Optimization of fermentation medium for xylanase-producing strain Xw2

To improve the fermentation yield of xylanase by optimizing the fermentation conditions for strain Xw2, a Plackett-Burman design was used to evaluate the effects of eight variables on xylanase production by strain Xw2. The steepest ascent （descent） method was used to approach the optimal response surface experimental area. The optimal fermentation conditions were obtained by central composite design and response surface analysis. The results showed that the composition of the optimal fermentation medium was corn cob ＋ 1.5% wheat bran （1：1）, 0.04% MnSO4, 0.04% K2HPO4. 3H2O, and an inoculum size of 6% in 50 mL liquid volume （pH = 6.0）. The optimal culture conditions were 28oc at 150 r/min for 54.23 h. The results of this study can serve as the basis for the industrial production and application of xylanase.     

Prokaryotic expression, purification and characterization of nattokinase

Nattokinase is a fibrinolytic enzyme that is considered to be a promising agent for thrombosis therapy. In this study, nattokinase was purified from fermentation broth of a Bacillus subtilis strain by ammonium sulfate salting-out, gel filtration chromatography, and hydrophobic interaction chromatography with a purification fold of 5.2 and at a yield of 46.3%. The purified enzyme has molecular mass of 28 kDa and fibrinolytic activity of 4 580 U/mg. Since the concentration of nattokinase on fermentation broth was quite low, we cloned nattokinase gene from B. subtilis and expressed it in E. coli BL21 (DE3). Nattokinase was actively expressed in the recombinant strain. The yield of nattokinase was increased significantly, but the activity of the protein produced by recombinant strain was low.     

液蜡发酵制取混合二元酸的研究

A mutant of Candida tropicalis FYD-2 was obtained from its parental strain SFP-1186 by ultraviolet treatments.On shaking flask,the yield of mixed dicarboxylic acid(DCA) by the mutant was 21.4% higher than that by its ancestor.The amount of mixed DCA reached 156g/L for 120h incubation in a 10 L autoconrolled fermentor where the culture medium contained 25% n-paraffin.The process of induced and screening mutant was introduced and the time course of fermentation in 10 L fermentor was discussed.     

Evaluation of Medium Components by Plackett-Burman Statistical Design for Lipase Production by Candida rugosa and Kinetic Modeling

Lipase production by Candida rugosa was carried out in submerged fermentation. Plackett-Burman statistical experimental design was applied to evaluate the fermentation medium components. The effect of twelve medium components was studied in sixteen experimental trials. Glucose, olive oil, peptone and FeCl3?6H2O were found to have more significance on lipase production by Candida rugosa. Maximum lipase activity of 3.8 u mL-1 was obtained at 50 h of fermentation period. The fermentation was carried out at optimized temperature of 30oC, initial pH of 6.8 and shaking speed of 120 r/min. Unstructured kinetic models were used to simulate the experimental data. Logistic model, Luedeking-Piret model and modified Luedeking-Piret model were found suitable to efficiently predict the cell mass, lipase production and glucose consumption respectively with high determination coefficient(R2). From the estimated values of the Luedeking-Piret kinetic model parameters, α and β, it was found that the lipase production by Candida rugosa is growth associated.     

Evaluation of Medium Components by Plackett-Burman Statistical Design for Lipase Production by Candida rugosa and Kinetic Modeling

Lipase production by Candida rugosa was carried out in submerged fermentation. Plackett-Burman statistical experimental design was applied to evaluate the fermentation medium components. The effect of twelve medium components was studied in sixteen experimental trials. Glucose, olive oil, peptone and FeCl3?6H2O were found to have more significance on lipase production by Candida rugosa. Maximum lipase activity of 3.8 u mL-1 was obtained at 50 h of fermentation period. The fermentation was carried out at optimized temperature of 30oC, initial pH of 6.8 and shaking speed of 120 r/min. Unstructured kinetic models were used to simulate the experimental data. Logistic model, Luedeking-Piret model and modified Luedeking-Piret model were found suitable to efficiently predict the cell mass, lipase production and glucose consumption respectively with high determination coefficient(R2). From the estimated values of the Luedeking-Piret kinetic model parameters, α and β, it was found that the lipase production by Candida rugosa is growth associated.     

Lactic acid production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing a <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> lactate dehydrogenase gene

This work demonstrates the first example of a fungal lactate dehydrogenase (LDH) expressed in yeast. A L(+)-LDH gene, ldhA, from the filamentous fungus Rhizopus oryzae was modified to be expressed under control of the Saccharomyces cerevisiae adh1 promoter and terminator and then placed in a 2μ-containing yeast-replicating plasmid. The resulting construct, pLdhA68X, was transformed and tested by fermentation analyses in haploid and diploid yeast containing similar genetic backgrounds. Both recombinant strains utilized 92 g glucose/l in approximately 30 h. The diploid isolate accumulated approximately 40% more lactic acid with a final concentration of 38 g lactic acid/l and a yield of 0.44 g lactic acid/g glucose. The optimal pH for lactic acid production by the diploid strain was pH 5. LDH activity in this strain remained relatively constant at 1.5 units/mg protein throughout the fermentation. The majority of carbon was still diverted to the ethanol fermentation pathway, as indicated by ethanol yields between 0.25–0.33 g/g glucose. S. cerevisiae mutants impaired in ethanol production were transformed with pLdhA68X in an attempt to increase the lactic acid yield by minimizing the conversion of pyruvate to ethanol. Mutants with diminished pyruvate decarboxylase activity and mutants with disrupted alcohol dehydrogenase activity did result in transformants with diminished ethanol production. However, the efficiency of lactic acid production also decreased. Electronic Publication     

甾体1，4-脱氢和11α-羟基化反应的两种不同微生物转化

Two kinds of micro-organism, Arthrobacter sp. AX86(1,4-dehdrogenator)and Absidia sp. A28(11α-hydroxylator) were used in this experiment. Two different fermentation techniques were performed to accomplish the multiple conversional reactions for producing 16β-methyl-11α, 17α, 21-trihydroxy-1,4-pregnadiene-3,20-dione (Ⅲ) from 16β-methyl-3β, 17α,21-trihydroxy-5α-pregnane-20-one-21-acetate(1) 1)To produce product(Ⅲ)by means of a two-step fermentation method which were independently performed first by Arthrobacter and next by Abslaia, and 2)the product was obtained by a sequential fermentation system of aforesaid two micro-organisms in a single fermentor without isolation of the intermediates from the mixture. Our results showed that in both fermentation systems high yield of product was obtained. However, according to the technical simplicity, shorter duration of fermentation cycle and efficient yield of product, the second method is better than the first one.     

Effect of nonionic surfactants on Rhizopus homothallicus lipase activity: a comparative kinetic study

Based on amino-terminal sequencing and mass spectrometry data on the Rhizopus homothallicus lipase extracted using solid (SSF) and submerged state fermentation (SmF) methods, we previously established that the two enzymes were identical. Differences were observed, however, in terms of the specific activity of these lipases and their inhibition by diethyl p-nitrophenyl phosphate (E600). The specific activity of the SSF lipase (10,700 μmol/min/mg) was found to be 1.2-fold that of SmF lipase (8600 μmol/min/mg). These differences might be the result of residual Triton X-100 molecules interacting with the SSF lipase. To check this hypothesis, the SmF lipase was incubated with submicellar concentrations of Triton X-100. The specific activity of the lipase increased after this treatment, reaching similar values to those measured with the SSF lipase. Preincubating SSF and SmF lipases with E600 at a molar excess of 100 for 1 h resulted in 80% and 60% enzyme inhibition levels, respectively. When the SmF lipase was preincubated with Triton X-100 for 1 h at a concentration 100 times lower than the Trition X-100 critical micellar concentration, the inhibition of the lipase by E600 increased from 60% to 80%. These results suggest that residual detergent monomers interacting with the enzyme may after the kinetic properties of the Rh. homothallicus lipase.     

Secretion expression of recombinant glucagon in Escherichia coli

A novel approach for the preparation of recombinant human glucagon was described. An expression vector pAGluT, containing phoA promoter, phoA signal peptide and glucagon gene, was constructed by means of genetic engineering. Escherichia coli strain YK537 was transformed with pAGluT. High-level secretory expression of recombinant human glucagon was achieved. The expression yield of recombinant human glucagon was found to be 80 mg/L, approximately 30% of the total proteins in supernatant. The biological activities and the physicochemical properties of the purified recombinant human glucagon were found to be the same as that of native glucagon. In addition, our results suggested that phoA expression system may be suitable for the expression of other small peptides.     

酵母菌SH2发酵产物增强干扰素抗病毒活性 及其有效成分的初步研究

The fermentation broth of yeast strain SH-2 (FSH-2) which could enhance the biological potency of human interferon alpha (huIFN-α) was detected. Its enhancing ratio was 1.64～6.86 fold. A group of proteins was seperated and purified from the fermentation supernatant by chromatography on Sephadex G-75 and HPLC. Each protein showed the similar band on PAGE and SDS-PAGE. Their molecular weights ranged from 52 to 72 kD. The proteins were stainded with periodic acid Schiff's agent. Lowry's method and sulfuric acid-phenol method were respectively used to determine the contents of proteins and neutral sugars. The results showed the ratio of protein to sugar was 3∶1. These results indicated that the proteins were extracellular glycoproteins (YEGPs) secreted by yeast strain SH-2. YEGPs could significantly enhance the biological potency of huIFN-α. It was veritied by the experiment of cytopathic effect inhibition in Wish cells. The enhancing ratios were 1.6～2.8 fold and 1.4～4.0 fold, respectively. The enhancing ratio of the elution protein of FSH-2 seperated by Sephadex G-75 was 2.01～5.68 fold. YEGPs alone could not enhance the potency of huIFN-α and had no biological activity as IFNs.     

Culture condition improvement for whole-cell lipase production in submerged fermentation by Rhizopus chinensis using statistical method

Rhizopus chinensis CCTCC M201021 was a versatile strain capable of producing whole-cell lipase with synthetic activity in submerged fermentation. In order to improve the production of whole-cell lipase and study the culture conditions systematically, the combination of taguchi method and response surface methodology was performed. Taguchi method was used for the initial optimization, and eight factors viz., maltose, olive oil, peptone, K2HPO4, agitation, inoculum size, fermentation volume and pH were selected for this study. The whole-cell lipase activity yield was two times higher than the control experiment under initial optimal conditions, and four significant factors (inoculum, olive oil, fermentation volume and peptone) were selected to test the effect on the lipase production using response surface methodology. The optimal fermentation parameters for enhanced whole-cell lipase yield were found to be: inoculum 4.25 x 10(8) spores/L, olive oil 2.367% (w/v), fermentation volume 18 mL/250 mL flask, peptone 4.06% (w/v). Subsequent experimental trails confirmed the validity of the model. These optimal culture conditions in the shake flask led to a lipase yield of 13875 U/L, which 120% increased compare with the non-optimized conditions.     

费希尔曲霉脂肪酶在毕赤酵母中的优化表达及高密度发酵

【背景】脂肪酶广泛应用于纺织、食品、药品、皮革等工业领域,其在微生物中的异源表达研究进一步促进了脂肪酶产品的生产和应用。【目的】实现来源于费希尔曲霉的脂肪酶在毕赤酵母中的高效异源表达,探究其合适的表达及发酵条件,提高产量,降低成本。【方法】对费希尔曲霉的脂肪酶编码基因进行密码子优化后,应用pPIC9k质粒整合到毕赤酵母GS115基因组上,构建高产脂肪酶Lip605的毕赤酵母工程菌;并通过响应面发酵条件优化、筛选最适伴侣蛋白和高密度发酵相结合的方法,综合提高脂肪酶表达量。【结果】确定高产脂肪酶毕赤酵母工程菌的最优摇瓶发酵产酶条件为:甲醇3.103%(体积比),生物素0.4 mg/L,酵母粉11.5 g/L,酵母基础氮源培养基(yeast nitrogen base,YNB) 13.4 g/L,初始pH 6.4,装液量50 mL/250 mL,转速220 r/min,温度24°C,培养时间40 h。优化后的胞外脂肪酶酶活达到72.34 U/mL,较优化前提高了5.8倍;进一步选择12个伴侣蛋白分别与脂肪酶Lip605进行共表达,其中共表达伴侣蛋白Rpl10(pPICZA-RPL10)效果最佳,可使Lip605表达量进一步提高46.8%;在此基础上,经过10 L发酵罐分批补料的高密度发酵,工程菌株发酵142 h,胞外脂肪酶酶活最高达到680 U/mL,蛋白浓度为15.89 g/L。【结论】应用复合策略有效提高了脂肪酶Lip605在毕赤酵母中的发酵产量,为其进一步工业化生产奠定了良好的基础。     

Use of evolutionary operation (EVOP) factorial design technique to develop a bioprocess using grease waste as a substrate for lipase production

The aim of the present work was to develop a bioprocess using EVOP-factorial design technique employing grease waste as a substrate for the production of lipase. A newly isolated fungal strain of Penicillium chrysogenum was explored for the fermentation process. Solid-state fermentation (SSF) was carried out using grease waste and Czapek-dox medium, supplemented with wheat bran. The yield of lipase was 38 U/ml when SSF was carried out at 32 °C for 8 days and grease:wheat bran:Czapek-dox media in 1:1:2 (w/w/v). Different physicochemical parameters affecting the production of lipase were optimized through evolutionary operation (EVOP) factorial design technique and after optimization yield was enhanced up to 46 U/ml at 30 °C, pH 7.0 with 1:1:2 (w/w/v) grease waste:wheat bran:Czapek-dox media. Industrial grease waste has never been reported before for the production of industrially important lipase enzyme.     

Production, properties and application to nonaqueous enzymatic catalysis of lipase from a newly isolated Pseudomonas strain

A potent bacterium for lipase production was isolated from soil and identified as Pseudomonas species. It produced lipase constitutively. A mutant of this strain with a lipase productivity 3.25-fold higher was obtained by treatment with ultraviolet (UV) and nitrosoguanidine (NTG). Its fermentation condition was optimized to a lipase yield of 87.5 U/ml. The lipase had maximum activity at pH 9.0 and 45 degrees C. It was stable at pHs from 7.0 to 11.0 and below 60 degrees C. The effects of metal ions, surfactants and bile salts were also studied. The lipase was 1,3-specific. In organic solvents, the thermal stability of the lipase was significantly enhanced. Its optimum temperature was also slightly increased. The optimum water activity was found between 0.5 and 0.6. The lipase was successfully applied in organic phase to catalyze the glycerolysis of palm oil for monoglyceride (MG) production, and the enantioselective esterification of (R,S)-2-octanol. The enantioselectivity of the lipase could be enhanced substantially by treatment with an amphipathic.     

Production of a microbial lipase by Staphylococcus carnosus (pLipMut2) in a bubble column reactor and a centrifugal field bioreactor

Extracellular lipase production by the recombinant strain Staphylococcus carnosus (pLipMut2) has been studied. First substrate optimization was carried out in shaken cultures. As a result, the best substrate yield of 20 units/g (peptone + yeast extract) and maximum lipase activity in the culture supernatant of 1.7 units/cm³ could be obtained by a nutrient rich complex medium consisting of 75 kg/m³ yeast extract, 15 kg/m³ tryptone, 5 kg/m³ glucose and 0.5 kg/m³ K₂HPO₄. Higher initial substrate concentration caused inhibition of growth. Antifoam agent at higher levels than 1 cm³/ dm³ resulted in a negative influence on lipase yield. Comparative fermentation studies have been carried out in a bubble column reactor and in a centrifugal field bioreactor. Direct proportionality between growth, lipase production and oxygen consumption was observed. In the bubble column reactor usual superficial air velocities (4 cm/s) caused intensive foam generation, thus fermentation was only possible after installation of a broader column head to allow coalescence. In the centrifugal field bioreactor higher productivities were obtained without foam problems at superficial gas velocities which were one order of magnitude lower than in the bubble column. Fermentations have been performed batchwise and without holding pH constant. Neither pH control nor glucose feeding could improve the substrate yield further. Compared to former fermentation studies with the strain S. carnosus (pLipPS1) lipase yield (lipase activity/cell density) could be improved by 300% and substrate yield (lipase activity/substrate concentration) by 600%.     

Production of protease and lipase by solvent tolerant Pseudomonas aeruginosa PseA in solid-state fermentation using Jatropha curcas seed cake as substrate

Deoiled Jatropha seed cake was assessed for its suitability as substrate for enzyme production by solid-state fermentation (SSF). Solvent tolerant Pseudomonas aeruginosa PseA strain previously reported by us was used for fermentation. The seed cake supported good bacterial growth and enzyme production (protease, 1818 U/g of substrate and lipase, 625 U/g of substrate) as evident by its chemical composition. Maximum protease and lipase production was observed at 50% substrate moisture, a growth period of 72 and 120 h, and a substrate pH of 6.0 and 7.0, respectively. Enrichment with maltose as carbon source increased protease and lipase production by 6.3- and 1.6-fold, respectively. Nitrogen supplementation with peptone for protease and NaNO(3) for lipase production also enhanced the enzyme yield reaching 11,376 U protease activity and 1084 U lipase activity per gram of Jatropha seed cake. These results demonstrated viable approach for utilization of this huge biomass by solid-state fermentation for the production of industrial enzymes. This offers significant benefit due to low cost and abundant availability of cake during biodiesel production.     

Lipase Expression in Pseudomonas alcaligenes Is Under the Control of a Two-Component Regulatory System

Preliminary observations in a large-scale fermentation process suggested that the lipase expression of Pseudomonas alcaligenes can be switched on by the addition of certain medium components, such as soybean oil. In an attempt to elucidate the mechanism of induction of lipase expression, we have set up a search method for genes controlling lipase expression by use of a cosmid library containing fragments of P. alcaligenes genomic DNA. A screen for lipase hyperproduction resulted in the selection of multiple transformants, of which the best-producing strains comprised cosmids that shared an overlapping genomic fragment. Within this fragment, two previously unidentified genes were found and named lipQ and lipR. Their encoded proteins belong to the NtrBC family of regulators that regulate gene expression via binding to a specific upstream activator sequence (UAS). Such an NtrC-like UAS was identified in a previous study in the P. alcaligenes lipase promoter, strongly suggesting that LipR acts as a positive regulator of lipase expression. The regulating role could be confirmed by down-regulated lipase expression in a strain with an inactivated lipR gene and a threefold increase in lipase yield in a large-scale fermentation when expressing the lipQR operon from the multicopy plasmid pLAFR3. Finally, cell extracts of a LipR-overexpressing strain caused a retardation of the lipase promoter fragment in a band shift assay. Our results indicate that lipase expression in Pseudomonas alcaligenes is under the control of the LipQR two-component system.     

强化底物利用酶系表达,提升地衣芽孢杆菌生产碱性蛋白酶性能

地衣芽孢杆菌2709由于易于培养、GRAS状态和完善的蛋白质分泌能力,是已经投入工业生产碱性蛋白酶的菌株.为改善该菌株的发酵生产性能,提高菌体对培养基成分的利用和碱性蛋白酶产量,对菌株的胞外分泌酶系进行完善.利用同源重组机制,在基因组复制起始位点附近引入了来源于短小芽孢杆菌的木聚糖酶基因xynA和在复制起始位点中心对称...     

产低温脂肪酶菌株CZW001发酵培养基优化研究

【目的】研究产低温脂肪酶菌株CZW001发酵培养基。【方法】在单因素试验的基础上, 采用Plackett-Burman (P-B)设计, Box-Behnken (B-B)设计和响应面试验设计(RSM), 在20 °C、pH 8.0、160?r/min发酵2 d条件下, 对发酵培养基进行优化。【结果】该菌株最适产酶培养基为(g/L): 葡萄糖7.68, 橄榄油21.93, 硫酸铵2.0, 磷酸二氢钾1.0, 硫酸镁0.27, 氯化钙0.3, 氯化钠20.0, 吐温-80 1.0。其最高酶活为62.8 U/mL, 比优化前提高了3.14倍。【结论】通过对产低温脂肪酶菌株CZW001发酵培养基优化研究, 明显提高低温脂肪酶活力。     

Improvement of lipase production by addition of catalase during fermentation

A batch fermentation process for lipase production with the recombinant strain Staphylococcus carnosus (pLipMut2) was studied in a bubble column. The rates of growth and lipase production in this type of fermentor were compared with results from shakeflasks. It was seen that cultivation in the bubble column resulted in a prolonged lag time and a reduced lipase activity in comparison to flask cultures. However, by addition of catalase during the fermentation in the bubble column this different behaviour could be avoided. Correspondence to: E. Wenzig