酿酒酵母by1.1b产D-(-)-扁桃酸脱氢酶的发酵条件优化

对一株产D-(-)-扁桃酸对映选择性脱氢酶的酿酒酵母菌(Saccharomyces cerevisiae sp. strain by1.1b)发酵产酶条件进行了优化。研究各种碳源、氮源及无机盐对产酶的影响, 应用正交试验优化发酵培养基组成, 结果为: 蛋白胨 60 g/L, 麦芽糖 30 g/L, MgSO4 0.5 g/L, ZnSO4 0.01 g/L, KCl 1.0 g/L。优化后酶产量提高了7.9倍(由2.56 U/mL增至20.21 U/mL)。摇瓶培养最佳条件为: 装液量40 %, 发酵pH 6.5, 接种量10 %, 发酵温度30 ℃。考察了细胞生长及产酶的时间进程, 最佳培养时间为25 h。     

啤酒糟固态发酵产β-葡萄糖苷酶的研究

利用啤酒糟为培养基对黑曲霉固态发酵产β-葡萄糖苷酶的工艺条件进行了优化和动力学研究。单因素试验表明,最适产酶温度、料液比和接种量分别为30℃、1∶5(啤酒糟∶水,g∶mL)和10%(mL/g);利用L9(34)正交试验优化反应条件,结果表明,在25℃,初始料水比为1:5,接种量10%的条件下,培养4d,β-葡萄糖苷酶的酶活可达10.85U/g。动力学研究表明,β-葡萄糖苷酶在96h进入产酶的高峰期,120h达到酶活最大值。     

产β-葡聚糖酶基因工程菌发酵条件的优化

目的以1株酶产量高、耐热性好的重组大肠埃希菌BL21为材料发酵生产β-葡聚糖酶。方法选用麸皮、豆粕等农副产品配制复合碳源、氮源,优化半合成发酵培养基,并通过正常试验确定最佳培养条件。结果研究得到摇瓶水平产β-葡聚糖酶的最佳培养基（g/L）为：麸皮6.7,玉米粉1.7,豆粕13.8,豆粉13.8,酵母粉7.0,NH4Cl 7.0,Na2HPO4.12H2O3.0,MgSO4.7H2O0.75,CaCl20.5,吐温80 0.8%。通过正交试验确定了产酶最佳初始pH为6.2,装样量为35 mL/250 mL,接种量为1%。采用优化后的工艺,在37℃200 r/min培养过夜,经乳糖诱导6 h后,最高酶活可达到830.7 U/mL,是初始产酶条件的3.4倍。结论该半合成培养基在重组大肠埃希菌产β-葡聚糖酶方面具有很大优势。     

对映选择性Geotrichum sp. GXU33脂肪酶的筛选、产酶及酶学性质研究

利用溴麝香草酚蓝作为反应指示剂,快速地筛选到产对映选择性脂肪酶菌株GXU33(Geotrichum sp.),此酶能够拆分外消旋扁桃酸甲酯产生(S)-扁桃酸.此菌株最适生长、产酶条件为橄榄油 10 g/L, 酵母粉 5 g/L, Na2HPO4·12H2O 3.5 g/L, KH2PO4 1.0 g/L, MgSO4·7H2O 0.2 g/L, pH 7.0,28℃,200 r/min.PMSF和蛋白酶K对菌株生长没有影响,PMSF显著抑制酶活,蛋白酶K具有保护酶活力的作用.该脂肪酶最适作用pH 为7.5,最适作用温度为30 ℃; Ca2 ,Mg2 ,Zn2 不同程度提高酶活性,Cu2 , Co2 ,Mn2 ,Fe2 ,Fe3 严重抑制酶活性.当以5% DMSO为助剂,消旋扁桃酸甲酯20 mg,GXU33 脂肪酶1500 U,25 mmol/L磷酸钠缓冲液(pH 7.5)加至总体积2 mL,32 ℃,100 r/min, 反应8h,得到最佳拆分效果:转化率为44.8%,(S)-扁桃酸对映过量值为83.5%.     

塔拉单宁水解酶产生条件的研究

微生物通过发酵产酶可以将植物单宁降解成小分子酚类化合物或其衍生物,但培养条件对其产酶影响很大。论文采用固态培养法,对黑曲霉产生塔拉单宁水解酶的条件进行了研究。结果表明,当培养液中塔拉单宁浓度为75 g/L、葡萄糖浓度为3 g/L、(NH4)2SO4浓度为0.2 g/L2、50 mL锥形瓶装液量为25 mL、惰性载体用量为5.6%(w/v)、起始pH为5.5、接种量为12%(v/v)、30℃培养72 h时,该黑曲霉产生的塔拉单宁水解酶活力可达到44.29 U/mL,是其自然条件下酶活力(24.09 U/mL)的1.84倍;没食子酸产率达到79.3%。研究结果对于揭示塔拉单宁生物降解的机理具有一定的参考价值。     

泡盛曲霉CAU33固体发酵产β-1,3-1,4-葡聚糖酶发酵条件优化

利用单因素实验法优化了泡盛曲霉(Aspergillus awamori)CAU33利用农业废弃物固体发酵产β-1,3-1,4-葡聚糖酶的发酵条件。产酶的条件包括碳源种类、初始水分含量、氮源种类、初始p H、表面活性剂、培养温度和发酵时间。进一步运用响应面分析法优化了其中主要因素,得到最佳产酶条件为:啤酒糟为碳源、含水量为81.6%、吐温60添加量20g/L、大豆蛋白胨添加量25g/L、自然p H、35℃下培养6d。在优化后的发酵条件下,最大产酶水平达到40 832.9U/g。泡盛曲霉固体发酵产β-1,3-1,4-葡聚糖酶的酶活力高,工业化生产和应用潜力大。     

羰基还原酶产生菌Candida ontarioensis制备(R)-2-氯-1-(3-氯苯基)乙醇

从实验室保藏的菌株中筛选获得Candida sp.PT2A,并通过18S rRNA鉴定为安大略假单胞菌Candida on-tarioensis。对C.ontarioensis不对称还原合成(R)-2-氯-1-(3-氯苯基)乙醇的发酵产酶条件和转化条件进行优化,确定了最适的发酵产酶条件和转化条件:温度30℃,初始pH 6.5,摇床转速180 r/min,菌体质量浓度200 g/L。采用2-氯-1-(3-氯苯基)乙酮质量浓度为10 g/L时,还原反应72 h,(R)-2-氯-1-(3-氯苯基)乙醇的e.e.值为99.9%,产率为99%;底物质量浓度提高至30 g/L时,产率下降为84.3%。采用十六烷基三甲基溴化铵(CTAB)对C.ontarioensis细胞进行通透性处理(CTAB g/L,4℃下处理20 min),在30 g/L底物下反应24 h,产物的e.e.和产率分别达到99.9%和97.5%。     

产γ-谷氨基甲酰胺合成酶菌株的筛选及产酶条件研究

以假单胞菌(Pseudomonas sp.)为出发菌株,通过紫外诱变筛选得到一株γ-谷氨基甲酰胺合成酶高产菌株UV-19,其酶活提高32.54%。以突变株UV-19为供试菌株,对γ-谷氨基甲酰胺合成酶的发酵条件进行优化。首先利用Plackett-Burman设计筛选出影响较大的4个因素:葡萄糖、蛋白胨、起始pH值、装液量。在此基础上再利用CCD响应面分析法进行优化,得到最佳产酶培养条件为(g/L):葡萄糖15、蛋白胨12、NaCl 5.0、MgSO4.7H2O 0.2、K2HPO4.3H2O 0.5、甲胺盐酸盐1.0g/L、起始pH值6.5、装液量72mL/250mL。该优化条件下进行产酶培养,假单胞菌发酵产γ-谷氨基甲酰胺合成酶酶活力可达32.68U/mL。     

海洋枯草芽孢杆菌产纤溶酶的诱变育种与发酵工艺优化*

目的:对海洋来源的具有产纤溶酶能力的枯草芽孢杆菌(Bacillus subtilis)LC6-1进行紫外诱变,得到高产且稳定的突变株PW6-3,对该突变株发酵产酶的条件进行优化。方法:采用单因素和正交试验进行发酵培养基组分和培养条件的优化。结果:突变株PW6-3的酶活力为(6 960.21 ± 85.51)U/mL,较原始菌株提高了30.48%。以PW6-3为出发菌株,采用单因素及正交试验的方法对菌株进行发酵培养基组分与培养条件优化,最终得到的最佳培养基组分是:玉米淀粉30 g/L,玉米浆干粉40 g/L,CaCl₂ 3 g/L;最佳发酵培养条件是:32℃,转速200 r/min,接种量3%,pH 6.5,种龄18 h,发酵培养时间66 h,最终菌株的酶活力稳定在(9 203.63 ± 67.85)U/mL。结论:发酵工艺优化后,菌株PW6-3纤溶酶产量较诱变之前的菌株LC6-1提高72.53%,且发酵工艺成本较低,具有较好的经济效益。     

施氏假单胞菌PS59产脂肪酶发酵条件及脂肪酶洗涤性能优化北大核心CSCD

旨在对施氏假单胞菌(Pseudomonas stutzeri)PS59产脂肪酶发酵培养基进行单因子优化及响应面分析,选择出最佳的碳氮源,并分别对加酶量、洗涤时间、洗涤温度、洗涤pH和表面活性剂添加量对脂肪酶洗涤效果的影响进行评估,确定各个因素最佳值。结果表明,实验室培养施氏假单胞菌产脂肪酶的最佳碳氮源分别为蔗糖和大豆蛋白胨。优化发酵培养基配方为(g/L):大豆蛋白胨22.39 g,蔗糖10 g,K_2HPO_4·3H_2O 1 g,MgSO_4·7H_2O 0.5 g,无水CaCl_2 0.05 g,橄榄油8.1 g,pH 8.1,培养温度30℃,培养36 h获得平均脂肪酶产量为36.12±1.32 U/mL,相对于起始酶活15.65±4.81 U/mL,产量提高了1.3倍。确定最佳洗涤效果加酶量为100 U,最佳洗涤温度为25℃,最佳洗涤时间为25 min,洗涤pH对该脂肪酶洗涤效果影响不大。在最佳洗涤条件下,加酶洗涤液的洗涤效率可以提高30%-40%。     

Serum-free culture of Japanese quail kidney cells regulation of vitamin D metabolism

Cells obtained from male quail kidneys by digestion with collagenase and hyaluronidase were plated and maintained in a chemically defined, serum-free medium. Culture dishes (35 mm) were inoculated with 1.5 · 10⁶ cells which became confluent in 5 days. The cells maintained an epithelial-like morphology over the entire culture period. During a 2 h incubation the cells metabolized 25–30% of the 10 nM 25-hydroxyvitamin D-3 (25-OH-D-3) provided. Seven metabolites were chromotographically separated on Sephadex LH-20. Three have been identified as 1α,25-dihydroxyvitamin D-3 (1,25(OH)₂D-3), 24,25-dihydroxyvitamin D-3 (24,25(OH)₂D-3) and 1α,24,25-trihhydroxyvitamin D-3 (1,24,25(OH)₃D-3). The activities of the 25-OH-D-3:1α- and 24-hydroxylases increased eight times faster than the cell number in 5 days. Preincubation of the cells with 10 nM 25-OH-D-3 or 1,25(OH)₂D-3 decreased 1,25(OH)₂D-3 synthesis, and increased both 24,25(OH)₂D-3 and metabolite IV synthesis. The decrease in 25-OH-D-3:1α-hydroxylase activity required a 2 h preincubation with 25-OH-D-3, while stimulation of 25-OH-D-3:24-hydroxylase activity and metabolite IV production required a 6 h preincubation. Incubations of cells for 1 h with parathyroid hormone resulted in a 30-fold increase in cyclic AMP in the medium. A 6 h preincubation with parathyroid hormone decreased 24,25-(OH)₂D-3 synthesis 50% relative to control cells. These results demonstrate the amenability of this system for studying the regulation of 25-OH-D-3 metabolism, as well as its use for other in vitro studies on renal cell function in a chemically defined culture system.     

Production of D-(--)-3-hydroxyalkanoic acid by recombinant Escherichia coli

Pathways for extracellular production of chiral D-(-)-3-hydroxybutyric acid (3HB) and D-(-)-3-hydroxyalkanoic acid (mcl-3HA) were constructed by co-expression of genes of beta-ketothiolase (phbA), acetoacetyl-CoA reductase (phbB) and 3-hydroxyacyl-ACP CoA transacylase (phaG), respectively, in Escherichia coli strain DH5alpha. The effect of acrylic acid and glucose on production of both 3HB and mcl-3HA was investigated. It was found that the addition of acrylic acid significantly increased production of 3HB and mcl-3HA consisting of 3-hydroxyoctanoic acid and 3-hydroxydecanoic acid in a ratio of 1:3 from 199 mg x l(-1) to 661 mg x l(-1) and from 27 mg x l(-1) to 135 mg x l(-1), respectively, in shake flask studies when glucose was present in the medium at the very beginning of fermentation. The timing of glucose addition had no effect on 3HB production. In contrast, mcl-3HA production was affected by glucose addition, an mcl-3HA concentration of 193 mg x l(-1) was obtained when glucose was added to the culture at 12 h. A more than seven-fold increase was obtained when compared with that in medium containing glucose at the beginning of fermentation. However, a decrease in production of 3HB and mcl-3HA was found when glucose was added at 12 h to the culture containing acrylic acid. The repressive effect of acrylic acid on acetic acid production was also evaluated and discussed.     

Production of ethanol from sugars in wood hydrolysate byFusarium oxysporum

Wood hydrolysate used for ethanol production by two strains ofFusarium oxysporum contained 2.3% (w/v) reducing sugars (xylose and glucose). Ethanol production at the optimum reducing sugar concentration of 54.8 g/l medium, at pH 5.5, and 30°C was 12.3 g/l and 11.7 g/l byF. oxysporum D-140 and NCIM-1072, respectively in shake flasks during 96 h fermentation. The maximum production of ethanol under optimum cultural conditions, and in the presence of yeast extract plus minerals, was 13.2 g/l medium byF. oxysporum D-140 over 108 h fermentation.

---

Résumé L'hydrolysat de bois utilisé pour la production d'éthanol par deux souches deFusarium oxysporum contenait 2.3% (poids/vol.) de sucres réducteurs (xylose et glucose). La production d'éthanol, à la concentration optimum en sucres réducleurs de 54.8 g par litre de milieu à pH 5.5 et à 30°C était de 12.3 g/l et 11.7 g/l respectivement chezF. oxysporum D-140 et NCIM-1072, en flacons agités pendant 96 h de fermentation. La production maximum d'éthanol, dans les conditions optimum de culture, et en prosence d'extrait de levure et de minéraux a mit de 13.2 g par litre de milieu chezF. oxysporum D-140 en 108 h de lermentation.

     

Production of long-chain polyunsaturated fatty acids by monoxenic growth of labyrinthulids on oil-dispersed agar medium

A novel method is proposed for the production of long-chain polyunsaturated fatty acids (LCPUFA) by labyrinthulids. The method comprises a monoxenic culture with Psychlobacter phenylpyruvicus, using agar medium in which oil was dispersed. Soybean oil (SBO) was selected as the optimum material for an oil-dispersed agar medium. The labyrinthulids showed three-dimensional growth and an anastomosing ectoplasmic network in the SBO-dispersed agar medium. The oil plate changed from an opaque culture to a more transparent culture, due to growth of the labyrinthulids. The optimum culture conditions were 25-30 degrees C, an initial pH of 6-10 and artificial seawater with a salt concentration of 50-100%. These conditions are close to those where these strains were isolated. The maximum LCPUFA production (0.59 g/l) and dry cell weight (4.93 g/l) was obtained using strain S3-2 (isolated from Ishigaki Island) with 1.5% SBO at 14 days. This value was about 30 times more than that using glucose instead of SBO. The method proposed is promising in terms of the production of LCPUFA from reproducible oils.     

Relationship between bone resorption and lysosomal enzyme release as demonstrated by the effect of 1α-hydroxy-vitamin D-3 in an organ culture system

The effect of 1α-hydroxy-vitamin D-3 on the release of calcium (⁴⁰Ca, ⁴⁵Ca), inorganic phosphate and lysosomal enzymes, on glucose consumption and lactate production was studied in a bone organ culture system using half calvaria from 6–7-day-old mice. 1α-Hydroxy-vitamin D-3 stimulated the mobilization of minerals and increased the release of β-glucuronidase, β-N-acetylglucosaminidase and acid phosphatase, while no effect on the release of lactate dehydrogenase was seen. 1α-Hydroxy-vitamin D-3 also caused a significant increase in the total activities of acid phosphatase in the bones after culture, indicating increased enzyme synthesis. The stimulatory effect of the release of P_i and β-glucuronidase was also obtained after a temporary exposure to 1α-hydroxy-vitamin D-3. The stimulation by 1α-hydroxy-vitamin D-3 on the release of Ca²⁺, P_i and β-glucuronidase was suppressed by a protein synthesis inhibitor cycloheximide. No effect by 1α-hydroxy-vitamin D-3 on glucose consumption and lactate production was registered, suggesting that increased mineral mobilization does not require increased lactate production. It is concluded that although the data in the present paper do not prove a cause-and-effect relationship between lysosomal enzyme release and bone resorption, they give further support to the concept that the processes are intimately associated.     

代谢改造克雷伯氏菌合成D-1,2,4-丁三醇

【背景】D-1,2,4-丁三醇(D-1,2,4-butanetriol,BT)是一种重要的四碳多元醇,应用范围广,以木糖为底物的四步生化反应是目前最高效的BT生物合成路线。但大肠杆菌宿主存在严重的碳代谢抑制,限制了工程菌在木糖葡萄糖混合糖下的生长和BT合成。然而克雷伯氏菌具有生长速度更快、葡萄糖木糖混合糖利用效果好等优点。【目的】在碳代谢抑制效应较弱的克雷伯氏菌中构建以木糖为底物的BT合成途径,以提高混合糖下BT合成能力。【方法】将来源于Clostridium crescenti的木糖脱氢酶基因xdh和来源于Lactococcus lactis的2-酮异戊酸脱羧酶基因kivD及来源于Escherichia coli W3110的木糖酸脱水酶基因yjhG克隆至KlebsiellapneumoniaeZG25,得到重组菌K.pneumoniae ZG25-BT,对重组菌进行培养条件和培养基优化,进一步敲除xylA以提高BT产量。【结果】在37°C、200 r/min、接种量1%、诱导时间2 h、添加10.0 g/L CaCO3控制pH条件下,敲除xylA的重组菌在1.5倍LB培养基中以30.0 g/L木糖和10.0 g/L葡萄糖为底物,BT的产量达到4.52 g/L,摩尔转化率为0.21mol/mol,收率为15%,较优化前分别提高150%、62%和67%。【结论】实现了BT在K.pneumoniaeZG25中的发酵生产,同时通过培养条件和培养基的优化及xylA的敲除提高了BT合成能力,为进一步实验奠定了基础。     

4-氯乙酰乙酸乙酯羰基还原酶产生菌的筛选及产酶条件研究

从实验室保藏的菌株中,筛选到一株立体选择性较高的产4-氯乙酰乙酸乙酯(COBE)羰基还原酶的菌株———出芽短梗霉(Aureobasidiumpullulans)SW0202,菌体产酶条件研究表明,最佳的发酵培养基配方为:麦芽糖30.0g/L,酵母膏20.0g/L,蛋白胨3.0g/L,(NH4)2SO45.0g/L,KH2PO42.0g/L,MgSO4.7H2O0.7g/L,最适发酵温度及初始pH分别为:28°C和pH6.0。该菌在此条件下发酵培养24h,产菌丝体生物量16.78g干菌体/L,COBE羰基还原酶酶活力达到1007U/L。在COBE的转化反应中,产物S-CHBE的浓度达到10.12g/L,光学纯度>97%e.e.。     

顺式环氧琥珀酸水解酶产生菌的筛选及产酶条件

从65株诺卡氏菌中,筛选到一株产顺式环氧琥珀酸水解酶(ESH)的菌株,经鉴定为酒石酸诺卡氏菌(Nocardia tartaricans)SW13-57。在14L发酵罐中通过诱导培养,能在细胞内产生ESH酶活力达120u/g。产酶条件研究表明用丙二醇作碳源,硫酸铵作氮源,顺式环氧琥珀酸作诱导剂,初始pH7.0,温度30℃,通过培养24～30h,产酶量最高。该产酶菌株已被用于固定化细胞方法连续生产L(+)酒石酸。     

Regiospecific hydroxylation of 3-(methylaminomethyl) pyridine to 5-(methylaminomethyl) -2 (1H) -pyridinone byArthrobacter ureafaciens

Microbial production of a 6-hydroxy-3-pyridylmethyl compound from 3-pyridylmethyl compound was investigated. The hydroxylation of 3-(methylaminomethyl)pyridine to 5-(methylaminomethyl)-2(1H)-pyridinone, tautomer of 2-hydroxy-5(methylaminomethyl)pyridine, by resting cells ofArthrobacter ureafaciens JCM3873 was found to proceed regio- and chemo-selectively with an almost quantitative yield. The addition of molybdate ion and nicotine as an inducer to the culture medium was required for the preparation of cells containing high hydroxylation activity. The optimal temperature and pH for the hydroxylation by using resting cells were 35°C and around 7, respectively. This hydroxylation enzyme does undergo inhibition by the substrate. The inhibitory effect could be eliminated by stepwise feeding of the substrate. Under adequate conditions, 23 mg/ml of 5-(methylaminomethyl)-2(1H)-pyridinone was produced with a molar yield of nearly 100% from 3-(methylaminomethyl)pyridine.     

Influence of cultivating conditions on the alpha-galactosidase biosynthesis from a novel strain of Penicillium sp. in solid-state fermentation

AIMS: The work is intended to achieve optimum culture conditions of alpha-galactosidase production by a mutant strain Penicillium sp. in solid-state fermentation (SSF). METHODS AND RESULTS: Certain fermentation parameters involving incubation temperature, moisture content, initial pH value, inoculum and load size of medium, and incubation time were investigated separately. The optimal temperature and moisture level for alpha-galactosidase biosynthesis was found to be 30 degrees C and 50%, respectively. The range of pH 5.5-6.5 was favourable. About 40-50 g of medium in 250-ml flask and inoculum over 1.0 x 10(6) spores were suitable for enzyme production. Seventy-five hours of incubation was enough for maximum alpha-galactosidase production. Substrate as wheat bran supplemented with soyabean meal and beet pulp markedly improved the enzyme yield in trays. CONCLUSIONS: Under optimum culture conditions, the alpha-galactosidase activity from Penicillium sp. MAFIC-6 indicated 185.2 U g(-1) in tray of SSF. SIGNIFICANT AND IMPACT OF THE STUDY: The process on alpha-galactosidase production in laboratory scale may have a potentiality of scaling-up.