胺转胺酶

研究了添加十六烷基三甲基溴化铵(CTAB)对吸水链霉菌(Streptomyces hygroscopicus)合成谷氨酰胺转胺酶的影响。结果表明,添加CTAB可以提高发酵过程中谷氨酰胺转胺酶的酶活,摇瓶培养中,CTAB的最佳添加时间和添加量分别为32h和1%,发酵终了时,谷氨酰胺转胺酶酶活最高达5.04u/mL,比对照提高了21.8%。初步研究表明,CTAB的主要作用是促使谷氨酰胺转胺酶的酶原转化为成熟酶,因此,在发酵过程中添加适当浓度的CTAB,可使酶原快速、完全地转化为成熟的MTG,解除酶原的产物抑制作用,促进了细胞产酶。     

添加CTAB促进吸水链霉菌产谷氨酰胺转胺酶

研究了添加十六烷基三甲基溴化铵（CTAB）对吸水链霉菌（Streptomyces hygroscopicus）合成谷氨酰胺转胺酶的影响。结果表明,添加CTAB可以提高发酵过程中谷氨酰胺转胺酶的酶活,摇瓶培养中,CTAB的最佳添加时间和添加量分别为32h和1%,发酵终了时,谷氨酰胺转胺酶酶活最高达5.04u/mL,比对照提高了21.8%。初步研究表明,CTAB的主要作用是促使谷氨酰胺转胺酶的酶原转化为成熟酶,因此,在发酵过程中添加适当浓度的CTAB,可使酶原快速、完全地转化为成熟的MTG,解除酶原的产物抑制作用,促进了细胞产酶。     

双酶偶联转化富马酸制备β-丙氨酸的工艺的建立与优化

酶转化法是生产β-丙氨酸的重要途径,但单一酶法转化存在底物价格较高的问题。通过构建双酶催化体系制备β-丙氨酸,即将来源于大肠杆菌的天冬氨酸酶(AspA)和来源于谷氨酸棒杆菌的L-天冬氨酸α-脱羧酶(PanD)偶联,以富马酸和氨为底物进行酶促反应合成β-丙氨酸。催化反应中AspA与PanD的最适加酶比例为1∶80,其中AspA的浓度为10μg/mL,转化温度为37℃,pH为7.0;浓度为100 mmol/L的富马酸可在8 h内被完全转化,转化率为100%,摩尔产率为90.9%,β-丙氨酸的产量为90 mmol/L,约为7 g/L;浓度为200 mmol/L的富马酸在反应8 h后,体系中β-丙氨酸的产量为126 mmol/L,约合9.8 g/L,继续延长反应时间,转化率并没有明显提高。根据该研究提出的双酶偶联转化工艺可将价格低廉的富马酸一步转化为具有高附加值的β-丙氨酸。     

连续糖化和乙醇发酵用酵母的耐热性改善法

乙醇连续糖化和发酵(SSF)工艺是将纤维素的酶法糖化和糖的连续酵母发酵在同一只容器中进行。其主要优点是节省了工艺中的容器设备以及提高纤维素水解率,后者的原因是代谢物(乙醇)的阻遏作用减轻了的缘故。要想使SSF工艺获得成功应当尽量使酶作用的最适pH,温度等与酵母发酵的相适应。但是普通酵母发酵温度往往与酶作用温度不一致,前者低,后者高。因此,提高酵母发酵温度,或者说其耐热性,是SSF工艺成功的关键。     

新一代高产甘油重组菌的构建及其初步发酵

产甘油假丝酵母(Candida glycerinogenes WL2002-5)是一株发酵生产甘油的工业化菌株。为进一步提高其产甘油能力,本研究利用前期研究中成功克隆的产甘油假丝酵母中甘油合成关键酶3-磷酸甘油脱氢酶基因CgGPD1,构建根癌农杆菌双元载体pCAM3300-zeocin-CgGPD1后,电击转化根癌农杆菌LBA4404,通过根癌农杆菌介导法(ATMT)转化产甘油假丝酵母,构建了产甘油假丝酵母重组菌。并从中筛选出一株酶活力和产甘油性能较好的产甘油假丝酵母重组菌株C.g-G8。以葡萄糖为底物摇瓶发酵96h后,重组菌C.g-G8的甘油产量比野生型菌株Candida glycerinogene提高18.06%,平均耗糖速率提高12.97%,平均酶活力提高27.55%。本研究成功利用ATMT法转化产甘油假丝酵母构建新一代高产甘油菌株。     

酶法合成生物柴油工业化研究进展

介绍了北京化工大学近年来酶法合成生物柴油工业化研究的结果。主要内容包括以下几个方面:高产脂肪酶菌株的选育、脂肪酶发酵工艺优化及放大、脂肪酶固定化方法、酶反应器放大、生物柴油分离精制及副产物甘油综合利用。该脂肪酶假丝酵母Candida sp.99-125在5 m3罐发酵活力不低于8 000 IU/mL,然后将该脂肪酶吸附固定在织物膜上并进行表面改性,用于搅拌罐式反应器生产每吨甲酯的需酶量仅为4.2 kg,产品经分离精制调质后,其各项指标完全符合德国生物柴油生产标准。副产物甘油可用于1,3-丙二醇发酵,30 L发酵罐中1,3-丙二醇的产量可达到76.1 g/L。     

信息库

1.由光滑球拟酵母从葡萄糖发酵生产丙酮酸 丙酮酸是代谢途径巾重要的有机酸。它是许多药物合成的原料和动物细胞培养的重要成分,它还是酶法合成L-色氨酸,L-酪氨酸,L-二羟苯丙氨酸等氨基酸的底物。许多酵母,如酵母属,球拟酵母属,毕赤酵母属,假丝酵母属和丝孢酵母属中,用羟基硫胺素(硫胺素类似物)的休眠细胞法都可以筛选到产丙酮酸的菌株。在初筛中,酵母属菌株产丙     

利用毕赤酵母系统直接分泌表达具有活性的谷氨酰胺转胺酶

使用异源表达系统直接分泌表达具有活性的微生物谷氨酰胺转氨酶(Microbial transglutaminase,MTG)是目前最具前景的MTG生产方法之一,但由于产量较低无法实现工业化生产.毕赤酵母是近年来发展出的高效蛋白表达系统.通过采用pro序列与成熟MTG基因共表达的策略,成功地实现了用重组毕赤酵母分泌表达具有活性的茂原链霉菌Streptomyces mobaraense MTG.进一步通过对pro序列和MTG基因拷贝数以及重组酵母培养条件的优化,最终使得MTG在1L发酵罐中高密度发酵的酶活达到7.3 U/mL,为MTG的工业化生产奠定了基础.     

构建可合成非天然辅酶的圆红冬孢酵母工程菌*

烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide,NAD)及其还原态是生物体通用的氧化还原辅酶和重要小分子,参与胞内众多代谢反应,因此调控NAD水平不仅难以选择性作用于代谢途径,还常常产生意外的生物学效应。最近研究发现利用非天然辅酶烟酰胺胞嘧啶二核苷酸(nicotinamide cytosine dinucleotide,NCD),可构建正交的氧化还原催化体系,为调控胞内代谢提供了新机遇。为实现在产油酵母圆红冬孢酵母中建立NCD介导的氧化还原代谢,采用农杆菌介导转化方法,在基因组整合表达密码子优化的NCD合酶(NcdS)编码基因NCDS,获得系列有效表达NcdS的工程菌株。酶偶联法分析发现,工程菌细胞裂解液NcdS酶活达8.1×10^-3 U/OD_{600 nm}。通过高效液相色谱法(HPLC)和超高分辨率质谱检测,确定细胞裂解液可催化合成NCD。在培养基内补加5.0 mmol/L烟酰胺核糖后,工程菌胞内合成NCD达41.6 μmol/L。对工程菌进行发酵和油脂提取,发现胞内表达NCD合酶未导致细胞产油性能降低,后续可通过表达其他NCD偏好性酶,有望在圆红冬孢酵母中建立受NCD调控的油脂合成代谢体系。     

Glutamine Production by Coupling with Energy Transfer: Employing Yeast Cells and Gluconobacter Glutamine Synthetase

A glutamine production process was established by combining alcoholic fermentation of baker's yeast cells with glutamine synthetase from the bacterium Gluconobacter suboxydans. The maximum amount of glutamine formed under optimum conditions was about 20 mM in 3 hr with 80% yield based on glutamate, substrate. The fermentation proceeded in two steps: the accumulation of energy in a form of fructose 1,6-diphosphate (FDP) by yeast fermentation of sugar based on the Harden-Young effect and the fermentation of FDP coupled with glutamine synthetase reaction (an endergonic reaction) through an ATP-ADP system. The following factors were found to be important: (a) the ratio of the activities of yeast fermentation of sugar and glutamine synthetase, (b) effect of contaminating enzyme(s) in glutamine synthetase preparation, and (c) enzymatic properties of glutamine synthetase.     

Isolation of a New Tobacco Constituent, 5,6-Dihydro-5-hydroxy-3,6-epoxy-β-ionol,from Japanese Domestic Suifu Tobacco

Glutamine production was investigated by coupling of glutamine synthetase from Gluconobacter suboxydans with a sugar fermentation system of baker's yeast (energy generating system). Under the optimum condition, 22 mM glutamine was formed in 3 hr, and the yield was 92% based on the substrate glutamate. The first step of the process was the accumulation of fructose 1,6-diphosphate (FDP) as a reservoir of fermentation energy, in the presence of a high concentration of inorganic phosphate; and the second step was accomplished by coupling the degradation of FDP with glutamine synthetase reaction through an ADP-ATP system. The effects of enzyme concentration, additives in the reaction mixture and others on glutamine formation were investigated, and the importance of three factors was pointed out: (a) the ratio of activity of energy generating system to utilizing system, (b) contaminated enzyme(s) in the energy utilizing system and (c) the enzymatic properties of the energy utilizing system.     

Characteristics and Efficiency of Glutamine Production by Coupling of a Bacterial Glutamine Synthetase Reaction with the Alcoholic Fermentation System of Baker’s Yeast

Glutamine production with bacterial glutamine synthetase (GS) and the sugar-fermenting system of baker’s yeast for ATP regeneration was investigated by determining the product yield obtained with the energy source for ATP regeneration (i.e., glucose) for yeast fermentation. Fructose 1,6-bisphosphate was accumulated temporarily prior to the formation of glutamine in mixtures which consisted of dried yeast cells, GS, their substrate (glucose and glutamate and ammonia), inorganic phosphate, and cofactors. By an increase in the amounts of GS and inorganic phosphate, the amounts of glutamine formed increased to 19 to 54 g/liter, with a yield increase of 69 to 72% based on the energy source (glucose) for ATP regeneration. The analyses of sugar fermentation of the yeast in the glutamine-producing mixtures suggested that the apparent hydrolysis of ATP by a futile cycle(s) at the early stage of glycolysis in the yeast cells reduces the efficiency of ATP utilization. Inorganic phosphate inhibits phosphatase(s) and thus improves glutamine yield. However, the analyses of GS activity in the glutamine-producing mixtures suggested that the higher concentration of inorganic phosphate as well as the limited amount of ATP-ADP caused the low reactivity of GS in the glutamine-producing mixtures. A result suggestive of improved glutamine yield under the conditions with lower concentrations of inorganic phosphate was obtained by using a yeast mutant strain that had low assimilating ability for glycerol and ethanol. In the mutant, the activity of the enzymes involved in gluconeogenesis, especially fructose 1,6-bisphosphatase, was lower than that in the wild-type strain.     

Regulation of glutamine synthetase from Saccharomyces cerevisiae by repression, inactivation and proteolysis

Glutamine synthetase activity is modulated by nitrogen repression and by two distinct inactivation processes. Addition of glutamine to exponentially grown yeast leads to enzyme inactivation. 50% of glutamine synthetase activity is lost after 30 min (a quarter of the generation time). Removing glutamine from the growth medium results in a rapid recovery of enzyme activity. A regulatory mutation (gdhCR mutation) suppresses this inactivation by glutamine in addition to its derepressing effect on enzymes involved in nitrogen catabolism. The gdhCR mutation also increases the level of proteinase B in exponentially grown yeast. Inactivation of glutamine synthetase is also observed during nitrogen starvation. This inactivation is irreversible and consists very probably of a proteolytic degradation. Indeed, strains bearing proteinase A, B and C mutations are no longer inactivated under nitrogen starvation.     

Characterization of theanine-forming enzyme from Methylovorus mays no. 9 in respect to utilization of theanine production

For development of theanine production from glutamic acid and ethylamine by coupling yeast sugar fermentation as an ATP-regenerating system, several strains were selected from among about 200 methylamine- and/or methanol-assimilating bacteria depending on the theanine-forming activity of their permeated cells. The amount of theanine formed by the cells of the selected strains was much larger than that by the cells of Escherichia coli AD494 (DE3) expressing Pseudomonas taetrolens Y-30 glutamine synthetase (GS), which has been found to be a usable enzyme for theanine production. A GS-like enzyme responsible for the theanine-forming reaction was obtained from an obligate methylotroph isolate, Methylovorus mays No. 9. The enzyme was induced by methylamine in the culture medium. A molecular mass of 410-470 kDa was obtained by gel filtration of the enzyme, and 51 kDa by SDS-PAGE analysis. The enzyme showed high activity toward methylamine rather than ammonia, which indicates that it is similar to known gamma-glutamylmethylamide synthetase. The isolated enzyme also had high reactivity to ethylamine in a neutral pH range, and formed theanine from glutamic acid and ethylamine in a reaction mixture containing a yeast sugar fermentation system for ATP-regeneration.     

Inactivation of Glutamine Synthetase by Ammonia Shock in the Gram-Positive Bacterium Streptomyces cattleya

In cultures of the gram-positive bacterium Streptomyces cattleya, a rapid inactivation of glutamine synthetase was seen after ammonia shock. pH activity curves for ammonia-shocked and control cultures are shown. A peak of glutamine synthetase activity was seen during fermentation for production of the antibiotic thienamycin.     

Cloning and expression of Methylovorus mays No. 9 gene encoding gamma-glutamylmethylamide synthetase: an enzyme usable in theanine formation by coupling with the alcoholic fermentation system of baker's yeast

Gamma-glutamylmethylamide synthetase (GMAS), found in an obligate methylotroph, Methylovorus mays No. 9, can form theanine from glutamic acid and ethylamine in a mixture in which yeast sugar fermentation is coupled for ATP regeneration. The internal and N-terminal amino acid sequences of GMAS had certain similarities to putative glutamine synthetase type III (GS III) of Methylobacillus flagellatus KT. From the M. mays No. 9 genomic DNA library, a clone containing a 6.5-kbp insertional DNA fragment was selected by the PCR screening technique with oligonucleotide primers specific for the GMAS gene. The fragment had an open reading frame of the GMAS gene encoding a protein of 444 amino acids (molecular mass, 49 kDa). The deduced amino acid sequence showed significant identity with that of Met. flagellatus KT GS III (78%). The isolated gene was ligated into an expression vector (pET21a) and expressed in Escherichia coli AD494 (DE3). Enzyme productivity in the expression system was about 23-fold higher than that in M. mays No. 9. Recombinant GMAS had the same properties as intrinsic GMAS, and it formed theanine by coupling the reaction with the ATP-regeneration system of yeast sugar fermentation.     

An association between glutamine synthetase activity and adipocyte differentiation in cultured 3T3-L1 cells

Confluent 3T3-L1 Swiss mouse fibroblasts acquired morphological and biochemical characteristics of adipocytes when maintained in medium containing 10% calf serum and added insulin. Identical cultures maintained in the absence of added insulin did not differentiate into adipocytes. Incubation of confluent cultures for 48 h with 0.25 μm dexamethasone and 0.5 mm 1-methyl-3-isobutylxanthine yielded subsequent adipocyte differentiation when the culture medium contained 10% fetal calf serum. In contrast, differentiation did not occur when similarly treated cultures were maintained in medium containing 10% calf serum. The increase in glutamine synthetase which occurred during adipocyte differentiation was closely associated with an increased rate of triglyceride synthesis from acetate, with increased protein, and with increases in the activities of glycerol-3-P dehydrogenase and glucose-6-P dehydrogenase. Glutamine synthetase activity remained undetectable in insulin-treated confluent 3T3-C2 cells maintained under conditions which yielded high glutamine synthetase activity in 3T3-L1 cells. (3T3-C2 cells did not differentiate into adipocytes.) Glutamine accumulated in the culture medium of 3T3-L1 adipocytes, but it did not accumulate in the medium from identically treated 3T3-C2 cells. A half-maximal increase in glutamine synthetase specific activity occurred at a culture medium insulin concentration of 10 ng/ml. Neither adipocyte differentiation nor the rise in glutamine synthetase activity were substantially altered by maintaining confluent cultures in medium lacking added glutamine. Incubation of confluent 3T3-L1 cultures with 3 mml-methionine sulfone, a reversible inhibitor of glutamine synthetase, increased by two-fold both the activity and the cellular content of glutamine synthetase. Incubation of confluent 3T3-L1 cultures with 4 mml-glutamine and l-methionine-dl-sulfoximine, an irreversible inhibitor of glutamine synthetase activity, decreased glutamine synthetase activity to less than 5% of the activity in control cultures; however, neither cellular content of the enzyme nor synthesis rate of the enzyme were substantially altered. In the presence of added glutamine, neither methionine sulfone nor methionine sulfoximine had a significant effect on phenotypic adipocyte conversion. By contrast, when confluent cultures were incubated with methionine sulfoximine and no added glutamine, glutamine synthetase remained absent and there was no evidence of adipocyte conversion. Our data indicate (1) that added insulin is required for adipocyte differentiation of 3T3-L1 cells maintained in medium containing calf serum, (2) that glutamine synthetase activity increases during adipocyte conversion regardless of the culture conditions employed to achieve differentiation, and (3) that glutamine synthetase activity may be required for adipocyte differentiation when cultures are maintained in medium lacking added glutamine.     

我国抗体药物产业化发展的策略探讨

随着重组抗体药物展示出良好的治疗效果和市场效益,抗体药物的研发逐渐成为生物医药产业发展的主要方向。但是目前国内动物细胞表达水平普遍较低和发酵工艺落后的现状制约了我国抗体药物产业化的发展。主要综述了国际抗体药物产业的发展态势,重点比较二氢叶酸还原酶和谷氨酰胺合成酶表达体系、连续灌注和流加培养发酵模式的各自优势,结合我们抗体药物表达、发酵方面的经验,对当前我国抗体药物产业化发展策略进行了探讨,提出抗体药物产业化模式应根据企业对抗体产率、产能和市场经济学的多重考虑选择发酵工艺和发酵规模,应用谷氨酰胺合成酶/CHO-K1表达系统和连续灌注培养工艺可能更适应目前中国抗体产业化的需要。