4-丁内酰胺水解酶法制备γ-氨基丁酸

以吡咯烷酮为唯一碳源,从采集的土样中分离、筛选得到具有4-丁内酰胺水解酶活性的菌株.采用响应面分析法对该菌株的产酶培养基组分进行了优化研究并对酶促转化反应条件也进行了研究.结果表明:编号为HHSW-16的菌株水解活性最高.优化后的培养基组成为:葡萄糖11.50 g·L-1,牛肉膏6.35 g·L-1,酵母粉5.58 g...     

Biosynthesis of γ-aminobutyric acid (GABA) using immobilized whole cells of <Emphasis Type="Italic">Lactobacillus brevis</Emphasis>

On an industrial scale, the production of γ-aminobutyric acid (GABA) from the cheaper sodium L-glutamate (L-MSG) is a valuable process. By entrapping Lactobacillus brevis cells with higher glutamate decarboxylase (GAD) activity into Ca-alginate gel beads, the biotransformation conditions of L-MSG to GABA were optimized with the immobilized cells. The cells obtained from a 60-h culture broth showed the highest biotransformation efficiency from L-MSG to GABA. The optimal cell density in gel beads, reaction pH and temperature were 11.2 g dry cell weight (DCW) l⁻¹, 4.4 and 40°C respectively. The thermal stability of immobilized cells was significantly higher than free cells. Under the optimized reaction conditions, the yield of GABA reached above 90% during the initial five batches and the yield still remained 56% in the tenth batch. Continuous production of GABA was realized with a higher yield by incorporating cell re-cultivation using the packed bed reactor.     

响应面法优化酿酒酵母产油脂条件

运用响应面法对酿酒酵母(Saccharomyces cerevisiae)产油脂以及发酵条件优化进行了研究。首先根据单因素实验结果,利用Plackett-Burman设计对影响其产油脂相关因素进行评估并筛选出具有显著效应的3个因素:柠檬酸,CaCl2和初始pH值。接着用最陡爬坡试验逼近以上3个因子的最大响应区域后,采用Box-Behnken设计以及响应面分析法,确定其优化后发酵条件为(w/v):葡萄糖15%,蛋白胨0.2%,酵母浸粉0.4%,柠檬酸0.471%,MgSO4·7H2O0.1%,ZnSO4·7H2O0.2%,CaCl20.025%,FeSO4·7H2O0.005%,初始pH值为6.74,180r/min,30°C培养96h。优化后的油脂产率(干重)达到14.55%,比在种子培养基中油脂产率4.76%提高了2倍左右。     

响应面分析优化产木聚糖酶毕赤酵母基因工程菌培养条件

研究不同碳源、氮源和无机盐对毕赤酵母AX181菌株产木聚糖酶的影响。实验表明,分别采用葡萄糖和玉米浆干粉为碳源和氮源可以明显提高木聚糖酶的产量。无机盐单因子优化实验显示添加适量的（NH4）2SO4、KH2PO4、MnSO4·H2O、FeSO4·7H2O也可以部分提高木聚糖酶产量。在此基础上利用响应面法优化毕赤酵母产木聚糖酶培养基,利用12次实验的Plackett—Burman设计实验筛选出影响产木聚糖酶的3个主要因素,即玉米浆干粉、MnSO4·H2O和FeSO4·7H20。并进一步通过最陡爬坡路径逼近最大响应区域,采用中心组合实验设计确定最佳条件。优化后的产木聚糖酶培养基组分为（g/L）：葡萄糖40．00,玉米浆干粉80．84,（NH4）2SO46．25,KH2PO41．25、MnSO4·H2O0．35,FeS04-7H2O1．31。培养基优化后,实际产酶2883．86u／mL,是优化前YPD培养基产酶的2．51倍。     

Optimization of glutamate concentration and pH for H production from volatile fatty acids by Rhodopseudomonas capsulata

AIMS: This study attempted to employ response surface methodology (RSM) to evaluate the effects of glutamate concentration and pH on H(2) production from volatile fatty acids by Rhodopseudomonas capsulata. METHODS AND RESULTS: A mixture of acetate, propionate and butyrate was used as a carbon source for the H(2) production by R. capsulata. The H(2) yield and H(2) production rate were strongly affected by the glutamate concentration, pH and their interaction. The predicted maximum H(2) yield of 0.534 was obtained when glutamate concentration and pH were 6.56 mmol l(-1) and 7.29 respectively. On the contrary, the maximum H(2) production rate of 18.72 ml l(-1) h(-1) was achieved at a glutamate concentration of 7.01 mmol l(-1) and pH 7.31. CONCLUSIONS: Taking H(2) yield and H(2) production rate together into account, a glutamate concentration of 6.56-7.01 mmol l(-1) and pH of 7.29-7.31 should be selected for H(2) production from a mixture of acetate, propionate and butyrate by R. capsulata. SIGNIFICANCE AND IMPACT OF THE STUDY: The RSM was a useful tool for maximizing H(2) production by photosynthetic bacteria (PSB).     

阿拉伯胶酶产生菌株的筛选及其发酵培养基的优化

从土壤中筛选产阿拉伯胶酶的微生物菌株,并通过紫外诱变选育后得到高产突变菌株ZHB05F,依据菌落和孢子形态特征初步鉴定为镰刀茵(Fusarium sp.).通过单因素试验,优化了产酶培养基的主要组分的浓度和pH值,得到最佳的产酶发酵培养基组成为:阿拉伯胶30 g/L,(NH4)2SO4 8 g/L,K2HPO4 1g/...     

毛头鬼伞子实体中甾类化合物的结构鉴定及其抑制肿瘤细胞增殖活性的研究

从毛头鬼伞子实体中分离得到4个甾类化合物,通过波谱分析,分别鉴定为麦角甾醇(1)、啤酒甾醇(2)、麦角甾醇葡萄糖甙(3)和tuberoside(4)。4个化合物均为首次从毛头鬼伞中得到。通过体外细胞毒性筛选试验,结果表明化合物4有较强的抑制人乳腺癌细胞MCF-7和狗肾细胞MDCK增殖的活性,其抑制增殖的IC50值分别为10.9μg/mL(18.4μmol/L)和5.8μg/mL(9.8μmol/L)。化合物3对MCF-7和MDCK的抑制作用则较弱,当其浓度为10.0μg/mL(17.9μmol/L)时,对MCF-7和MDCK的增殖抑制率分别为12.5%和7.5%。     

响应面分析法优化香菇多糖发酵培养基

香菇多糖是香菇中分离出的一种重要的具有生理活性的物质。采用深层发酵技术培养香菇菌丝体生产多糖,可利用农副产品作原料,成本低,周期短,易于大规模生产。采用响应面优化香菇Lentinula edodes135发酵培养基产多糖,首先Plackett-Burman设计对培养基8因素进行筛选,获得影响多糖产量的3个主要因素:葡萄糖,鱼粉和KH2PO4;然后用最陡爬坡路径逼近最大响应区域;最后通过Box-Behnken设计及响应面分析确定了主要影响因素的最佳浓度:葡萄糖2.2533%,鱼粉0.9756%,VB10.003%,NaCl0.8%,MgSO4·7H2O0.1%,FeSO4·7H2O0.04%,KH2PO40.0993%,初始pH值5.5。在优化后的培养基中,粗多糖产量为2.33g/L,实测值与预测值的误差为+3.61%,比初始培养基多糖产量提高1.91倍。     

Statistical optimization of medium components for the production of biosurfactant by Bacillus licheniformis K51

The nutritional medium requirement for biosurfactant production by Bacillus licheniformis K51 was optimized. The important medium components, identified by the initial screening method of Plackett-Burman, were H3PO4, CaCl2, H3BO3, and Na-EDTA. Box-Behnken response surface methodology was applied to further optimize biosurfactant production. The optimal concentrations for higher production of biosurfactants were (g/l): glucose, 1.1; NaNO3, 4.4; MgSO4 x 7H2O, 0.8; KCl, 0.4; CaCl2, 0.27; H3PO4, 1.0 ml/l; and trace elements (mg/l): H3BO3, 0.25; CuSO4, 0.6; MnSO4, 2.2; Na2MoO4, 0.5; ZnSO4, 6.0; FeSO4, 8.0; CoCl2, 1.0; and Na-EDTA, 30.0. Using this statistical optimization method, the relative biosurfactant yield as critical micelle dilution (CMD) was increased from 10x to 105x, which is ten times higher than the non-optimized rich medium.     

Robust production of gamma-amino butyric acid using recombinant Corynebacterium glutamicum expressing glutamate decarboxylase from Escherichia coli

Gamma-amino butyric acid (GABA) is a component of pharmaceuticals, functional foods, and the biodegradable plastic polyamide 4. Here, we report a simple and robust system to produce GABA from glucose using the recombinant Corynebacterium glutamicum strain GAD, which expresses GadB, a glutamate decarboxylase encoded by the gadB gene of Escherichia coli W3110. As confirmed by HPLC analysis, GABA fermentation by C. glutamicum GAD cultured at 30°C in GABA Production 1 (GP1) medium containing 50 g/L glucose without the addition of glutamate yielded 8.07 ± 1.53 g/L extracellular GABA after 96 h. Addition of 0.1mM pyridoxal 5'-phosphate (PLP) was found to enhance the production of GABA, whereas Tween 40 was unnecessary for GABA fermentation. Using the optimized GABA Production 2 (GP2) medium, which contained 50 g/L glucose and 0.1mM PLP, fermentation was performed in a flask at 30°C with 10% (v/v) seed culture of C. glutamicum GAD. GABA was produced in the culture supernatant with a yield of 12.37 ± 0.88 g/L after 72 h with a space-time yield of 0.172 g/L/h, which is the highest yield obtained to date for GABA from fermentation with glucose as a main carbon source.     

L-脯氨酸-4-羟化酶的异源表达和合成反式-4-羟基-L-脯氨酸的条件优化

L-脯氨酸-4-羟化酶(L-Proline-4-hydroxylase,P4H)是依赖α-酮戊二酸(α-KG)和Fe2+的双加氧酶成员之一,在反式-4-羟基-L-脯氨酸(trans-4-hydroxy-L-proline,t-4Hyp)等重要手性化合物的生物合成中发挥关键作用。本研究构建了来源于Bradyrhizobium japonicum USDA 6的P4H重组大肠杆菌Escherichia coli BL21(DE3)/p ET-28b-p4h BJ,SDS-PAGE和酶活检测结果表明,该菌株具有表达可溶性P4H和催化合成t-4Hyp的能力。通过优化,确定了该重组菌全细胞催化合成t-4Hyp较优的反应体系和条件:10 m L p H 6.5 80 mmol/LMES缓冲液、9 mmol/L L-Pro,6 mmol/L L-抗坏血酸,6 mmol/Lα-KG,0.8 mmol/L Fe SO4·7H2O,反应温度为35℃;在20 g/L湿细胞的催化反应中,t-4Hyp的合成量达到34.86 mg/L,比优化前(17.53 mg/L)提高了98.86%。该工作为进一步利用P4H生物催化法合成t-4Hyp奠定了一定的技术基础。     

Isolation and some properties of a mesophilic and mixotrophic iron-oxidizing bacterium, OKM-9

An iron-oxidizing bacterium strain, OKM-9, isolated from mud obtained from the bottom of a pond, Minamikata Ohike, in Okayama prefecture, Japan, grew well in an FeSO4 x 7H2O (3%)-medium (pH 2.5) with 0.03% yeast extract. However, the strain could not grow either in an FeSO4 x 7H2O (3%)-medium without yeast extract or in a yeast extract (0.03%)-medium (pH 2.5) without Fe2+. The strain did not use elemental sulfur as an energy source and did not have the activity to fix carbon dioxide. Strain OKM-9 could grow in an FeSO4 x 7H2O (3%)-medium with twenty different L-amino acids instead of yeast extract. Incorporation of [U-14C] glutamic acid into the cells was dependent on the energy produced by the oxidation of Fe2+. Strain OKM-9 did not grow heterotrophically using amino acids and hexoses as a sole energy and carbon source. The results that strain OKM-9 absolutely required ferrous iron (Fe2+) as a sole energy source and yeast extract or L-amino acids as a carbon source for growth strongly suggest that the strain is a mixotrophic iron-oxidizing bacterium. Strain OKM-9 was a gram-negative and rod-shaped bacterium (0.4-0.6 x 1.6-2.2 microm) and the mean G + C content of the DNA of the bacterium was 59.6 mol%. The optimum temperature and pH for growth were 30 degrees C and 2.1, respectively. However, the strain could not grow at temperatures above 45 degrees C. Iron-oxidizing activities of strain OKM-9 measured with intact cells and the plasma membrane were 14.3 and 5.7 microl O2 uptake/mg protein/min, respectively. The pyridine ferrohemochromes prepared from the plasma membrane of this strain showed absorption peaks characteristic of alpha-bands of heme a and b, but not heme c, at 587 and 557 nm, respectively. The results suggest that the cytochromes composing an iron-oxidation system of strain OKM-9 are different from those of the well-known mesophilic iron-oxidizing bacteria Thiobacillus ferrooxidans and Leptospirillum ferrooxidans.     

Statistical optimization of a high maltose-forming,hyperthermostable and Ca2+-independent alpha-amylase production by an extreme thermophile Geobacillus thermoleovorans using response surface methodology

AIM: Statistical optimization for maximum production of a hyperthermostable, Ca2+-independent and high maltose-forming alpha-amylase by Geobacillus thermoleovorans. METHODS AND RESULTS: G. thermoleovorans was cultivated in 250 ml flasks containing 50 ml of chemically defined glucose-arginine medium (g l(-1): glucose 20; arginine 1.2; riboflavin 150 microg ml(-1); MgSO4. 7H2O 0.2; NaCl 1.0; pH 7.0). The medium was inoculated with 5 h-old bacterial inoculum (1.8x10(8) CFU ml(-1)), and incubated in an incubator shaker at 70 degrees C for 12 h at 200 rev min(-1). The fermentation variables optimized by 'one variable at a time' approach were further optimized by response surface methodology (RSM). The statistical model was obtained using central composite design (CCD) with three variables: glucose, riboflavin and inoculum density. An over all 24 and 70% increase in enzyme production was attained in shake flasks and fermenter because of optimization by RSM, respectively. A good coverage of interactions could also be explained by RSM. The end products of the action of alpha-amylase on starch were maltose (62%), maltotriose (31%) and malto-oligosaccharides (7%). CONCLUSIONS: RSM allowed optimization of medium components and cultural parameters for attaining high yields of alpha-amylase, and further, a good coverage of interactions could be explained. The yield of maltose was higher than maltotriose and malto-oligosaccharides in the starch hydrolysate. SIGNIFICANCE AND IMPACT OF THE STUDY: By applying RSM, critical fermentation variables were optimized rapidly. The starch hydrolysate contained a high proportion of maltose, and therefore, the enzyme can find application in starch saccharification process for the manufacture of high maltose syrups. The use of this enzyme in starch saccharification eliminates the addition of Ca2+.     

假丝酵母YQ5产S-腺苷甲硫氨酸的发酵培养基优化

利用单因子实验和正交实验对假丝酵母(Candida sp.)突变菌株YQ5摇瓶发酵产生S-腺苷甲硫氨酸的培养基成分进行了优化。单因子实验结果表明, 发酵最适pH值为6.0, 最佳碳源为 8%蔗糖, 最佳氮源为1.5%胰蛋白胨, 酵母粉最适浓度为2%, MgSO4·7H2O、CaCl2、FeSO4·7H2O、CoCl2、CuSO4·5H2O、H3BO3等作为无机离子对胞内S-腺苷甲硫氨酸的积累均有促进作用。利 用正交实验获得了最终的发酵培养基配方。在最适条件下发酵48 h, S-腺苷甲硫氨酸含量可达1740.0 mg/L。     

Optimization of medium compositions favoring butanol and 1,3-propanediol production from glycerol by Clostridium pasteurianum

Medium compositions favoring butanol and 1,3-propanediol (1,3-PDO) production from glycerol by Clostridium pasteurianum DSM525 were investigated using statistical experimental designs. Medium components affecting butanol and 1,3-PDO production were screened using a fractional factorial experimental design. Among the six tested variables (phosphate buffer, MnSO4·H2O, MgSO4·7H2O, FeSO4·7H2O, (NH4)2SO4, and yeast extract), FeSO4·7H2O, (NH4)2SO4, and yeast extract were found to be significant variables for further optimization of medium using a Box-Behnken design. Optimal butanol (0.98 g/L/h) and 1,3-PDO (1.19 g/L/h) productivities were predicted by the corresponding quadratic model for each product and the models were validated experimentally under optimized conditions. The optimal medium composition for butanol production was significantly different from that for 1,3-PDO production (0.06 vs. 0 g/L for FeSO4·7H2O, 7.35 vs. 0 g/L for (NH4)2SO4, and 5.08 vs. 8.0 g/L for yeast extract), suggesting that the product formation from glycerol by C. pasteurianum DSM525 can be controlled by changing medium compositions.     

人己糖胺酶D的原核表达、纯化及酶学特性研究

糖基化修饰是一种重要的蛋白质翻译后修饰,参与生物体中的信号传导、细胞识别等多种细胞活动,糖基缀合物的正常水解是生物体代谢的必需途径.人己糖胺酶D( Hexosaminidase D)是新发现的一种存在于人细胞质中的切除GalNAc糖基化修饰的外切酶,但该酶的酶学特性尚不清楚.利用PCR的方法,将Hex D的cDNA序列构建到质粒pET3C中,重组质粒转化大肠杆菌BL21( DE3) plysS后,通过优化异丙基-β-D-硫代吡喃半乳糖苷(IPTG)浓度(0.1mmol/L)和诱导时间(10 h)获得了高可溶性表达的重组蛋白酶.采用Ni-NTA亲和层析对重组蛋白进行了纯化,SDS-PAGE检测分子量的大小(58 kDa)和纯度(95％以上).以4-甲基伞形酮-2-乙酰氨基-2-脱氧半乳糖(4-MU-O-GalNAc)为荧光底物,测定该酶的最适反应pH值为5.5,最适反应温度为37℃,且该酶的热稳定性较好,在50℃下放置半小时仍有较高活性,1mmol/L的金属离子(CuSO4、FeSO4·7H2O、MgCl2· 6H2O、CaCl2、NiSO4·6H2O、AlCl3·6H2O、ZnSO4·7H2O、MnCl2)及EDTA对该酶活性影响不大,10mmol/L AlCl3、CuSO、FeSO4·7H2O对该酶有不同程度的抑制.在最适条件下(pH 5.5,37℃)下,该酶的Km为0.16mmoL/L,最大反应速率为3.06 μmol/( min·mg).     

嗜中高温嗜酸古菌Ferroplasma thermophilum的培养条件优化

在中高温和较低pH条件下, Ferrroplasma spp. 是进行硫化矿生物浸出的重要微生物。Ferroplasma spp.为古菌,无细胞壁, 对矿浆浓度、搅拌剪切力以及溶液中的重金属离子等敏感, 很难得到高密度的纯培养, 给大规模的工业应用带来了一定难度。研究了F. thermophilum摇瓶培养时的最佳生长条件, 单因素考察结果表明最适培条件为: 温度50oC, 初始pH 0.5, 250 mL的摇瓶装液量为50 mL, 无机氮源(NH4)2SO4。通过正交试验确定了FeSO4·7H2O、酵母粉和蛋白胨最适组合为FeSO4·7H2O 40 g/L, 酵母粉0.3 g/L, 蛋白胨0.2 g/L。优化培养后, F. thermophilum 浓度达到了6.3×107个/mL, 40 g/L的FeSO4·7H2O在72 h内全部氧化完全。该结果可为该类古菌的扩大培养以及工业应用提供参考。     

嗜中高温嗜酸古菌Ferroplasma thermophilum的培养条件优化

在中高温和较低pH条件下, Ferrroplasma spp. 是进行硫化矿生物浸出的重要微生物。Ferroplasma spp.为古菌,无细胞壁, 对矿浆浓度、搅拌剪切力以及溶液中的重金属离子等敏感, 很难得到高密度的纯培养, 给大规模的工业应用带来了一定难度。研究了F. thermophilum摇瓶培养时的最佳生长条件, 单因素考察结果表明最适培条件为: 温度50oC, 初始pH 0.5, 250 mL的摇瓶装液量为50 mL, 无机氮源(NH4)2SO4。通过正交试验确定了FeSO4·7H2O、酵母粉和蛋白胨最适组合为FeSO4·7H2O 40 g/L, 酵母粉0.3 g/L, 蛋白胨0.2 g/L。优化培养后, F. thermophilum 浓度达到了6.3×107个/mL, 40 g/L的FeSO4·7H2O在72 h内全部氧化完全。该结果可为该类古菌的扩大培养以及工业应用提供参考。     

On the application of the Clark oxygen electrode to the study of enzyme kinetics in apolar solvents: the catalase reaction.

A method for recording O2 concentrations in nonconducting organic media with the Clark oxygen electrode was developed. Spontaneous oxidation of Na2S2O4 and the enzymatic reduction of NaBO3 or H2O2 by bovine liver catalase trapped in hydrated micelles of dioctylsulfosuccinate (AOT)/toluene were used as model systems. O2 titration with the above systems showed that air-saturated 1.6 M H2O/0.2 M AOT/toluene media contain seven times more O2 (1.4 mM) than aqueous solutions (0.2 mM). The measured Km values of catalase for NaBO3 and H2O2 in organic media were Kmov = 15 and 17 mM, respectively, whereas in aqueous buffer the values were 45 and 54 mM. In the toluene media, catalase activity increased with the W0 (H2O/AOT molar ratio) of the micellar preparation, reaching maximal activity at W0 = 10-12; under this condition, the catalytic center activity (Kp) of H2O2 was 7 x 10(6) min-1, similar to that obtained in the aqueous buffer (H2O2 = 7 x 10(6) min-1). It was found that the optimal pH for catalase in toluene media (pH 8.0) was shifted 1.0 unit compared to that in the aqueous buffer (pH 7.0). On the other hand, catalase was severely inhibited by NaN3 in both media. Thus, polarography based on the Clark oxygen electrode seems to be an easy, rapid, and sensitive technique for studying enzyme reactions consuming or evolving O2 in apolar media.