链霉菌Z94-2碱性脂肪酶产生条件及酶学性质

在１５２ 株脂肪酶产生菌中,链霉菌Ｚ９４２ 产脂肪酶活力为５９６ｕ／ ｍＬ,其最适培养基（ｇ／Ｌ） 为：糊精１０ 、黄豆饼粉３０ 、尿素１０ 、Ｋ２ＨＰＯ４ ０－５ 、ＭｇＳＯ４ ０－５ 、ＮａＣｌ １ 和ＡＥＯ９ ０ ．５ ,产酶的最适条件为：初始ｐＨ９ ．５ ～１０－０ ,在２６ ℃培养４８ｈ 。用ＰＶＡ 橄榄油乳化系统测定该酶的最适ｐＨ９ ．８ ,最适温度３７ ℃,在ｐＨ８－６ ～１０－２ 于５ ℃存放２４ ｈ ,酶活力不变。０－１４ｍｏｌ／Ｌ 的氯化钙有较大的激活作用。     

链霉菌Z94-2碱性脂肪酶产生条件及酶学性质

在１５２株脂肪酶产生菌中,链霉菌Ｚ９４－２产脂肪酶活力为５９６ｕ／ｍＬ,其最适培养基（ｇ／Ｌ）为：糊精１０、黄豆饼粉３０、尿素１０、Ｋ２ＨＰＯ４０．５、ＭｇＳＯ４０．５、ＮａＣｌ１和ＡＥＯ９０．５,产酶的最适条件为：初始ｐＨ９．５～１０．０,在２６℃培养４８ｈ。用ＰＶＡ橄榄油乳化系统测定该酶的最适ｐＨ９．８,最适温度３７℃,在ｐＨ８．６～１０．２于５℃存放２４ｈ,酶活力不变。０．１４ｍｏｌ／Ｌ的氯     

紫菀花序芽培养及植株再生

１植物名称紫菀（Ａｓｔｅｒｔａｔａｒｉｃｕｓ）。２材料类别花序芽。３培养条件愈伤组织、不定芽诱导及增殖培养基：（１）ＭＳ＋６－ＢＡ０．５～２．０ｍｇ·Ｌ－１（单位下同）;生根培养基：（２）ＭＳ＋ＮＡＡ０．５～２．０＋６－ＢＡ０．２,（３）１／２ＭＳ（大量元素减半）＋ＮＡＡ０．５～２．０＋６－ＢＡ０．２。上述培养基均加０．８％琼脂和３％蔗糖,高温高压灭菌前ｐＨ值调至５．８。培养温度为（２３１２）℃,每天光照１２ｈ,光照度为１５００～２０００ｌｘ４生长与分化情况４．１愈伤组织及不定芽的诱导剪取长约０…     

青蒿毛状根合成青蒿素的培养条件研究

对影响青蒿（ＡｒｔｅｍｉｓｉａａｎｎｕａＬ．）毛状根生长及青蒿素合成的培养条件进行了研究,确定最适的培养条件为：初始ｐＨ５．８～６．０,摇瓶转速１３０～１５０ｒ／ｍｉｎ,摇瓶装液量体积分数为２５％,光照周期为１６ｈ／ｄ,温度为３０℃。在此条件下,经过２５ｄ培养获得青蒿素产量为２２３．３ｍｇ／Ｌ。     

菌根真菌——赭丝膜伞原生质体分离与再生研究

振荡培养４８ｈ的赭丝膜伞（Ｃｏｒｔｉｎａｒｉｕｓｒｕｓｓｕｓ）菌丝体经０．５％巯基乙醇预处理后,离心法收集,以０．６ｍｏｌ／ＬＭｇＳＯ４作高渗稳定剂,ｐＨ５．８,采用纤维素酶,离析酶和浸解酶联合处理,在三个温度（２４℃,３１℃３５℃）下经１１～１６ｈ反应均可获得较高的原生质体产量,最高产量达８×１０＾７个／ｇ鲜重菌丝,分离得到的原生质体表现了快速的再生能力,在ＭＭＮＣ固体培养基上可达到２０％以上的     

角质蛋白酶固态发酵工艺及酶解条件的研究

曲霉Ａ２８－８是一株优良的角蛋白酶分泌突变菌株。其最佳固体发酵培养基为：２０％羽毛粉,８０％麸皮和微量无机氮（每克培养基中加０．５ｍｇ）;最适产酶条件为：起始ｐＨ７．５～８．０,温度２８℃～３０℃,时间为６０～７０小时,酶活高达２５００ＫＵ／ｇ曲;最适酶解条件为ｐＨ７．０～９．０,温度４５℃～５０℃。     

球形红杆菌W1分批发酵和恒化培养的研究

本文对球形红杆菌Ｗ１的培养方式进行了研究。由分批培养确定了适于常规发酵的培养基及培养条件。恒化培养该菌的最大生长速率Ｐ＝３．８ｇ／ｌ．ｈ;最适稀释率Ｄ＝０．１５０ｈ＾－１;最大比生长速率为０．２０ｈ＾－１;饱和常数Ｋｓ＝２．８２ｇ／ｌ。恒化培养可获得比分批培养高的生长速率。     

细菌产木聚糖酶发酵条件的研究*

研究了碳源、氮源以及其他因子对木聚糖酶高产菌ＷＬＵＮ０２４（Ｐｓｅｕｄｏｍｏｎａｓ ｓｐ．）产酶的影响,结果表明在麸皮６ｇ／Ｌ、（ＮＨ４）２ＳＯ４ ０．８ｇ／Ｌ、Ｋ２ＨＰＯ４ ０．４ｇ／Ｌ、接种量５％－１０％的条件下,３７℃培养３６ｈ,其木聚糖酶活力可达６００ＩＵ／ｍＬ。同时研究了在较优条件下该菌的摇瓶产酶曲线。     

肉碱脱水酶突变株的获得及其休止细胞反应条件的优化

对一株产肉碱脱水酶的菌株用溴化乙锭进行诱变,并经肉碱选择性培养基进行筛选,获得了一株高活性酶活的菌株,并进行了休止细胞转化巴豆甜菜碱为Ｌ－内碱的反应条件的研究。实验确定休止细胞的最适反应温度为３０℃～３２℃,ｐＨ８．１、０．０１ｍｏｌ／Ｌ磷酸缓冲液,时间８．５～１０ｈ。最适底物浓度为６０ｍｇ／ｍｌ,最适休止细胞浓度为６０ｍｇ／ｍｌ（湿重）,产率４０％（摩尔比）。建立了休止细胞酶反应的表观米氏方程,     

无花果蛋白酶在阴离子交换树脂上的固定化

无花果蛋白酶通过８％戊二醛活化载体,共价结合到聚苯乙烯阴离子交换树脂ＧＭ２０１上,固定化作用在ｐＨ７．７,酶浓度０．８ｍｇ／ｇ树脂,４℃下进行６ｈ。得到的固定化酶表观Ｋ_ｍ值（酪蛋白,１．１１×１０~（－４）ｍｏｌ／Ｌ）小于溶液酶Ｋ_ｍ值（１．９６×１０~（－４）ｍｏｌ／Ｌ）;固定化酶活性在ｐＨ６～８保持稳定,溶液酶最适ｐＨ为７．２;固定化酶最适温度由溶液酶的５０～６０℃移至３７℃;固定化酶２５℃保持７ｄ,重复水解酪蛋白７次后,保留８３．３％活性。固定化酶对酪蛋白水解度达４７．５％,对大豆球蛋白达１１．６％。     

EFFICIENT PRODUCTION OF Staphylococcus simulans LYSOSTAPHIN IN A BENCHTOP BIOREACTOR BY RECOMBINANT Escherichia coli

Lysostaphin is an enzyme with bactericidal activity against Staphylococcus aureus and other staphylococcal species. In spite of many advantages and promising results of preliminary research, the enzyme is still not widely used in medicine, veterinary medicine, or as a food preservative. One of the most important factors limiting application of the enzyme in clinical or technological practice is the high cost of its production. In this study we have determined the optimal conditions for lysostaphin production in a 5-L batch bioreactor. The enzyme production was based on a heterologous, Escherichia coli expression system designated as pBAD2Lys and constructed earlier in our laboratory. An evident influence of physicochemical conditions of the process (areation, pH and temperature) and composition of the growing media on the amount and activity of produced enzyme was noticed. Efficiency of production of about 13,000 U/L has been achieved in the optimal conditions of the production process: low aeration (400 rpm of mechanical stirrer), pH 6, and temperature 37°C in classical LB medium. Further, about twofold improvement in the production efficiency of the enzyme was achieved as a result of modification of composition of growing media. Finally, more than 80,000 units of lysostaphin were obtained from one (batch) bioreactor with 3 L of culture of E. coli TOP10F’ transformed with pBAD2Lys plasmid. To the best of our knowledge, this is the most efficient method of production of recombinant lysostaphin in E. coli expression systems described to date.     

<Emphasis Type="Italic">Staphylococcus simulans</Emphasis> recombinant lysostaphin: Production,purification, and determination of antistaphylococcal activity

Staphylococcus simulans lysostaphin is an endopeptidase lysing staphylococcus cell walls by cleaving pentaglycine cross-bridges in their peptidoglycan. A synthetic gene encoding S. simulans lysostaphin was cloned in Escherichia coli cells, and producer strains were designed. The level of produced biologically active lysostaphin comprised 6-30% of total E. coli cell protein (depending on E. coli M15 or BL21 producer) under batch cultivation conditions. New methods were developed for purification of lysostaphin without affinity domains and for testing its enzymatic activity. As judged by PAGE, the purified recombinant lysostaphin is of >97% purity. The produced lysostaphin lysed cells of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates. In vitro activity and general biochemical properties of purified recombinant lysostaphin produced by M15 or BL21 E. coli strains were identical to those of recombinant lysostaphin supplied by SigmaAldrich (USA) and used as reference in other known studies. The prepared recombinant lysostaphin represents a potential product for development of enzymatic preparation for medicine and veterinary due to the simple purification scheme enabling production of the enzyme of high purity and antistaphylococcal activity.     

溶葡球菌酶的纯化和某些性质

1.应用本实验室构建的克隆菌株枯草杆菌0044进行了溶葡球菌酶的发酵生产,产量为150—200mg/L; 2.通过DEAE-纤维素,CM-纤维素和Sephadex G-50层析纯化了该酶;并以NaCl盐析方式,首次获得了该酶结晶; 3.测定了溶葡球菌酶的某些性质; 4.观察并讨论了溶葡球菌酶与溶菌酶等在溶菌作用上的相互加强。     

Random Mutagenesis Identifies Novel Genes Involved in the Secretion of Antimicrobial, Cell Wall-Lytic Enzymes by Lactococcus lactis

Lactococcus lactis is a gram-positive bacterium that is widely used in the food industry and is therefore desirable as a candidate for the production and secretion of recombinant proteins. Previously, we generated a L. lactis strain that expressed and secreted the antimicrobial cell wall-lytic enzyme lysostaphin. To identify lactococcal gene products that affect the production of lysostaphin, we isolated and characterized mutants generated by random transposon mutagenesis that had altered lysostaphin activity. Out of 35,000 mutants screened, only one with no lysostaphin activity was identified, and it was found to contain an insertion in the lysostaphin expression cassette. Ten mutants with higher lysostaphin activity contained insertions in only four different genes, which encode an uncharacterized putative transmembrane protein (llmg_0609) (three mutants), an enzyme catalyzing the first step in peptidoglycan biosynthesis (murA2) (five mutants), a putative regulator of peptidoglycan modification (trmA) (one mutant), and an uncharacterized enzyme possibly involved in ubiquinone biosynthesis (llmg_2148) (one mutant). These mutants were found to secrete larger amounts of lysostaphin than the control strain (MG1363[lss]), and the greatest increase in secretion was 9.8- to 16.1-fold, for the llmg_0609 mutants. The lysostaphin-oversecreting llmg_0609, murA2, and trmA mutants were also found to secrete larger amounts of another cell wall-lytic enzyme (the Listeria monocytogenes bacteriophage endolysin Ply511) than the control strain, indicating that the phenotype is not limited to lysostaphin.     

Enhanced production of recombinant Staphylococcus simulans lysostaphin using medium engineering

Abstract

Staphylococcus aureus, among other staphylococcal species, developed multidrug resistance and causes serious health risks that require complex treatments. Therefore, the development of novel and effective strategies to combat these bacteria has been gaining importance. Since Staphylococcus simulans lysostaphin is a peptidoglycan hydrolase effective against staphylococcal species, the enzyme has a significant potential for biotechnological applications. Despite promising results of lysostaphin as a bacteriocin capable of killing staphylococcal pathogens, it is still not widely used in healthcare settings due to its high production cost. In this study, medium engineering techniques were applied to improve the expression yield of recombinant lysostaphin in E. coli. A new effective inducible araBAD promoter system and different mediums were used to enhance lysostaphin production. Our results showed that the composition of autoinduction media enhanced the amount of lysostaphin production 5-fold with the highest level of active lysostaphin at 30?°C. The production cost of 1000?U of lysostaphin was determined as 4-fold lower than the previously proposed technologies. Therefore, the currently developed bench scale study has a great potential as a large-scale fermentation procedure to produce lysostaphin efficiently.     

Gene and Protein Sequence Optimization for High-Level Production of Fully Active and Aglycosylated Lysostaphin in Pichia pastoris

Lysostaphin represents a promising therapeutic agent for the treatment of staphylococcal infections, in particular those of methicillin-resistant Staphylococcus aureus (MRSA). However, conventional expression systems for the enzyme suffer from various limitations, and there remains a need for an efficient and cost-effective production process to facilitate clinical translation and the development of nonmedical applications. While Pichia pastoris is widely used for high-level production of recombinant proteins, there are two major barriers to the production of lysostaphin in this industrially relevant host: lack of expression from the wild-type lysostaphin gene and aberrant glycosylation of the wild-type protein sequence. The first barrier can be overcome with a synthetic gene incorporating improved codon usage and balanced A+T/G+C content, and the second barrier can be overcome by disrupting an N-linked glycosylation sequon using a broadened choice of mutations that yield aglyscosylated and fully active lysostaphin. The optimized lysostaphin variants could be produced at approximately 500 mg/liter in a small-scale bioreactor, and 50% of that material could be recovered at high purity with a simple 2-step purification. It is anticipated that this novel high-level expression system will bring down one of the major barriers to future development of biomedical, veterinary, and research applications of lysostaphin and its engineered variants.     

Antimicrobial activity of lysostaphin and a Listeria monocytogenes bacteriophage endolysin produced and secreted by lactic acid bacteria

The expression and secretion signals of the Sep protein from Lactobacillus fermentum BR11 were used to direct export of two peptidoglycan hydrolases by Lb. fermentum BR11, Lactobacillus rhamnosus GG, Lactobacillus plantarum ATCC 14917 and Lactococcus lactis MG1363. The production levels, hydrolytic and bacteriocidal activities of the Listeria monocytogenes bacteriophage N-acetylmuramoyl-l-alanine amidase endolysin Ply511 and the glycylglycine endopeptidase lysostaphin were examined. Buffering of the growth media to a neutral pH allowed detection of Ply511 and lysostaphin peptidoglycan hydrolytic activity from all lactic acid bacteria. It was found that purified Ply511 has a pH activity range similar to that of lysostaphin with both enzymes functioning optimally under alkaline conditions. Supernatants from lactobacilli expressing lysostaphin reduced viability of methicillin resistant Staphylococcus aureus (MRSA) by approximately 8 log(10) CFU/ml compared to controls. However, supernatants containing Ply511 were unable to control L. monocytogenes growth. In coculture experiments, both Lb. plantarum and Lb. fermentum synthesizing lysostaphin were able to effectively reduce MRSA cell numbers by >7.4 and 1.7 log(10)CFU/ml, respectively, while lactic acid bacteria secreting Ply511 were unable to significantly inhibit the growth of L. monocytogenes. Our results demonstrate that lysostaphin and Ply511 can be expressed in an active form from different lactic acid bacteria and lysostaphin showed superior killing activity. Lactobacilli producing lysostaphin may have potential for in situ biopreservation in foodstuffs or for prevention of S. aureus infections.     

Cloning of lysozyme and lysostaphin genes of Staphylococcus aureus and their expression in Bacillus subtilis cells

The gene of microbial lysozyme (lyz) of S. aureus 118 and the gene of lysostaphin (lzf) of S. aureus RN 3239 were cloned and their expression in B. subtilis cells was shown. Lysozyme production in B. subtilis recombinant clone pLF14-Lyz, obtained as the result of cloning, was 2.5-fold greater than lysozyme production in S. aureus wild strain 118. Lysostaphin production in B. subtilis recombinant strain pLF14-Lzf which had inherited the cloned genes was approximately equal to lysostaphin production observed in S. aureus initial strain RN 3239. The production of lysozyme and lysostaphin in the cells of B. subtilis recombinant strains was observed at 30 degrees C and pH 5.5, while in S. aureus initial strains 118 and RN 3239 bacteria produced lysozyme and lysostaphin at 37 degrees C and pH 7.5 respectively.     

Comparative In Vitro Activities of Lysostaphin and Other Antistaphylococcal Antibiotics on Clinical Isolates of Staphylococcus aureus

The in vitro activity of lysostaphin against clinical isolates of Staphylococcus aureus was determined by conventional tube-dilution methods. For comparison, minimal inhibitory concentration (MIC) values were also determined for penicillin G, ampicillin, methicillin, ristocetin, vancomycin, and erythromycin. Phage type and penicillinase and coagulase production were determined for each isolate. The MIC values for lysostaphin ranged from <0.047 to 12.5 μg/ml; 96% of the penicillinase-positive strains were inhibited by 1.56 μg/ml of lysostaphin, whereas 3.12 μg/ml of vancomycin and methicillin were required to attain the same degree of inhibition.     

Effects of recombinant lysostaphin on cytotoxicity and interleukin-8 level in normal human epidermal keratinocytes cell line

The normal human epidermal keratinocyte (NHEK) was used to evaluate the cytotoxicity of recombinant lysostaphin. As determined with the Neutral Red (NR) cytotoxicity assay, the midpoint toxicity value (NR50) after 48 h exposure was 16 ± 0.4 g lysostaphin/l. Lysostaphin cytotoxicity effect is much less than the surface active agent, sodium laurate. However, the NR50 value after 48-h exposure was 1.9 ± 0.02 g/l for S. aureus lysate derived from the bacterial lytic action of lysostaphin. A linear increase in interleukin-8 (IL-8) level in NHEK cells from resting levels of 65 ± 3 pg/ml to peak of 760 ± 15 pg/ml during the first 9 hours was noted for the cells treated with 800 mg lysostaphin/l. S. aureus lysate has the same effect on the induction of IL-8 levels. The induced rises in IL-8 were lysostaphin and S. aureus lysate concentration dependence. © Rapid Science Ltd. 1998