高产酸性普鲁兰酶工程菌株种子强化工艺

为进一步提高酸性普鲁兰酶工程菌株(Baclicus subtilis F1147)的产酶水平,开展了工程菌株种子强化工艺的研究。采用小米为载体,制备菌株接触面大、通气量好的小米种培养基,小米种摇瓶发酵最佳接种量为0.33%,其发酵酶活较二代斜面种高64.5%,较摇瓶种高21.4%。30 L罐补料分批发酵,小米种最佳接种量为0.33%,种子罐周期约为8 h,较摇瓶种种子罐提前1 h,发酵最高酶活达1 209 U/m L,较摇瓶种罐高73.7%,生长及产酶周期较摇瓶种罐延长4 h,最高菌浓也较摇瓶种罐高出22.1%,显著促进菌株生长及延长产酶周期。并且小米种发酵有利于改善发酵液特性,提高陶瓷膜除菌效率,其除菌率达100%,滤液浓缩4倍,酶活力回收率可达90.4%,体现了较好的浓缩效果,为工程菌株的工业化放大应用奠定了良好基础。     

紫外线诱变选育高产柠檬酸菌株及发酵条件研究

对1株初筛产柠檬酸黑曲霉进行紫外线诱变处理,并对选育的优势菌进行了发酵条件实验。研究结果表明,选育出的（Aspergillus niger）UV-5是一株优良高产菌株,该菌株的产酸率达到11.54%,比初始菌株（YZ-35）产酸率提高了24.41%;以红薯为发酵原料,在初始糖浓度15%、温度为35℃、起始pH值为5.5,转速140r·min-1、发酵84h的优化条件下,摇瓶发酵平均产酸率可达到13.15%。     

基因工程菌毕赤酵母发酵产纤维二糖酶条件研究

本文对基因工程菌毕赤酵母产纤维二糖酶活的发酵条件进行研究,结果表明:确定最优摇瓶培养基为小麦B浆4%,硫酸镁0.05%,玉米浆1%。其摇瓶最佳产酶条件为诱导时间12h,诱导前适宜的增殖时间为16h,接种量10%,转速220r/min,发酵pH值为6。在此基础上进行了50L发酵罐发酵中试,50L发酵罐诱导产酶高达125U。     

十二碳二元酸生产菌培养基碳源与氮源的优化

以热带假丝酵母菌(Candida tropicalis)为试验菌株,在摇瓶发酵培养基础上,对种子培养基及发酵产酸培养基的碳源、氮源进行优化。结果表明:种子培养基最适碳源为葡萄糖,最适氮源为酵母浸粉FM902;菌体产酸培养基最适碳源为蔗糖(w)2%、葡萄糖(w)0.5%,比仅有2%蔗糖的培养基产酸量提高14.5%;最适氮源为0.08%(w)酵母浸粉FM902,比只含酵母浸膏LM902的培养基产酸量提高35.3%。     

一株产2-酮基-D-葡萄糖酸菌的筛选鉴定及其发酵优化

以(NH4)2SO4为底物氮源,采用平板变色圈法从土壤中分离筛选出一株2-酮基-D-葡萄糖酸生产菌株Serratia sp. FMME043,对其碳源、氮源、无机源和摇瓶类型等产酸条件进行优化,确定以(NH4)2SO4为氮源的摇瓶产酸最佳条件为葡萄糖180 g/L, (NH4)2SO4 2.0 g/L, KH2PO4 1 g/L,在初始pH 7.0及750 mL双刺摇瓶装液量10%、培养温度30℃、摇床转速200 r/min条件下发酵48 h,2-酮基-D-葡萄糖酸产量达169.5 g/L,得率为0.87 mol/mol,并在7 L发酵罐中进行了验证.     

微生物发酵法1,3-丙二醇克雷伯氏菌的诱变育种

克雷伯氏菌是1,3-丙二醇(1,3-PD)生产菌。以产酸克雷伯氏菌2-1为出发菌株,经硫酸二乙酯(DES)诱变处理,运用质子自杀法选育,从含0.2 mol/L NaBr-NaBrO3的初筛平板上选出48株单菌落,然后结合培养基优化后的摇瓶发酵复筛,获得6个产酸突变株,其中Y-37具有较高的1,3-丙二醇转化率。经过初筛、复筛和传代实验,表明其是稳定的突变株。对菌株Y-37在自动发酵罐上进行批式发酵,结果显示:突变株产酸量大幅降低,而1,3-丙二醇的产量则显著增加。Y-37的1,3-丙二醇产量由出发菌株的10.67g/L升至18.23g/L。     

响应面法优化壳聚糖酶产生菌Mitsuaria sp.K1的产酶发酵条件

壳聚糖（chitosan）及其降解产物因具有优良的物理特性和生物活性而被广泛关注。通过平板透明圈初筛、摇瓶复筛方法,从青岛海岸土壤中分离筛选到1株产壳聚糖酶活性较高的细菌Mitsuaria sp.K1,并对其产酶发酵条件进行了单因素试验和响应面优化分析试验。结果表明：在最适培养基组成（1%粉末壳聚糖、0.5%硝酸钾、0.22%KH2PO4、0.1%Na2HPO4、0.15%KCl、0.05%MgSO4?7H2O）和最佳培养条件（培养温度25.2 ℃,培养时间25.4 h,起始pH值6.5,接种量3%,装液量100 mL/500 mL摇瓶,160 r/min）下,Mitsuaria sp.K1的发酵粗酶液最高酶活平均达11.56 U/mL,比优化前的2.17 U/mL提高了4.32倍。与前人研究结果相比,该菌发酵产酶温度降低了5～10 ℃,产酶周期缩短了23～47 h,因此具有工业发酵应用价值。     

海洋微生物溶菌酶的发酵优化与中试生产

以海洋细菌S-12-86为试验菌株,采用摇瓶发酵优化的方式,研究培养基组分(碳源、氮源、碳源与氮源的比例、金属离子)与发酵条件(培养温度、接种体积分数、装液体积分数、起始pH值、产酶周期)对海洋微生物溶菌酶产量的影响,并进行中试放大试验。结果表明:该菌产酶最佳培养基组分为:葡萄糖10 g/L,蛋白胨5 g/L,MgSO45 g/L,CaCl22 g/L;最适发酵培养温度为30℃,接种体积分数为4.0%,装液体积分数为10.0%,起始pH值为8.0,发酵周期24 h。海洋细菌S-12-86发酵优化后的产酶量(25636.8 U/mL)较优化前的产酶量(14454.4 U/mL)提高了75.4%。海洋微生物溶菌酶中试发酵的产酶量达26697.87 U/mL。说明摇瓶发酵优化条件可以应用于海洋微生物溶菌酶中试生产上。     

美伐他汀产生菌的原生质体诱变育种

以美伐他汀产生菌SIPI-8915为出发菌,研究了影响原生质体形成和再生的各种因素并选出最佳条件,其再生频率达到23.5%;并对原生质体进行紫外诱变育种,筛选到变异株.该菌株摇瓶发酵效价比对照提高30%,传代稳定好.     

一株污泥减量菌喷雾干燥条件的优化

使用摇瓶发酵制备解淀粉芽孢杆菌,喷雾干燥将其制备成菌粉。采用单因子实验首先对影响摇瓶发酵制备解淀粉芽孢杆菌的条件因素进行优化,然后采用单因子实验和正交实验,对影响喷雾干燥产物指标的因素进行优化。优化后的摇瓶发酵条件为：5g/L可溶性淀粉、10g/L氮源（酵母粉与蛋白胨体积比为2：1）、接种量7%、摇瓶装液量40%,摇床200r/min、30℃条件下摇瓶发酵48h,菌液活菌数为8.3×10⁸CFU/mL,优于优化前的3.8×10⁸CFU/mL。优化后的操作条件为：进风温度180℃、热风流量315m³/h、进样速度500mL/h、麦芽糊精质量分数5%。各因素对其喷雾干燥工艺的影响程度为：麦芽糊精浓度 > 进料速度 > 进风温度 > 空气流量。在此条件下,解淀粉芽孢杆菌菌粉活菌数可达1.28×10¹⁰CFU/g。     

Effective microbial production of poly(4‐hydroxybutyrate) homopolymer by Ralstonia eutropha H16

The effective microbial production of copolyesters of 3‐hydroxybutyrate (3HB) and 4‐hydroxybutyrate (4HB) with high mole fractions of 4HB units by a wild‐type strain of Ralstonia eutropha H16 was investigated in culture solutions containing 4‐hydroxybutyric acid (4HBA) and various carbon substrates in the presence of a nitrogen source such as ammonium sulfate. The addition of glucose or acetic acid to the culture solution containing 4HBA in the presence of ammonium sulfate resulted in the production of random copolymers of P(3HB‐co‐4HB) with compositions of up to 82 mol% 4HB, but the yield of copolymers was less than 7 wt% of dried cell weights. In contrast, when n‐alkanoic acids such as propionic acid, butyric acid, valeric acid and hexanoic acid, being subject to β‐oxidation metabolism in the cell, were used as the co‐substrates of 4HBA in the presence of ammonium sulfate, a mixture of copolymers with two different 4HB compositions was produced, and copolyesters with compositions of 93–100 mol% 4HB were isolated from chloroform–n‐hexane insoluble fractions in the mixture of copolymers. Especially, when this wild‐type Ralstonia eutropha H16 was cultivated in a medium containing 4HBA (15 g litre⁻¹), propionic acid (5 g litre⁻¹) and ammonium sulfate (5 g litre⁻¹), namely C/N (mol/mol) = 10, the P(4HB) homopolymer was produced at maximally 34 wt% of dry cell weight (7.8 g litre⁻¹), and the conversion yield of 4HBA to P(4HB) homopolymer resulted in values as high as 21 mol%. © 1999 Society of Chemical Industry     

Effective biosynthesis of poly(3‐hydoxy‐ butyrate‐co‐4‐hydroxybutyrate) with high 4‐hydroxybutyrate fractions by Wautersia eutropha in the presence of α‐amino acids

BACKGROUND: Biopolymers produced by microbes are in demand as their biodegradable and biocompatible properties make them suitable for disposable products and for potential use as biomaterials for medical applications. The effective microbial production of copolyesters of 3‐hydroxybutyrate (3HB) and 4‐hydroxybutyrate(4HB) with high molar fractions of 4HB unit by a wild‐type Wautersia eutropha H16 was investigated in culture media containing 4‐hydroxybutyric acid (4HBA) and different carbon substrates in the presence of various α‐amino acids. RESULTS: The addition of carbon sources such as glucose, fructose and acetic acid to the culture medium containing 4HBA in the presence of α‐amino acids resulted in the production of random poly(3HB‐co‐4HB) with compositions of up to 77 mol% 4HB unit, but the yields of copolyesters with 60–77 mol% 4HB units were less than 15 wt% of dried cell weights. In contrast, when carbon sources such as propionic acid and butyric acid were used as the co‐substrates of 4HBA in the presence of α‐amino acids, poly(3HB‐co‐4HB) copolyesters with compositions of 72–86 mol% 4HB were produced at maximally 47.2 wt% of dried cell weight (11.3 g L^?1) and the molar conversion yield of 4HBA to 4HB fraction in copolyesters was as high as 31.4 mol%. Further, poly(3HB‐co‐4HB) copolyesters with compositions of 93–96 mol% 4HB were isolated at up to 35.2 wt% of dried cell weights by fractionation of the above copolymers with chloroform/n‐hexane. CONCLUSION: The productivity of copolyesters with over 80 mol% 4HB fractions was as high as 0.146 g L^?1 h^?1 (3.51 g L^?1 for 24 h) by flask batch cultivation. Copyright © 2007 Society of Chemical Industry     

Effects of supplementation of succinic acid on the production and molecular weight distribution of exopolysaccharides by Antrodia camphorata in batch cultures

The effects of organic acid supplementation on both yields and molecular weight distributions of exopolysaccharide (EPS) of Antrodia camphorata were investigated in shaker flasks and air‐lift bioreactors. In the shaker flask study, five out of six organic acid‐supplemented cultures showed negative effects on cell growth, the exception being pyruvic acid‐supplemented culture; lower number average molecular weights (Mn) of EPS were obtained in all the supplemented cultures. EPS production was enhanced by 31% due to the addition of succinic acid. Optimum product yield was obtained between 2.0 and 3.0 g dm^?3 succinic acid; however, the specific production of EPS increased monotonically as succinic acid concentration was increased from 0 to 5 g dm^?3. Enhancement of EPS yield by 28% and a higher Mn of EPS (around 310 kDa) due to the addition of succinic acid were also demonstrated in an air‐lift bioreactor. In addition, a novel fermentation process resistant to EPS degradation is proposed, based on the inhibition of β‐glucanase activity by the supplementation with succinic acid. Copyright © 2004 Society of Chemical Industry     

微波诱变选育纤维素酶高产木霉

利用微波诱变的方法,对一株产纤维素酶的木霉进行了改造,提高了其对纤维素的降解能力。通过微波诱变筛选出5株纤维素酶高产菌株,其在摇瓶试验中的平均纤维素酶活力为野生型菌株的1.35倍,其中酶活力最高的菌株MW6,其纤维素酶产量为野生型菌株的1.4倍。固体发酵试验结果显示野生型菌株及诱变后的突变菌株在固体发酵中的酶活力均略低于摇瓶培养的酶活力,酶活力提高最快的菌株MW6,其固体培养的酶活力与液体摇瓶培养的差距最小。     

通过抑制酿酒酵母乙醇发酵中的甘油产率提高乙醇产率

In ethanol fermentation of Saccharomyces cerevisiae （S. cerevisiae）, glycerol is one of the main by-products. The purpose of this investigation was to increase ethanol yield through minimizing glycerol yield by using mutants in which FPS1 encoding a channel protein that mediates glycerol export and GPD2 encoding one of glycerol-3-phosphate dehydrogenase were knocked-out using one-step gene replacement. GLT1 and GLN1 that encode glutamate synthase and glutamine synth.etase, respectively,were overexpressed using two-step gene replacment in fpsl△gpd2△ mutant.The fermentation properties of ZAL69（fpsl△：：LEU2 gpd2△：：URA3） and ZAL808 （fps1△：：LEU2 gpd2△：：URA3 PPGK1-GLT1 PPGK1-GLN1） under microaerobic conditions were investigated and compared with those of wild type（DC124）. Consumption of glucose, yield of ethanol, yield of glycerol, acetic acid, and pyruvic acid were monitored. Compared with wild type, the ethanol yield of ZAL69 and ZAL808 were improved by. 13.17% and 6.66 %, respectively, whereas glycerol yield decreased by 37.4 % and 41.7 %. Meanwhile, acetic acia yield and pyruvic acid yield aecreasea aramatlcally comparea to wild type. Our results indicate that FPS1 and GPD2 deletion of S. cerevisiae resulted in reduced glycerol yield and increased ethanol yield, but simultaneous overexpression of GLT1 and GLN1 infps1△gpd2△ mutant did not have a higher ethanol yield thanfps1△gpd2△ mutant.     

磷脂酶D的紫外诱变选育及发酵条件的优化

磷脂酶D在催化磷脂酰基交换反应中具有重要的应用价值。本文对产磷脂酶D野生链霉菌株进行紫外诱变,筛选得到一株产酶活力提高42.5%的变异株。通过摇瓶培养,对发酵条件进行探究。确定的最佳培养基组成为：葡萄糖10.0 g/L,牛肉膏和蛋白胨各5.0 g/L,MgSO4?7H2O 1.0 g/L,CaCl2 3.0 g/L,NaCl 2.0 g/L;表面活性剂Tween80对产酶有促进作用,其适宜浓度为0.60 g/L。在上述条件下,摇瓶发酵产酶活力达3.23 U/mL。     

采用紫外线和微波处理法选育万古霉素高产菌株

以东方拟无枝酸菌（Amycolatopsisoriental）V30为出发菌株,经过紫外线、微波复合诱变,选育得到万古霉素高产菌株V311-04,较出发菌摇瓶单位提高7%以上,并且有良好的遗传稳定性。     

Glucobacter oxydans全细胞直接催化秸秆稀酸预处理水解液产木糖酸

以长期驯化选育得到的耐抑制物氧化葡萄糖酸杆菌Glucobacter oxydans NL71为催化菌株,对全细胞直接催化木质纤维(麦秆)稀酸预处理水解液产木糖酸的工艺进行研究。结果表明:未脱毒水解液对催化反应生产木糖酸具有抑制作用,且随浓度提高,抑制作用增强。提高细胞浓度和提高供氧能力可以有效抵抗麦杆稀水解液的抑制效应。在摇瓶体系中以2 g/L细胞接种量直接催化未经脱毒的麦秆稀酸水解浓缩液,当初始木糖质量浓度超过100 g/L时,细胞的催化性能受到严重抑制,木糖酸得率均低于50%;当细胞接种量提升至8 g/L时,木糖酸得率可以达到85.7%;在机械搅拌式通氧加压反应体系中,采用密封加氧技术对100 g/L木糖浓度的麦秸稀酸水解浓缩液全细胞催化24 h的细胞接种量8 g/L,木糖酸终质量浓度达到103.1 g/L,木糖酸得率可达到93.1%,产生速率为摇瓶体系的1.6倍。     

产(R)-α-基苯乙酸的芽抱杆菌B26的鉴定与诱变育种(英文)

A bacterium strain B26 capable of producing(R)-α-hydroxyphenylacetic acid [(R)-HPA](yield,47.5%;enantiomeric excess,99.1%) from phenylglyoxylic acid(PGA) with high optical purity was isolated and identified as Bacillus sp.B26 by 16S rDNA(ribosomal DNA) sequencing.Phylogenic analysis showed that the strain was most similar to Bacillus sp.enrichment culture clone SYW5(FJ601635.1) and Bacillus cereus strain FM-4(EU794727.1).Efforts were made to further improve HPA-production and PGA-tolerance by UV irradiation and UV-LiCl cooperative mutagenesis.Among viable mutants,B.sp.UV-38 and B.sp.ULi-11 exhibited better productivities than the wild type.Comparisons of HPA production and time course among wild strain and two mutants showed that B.sp.ULi-11 was more competent than B.sp.UV-38.HPA production was increased by 39.1% with B.sp.ULi-11(yield,65.4%) compared to that with B.sp.B26(yield,47.0%) when cultured in fermentation broth(pH 7.2) at 32℃ with an agitation speed of 180 r·min-1 and PGA final concentration of 15 mmol·L-1 for 25 h.