胞外青霉素酰化酶产生菌的自然分离

利用菌种的自然突变进行“出发菌株Ⅱ”的自然分离,从用肉眼选出的22株菌中,经初筛选选出650u／10ml以上高产菌株8株,再经过复筛选出780u／100ml的活力菌株2株,命名为“6‘＃、8’＃”。用于胞外酶的生产,月平均酶活5975u／100ml。经验证,“6‘＃、8‘＃”高产菌产菌适用于胞外青霉素酸化酶的生产。     

耐低温淀粉酶产生菌Y89微波诱变及发酵工艺

为进一步提高耐低温淀粉酶产生菌的发酵生产水平,以前期筛选得到的1株耐低温兼性厌氧淀粉酶产生菌Y89为出发菌株,对其进行微波诱变处理,通过酶产量及遗传性能稳定检测筛选高活力突变株,并采取单因素优化法对菌株的培养基和培养条件进行优化。获得1株遗传稳定的高活力突变株Y89-11,淀粉酶产量达750.2 U/m L,是原出发菌株的1.94倍。采用单因素实验确定该突变株的最佳发酵条件:最适生长及产酶温度为16℃,最佳产酶时间60 h,最优碳源为可溶性淀粉,氮源为酵母膏,培养基中添加Ca2+可显著提高产酶量。经诱变选育出的突变株Y89-11与原菌株相比产酶量提高了94%,所产淀粉酶为中低温酶,最适反应温度30℃,耐低温效果较好,应用前景广阔。     

果胶酶CP-85211菌株的选育及其液体发酵条件的研究

黑曲霉(Aspergillus niger) CP-831经0.03％亚硝基呱在40℃处理30分钟,获得一株高产果胶酶突变株CP-85211。该突变株发酵滤液,当以果胶为底物时,酶活力为308u／m’;当以多聚半乳糖醛酸为底物时,酶活力为842u／ml。产酶活力水平约为出发菌种的5倍。产酶最适培养基组成为：8％麦麸,2％桔皮粉和2％硫酸铵。最适培养条件为：起始pH 3．5,如℃,72小时。     

产适冷木聚糖酶的海洋产青霉的筛选和诱变

从黄海深层海底泥样中分离到1株产低温木聚糖酶的青霉,经EMS诱变得到11株酶活性提高的菌株,对其中1株产低温木聚糖酶活力最高的菌株产酶性质进行了初步研究。所产木聚糖酶在pH4．6,45℃时酶活可达25．8u／ml,比出发菌株提高126％。诱变后菌株所产木聚糖酶在0℃仍有显著酶活性,达8．2u／ml。     

高温α-淀粉酶生产菌种选育的研究

以地衣芽孢杆菌B．198为出发株,经不同诱变剂反复诱变和自然选育,得到突变株A.404l,其产高温α-淀粉酶为出发株的100倍。在摇瓶培养条件下,酶活力达200u／ml．是一株无孢子并带有Rifr、Met-、Arg-标记的抗分解代谢阻遏突变株。初步纯化的A1041α-淀粉酶最适反应pH为5—7,晟适反应温度90—95℃,于90℃、60分钟处理不失活,表明突变株A．4041所产α-淀粉酶是高温淀粉酶,具有工业生产价值。     

高温α-淀粉酶生产菌种选育的研究

以地衣芽孢杆菌B．198为出发株,经不同诱变剂反复诱变和自然选育,得到突变株A.404l,其产高温α-淀粉酶为出发株的100倍。在摇瓶培养条件下,酶活力达200u／ml．是一株无孢子并带有Rifr、Met-、Arg-标记的抗分解代谢阻遏突变株。初步纯化的A1041α-淀粉酶最适反应pH为5—7,晟适反应温度90—95℃,于90℃、60分钟处理不失活,表明突变株A．4041所产α-淀粉酶是高温淀粉酶,具有工业生产价值。     

真空微波诱变结合化学诱变选育酸性纤维素酶高产菌株

以酸性纤维素酶产生菌绿色木霉（Trichoderma viride）WL0512作为原始出发菌株,首先经自然分离筛选出一株产酶较稳定的菌株TVN-18,其羧甲基纤维素酶活（CMC酶活）达2765．8U／g,滤纸酶活（FPA酶活）达48．5U／g。再经真空微波和甲基磺酸乙酯（EMS）逐级诱变处理,获得了一株高产、稳产酸性纤维素酶的E6—1菌株,其CMC酶活达4396．6U／g,FPA酶活达126．0U／g,分别是菌株TVN-18的1．59倍和2．60倍。通过对固态发酵培养基麸皮和稻草比例、料水比以及初始pH值的优化,突变株的产酶能力进一步得到提高,其产的CIVIC酶活和FPA酶活分别提高了22．3％和22．4％。     

用快中子法选育细菌脂肪酶高产菌株*

研究了从土壤中筛选产脂肪酶的菌株,利用紫外线、快中子、快中子和磁场复合、γ射线、γ射线和磁场复合诱变,以酶活为筛子进行诱变育种。结果,出发菌酶活较低的一株得到了一株酶活为３９６．２２Ｕ／ｍＬ的诱变株,此酶活比出发菌株高９２倍,并发现此菌对紫外线和快中子比较敏感;而出发菌酶活较高的一株得到了酶活为４２４．６０Ｕ／ｍＬ发酵液的诱变株,此酶活为出发菌株３．０倍。在此基础上,初步探讨了快中子、γ射线及磁场复合处理在产脂肪酸菌种诱变中的作用,并认为,在产脂肪酸菌株的诱变中快中子诱变更为有效。     

产纤溶酶少根根霉菌株的诱变筛选

目的:通过对自南方小酒药中筛选得到的1株产纤溶酶的少根根霉Or株的诱变筛选,提高原有菌株的产酶能力。方法:以Or为出发菌株,进行亚硝基胍、紫外线、Co60诱变,以血纤维蛋白平板法为检测方法,筛选高产酶突变株。结果:经诱变传代后得到高产突变株8B,其产酶活性稳定为291.05U/mL,为原菌株产酶活力的6.34倍。该菌在血琼脂平板上不产生溶圈,诱变后孢子成熟提前8h。结论:物理诱变和化学诱变交替应用,能显著提高少根根霉Or株单位体积发酵液的产酶量,并能缩短孢子的成熟周期。该菌不具有溶血性,与已有报道的产纤溶酶的菌株不同。     

产果胶酶的菌种选育及发酵条件

炭黑曲霉(Aspergillus carbonarius)AS 3.396经亚硝基胍和Co60r-射线诱变,获得一株高产果胶酶突变株G5512。该菌株的发酵滤液,以高酯果胶为底物的酶活力为860u／ml;以低酯果胶为底物的酶活力为1227u／ml。产酶活力水平约为原出发菌株的2—6倍。产酶最适培养条件为：起始pH4.0—4.5,30℃,72—90小时。酶作用最适条件为：高酯果胶为底物时,pH3.5,50℃;低酯果胶为底物时,pH4.5,50℃。pH稳定范围为2．0-6．5(高酯果胶)和4.5—5.0(低酯果胶)。酶在60℃保温15分钟,高酯果胶为底物的酶剩余活力71％,低酯果胶为底物的酶活力仅剩余1％。     

High expression of the penicillin G acylase gene (pac) from Bacillus megaterium UN1 in its own pac minus mutant

By marker exchange mutagenesis, Bacillus megaterium strain UN-1 (Bm-UN1) was used to prepare a mutant strain B. megaterium UN-cat (Bm-UNcat) lacking the penicillin G acylase gene (pac). The pac gene from Bm-UN1 was subcloned into pTF6 and the resultant plasmid, pBA402, was introduced into Bm-UNcat and Bacillus subtilis. Bm-UNcat harbouring pBA402 produced high penicillin G acylase (PAC) activity of 13.7, 19.5 and 20.4 U ml(-1) at 24, 36 and 48 h of culture, respectively. This was two- to fivefold higher than PAC produced by B. subtilis harbouring pBA402 and about 20-fold higher than PAC produced by the parent strain, Bm-UN1.     

聚丙烯腈纤维固定化青霉素酰化酶性质的研究

将巨大芽孢杆菌（Bacillusmegaterium）青霉素酞化酶连接到聚丙烯腈纤维载体上,制成固定化青霉素酰化酶。其表现活力约为2000u／g。水解青霉素G的最适温度为50℃;最适PH为9．0;在PHS．5～10．3、温度50℃以下酶的活力稳定;表观米氏常数Ka为1．33×10-8mol／L;最大反应速度Vm为2．564mmol·min-1;苯乙酸为竞争性抑制剂,抑制常数为0．16mol／L。水解10％的青霉素G钾盐溶液,使用20批,保留酶活力80％。     

Mutagenic effect of acridine orange on the expression of penicillin G acylase and beta-lactamase in Escherichia coli

AIMS: The present work aimed to improve the production of penicillin G acylase (PGA) and reduce the beta-lactamase activity through acridine orange (AO) induced mutation in Escherichia coli. METHODS AND RESULTS: Three wild E. coli strains BDCS-N-FMu10, BDCS-N-S21 and BDCS-N-W50, producing both the enzymes PGA and beta-lactamase were treated by AO. Minimum inhibitory concentration of AO was 10 microg ml(-1) and it was noted that bacterial growth was gradually suppressed by increasing the concentration of AO from 10 to 100 microg ml(-1). The highest concentration that gave permissible growth rate was 50 microg ml(-1). The isolated survivals were screened on the bases of PGA and beta-lactamase activities. Among the retained mutants, the occurrence of beta-lactamase deficient ones (91%) was significantly higher than penicillin acylase deficient ones (27%). CONCLUSIONS: In seven of the mutants, PGA activity was enhanced with considerable decrease in beta-lactamase activity. One of the mutant strains (BDCS-N-M36) exhibited very negligible expression of beta-lactamase activity and twofold increase in PGA activity [12.7 mg 6-amino-penicillanic acid (6-APA) h(-1) mg(-1) wet cells] compared with that in the wild-type strain (6.3 mg 6-APA h(-1) mg(-1) wet cells). SIGNIFICANCE AND IMPACT OF THE STUDY: The treatment of E. coli cells with AO resulted in mutants with enhanced production of PGA and inactivation of beta-lactamase. These mutants could be used for industrial production of PGA.     

Cloning and expression of penicillin acylase genes from overproducing strains of Escherichia coli and Bacillus megaterium

Summary Penicillin acylase genes from Escherichia coli 194, of an overproducing mutant (194-3) of this strain, and of a similar overproducing mutant of Bacillus megaterium UN1 were cloned in E. coli DH1 on the plasmid vector pACYC184. The sizes of chromosomal DNA fragments essential for penicillin acylase production were found by Tn1000 mutagenesis and in vitro deletions to be between 2.2 and 2.5 kb in the case of both E. coli genes and between 2.3 and 2.7 kb in the case of the mutant Bacillus gene. Restriction mapping indicated substantial sequence differences between the E. coli and B. megaterium penicillin acylase genes. Enzyme production in E. coli recombinants from both overproducing mutants was found to be constitutive and higher than in the original strains. The Bacillus penicillin acylase was produced intracellularly in E. coli recombinants, which is in contrast to the normal extracellular production of this enzyme in B. megaterium. Recombinant plasmids containing penicillin acylase genes from either source were found to be unstable in the absence of selection pressure for retention of the vector.     

紫外线诱变选育碱性蛋白酶高产菌株

本实验以枯草杆菌A4－3为出发菌株,发酵产生碱性蛋白酶,其野生型菌株产酶为2412u／ml。采用孢子热处理方法处理出发菌株孢子,得到变异株As—4,产酶2732．8u／ml。对As—4菌株进行紫外线绣变处理1min,得变异菌株AX—46,产酶为3213．8u／ml,且产酶能力稳定。     

聚丙烯腈纤维固定化青霉素酰化酶合成头孢氨苄的研究

将巨大芽孢杆菌胞外青霉素酰化酶通过共价键结合到聚丙烯腈纤维的衍生物上。制成的丝状固定化青霉素酰化酶表现活力达 1 5 3U g(湿重 )。固定化酶合成头孢氨苄的最适pH为 6 5 ,最适温度为 40℃。 7 ADCA的投料浓度以 4%为好 ,7 ADCA与PGME的投料量比率为1∶2 ,最佳用酶量为 1 70U g 7 ADCA。在pH6 5、温度 3 0℃时 ,固定化酶对 7 ADCA的表观米氏常数K7 ADCA为 0 1 6 2mol L ,对PGME的表观米氏常数KPGME为 0 3 6 4mol L ,最大反应速度Vmax为0 0 4 6 2mol·L- 1·min- 1,用固定化酶合成头孢氨苄 ,使用 5 0次保留酶活力 83 9%     

Genome sequences of the biotechnologically important Bacillus megaterium strains QM B1551 and DSM319

Bacillus megaterium is deep-rooted in the Bacillus phylogeny, making it an evolutionarily key species and of particular importance in understanding genome evolution, dynamics, and plasticity in the bacilli. B. megaterium is a commercially available, nonpathogenic host for the biotechnological production of several substances, including vitamin B(12), penicillin acylase, and amylases. Here, we report the analysis of the first complete genome sequences of two important B. megaterium strains, the plasmidless strain DSM319 and QM B1551, which harbors seven indigenous plasmids. The 5.1-Mbp chromosome carries approximately 5,300 genes, while QM B1551 plasmids represent a combined 417 kb and 523 genes, one of the largest plasmid arrays sequenced in a single bacterial strain. We have documented extensive gene transfer between the plasmids and the chromosome. Each strain carries roughly 300 strain-specific chromosomal genes that account for differences in their experimentally confirmed phenotypes. B. megaterium is able to synthesize vitamin B(12) through an oxygen-independent adenosylcobalamin pathway, which together with other key energetic and metabolic pathways has now been fully reconstructed. Other novel genes include a second ftsZ gene, which may be responsible for the large cell size of members of this species, as well as genes for gas vesicles, a second β-galactosidase gene, and most but not all of the genes needed for genetic competence. Comprehensive analyses of the global Bacillus gene pool showed that only an asymmetric region around the origin of replication was syntenic across the genus. This appears to be a characteristic feature of the Bacillus spp. genome architecture and may be key to their sporulating lifestyle.     

Construction and hyperproductivity of engineered strain QE79 bearing recombinant plasmid containing penicillin G acylase gene from Escherichia coli strain AS1.76

Summary An engineered strain QE79 bearing a recombinant plasmid containing the penicillin G acylase gene from E.coli strain AS1.76 was constructed. Formation conditions of the penicillin G acylase were studied. The activity of the enzyme reached over 200 units per 100ml of the culture when the strain QE79 was grown in the medium consisting of mineral salts with supplementary glucose or sucrose at 28°C for 32 hr on shaker. The productivity of the engineered strain QE79 was nearly nine times higher than that of the original strain.     

Cloning and expression of keratinase gene in Bacillus megaterium and optimization of fermentation conditions for the production of keratinase by recombinant strain

AIMS: Cloning and expression of keratinase gene in Bacillus megaterium and optimization of fermentation conditions for the production of keratinase by recombinant strain. METHODS AND RESULTS: The keratinase gene with and without leader sequence from the chromosomal DNA of Bacillus licheniformis MKU3 was amplified by PCR and cloned into pET30b and transferred into Escherichia coli BL21. The ker gene without leader sequence only expressed in E. coli and the recombinant strain produced an intracellular keratinase activity of 74.3 U ml(-1). The ker gene was further subcloned into E. coli-Bacillus shuttle vector, pWH1520. Bacillus megaterium ATCC 14945 carrying the recombinant plasmid pWHK3 expressed the ker gene placed under xylA promoter and produced an extracellular keratinase activity of 95 U ml(-1). Response surface methodology (RSM) was employed to optimize the fermentation condition and to improve the level of keratinase production by the recombinant strain. A maximum keratinolytic activity of 166.2 U ml(-1) (specific activity, 33.25 U mg(-1)) was obtained in 18 h of the fermentation carried out with an initial inoculum of 0.4 OD600 nm and xylose concentration of 0.75% w/v. CONCLUSIONS: Bacillus licheniformis keratinase was cloned and successfully expressed using T7 promoter in E. coli and xylose inducible expression system in B. megaterium. Response surface methodology was employed to optimize the process parameters, which resulted in a three-fold higher level of keratinase production by the recombinant B. megaterium (pWHK3) than the wild type strain B. licheniformis MKU3. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that B. megaterium is a suitable host for the expression of cloned genes from heterologous origin. Optimization of fermentation conditions improved the keratinase production by B. megaterium (pWHK3) and suggested that this recombinant strain could be used for the production of keratinase.