Evolutionary operation (EVOP) to optimize three-dimensional biological experiments

Evolutionary operation (EVOP) is used as an efficient technique for optimization of three variable experimental parameters using two-level factorial designs with center points. For metabolic function like enzyme synthesis by microorganisms, small amounts of metal ions, surfactants, and vitamins act as inducers. The desired quantities of these chemicals are, however, species dependent and have to be found out or each microorganism. By employing the EVOP technique, the requirements of these chemicals have been optimized for the production of protease by Rhizopus oryzae (RO, IIT KGP). (c) 1993 John Wiley & Sons, Inc.     

Evolutionary operation (EVOP) to optimize protease biosynthesis by Rhizopus oryzae

To optimize the operating conditions of any microbial process, evolutionary operation (EVOP) technique can be applied as a tool. This is very efficient to optimize the combined effect of two or three variables and their interaction on a biological process. The application of EVOP methodology to optimize the environmental conditions for any biological system has not yet been reported in the literature. In this paper firstly EVOP methodology for optimizing two-variable experiments has been outlined and then applied to optimize protease biosynthesis by Rhizopus oryzae (RO, IIT KGP).     

Production of tannase by solid-state fermentation

An attempt has been made to optimize the production of enzyme tannase by solid state fermentation (SSF) using the organism Rhizopus oryzae. The best favourable conditions for enzyme production include initial pH 5 with 4 days of incubation period at 40°C and 72% humidity, and 10 g wheat bran soaked in 2.5% tannic acid.     

Microbial transformation of tannin-rich substrate to gallic acid through co-culture method

Modified solid-state fermentation (MSSF) of tannin-rich substrate yielding tannase and gallic acid was carried out using a co-culture of the filamentous fungi, Rhizopus oryzae (RO IIT RB-13, NRRL 21498) and Aspergillus foetidus (GMRB013 MTCC 3557). Powdered fruits of Terminalia chebula and powdered pod cover of Caesalpinia digyna was used in the process and the different process parameters for maximum production of tannase and gallic acid by co-culture method were optimized through media engineering. MSSF was carried out at the optimum conditions of 30 degrees C and 80% relative humidity. The optimal pH and incubation period was 5.0 and 48 h respectively. Through the co-culture technique the maximum yield of tannase and gallic acid was found to be 41.3 U/ml and 94.8% respectively.     

Optimization of multiple inducers effect on protease biosynthesis by Rhizopus oryzae

Effect of additional chemicals at different culture conditions was studied in detail on newly isolated Rhizopus oryza (RO, IIT KGP) to maximize protease synthesis. The surfactants and metal ions when added to the basal medium individually were acted as inducers. The combined effect of these inducers on protease production was also studied in detail and a semiempirical mathematical model based on the data obtained was proposed to optimize their effects. A 2.5 fold increase in the protease production was possible by optimizing the two variable inducers system.     

Efficient and selective microbial esterification with dry mycelium of Rhizopus oryzae

The use of dry mycelium of Rhizopus oryzae as biocatalyst for ester production in organic solvent has been studied. Mycelia with notable carboxylesterase activity were produced when different Tweens (20, 40, 60 and 80) were employed as main carbon source for the growth. Dry mycelium of four strains of Rhizopus oryzae proved effective for efficiently catalysing the synthesis of different flavour esters (hexylacetate and butyrate, geranylacetate and butyrate) starting from the corresponding alcohol and free acid, including acetic acid. The esterification of the racemic mixture of 2-octanol and butyric acid proceeded with high enantioselectivity (R-ester produced with enantiomeric excess > or =97%) when Rhizopus oryzae CBS 112.07 and Rhizopus oryzae CBS 260.28 were employed.     

Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit

We have isolated a lipolytic strain from palm fruit that was identified as a Rhizopus oryzae. Culture conditions were optimized and highest lipase production amounting to 120 U/ml was achieved after 4 days of cultivation. The extracellular lipase was purified 1200-fold by ammonium sulfate precipitation, sulphopropyl-Sepharose chromatography, Sephadex G 75 gel filtration and a second sulphopropyl-Sepharose chromatography. The specific activity of the purified enzyme was 8800 U/mg. The lipolytic enzyme has a molecular mass of 32 kDa by SDS-polyacrylamide gel electrophoresis and gel filtration. The enzyme exhibited a single band in active polyacrylamide gel electrophoresis and its isoelectric point was 7.6. Analysis of Rhizopus oryzae lipase by RP-HPLC confirmed the homogeneity of the enzyme preparation. Determination of the N-terminal sequence over 19 amino acid residues showed a high homology with lipases of the same genus. The optimum pH for enzyme activity was 7.5. Lipase was stable in the pH range from 4.5 to 7.5. The optimum temperature for lipase activity was 35 degrees C and about 65% of its activity was retained after incubation at 45 degrees C for 30 min. The lipolytic enzyme was inhibited by Triton X100, SDS, and metal ions such as Fe(3+), Cu(2+), Hg(2+) and Fe(2+). Lipase activity against triolein was enhanced by sodium cholate or taurocholate. The purified lipase had a preference for the hydrolysis of saturated fatty acid chains (C(8)-C(18)) and a 1, 3-position specificity. It showed a good stability in organic solvents and especially in long chain-fatty alcohol. The enzyme poorly hydrolyzed triacylglycerols containing n-3 polyunsaturated fatty acids, and appeared as a suitable biocatalyst for selective esterification of sardine free fatty acids with hexanol as substrate. About 76% of sardine free fatty acids were esterified after 30 h reaction whereas 90% of docosahexaenoic acid (DHA) was recovered in the unesterified fatty acids.     

Kinetic studies of Rhizopus oryzae lipase using monomolecular film technique

Rhizopus oryzae lipase (ROL) was found to be a true lipase. This enzyme presents the interfacial activation phenomenon. The N-terminal amino acid sequence of ROL was compared to those of rhizopus lipases. Purified ROL possesses the same N-terminal sequence as the mature Rhizopus niveus lipase (RNL). This sequence was found in the last 28 amino acids of the propeptide sequence derived from the cDNA of Rhizopus delemar lipase (RDL). Using the baro-stat method, we have measured the hydrolysis rate of dicaprin films by ROL as a function of surface pressure. Our results show that Rhizopus oryzae lipase is markedly stereoselective of the sn-3 position of the 2,3 enantiomer of dicaprin. Polyclonal antibodies (PAB) directed against ROL have been produced and purified by immunoaffinity. The effects of these PAB on the interfacial behavior of ROL were determined. The immunoblot analysis with polyclonal antibodies anti-ROL (PAB anti-ROL) and various lipases shows a cross-immunoreactivity between the lipase from the rhizopus family (Rhizopus delemar lipase and Rhizopus arrhizus lipase).     

米根霉乙醇脱氢酶突变株的筛选及其锌镁离子的调控研究

利用亚硝基胍(NTG)对米根霉As3.3461进行诱变,在含丙烯醇0.6%的YPD平板上筛选获得21株乙醇含量降低的突变株,其中突变株HBF-12乳酸产量最高。与出发菌株相比,突变株HBF-12的乙醇产量和乙醇脱氢酶(ADH)活力分别降低了73.6%和76%,乳酸产量和乳酸脱氢酶(LDH)活力分别提高了41.2%和19.6%。研究Zn2 与Mg2 对HBF-12中ADH与LDH活性的调控,结果显示Zn2 对ADH有强烈的激活作用,但抑制LDH活性;Mg2 则轻微抑制ADH活性,促使LDH活性增强。考察两种离子影响末端产物乙醇与乳酸形成的实验说明:培养基中Zn2 浓度与乳酸积累基本上呈负相关性,与乙醇积累呈正相关性,浓度降低有利于生物量积累;Mg2 浓度增加可以促进乳酸积累和生物量增加,对于乙醇积累无明显作用。发酵培养基中添加0.01%Zn2 、0.04%Mg2 ,突变株产酸可达96.21g/L。     

Expression and characterization of fumarase (FUMR) from Rhizopus oryzae

Fumarase catalyzes the reversible hydration of fumarate to l-malate in Rhizopus oryzae. A recombinant pET22b-fumR harboring a fumarase gene (fumR) from R. oryzae was constructed for high level expression in E. coli BL21 (DE3). The FUMR activity was optimal at 30°C and pH 7.2. The enzyme was stable below 45°C and at pH 3.0-9.0. No effects of Zn(2+), Fe(2+), or EDTA were observed on enzyme activity. A slight inhibition of FUMR activity was seen with Mg(2+), while Ca(2+) had a small stimulatory effect. The K(m) for l-malic acid and fumaric acid were 0.46 mM and 3.07 mM, respectively. The activity of FUMR catalyzing hydration of fumarate to l-malate was completely inhibited by 2mM fumaric acid. The unique enzymatic properties suggested that overexpression of FUMR could enhance fumaric acid accumulation in R. oryzae.     

离子注入选育高产L-乳酸的米根霉突变株及其发酵特性研究

通过氮离子注入获得米根霉突变株RQ4012,其利用木糖的能力比出发菌株提高了1.6倍;通过多次传代,证明其具有良好的遗传稳定性。试验测定菌株RQ4012发酵木糖生产L-乳酸的最佳发酵条件:木糖10%,生理盐水浸泡孢子9 h,(NH4)2SO43 g/L,接种量4%,CaCO3添加量6%,装液量20%,温度37℃,转速200 r/min,在此条件下,乳酸产量达到79.51 g/L。对混合糖的发酵进行了探索,结果表明该菌能高效利用混合糖生产L-乳酸,在利用植物纤维素水解液生产L-乳酸上有良好的应用前景。     

Production of proteolytic enzyme by Rhizopus oryzae in a bubble column bioreactor

Production of proteolytic enzyme in a bubble column bioreactor was carried out using the microorganism Rhizopus oryzae. Optimization of the production conditions were done adopting the EVOP (evolutionary operation technique). Studies on two variable and three variable parameters were carried out. The variable parameters were air flow rate, aeration time and incubation period. 8 hours aeration time per day, 1.66 vvm air flow rate and 2 days incubation was found to be optimum.     

Purification and characterization of protease from a newly isolated Rhizopus oryzae

A newly isolated Rhizopus oryzae was found to exhibit some unusual phenomenon of secreting alkaline protease which was purified and characterized. The molecular weight was determined to be 28,600 dalton in gel electrophoresis. The enzyme is stable in the pH range from 3 to 11 and most active at pH 8. The temperature optimum of this thermostable biocatalyst is at 60 °C. The enzyme is sensitive to metal chelators, most of the metal ions (excepting a few monovalent cations) and inhibitor like PMSF. This indicates that the protease of isolated Rhizopus oryzae falls under alkaline serine group.     

Production of gallic acid by immobilization of Rhizopus oryzae

Production of gallic acid using the immobilized cells of Rhizopus oryzae has been studied. It was observed that 2% tannic acid concentration, 200 numbers of calcium alginate beads of spore concentration 2?×?10⁵/ml and initial pH 5.0 gave the maximum gallic acid production. The % of tannin conversion was 78.5% whereas in free cell culture, the % conversion was 83.5% in 4 days of incubation period. The beads were used for 3 times successfully. A drastic fall in the hydrolysis process was observed when the beads were treated with glutaraldehyde.     

利用天然纤维废弃物发酵生产L-乳酸的研究

为了降低L-乳酸的生产成本,更好的实现生物质秸秆的资源化,利用天然纤维素依次接种经离子注入诱变处理的木聚糖酶高产菌黑曲霉P602和米根霉RL6041高产菌进行固、液体二次发酵的方法,将其转化成用于工业生产的L-乳酸。结果表明:本实验条件下,未经过任何化学预处理的秸秆等物质接种黑曲霉P602进行固体发酵,产生的木聚糖酶活力为6 320 IU/g干(培养)基,纤维素酶活力为29 IU/g干基;加入100 mL水浸提后,产生的还原糖浓度为14.07 g/L,纤维物质糖化率为79.45%。取滤液接入米根霉RL6041进行液体发酵后,生成乳酸的量为7 g/L,糖酸转化率为47.6%,以(NH4)2SO4作为氮源时,最佳氮源浓度为3 g/L。     

Microbial production of gallic acid by modified solid state fermentation

Bioconversion of tannin to gallic acid from powder of teri pod (Caesalpinia digyna) cover was achieved by the locally isolated fungus, Rhizopus oryzae, in a bioreactor with a perforated float for carrying solid substrate and induced inoculum. Modified Czapek-Dox medium, put beneath the perforated float, with 2% tannic acid at pH 4.5, temperature 32°C, 93% relative humidity, incubated for 3 days with 3-day-old inoculum was optimum for the synthesis of tannase vis-à-vis gallic acid production. Conversion of tannin to gallic acid was 90.9%. Diethyl ether was used as the solvent for extraction of gallic acid from the fermented biomass. Received 14 December 1998/ Accepted in revised form 17 June 1999     

米根霉诱导因子对紫草细胞培养中紫草宁色素分泌的影响

在紫草细胞培养中,加入采根霉粗提物可显著提高紫草宁色素产量,并可加快胞内色素分泌到培养液中的速率和数量。在细胞培养的第6天加入米根霉诱导因子时,其促进紫草宁色素分泌的作用最大,培养液中紫草宁色素含量是对照的2．24倍。此外,同时加入正十六烷和米根霉诱导因子对紫草宁色素的分泌具有协同作用。     

米根霉菌ldhL基因的克隆及其在大肠杆菌中的表达

以米根霉菌基因组DNA为模板,根据GenBank上已公布的米根霉L-乳酸脱氢酶基因(ldhL)序列设计特异性引物,PCR扩增得到963 bp的DNA片段,经序列分析后将其亚克隆到原核表达载体pET30a上,构建成重组质粒pET30a-ldhL.将pET30a-ldhL转化到BL21感受态细菌中,经IPTG诱导表达后进行SDS-PAGE分析,可见约43 kD的与预期大小一致的目的蛋白条带,结果表明ldhL基因在大肠杆菌中进行了表达,经酶活分析产物的酶活力为98 U/mL,证明了表达产物具有预期的酶活性,这为进一步研究利用乳清为发酵原料高产L-乳酸的米根霉基因工程菌株奠定了基础.     

Strain improvement for tannase production from co-culture of Aspergillus foetidus and Rhizopus oryzae

Spores from the co-culture of Aspergillus foetidus and Rhizopus oryzae were subjected to UV, heat and NTG (3-nitro,5-methylguanidine) mutagenesis. A few colonies were screened from the selected media for tannase study. Amongst all, the best mutant isolated from the heat treatment (60 degrees C for 60 min) was SCPR 337. The maximum yield of gallic acid and tannase in case of mutant strain was 95.2% and 53.6 U/ml with an incubation period of 30 h as compared to wild strain where the incubation period was 48 h with an enzyme activity of 44.2 U/ml and gallic acid yield of 94%, respectively. The mutant was sensitive to tetracycline and was also an over-producer of protease and amylase.