Mutanase induction in Trichoderma harzianum by cell wall of Laetiporus sulphureus and its application for mutan removal from oral biofilms

The cell wall material from fruiting bodies of Laetiporus sulphureus has been suggested as a new alternative to mutan for the mutanase induction in Trichoderma harzianum. Structural analyses revealed that the alkali-soluble wall fraction from this polypore fungus contained 56.3% of (1-->3)-linked alpha-glucans. When the strain T. harzianum F-340 was grown on a cell wall preparation from L. sulphureus, the maximal enzyme productivity obtained after 3 days of cultivation was 0.71 U/ml. This yield was about 1.8-fold higher than that achieved on mutan, known so far as the best, but expensive and inaccessible, inducer of mutanase production. Cell-wall-induced mutanase showed a high hydrolytic potential in reaction with a dextranasepretreated mutan, where maximal degrees of saccharification and solubilization of this biopolymer (80% and 100%, respectively) were reached in 3 h at 45oC. The mutanase preparation was also effective in degradation of streptococcal mutan and its removal from oral biofilms, especially in a mixture with dextranase.     

Selection of strain and optimization of mutanase production in submerged cultures of Trichoderma harzianum

Nineteen fungal strains belonging to different genera were tested for extracellular mutanase production in shaken flasks. The optimal enzymatic activity was achieved by Trichoderma harzianum F-470, a strain for which the mutanase productivity has not yet been published. Some of factors affecting the enzyme production in shaken flasks and aerated fermenter cultures have been standardized. Mandels mineral medium with initial pH 5.3, containing 0.25% mutan and inoculated with 10% of the 48-h mycelium, was the best for enzyme production. A slight mutanolytic activity was also found when sucrose, raffinose, lactose and melibiose were carbon sources. Application of optimized medium and cultural conditions, as well as use of a fermenter with automatic pH control set at pH 6.0 enabled to obtain a high mutanase yield (0.33 U/ml, 2.5 U/mg protein) in a short time (2-3 days). The enzyme in crude state was stable over a pH range of 4.5-6.0, and at temperatures up to 35 degrees C; its maximum activity was at 40 degrees C and at pH 5.5.     

Development of a stirred tank reactor system for the production of lignin peroxidases (ligninases) byPhanerochaete chrysosporium BKM-F-1767

Summary Lignin peroxidases produced byPhanerochaete chrysosporium have several important potential industrial applications based on their ability to degrade lignin and lignin-like compounds. A stirred tank reactor system for the production of lignin peroxidases is described here. Included in this study is an examination of the mechanics of pellet biocatalyst formation and the optimization of an acetate buffered medium. Higher levels of lignin peroxidase were obtained with acetate buffer compared to the other buffer systems tested. Concentrations of 0.05% (w/v) Tween 80 and 0.4 mM veratryl alcohol gave optimal lignin peroxidase activity in acetate buffered medium. In shake flask cultures, mycelial fragments in the inoculum aggregated into pellets during the first eight hours of incubation and thereafter increased in size through the eighth day. The agitation rate in shake flask cultures affected pellet size, the number of pellets formed, and lignin peroxidase activity. Transfer of fungal pellets from shake flask culture to a continuously oxygenated baffled stirred tank reactor (STR) resulted in production of high lignin peroxidase titres comparable to those of shake flask cultures when the agitation rate, oxygen dispersion and foaming were closely controlled.     

重组巴斯德毕赤酵母产米根霉α-淀粉酶发酵条件的研究

为了提高重组菌的淀粉酶表达量,以可分泌表达米根霉α-淀粉酶的甲醇快速利用型巴斯德毕赤酵母重组菌为基础,采用摇瓶发酵方式对影响重组菌表达淀粉酶的多个因素进行了研究和优化。摇瓶发酵条件确定为:温度为30℃,pH值为6.0,接种量为2.0(OD_(600)),甲醇补加方式采用前72 h发酵时间内每隔12h添加至终浓度为1.0%,72 h以后每隔24 h添加至终浓度为1.0%,在此条件下获得的淀粉酶最高表达量为47.5 U/mL,且在无机盐培养基中和有机氮源培养基中获得的淀粉酶发酵单位相当。以摇瓶发酵数据为基础确定15 L发酵罐放大实验条件为:无机盐培养基,温度为30℃,pH值为6.0,接种量为10%,甲醇流加方式采用DO—Start法控制,在此发酵条件下获得的淀粉酶表达量为440 U/mL,约为摇瓶发酵方式获得的淀粉酶表达量的9倍。     

Optimization of cellulase production in batch fermentation byTrichoderma reesei

Maximum cellulase production was sought by comparing the activities of the cellulases produced by differentTrichoderma reesei strains andAspergillus niger. Trichoderma reesei Rut-C30 showed higher cellulase activity than otherTrichoderma reesei strains andAspergillus niger that was isolated from soil. By optimizing the cultivation condition during shake flask culture, higher cellulase production could be achieved. The FP (filter paper) activity of 3.7 U/ml and CMCase (Carboxymethylcellulase) activity of 60 U/ml were obtained from shake flask culture. When it was grown in 2.5L fermentor, where pH and DO levels are controlled, the Enzyme activities were 133.35 U/ml (CMCase) and 11.67 U./ml (FP), respectively. Ammonium sulfate precipitation method was used to recover enzymes from fermentation broth. The dried cellulase powder showed 3074.9 U/g of CMCase activity and 166.7 U/g of FP activity with 83.5% CMCase recovery.     

L-lysine-alpha-oxidase activity of some Trichoderma species

Trichoderma cultures were tested for their ability to produce L-lysine-alpha-oxidase. The highest enzyme activity was manifested by T. harzianum (MGU), T. longibrachiatum Rifai VKM F-2025 and T. aureoviride Rifai VKM F-2026. The biosynthesis of the enzyme did not depend on the growth of the cultures and did not vary among the species.     

Genome shuffling enhanced L-lactic acid production by improving glucose tolerance of Lactobacillus rhamnosus

Genome shuffling is a powerful strategy for rapid engineering of microbial strains for desirable industrial phenotypes. Here we applied the genome shuffling to improve the glucose tolerance of Lactobacillus rhamnosus ATCC 11443 while simultaneously enhancing the L-lactic acid production. The starting population was generated by ultraviolet irradiation and nitrosoguanidine mutagenesis and then subjected for the recursive protoplast fusion. The positive colonies from library created by fusing the inactivated protoplasts were more likely to be screened on plates containing different concentrations of high glucose and 2% CaCO(3). Characterization of all mutants and wild-type strain in the shake flask indicated the compatibility of two optimal phenotypes of glucose tolerance and lactic acid enhancement. The lactic acid production, cell growth and glucose consumption of the best performing strain from the second round genome shuffled populations were 71.4%, 44.9% and 62.2% higher than those of the wild type at the initial glucose concentration of 150 g/l in the 16l bioreactor. Furthermore, the higher lactic acid concentrations were obtained when the initial glucose concentrations increased to 160 and 200 g/l in batch fermentation.     

Different functional subsets of cultured murine T cells express characteristic levels of adenosine deaminase activity

The level of adenosine deaminase (ADA) activity in mouse T-lymphocyte cultures was studied under different growth-supporting conditions and in mixed lymphocyte culture-derived long-term T-cell lines and clones. Early after the initiation of in vitro culture, the levels of ADA (2000 U/mg) were similar in bulk cultures either depleted or not depleted in Lyt-2+ T cells. Enrichment for cytolytic T lymphocytes (CTL) obtained by addition of exogenous interleukin 2 (IL-2), was accompanied by a net decrease of ADA activity (110 +/- 15 U/mg). All the tested CTL-A lines derived from such cultures were also characterized by a low or undetectable level of this enzyme (at best 160 +/- 70 U/mg) as previously observed. In contrast, "Lyt-2-" T-cell bulk cultures grown, without addition of exogenous IL-2, in the presence of gamma-irradiated H-2d stimulators maintained a constant level of ADA activity (1770 +/- 340 U/mg) for at least 3 months. Functionally distinct types of Lyt-2- T-cell lines were also analyzed: T-cell lines competent to activate B lymphocytes to growth and terminal maturation as well as others devoid of detectable functions showed a stable ADA level comparable to that expressed by the original bulk culture 1685 +/- 620 U/mg). The present results demonstrate that, like tumor cell lines, most normal T lymphocytes express a high level of ADA activity in culture, which strongly suggests that the low level of ADA activity exhibited by CTL is a characteristic of this functional subset.     

Scaleup of ajmalicine production by plant cell cultures of Catharanthus roseus

The effect of scaleup on he production of ajmalicine by a Catharanthus roseus cell suspension culture in a selected induction medium were studied. In preliminary experiments it was observed that the culture turned brown and the production was inhibited upon transfer from a shake flask to a stirred bioreactor with forced aeration. Two factors were recognized as the potential origin of the differences between shake flask and bioreactor cultures: gas composition and mechanical shear forces. These factors were studied separately.By recirculating a large part of the exhaust gas, a comparable gas regime was obtained in a bioreactor as occurred in a shake flask cultures. This resulted in the absence of browning and a similar pattern of ajmalicine production as observed in shake flasks. The effect of shear forces could not be demonstrated. However, the experiments showed that the culture may be very sensitive to liquid phase concentrations of gaseous compounds. The effects of k(L)a, aeration rate, CO(2) production rate, and influent gas phase CO(2) concentration on the liquid phase CO(2) concentration are discussed. (c) 1993 John Wiley & Sons, Inc.     

Cellulase and Xylanase Expression in Response to Different pH Levels of Penicillium echinulatum S1M29 Medium

The effect of pH on the production of cellulases and xylanases by Penicillium echinulatum S1M29 was evaluated in a shake flask and in a bioreactor. To control the pH in a shake flask, a buffer made with citric acid and disodium phosphate was used. The buffer was capable of maintaining the culture pH values for the first 48 h. In the bioreactor, the pH was controlled automatically by the addition of NaOH and H₂SO₄. In the shake flask, the highest activities of xylanases (18.5 IU/mL) and endoglucanases (8.2 IU/mL), as well as the highest filter paper activity (FPA) (0.9 IU/mL), were obtained at initial pH values of between 6.0 and 7.0. In the bioreactor, the highest activities of these enzymes were obtained in a pH range of 5.5 to 6.5. Different isoforms of the endoglucanases were found in the various cultures depending on the pH. More acidic pH ranges favored the production of β-glucosidases in both the shake flask and the bioreactor.     

麦芽糖诱导软化芽孢杆菌α-环糊精葡萄糖基转移酶在枯草杆菌中的表达

通过PCR扩增软化芽孢杆菌α-环糊精葡萄糖基转移酶基因,将基因片段克隆到大肠杆菌-枯草杆菌穿梭载体pGJ103中,转化枯草杆菌WB600得基因工程菌进行外源表达。在1.5%的麦芽糖初始发酵培养基上摇瓶培养,48 h后重组枯草杆菌产酶活性为6.1U/ml。通过单因素分析和响应面分析对重组枯草杆菌产CGT酶摇瓶发酵条件进行优化。分析得到培养基关键组分麦芽糖,玉米淀粉和酵母粉三者最佳浓度分别为:15.5g/L,13g/L和20g/L。在此条件下,摇瓶培养36h后α-CGT酶活性为17.6U/ml,5L罐分批发酵30h后酶活达到20U/ml (水解活性为1.4×10⁴ IU/ml)。     

Biofilm-specific cross-species induction of antimicrobial compounds in bacilli

An air-membrane surface (AMS) bioreactor was designed to allow bacteria to grow attached to a surface as a biofilm in contact with air. When Bacillus licheniformis strain EI-34-6, isolated from the surface of a marine alga, was grown in this reactor, cells produced antimicrobial compounds which they did not produce when they were grown in shake flask cultures. An unidentified red pigment was also produced by surface-grown cells but not by planktonically grown cells. Glycerol and ferric iron were important for the production of antimicrobial compounds and the red pigment. Release of these secondary metabolites was not due to the onset of sporulation. Cell-free spent medium recovered from beneath the reactor membrane could induce production of antimicrobial compounds and red pigment in shake flask cultures. Neither glycerol nor ferric iron was required for production of these inducer compounds. Spent medium from beneath the membrane of an AMS bioreactor culture of Bacillus subtilis strain DSM10(T) and Bacillus pumilus strain EI-25-8 could also induce production of antimicrobial compounds and a red pigment in B. licheniformis isolate EI-34-6 grown in shake flask cultures; however, the corresponding spent medium from shake flask cultures of DSM10(T) and EI-25-8 could not. These results suggest that there is a biofilm-specific cross-species signaling system which can induce planktonically grown cells to behave as if they were in a biofilm by regulating the expression of pigments and antimicrobial compounds.     

重组毕赤酵母生产β-葡萄糖苷酶发酵条件初步研究

为提高重组毕赤酵母(P.pastoris KM71/pPIC9K-bgl)生产β-葡萄糖苷酶的产量,在摇瓶条件下对重组P.pastoris产β-葡萄糖苷酶的发酵过程进行了优化,得到最佳的条件:生长阶段甘油浓度为30 g/L,接种量为10%,诱导阶段甲醇的初浓度为4%,过程补加甲醇0.5%,诱导温度30℃,pH7.5,诱导周期120 h,酶活可达到245 U/mL。在此基础上,在3 L发酵罐上进行初步放大,流加甘油提高细胞密度至OD_(600)为170,开始流加甲醇诱导,最终BGL酶活达到1 175 U/mL。比摇瓶提高了4.8倍,为β-葡萄糖苷酶工业化生产打下了坚实的基础。     

Multiple forms of endo-1,4-beta-D-glucanase in the extracellular cellulase of Penicillium pinophilum

The influence of the composition of the growth medium on the production of endo-1,4-beta-D-glucanase (CM-cellulase) activity by P. pinophilum was studied in shake flask cultures using Avicel PH101 as the carbon source. It was observed that the culture conditions had a profound effect on the level of endoglucanase (CM-cellulase) produced by P. pinophilum. However, isoelectric focusing of the endoglucanase activity obtained from shake flask and fermenter cultures using the same growth medium revealed that the enzyme system found in both cultures was identical qualitatively, and contained seven or eight different endoglucanase components. All the endoglucanase components appeared simultaneously in the early stages of culture and prolonged incubation resulted only in an increase in the concentration of these enzymes. Protease levels were found to be low in both types of culture but were particularly so in the growth medium which contained corn steep liquor. The proteases were unable to release low molecular weight peptides when P. pinophilum cellulase protein was used as a substrate. The results were interpreted to indicate that the multiplicity of endoglucanase components found in cultures of P. pinophilum is most likely the result of expression of a number of specific genes rather than by post-secretional modification of one or more endoglucanase(s) synthesized by the fungus.     

Production and biotransformation of 6-pentyl-α-pyrone by Trichoderma harzianum in two-phase culture systems

The final concentration of 6-pentyl-a-pyrone (6PP) produced in cultures of Trichoderma spp. is limited by the fact that inhibition of biomass growth occurs at 6PP concentrations as low as 100 mg/l. The aim of this work was to evaluate liquid-liquid extractive fermentation systems as an alternative to overcome the toxicity problems and to increase the production of 6PP by this fungus. Two alkanes (n-decane and n-hexadecane) and two dicarboxylic esters (dibutyl phthalate and dioctyl phthalate) were evaluated in shake flask cultures. The highest 6PP production (173 ppm) was achieved when n-hexadecane was used, being 3.5-fold the maximum 6PP concentration of a culture without the solvent. Cultivation of Trichoderma harzianum in a 10-1 bioreactor with n-hexadecane yielded 6PP production ninefold higher than that from control cultures. However, 6PP production in the bioreactor (83 ppm) was lower than in shake flasks. Differences in the power drawn to the fluid at each scale could account for such behavior. Even in the presence of the solvent, 6PP content decreased after reaching its maximal concentration.     

Meetings & Symposia

Abstract

The strain Paenibacillus curdlanolyticus MP-1 was used to obtain mutan-hydrolyzing enzymes. Different methods of precipitation and concentration of the post culture liquid were tested. All these methods produced satisfactory results in regard to the overall activity of mutanase and yielded active preparations of the enzyme. The best precipitation was obtained with propanol –98% of the initial enzyme activity was preserved with a purification of 2-fold. Salting out with ammonium sulfate at 50% saturation gave mutanase recovery of 77% and a purification of around 2-fold. Ultrafiltration yielded an about 10-fold concentrated preparation of the enzyme with a yield of 98%. Lyophilization and concentration of the culture broth (in the range from 5 to 20 times) in a vacuum evaporator yielded active crude preparations with mutanase recovery of 97%.     

Enhancement of gentamicin production by mutagenesis and non-nutritional stress conditions in Micromonospora echinospora

Gentamicin producing strain of Micromonospora echinospora was treated with chemical mutagens like EtBr and MNNG and physical mutagens such as UV was carried out to obtain a mutant with enhanced production of gentamicin. After inducing mutations screening for penicillin and kanamicin resistant mutants was done. M. echinospora EtBr-22 strain was obtained by mutations and its gentamicin production in shake flask reaches 1354 mg l⁻¹ which is 1.53-fold higher than that of the parent strain. Application of different stress conditions like heat shock, feeding high ethanol and high NaCl concentrations during fermentation has found to be effective for the increased production of gentamicin. Production of gentamicin was increased to 1.26-fold in medium supplemented with 0.6% NaCl to 48-h-old culture.     

Methods for obtaining active mutanase preparations from Paenibacillus curdlanolyticus

The strain Paenibacillus curdlanolyticus MP-1 was used to obtain mutan-hydrolyzing enzymes. Different methods of precipitation and concentration of the post culture liquid were tested. All these methods produced satisfactory results in regard to the overall activity of mutanase and yielded active preparations of the enzyme. The best precipitation was obtained with propanol -98% of the initial enzyme activity was preserved with a purification of 2-fold. Salting out with ammonium sulfate at 50% saturation gave mutanase recovery of 77% and a purification of around 2-fold. Ultrafiltration yielded an about 10-fold concentrated preparation of the enzyme with a yield of 98%. Lyophilization and concentration of the culture broth (in the range from 5 to 20 times) in a vacuum evaporator yielded active crude preparations with mutanase recovery of 97%.