复合诱变黑曲霉选育β-葡萄糖苷酶高产菌株*

对黑曲霉原生质体进行紫外、ＬｉＣｌ复合诱变,观察诱变后原生质体再生菌落,发现了抱子颜色变异较大的菌株,且其变化与酶产量相关。经发酵筛选,获得卜葡萄糖昔酶活较高菌株,其酶活由出发菌株的１０ｕ／ｍｌ提高到１４．７ｕ／ｍｌ。再对高产突变株进行氮离子注入,酶活又提高２０％（达１７ｕ／ｍｌ）。     

复合诱变黑曲霉选育β-葡萄糖苷酶高产菌株

对黑曲霉原生质体进行紫外、ＬｉＣｌ复合诱变,观察诱变后原生质体再生菌落,发现了抱子颜色变异较大的菌株,且其变化与酶产量相关。经发酵筛选,获得卜葡萄糖昔酶活较高菌株,其酶活由出发菌株的１０ｕ／ｍｌ提高到１４．７ｕ／ｍｌ。再对高产突变株进行氮离子注入,酶活又提高２０％（达１７ｕ／ｍｌ）。     

复合诱变黑曲霉选育β—葡萄糖苷酶高产菌株

对黑曲霉原生质体进行紫外、ＬｉＣｌ复合诱变,观察诱变后原生质体再生菌落,发现了孢子色变异较大的菌株,且其变化与酶产量相关。经发酵筛选,获得β－葡萄糖苷酶活较高菌株,其酶活由出发菌株的１０ｕ／ｍｌ提高到１４．７ｕ／ｍｌ。再对高产突变株进行氮离子注入,酶活又提高２０％（达１７ｕ／ｍｌ）。     

几种诱变因子对龟裂链霉菌原生质体的影响

作者应用通电、ＵＶ、ＮＴＧ和亚硫酸氢钠等诱变因子对土霉素产生菌——龟裂链霉菌１３８—ｌ原生质体进行处理。结果表明,各种诱变因子在对原生质体致死率５０％左右时具有较好的诱变效应。经亚硫酸氢钠＋ＬｉＣｌ处理获得的Ｃｌ１８菌株通过１６５００１发酵罐试验,最高发酵水平达３３３３０ｕ／ｍｌ,平均发酵水平为３０５４２．５ｕ／ｍｌ,比出发菌株１３８—１提高２０．６％。     

碱性普鲁兰酶产生菌的原生质体制备与诱变选育

研究报道了Bacillus sp SX—12原生质体制备与再生最佳条件。实验表明,在液体完全培养基中加入2％的甘氨酸培养10h,原生质体制备最佳条件为：溶菌酶浓度0．5mg／mL,酶解温度37℃,酶解时间1．5h时原生质体形成率为93．8％。原生质体形成最佳高渗稳定剂为甘露醇,再生率26．4％。在原生质体制备的最佳条件下,用紫外线诱变技术选育产碱性普鲁兰酶的高产菌株。筛选到1株高产菌株SX—12C87,酶活由出发菌株的2．42μ／mL提高到6．87μ／mL,提高了约1．8倍。     

植酸酶高产菌的诱变选育

以黑曲霉ＭＡＯ２１为出发菌株,经紫外线,、亚硝基胍单独处理和复合处理,获得一植酸酶高产菌株ＵＮ－１２－１０。该菌株具有稳定的遗传性能,在经过优化的摇瓶培养基上发酵１０８ｈ,其植酸酶活力达到２９５０－３０１５ｕ／ｍｌ,是原始出发菌株的３．６倍。     

应用原生质体技术选育天兰淡红链霉菌

本文报道柔红霉素产生菌——天兰淡红链霉菌原生质体的制备和再生方法,并从再生菌落中筛选得到摇瓶发酵单位提高５％的高产菌株３０＃。将再生高产株经铜蒸气激光辐照,得到在生产罐中发酵水平比对照出发菌株提高１２．９％的高产菌株８２＃。     

紫外诱变原生质体选育赖氨酸高产菌株

以钝齿棒杆菌102S₂-58为出发菌株,在原生质体形成及再生的最佳条件下制备原生质体,并对原生质体进行紫外诱变处理,对大量的再生突变株进行发酵筛选．获得了高产稳定株102－100号,其发酵液经氨基酸自动分析仪测定L－赖氨酸积累量由出发菌株的5O．Omg／ml提高到80．8mg／ml,糖转化率达到63．88％．发酵液中主要副产酸——纈氨酸和蛋氨酸的量明显降低。     

灵芝多糖对人脐血LAK细胞活性的影响

本文研究了灵芝多糖（ＧＬＰ）对人脐血ＬＡＫ（ＣＢ—ＬＡＫ）细胞活性的影响,结果发现,单独ＧＬＰ能刺激人脐血单个核细胞（ＣＢＭＣ）增殖,但不能诱导ＬＡＫ活性,当与５０ｕ／ｍｌｒＩＬ—２伍用时,可增殖ＣＢ—ＬＡＫ细胞诱导活性,不同剂量ＧＬＰ（０．５—１００μｇ／ｍｌ）影响作用不同,以１０μｇ／ｍｌ浓度最好．在不同浓度ｒＩＬ—２（１０—１００ｕ／ｍｌ）诱导ＣＢ—ＬＡＫ细胞过程中加入ＧＬＰ（１０μｇ／ｍｌ）,可明显提高细胞增殖能力,减少ｒＩＬ—２用量。ＧＬＰ亦能促进效应阶段ＣＢ—ＬＡＫ细胞对Ｒａｊｉ肿瘤靶细胞的杀伤作用（Ｐ＜０．００１）。由此看出,ＧＬＰ具有增强ＣＢ—ＬＡＫ细胞活性的作用,是一很好的生物反应调节剂（ＢＲＭ）,有必要进行深入的研究。     

高活力碱性淀粉酶菌种的选育及培养条件研究

通过对产碱性淀粉酶芽胞杆菌Ｂ－９５４５进行紫外线、原生质体亚硝基胍复合诱变等反复处理,获得一株具有较高碱性淀粉酶活力的变异株ＰＮ－６。产酶活力从５４０ｕ／ｍｌ提高到７８０ｕ／ｍｌ,确定的最佳培养条件是：ｐＨ８－１０,３０℃培养４８ｈ。从变异株和出发菌株的生长及产酶曲线看到,变异株的生长速度低于出发菌株,其产酶高峰与出发菌株相比略拖后一段时间,但是酶活力要远大于出发菌株。     

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。     

"Marker augmentation" phenomenon in the plasmid transformation and transduction of Bacillus subtilis

Transformation of Bacillus subtilis cells carrying pUB110 plasmid by DNA of homologous plasmid pBD12 results in the significant increase in the number of plasmid transformants. This phenomenon named "augmentation" was not observed when instead of intact cells, regenerating protoplasts were used, or if pBD12 DNA was introduced into the cells via transduction.     

Transfer of phospholipids between mesosomes and protoplasts from Bacillus subtilis

1. Phospholipids were more intensively labelled from ammonium [1-14C]glycerophosphate in mesosomes than in protoplasts isolated from Bacillus subtilis. 2. When mesosomes, containing phospholipids labelled from sodium-[32P] phosphate were incubated with non radioactive protoplasts, labelled phospholipids were recovered in protoplasts after incubation. 3. This transfer of phospholipids from mesosomes toward protoplasts is time-dependent. 4. Soluble proteins obtained from Bacillus subtilis after ammonium sulphate precipitation were separated on a Sephadex G-100 column. 5. The two main fractions were able to accelerate the transfer of phospholipids from mesosomes toward protoplasts. 6. The first peak stimulated more actively the transfer of phosphatidylethanolamine whereas the second one preferentially accelerated the transfer of phosphatidylglycerol and diphosphatidylglycerol.     

The role of DNA-gyrase from Bacillus subtilis during intermolecular irregular recombination]

The location of oxolinis acid-induced gyrase cleavage sites on pBR322 and pUB110 plasmid DNA in Bacillus subtilis cells has been studied and established. The treated Bacillus subtilis protoplasts were used in the study. Coordinates of the gyrase cleavage sites were compared to the location of the illegitimate recombination sites precisely mapped on the plasmid genomes. The obtained data indicate involvement of the DNA gyrase in formation of a fraction of recombinants in Bacillus subtilis.     

Interspecific transformation of Bacillus subtilis competent cells by chromosomal DNA in lysates of protoplasts of Bacillus amyloliquefaciens

Competent cells of Bacillus subtilis were transformed with chromosomal DNA in lysates of protoplasts of B. subtilis or B. amyloliquefaciens. The interspecific transformation frequency of B. subtilis by cysA in a conserved region was 3.1 x 10(4) transformants per microg DNA, 60 times higher than that for conventional transformation using purified DNA. Increased interspecific transformation frequencies of B. subtilis were also observed for arg-1, lys-1, leuB, aroG, thr-5, hisH, or metC markers outside the conserved region (3.1 x 10 approximately 5.2 x 10(2) transformants per microg DNA). An interspecific cotransformation ratio (33-50%) as high as an intraspecific one (46%) using purified DNA was also detected between cysA and rpsL markers, which are separated by 16 kb on the B. subtilis chromosome. Interspecific double transformation of the cysA-arg-1 or cysA-metC marker was observed, which have not been detected for conventional transformation. The involvement of mutS in the interspecific transformation was not significant.     

Production of protoplasts by autolytic induction in Bacillus thuringiensis: Transformation and interspecific fusion

Abstract A highly efficient method for the production of Bacillus thuringiensis protoplasts was developed. Formation of protoplasts was due to the activation of autolytic factors. In order to achieve induction of this autolytic system in a rapid and efficient manner, the appropriate conditions for growth and treatment were determined and optimized. Protoplast preparations obtained in this way were subjected to transformation with plasmid DNA and to interspecific fusion with a Bacillus subtilis rec⁻ strain.
Results indicated that these protoplasts could efficiently take up exogenous plasmid DNA as well as act as DNA donors in fusion experiments.     

Regeneration of protoplasts of Bacillus subtilis 168 and closely related strains

Regeneration of protoplasts to bacilli was attempted in several strains of Bacillus closely related to Bacillus subtilis 168. On DM3 and similar media using succinate as osmotic support, only B. subtilis 168 and Bacillus natto ATCC 15245 were able to regenerate. Media containing mannitol as osmotic support, and agar as gelling agent gave rise to L-form colonies with Bacillus licheniformis NCTC 6346. Many of the L-form colonies were able to regenerate to the bacillary form when plated on the mannitol medium solidified with gelatin. All of the Bacillus species tested were able to regenerate on the latter medium at rates sufficient to allow protoplast transformation and fusion experiments.     

Transformation of Bacillus subtilis by single-stranded plasmid DNA.

The single-stranded form of a pE194-based plasmid transformed Bacillus subtilis protoplasts at least as efficiently as did the double-stranded plasmid, but the single-stranded form did not detectably transform B. subtilis competent cells.     

Studies on transfection and transformation of protoplasts of Bacillus larvae, Bacillus subtilis, and Bacillus popilliae

Protoplasts of Bacillus larvae NRRL b-3555 and Bacillus subtilis RM125 (restrictionless, modificationless mutant) were transfected with DNA from the B. larvae bacteriophage PBL1c in the presence of polyethylene glycol. B. subtilis 168 and Bacillus popilliae NRRL B-2309M protoplasts could not be transfected with PBL1c DNA. Protoplasts of B larvae NRRL B-3555 were transformed with plasmids pC194 and pHV33 in the presence of polyethylene glycol. The frequency of transformation was much higher when the plasmids were isolated from B. larvae NRRL B-3555 transformants than when they were isolated from B. subtilis 168. These results indicate that the restriction-modification systems found in B. larvae NRRL B-3555 and B. subtilis 168 may be different. Conditions for protoplast formation and cell wall regeneration were developed for B. popilliae NRRL B-2309S. However, no transformation occurred with plasmids pC194 and pHV33 (isolated from B. subtilis 168).     

Studies on transfection and transformation of protoplasts of Bacillus larvae, Bacillus subtilis, and Bacillus popilliae.

Protoplasts of Bacillus larvae NRRL b-3555 and Bacillus subtilis RM125 (restrictionless, modificationless mutant) were transfected with DNA from the B. larvae bacteriophage PBL1c in the presence of polyethylene glycol. B. subtilis 168 and Bacillus popilliae NRRL B-2309M protoplasts could not be transfected with PBL1c DNA. Protoplasts of B larvae NRRL B-3555 were transformed with plasmids pC194 and pHV33 in the presence of polyethylene glycol. The frequency of transformation was much higher when the plasmids were isolated from B. larvae NRRL B-3555 transformants than when they were isolated from B. subtilis 168. These results indicate that the restriction-modification systems found in B. larvae NRRL B-3555 and B. subtilis 168 may be different. Conditions for protoplast formation and cell wall regeneration were developed for B. popilliae NRRL B-2309S. However, no transformation occurred with plasmids pC194 and pHV33 (isolated from B. subtilis 168).