Metabolic activity of inorganic nitrogenous compounds affects not only microbial growth but also metabolite production in fermentation technology. We have worked on the enzymes participating in ammonia assimulation of some fermentation bacteria. This paper summarizes the results on glutamine synthetase and its application in practical field. Glutamine synthetase (L-glutamate:ammonia ligase, EC. 6.3.1.2) catalyzes the formation of glutamine from glutamate and ammonia at the expense of cleavage of ATP and inorganic phosphate. The enzyme plays a dual role in nitrogen metabolism in bacteria; it is a key enzyme not only in the biosynthesis of various compounds through glutamine but also in the regulation of synthesis of some enzymes involved in the metabolism of nitrogenous compounds. The detailed works with the Eschericia coli and other enterobacterial enzymes revealed that glutamine synthetase is controlled by the following complex of mechanisms: (a) feedback inhibition by end products, (b) repression and derepression of enzyme synthesis, (c) modulation of enzyme activity in response to divalent cation and (d) covalent modification of enzyme protein by adenylylation and its cascade control. Comparative studies have also been made on the enzymes from other organisms.

이온성 계면활성제 존재하에서 유기용매에 물을 첨가하면 물이 계면활성제에 약해 둘러싸이면서 유기용매에 용해되어 의사 이상계가 형성된다. 이러한 계를 역미셀 또는 W/O microemulsion이라고 하며 계면활성제로 둘러 쌓인 물분자 집하체를 water pool이라고 한다. 그런데, 10여년 전 water pool에 bipolymer를 용해시킬 수 있다는 사실이 밟혀짐에 따라 이러한 체계를 생체막을 단순화시킨 모형막으로서 막을 통해 일어나는 여러 가지 현상의 규명에 이용하거나 물에 불용성인 기질의 효소 촉매반응의 반응계로 이용하는 연구가 꾸준히 이루어져 있다. 본 강연은 역미셀계에 리파제를 용해시켜 유지의 가수분해를 유도함으로써 지방산을 생산하는 방법에 관한 연구이다. 역미셀계에서 리파제의 특성은 에멀전계와 비교했을 때 큰 차이가 없었으며 물과 계면활성제의 몰 비율(R값)은 효소의 초기반응 속도에 커다란 영향을 끼치는 인자로 나타났다. 올리브유 농도가 5%(v/v), AOT농도가 0.1M, 초기 물 농도가 1.0M의 조건에서 유지의 회분식 가수분해 실험을 행한 결과 이 기질은 거의 완전히 가수분해되었으며, 이 반응계에서 R값과 초기 물 농도는 반응의 평형에 커다란 영향을 끼치는 것으로 나타났는데 초기 물 농도가 증가할수록 평형 가수분해율은 증가하였단 이러한 결과를 반응속도론 측면에서 분석한 결과 역미셀계에서 리파제 반응은 에멀전계에서와는 달리 2차 반응을 따르는 것으로 나타났다. 물 농도가 평형 가수 분해율과 속도 변수에 끼치는 영향을 수학적으로 표시하기 위하여 2차 가역적 반응 속도론에 근거하여 가수분해율, 평형상수, 속도상수 둥을 나타내는 식을 유도하였고 이를 바탕으로 여러 가지 실험 조건 하에서 리파제 반응의 반응 시간에 따른 가수분해율을 예측한 결과는 실제 실험 결과와 잘 일치하였다(편차는 5%). 또한 속도상수와 R값과의 관계식 및 유도한 방정식을 이용하여 추정한 초기속도와 평형 가수분해율을 최대화하는 R값은 각각 10.4 와 11.4 였다.

The gene for iso-1-cytochrome c for Saccharomyces cerevisiae was recloned into a pSP65 vector containing an active bacteriophage SP6 promoter. The iso-1-cytochrome c gene was cloned as an 856 bp Xho 1-Hind III fragment. When the resulting plasmid was digested at the Hind 111 site 279 bases downstream from the termination codon of the gene and transcribed in vitro using SP6 RNA polymerase, full length transcripts were produced. The SP6 iso-1-cytochrome c mRNA was translated using a rabbit reticulocyte lysate system and the protein products analyzed on SDS polyacrylamide gels. One major band was detected by autofluorography. This band was found to have a molecular weight of 12,000 Da and coincided with the Coomassie staining band of apocytochrome c from S. cerebisiae. The product was also shown to be identical with that of standard yeast apocytochrome c on an isoelectric focusing gel. The in vitro synthesized iso-a-cytochrome c was methylated by adding partially purified S-adenosyl-L-methionine . protein-lysine N-methyltransferase (Protein methylase III; EC 2.1.1.43) from S. cerevisiae along with S-adenosyl-L-methionine to the in vitro translation mixtures. The methylation was shown to be inhibited by the addition of the methylase inhibitor S-adenosyl-L-homocysteine or the protein synthesis inhibitor pu omycin. The methyl derivatives in the protein were identified as $\varepsilon$-N-mono, di and trimethyllysine by amino acid analysis. The molar ratio of methyl groups incorporated to that of cytochrome c molecules synthesized showed that 23% of the translated cytochrome c molecules were methylated by protein methylase III.

L-Azetidine-2-carboxylic acid (A-2-C), a four-membered cyclic imino acid has been identified in certain plants, and the microorganism Actinoplanes ferrugineus. The imino acid A-2-C has a physiological significance as an antgaonist of proline during peptide synthesis. The biosynthetic mechanism for the formation of A-2-C has not been studied in any detail. By using various amino acids such as methionine and S-adenosyl-L-methionine labeled with deuterium or carbon-14, the details of the biosynthetic pathway and a possible mechanism for the formation of L-A-2-C in .4. ferrugineus have been unravelled, Both in vivo and in vitro experimental results suggest the biosynthesis of L-A-2-C is mediated by a confactor containing a carbonyl group, probably pyridoxal Phosphate. S-Adenosyl-L-methionine, which seems to be the direct biosynthetic substrate, has undergone a f-displacement by an ${\alpha}$-amino group of the amino acid portion of the substrate S-adenosyl-L-methionine potentially via a vinylglycine intermediate. The overall stereochemical events at the ${\beta}$-carbon of the substrate have been shown to inversion of configuration. The overall stereochemical events at the -position of the sub- strate have also been shown to occur with inversion of configuration. The ${\beta}$, ${\gamma}$-elimination reaction of the substrate seems to follow a cisoidal-type mechanism and the addition portion of the reaction a transoidal-type mechanism . The assignment of the proton NMR of A-2-C has been deduced by apply- ing NOE difference experiments, Gd(III) line-broadening experiments and 2D-NOESY experiments of regio-and stereospecificially deuterated A-2-C's.

Recent advances in recombinant DNA techniques have provided a tool for breeding of microorganisms of hyper production. Enzyme production by cloned microorganism has some advantages. They are ⅰ) Enzymes can be produced by a microorganism easily cultured ⅱ) Hyper production. ⅲ) In some cases, such as thermophilic enzyme gene is cloned in a mesophilic bacteria, the enzyme purification procedure can be simplified. One example, production of thermophilic ${\beta}$-galactosidase in B. subtilis will be presented. Bacillus stearothermophilus IAM 11001 produced three ${\beta}$-galactosidases, ${\beta}$-galactosidase I, II and III (${\beta}$-gal-I, II and III). By connecting restriction fragments of the chromosomal DNA to plasmid vector, followed by transformation of Escherichia coli, two ${\beta}$-galactosidase genes (bgaA and bgaB) located close to each other on the chromosome were cloned.

Owing to the growing interest in the production of fuels and chemicals from biomass the well-know butanol-acetone fermentation as carried out by Clostridium acetobutylicum has been intensely studied again in recent years. Several solvent-yielding fermentation processes were established which are operated by using batch cultures or continuous cultures. 1 could be shown that under conditions of phosphate limitation an asporogenous mutant of C. acetobutylicum establishes itself in a chemostat which produces the solvents continuously. Attempts have been made to change the butanol/acetone ratio in favor of butanol production. A corresponding shift of the product spectrum can be achieved by carbon monoxide addition to the head space of the fermentation (B.H. Kim et al., App. Envioron. Microbiol. 48, 764-770 1984) or by iron limitation. Progress has been made in understanding the mechanism underlying the shift from acid to solvent prodcction. Experimental results are in agreement with the view that intracellular accumulation of acetic and butyric acid results in a shortage of phosphate and coenzyme A. This shortage may serve then as signal for the synthesis of the enzymes involved in the formation of acetone and butanol.

Many enzymes require the participation of readily dissociable coenzymes as NAD for thir catalytic activities. The continuous utilization of the enzymes requires the retention and regeneration of the coenzymes. For this purpose, several kinds of macromolecular NAD derivatives have been prepared by covalently attaching NAD to watersoluble polymers. We have prepared poly (ethylene glycol)-bound NAD (PEG-NAD) by coupling N$\^$6/-(2-carboxyethyl)-NAD to one terminal of ${\gamma}$ $\omega$-diaminoly (ethylene glycol) (Mr 3000) with water-soluble carbodiimide. PED-NAD thus obtained has one NAD moiety located at a terminal of the linear, flexible and hydrophilic chain of poly (ethylene glycol). PED-NAD has good coenzyme activity for various dehydrogenases and is applicable in a continuous enzyme reactor. To use these macromolecular NAD derivatives in an enzyme reactor, it si necessary to understand the behavior of the system in which the reactions of dehydrogenases are coupled by the recycling of the NAD derivative. We investigated the kinetic properties of a continuous enzyme reactor containing lactate dehydrogenase, alcohol dehydrogenase and PEG-NAD. The steady-state behavior of the enzyme reactor is explained by a simple kinetic model.

The biotechnology including genetic engineering is expected to be applied to various fields of wastewater treatments in order to promote biological reaction rate, to grade up the effluent quality and to advance the stability of microorganisms against temperature, pH and toxic substances. The current topics in Japan on application of biotechnology to wastewater treatment will be reviewed at the beginning of the presentation. Next, the research of Biochemical Engineering Laboratory is to be presented, especially focused on the following subjects ; (1) Application of genetic engineering to the investiation of heavy metal uptake by the resistant bacteria of Hg Cd or Zn. (2) Relationship between sulfate reducing bacteria and wastewater treatment, offensive odor and corrosion of sewer tranks.

The cdd gene of Bacillus subtilis, encoding the deoxycytidinecytidine deaminase of pyrimidine nucleotide biosynthesis has been cloned into the EcoRl site of pBR322. The recombinant plasmid, pSol, promoted the synthesis of 100-140 fold elevated levels of the enzyme. A comparison of the polypeptides encoded by cdd complementing and non-complementing plasmids in the mini cell showed the gene product to have a molecular mass of approximately 14 kDa. The nucleotide sequence of the gene and 460 base pairs upstream from the coding region was determined. An open-reading frame, encoding a protein with a calculated molecular mass of 14337 Da, was deduced to be the coding region for cdd. However, the enzyme has an apparent molecular mass of 54 kDa as determined by gel filteration, whereas sucrose density gradient centrifugation shows 58 kDa. It means that the enzyme could be forming a tetramer in a physiological state. About 28 amino acids of the N-tetramer in a physiological state. About 28 amino acids of the N-terminal presumably form a signal for membrane translocation and six cystein residues are contained in the structure. S1 nuclease mapping indicated that transcription of cdd is initiated 17 base pairs upstream from the translational start. The structural characterization of the odd gene was performed.

A mutant of bacillus subtilis with a defective cdd gene encoding deoxycytidine-cytidine deaminase(EC 3.5.4.5.) has been characterized genetically. The genetic lesion causing the altered deoxycytidine-cytidine deaminase, cdd, was mapped at 225 min on the linkage map of B.subtilis by AR9 transduction Transductional analysis of the cdd region established the gene order as trp-lys-dnaE-cdd-aroD. The cdd gene was linked 72% with the aroD and 20% with the lys.

Hyphantria Cunea NPV는 (흰불나방 바이러스) 유전적 기능을 알기 위하여 돌연변이 유발물질인 Nitrosoguanidine를 처리하여 돌연변이를 유발시킨 후 허용온도인 $25^{\circ}C$와 제한온도인 32$^{\circ}C$에서 Plaque assay하여 온도민감성 돌연변이체 13균주를 (ts-N220, -N 258, -N606, -N649 -N877 -N1420, -N1488, -N1491, -N1498, -N1512, N-2065,-N2076, -N2085) 를 분리 하였다.

용마산에서 채취한 토양에서 Bacillus Thuringiensis HL-1과 HL-2를 분리하여 위상차현미경상에서 아포와 내독소결정체를 확인 촬영하였고 34개 생화학적 성상을 조사하였다.

유산균을 종균으로 하여 유제품을 만드는 경우에 종균의 취급관리는 매우 중요한 일이다. 종균을 취급하는 과정에서 어떤 원인으로 인하여 종균의 활력 특히 산생성력이 감퇴되어 종균으로 사용할 수 없는 경우가 발생한다. 이러한 경우의 원인을 분석하여 그 예방책을 수립하기 위한 기초 연구로써 발효유의 종균으로 사용하고 있는 L. casei YIT9018의 spontaneous lac 변이주의 발생빈도 및 이러한 Lac 변이주의 특성을 검토하였다. parent colony에서의 Lac의 발생빈도는 MRS 배지에서 400 coloney중에서 1개, 10% 환원 탈지분유에서는 984개중 1개의 배율로 나타났다. 분리된 Lac 변이주중에는 Lac Gal도 검출되었다 대부분의 Lac를 탈지유배지에 배양할 경우 10일이 지나면 우유를 응고시켰으나 Lac Gal는 30일이 지난뒤에 응고시켰다. Lac변이주를 MRS-lactose 배지의 plate에 도말하여 배양하면 4일 이후부터는 revertant가 나타났으나 Lac Gal Cell에서는 인정되지 않았다. 얻어진 Lac 변이주의 plasmid를 분리하여 parent와 비교한 결과 plasmid DNA가 소실되었음을 확인하였다.

토양으로부터 dextranase를 분비하는 균을 분리하였으며, 그 균이 Flavobacterium multivorum으로 동정되었다. 이 F. multivorum의 배치조건에 따른 dextran 이용과 dextranase 분비능을 조사하였고 아울러 성장조건도 알아보았다. 한편 위의 균은 plasmid를 갖고 있지 않았으며, 그에 따라 chromosomal DNA를 추출하여 Sau3Al 절단한 후

Since sisomicin which is produced by Micromonospora inyoensis is an intracellular antibiotic, the final antibiotic titer to be attained depends singnificantly on the cell mass in fermentation broth. Cobalt ion in medium was indispensable for getting a high antibiotic titer. However, in the presence of cobalt ion in medium, the antibiotic production proceeded up to about 4 days and thereafter stopped. From the experiments on theaddition of cobalt ion to culture medium, it was shown that the antibiotic production stopped due to the other physiological properties of cells rather than the accumulation of antibiotic in cells.

λP$_{L}$ promoter와 Infiuenza virus의 NS1 Structural gene이 있는 pASl EH801 plasmid를 E. coli host N5' 과 AR120에 각각 transformation하여 온도와 nalidixic acid로 각각 induction 하여 보았다 N5151의 경우, O.D.600 1.2에서 42$^{\circ}C$로 induction하였을 때 maximum productivity를 보였으며 AR120의 경우는 O. D. 600 1.2, 37$^{\circ}C$, 40$\mu\textrm{g}$ nalidixic acid/$m\ell$ induction 하였을 때 maximum yield를 보여주었다. 이때 pH, DO, temperature, $O_2$%, $CO_2$%를 A/D converter통해 computer에 연결시켜 data acquision을 한 결과, 접종 후 ON-line induction이 가능함을 알 수 있었다.

Thermostable tryptophanase was extracted from a thermophilie bacterium, strain T which was absolutely symbiotic with strain 5. The enzyme was purified 14.7 fold with 5.8% yield by chromatographies using ion exchange, gel filtration, and hydrophobic interaction columns, followed by high performance liquid chromatography on hydroxyapatite column. The purified enzyme has a molecular weight of approximately 210,000 estimated by gel filtration column chromatography, and the molecular weight of subunit was determined by SDS polyacrylamide gel electrophoresis to be 46,000, which indicates that the native enzyme is made of four homologous subunits. The tryptophanase was stable at 65o0 and the optimum temperature for the enzyme activity for 20 min reaction was 70$^{\circ}C$. The purified enzyme activity for 20 min ieaction was 70$^{\circ}C$. The purified enzyme catalyzed the degradation of L-tryptophan into indole, pyruvate and ammonia in the presence of pyridoxal phosphate. 5-Hydroxy-Ltryptophan, 5-methyl-DL-tryptophan, L-cysteine, S-methyl-L-cysteine, 5-methyl-DL-tryptophan, L-cysteine, S-methyl-Lcysteine, and L-serine were also used as substrates to form pyruvate. The amino acid composition of the tryptophanase was determined, and found to contain a high percentage of hydrophobic amino acids, especially in the proline content, which was much higher than that of Escherichia coli tryptophanase. In addition, the 35N-terminal amino acid sequence of the tryptophanase was completely different from that of E. coli tryptophanase.

B. sphaericus 1593 K-5 synthesize a potent entomicidal toxin against mosquito larvae. B. sphaericus EcoRl DNA fragment carrying the biocidal activity was clonied and expression in E. coli JM83. For the construction of a recombinant plasmid bearing the toxin activity the DNA of B. sphaericus was partially digested by the EcoRl. The EcoRl DNA fragments were ligated to plasmid pUC 8-EcoR1 site. The transformants were selected on LB plates containing X-gall and ampicilline. The transformants were bioassayed against mosquito larvae of which two clones showed biocidal activity. The two clones were redigested with Eco R1 and analyzed by 0.7% agarose gel electrophorsis.

Kajami Shik-hae processing consists of the fermentation of low salted Kajami (6% NaCl maximum) coated with a vegetable mixture, composed with cooked millet, red pepper, garlic and ginger. Lactic bacteria are the main component of the microflora. In order to determine their eventual selective role on the microflora the antimicrobial activity of garlic and red pepper was tested with some strain of bacteria and molds isolated from Shik-hae and Shik-hae raw materials. And the influence of their concentration in Kajami Shik-hae on the microflora was also checked. At the concentration of 10% garlic have no inhibitory activity against lactic bacteria but on strains of Bacillus, Micrococcus and Aspergillus niger. At the concentration of 20% red pepper showed a slight inhibitory activity on two strains of Bacillus. These results shows that red pepper and garlic are not only flavoring ingredients but they might play an important role in the control of the microflora growth and composition during Kajami Shik-hae fermentation.

DNA subfragments, sopA, sopB, and sopC supporting stable maintenance of an oriC plasmid, were derived from mini-F plasmid DNA (EcoRI restriction fragment, f5) after digestion with restriction endonucleases, and cloned in vector plasmid pBR322. The recombinant plasmid obtained were introduced into E. coli KY7231 and E. coli CSR603, and proteins specified by the mini-F fragments were analysed by SDS-polyacrylamide gel electrophoresis. Two proteins encoded by the F fragments were detected, having molecular weights of 41,000 and 37.000. The sopA protein (41K) encoded by a plasmid pXX288 was observed in the cytoplasm, whereas the sopB protein (37K) encoded by a plasmid pXX157 was in the membrane fraction. There was no novel protein band detected in the cell with a plasmid pXX300, which contained sopC fragment. Gene products of a plasmid pXX167, which is comprised of sopA, sopB, and sopC, were not detectable. Fluorography after one and two dimensional gel electrophoresis of the lysates showed that these two proteins were overproduced in the cells which were allowed to incorporate radioactive amino acid after plasmid amplification by chloramphenicol treatment. The isoelectric points of the sopA and sepB proteins were 6.6 and 7.0, respectively.

고온 호알카리성 Bacillus K-17의 Protease gene의 구조해명과 성질을 알기 위해서, E. coli HB101에 pER 322를 Vector로 하여 Protease gene을 Cloning하여 형질전환 된 균주를 선정하였다. 선정균주의 pretense activity를 Bacillus K-17의 상대활성도와 매우 유사하였으며 균체외에 보다 많은 효소 활성도를 지니고 있었다. 제한효소 Hind III로 절단하면 약 1.8kb와 0.4kb의 2개의 fragments 가 생성되었으며 Southern hybridization 결과 Cloning 된 gene이 Bacillus K-17에서 유래된 것임이 확인되었다.

The regulation of 3 ammonia assimilatory enzymes GDH(glutamate dehydrogenase), GS(glutamine synthetase) and GOGAT (glutamate synthase), have been examined in C. glutamicum for the biosynthesis of glutamate and glutmine. The cell free extracts of 3 kinds of arg, his and trp auxotrophs were investigated the activities of -ketoglutarate dehydrogenase, GDH, GS, and GOGAT on the media cultured with nitrogen excess and limiting conditions. Trp and his howed higher level of glutamate and glutamine than that of parental strain. The inhibition of GS activities by ADP suggested that GS is regulated by energy charge in C. glutamicum. The results with his, trp, glyc, ala, ser, and GMP implied that a system of feedback inhibition were effective. Three enzyme biosynthesis is repressed by nitrogen sources such as trp, pro, glyc, ala, ser and tyrosine.

국내 염장식품 및 염전으로부터 세균을 분리하여, 호염성 세균의 NaCl 농도에 따른 성장범위, 생리적 및 효소학적 특성을 조사하고자 했다. 염전으로부터 NaCl 20%배지에서 14주와 총 16종류의 젓갈류에서 NaCl 10% 배지로 56균주의 호염성 세균을 분리하여 0, 5, 10, 15, 20, 25% NaCl농도에서 성장률을 조사하고 최적온도 및 배지조성과 함께 동정에 필요한 생리실험을 하였다. 또한 세포의 효소로서 Lactate dehydrogenase, Glucokinase, Glucose-6-phosphate dehydrogenase, Alanine dehydrogenase, Isocitrate dehydrogenase 등의 특성도 조사하였다. 선별한 균주중 Acinetobacter sp, 등이 관찰 조사되었으며 최적 성장 NaCl농도는 10%이고, 최적온도는 3$0^{\circ}C$이며, 25% NaCl, 45$^{\circ}C$에서 자란 Halobacterium sp. 등이 분리되었다. 그중 Acinetobacter strain H6는 단백분해효소와 탄수화물 분해효소의 생성능이 15>10>20% NaCl순이며, 특히 Lactate dehydrogenase 활성은 2>3>1>OM NaCl 순으로 나타났고, NaCl 대신 KCl을 사용했을 때는 3>2>1> OM순으로 활성이 나타났다.

광합성 세균의 수소생성 기작에 대한 연구의 일환으로 광합성 세균에서 생성되는 색소의 성분을 조사하였다. 분리한 광합성 세균 K-7은 수소생성능이 뛰어난 균주로 조사된 홍색 비유황세균으로서 생리, 형태 및 배양학적 조사에 의하여 Rhodopseudomonas spheroides로 분류하였다. Type culture인 Rhodopseudomonas spheroides NCIB 8253과 비교 연구하였으며, SEM(Scanning Electron Microscope)하에서 기존균주와 분리균주의 형태학적인 특징을 확인하였다. 군 동정의 주요열쇠가 되는 색소성분을 조사한 결과, 균주 K-7에서 추출된 carotenoids로는 spheroidene의 산화형태인 OH-spheroidenone이 주성분이었고, Neurosporene, Lycopene, Anhydro- rhodovibrin, Rhodovibrin등이 동정되었다.

토양으로부터 분리한 질소고정균인 Klebsiella pneumoniae NFB320의 chromosomal DNA를 BamHI으로 절단하여 동일한 제한효소로 절단한 pBR322에 ligation시켜 E. coli HB101에 형질전환을 행하여 pullulanase activity를 나타내는 clone을 얻어내었다. 이 형질 전환체로부터 분리한 pullulanase 유전자가 재조합된 plasmid DNA는 약 10kb의 DNA단편을 가지고 있었으며, 재조합된 plasmid로부터 생산되는 pullulanase의 특성은 최적 활성 pH가 6.0이며, 효소의 pH안정성은 5-10이었다. 또한 형질 전환체로부터 생산되는 pullulanase의 localization,효소활성에 영향을 미치는 온도안정성 둥을 조사하였다.

Lactobccillus acidophilus와 Saccharomyces cerevisiae를 대두유에서 혼합 배양시켜 최적 젖산발효조건을 검토하였다. 대두유에서의 혼합배양시 20시간 이상 배양했을 때 산생성이 촉진되었고 배양온도는 34$^{\circ}C$ 부근으로 했을 때 가장 좋았다. 대두유에 sucrose와 skim milk가 젖산발효에 미치는 영향을 조사한 결과 sucrose 는 1.5% 첨가시 skim milk는 2.0% 첨가시 첨가하지 않고 배양할 때 보다 2배 정도의 산생성을 보였다.

고온 호알카리성 Bacillus K-17의 xylanase유전자의 구조해명과 대량 생산 균주를 개발하기 위채 Bacillus K-17의 염색체를 pER 322를 사용하여 E. coli에 형질전환시켜 xylanase 활성을 나타내는 형질전환체를 얻었다. 이 형질전환체에서 hybrid plasmid를 분리하여 제한효소로 mapping하였고 이 유전자가 Bacillus K-17유래인가를 hybridization에 의해 확인하였다. Recombinant plasmid pAX 1113은 5.1kb HindIII 절편을 가졌으며 BgIII site가 두곳, ECoRI과 pst site가 한곳이었으며 효소를 정제한 결과 Bacillus K-17이 생산하는 두 가지 xylanase중에서 xylanase I과 동일하였다.

Endo-$\beta$-1,3-glucanase는 barley glucan, laminarin등에 특이적으로 작용하는 효소로서 Malting process, Brewing process에 중요한 효소이다. 본 연구에서는 산업적으로 이용하기 위한 기초자료를 얻기 위하여 국산맥주맥으로 발아한 Green Malt를 Sample로 하여 Endo-$\beta$-1,3-glucanase를 추출하여 (DEAE Sephadex A-50, CM sephadex C- 50 Sephadex G-75)등을 이용하여 정제하여 이들 정제효소의 효소학적 성질등을 검토하였다.

Ames test와 포유동물 배양세포를 이용한 염색체 이상시험을 통하여 유전자 조작된 대장균이 생산한 알파 인터페론의 변이원성 유무를 조사하였다. rHu IFN-$\alpha$ A는 S. typhimurium (TA 1535, TA 1537, TA 1538, TA 98, TA 100) 에 30, 3000, 30,000 IU/plate의 농도에서 변이원성을 나타내지 않았으며, rat(Sprague-Dawley)의 골수세포를 이용한 생체내 염색체 이상시험을 실시하여 양성대조군인 nitroso-guanidine과 rHu IFN-$\alpha$A 각각에 대한 염색체 구조 이상의 출현율을 조사하였다.

L-Lysine 생성균인 Corynebacterium glutamicum의 동종간 융합주 RS 99를 담체인 Alginate와 여기에 TiCl$_4$로부터 제조된 Titanium hydroxide를 혼합하여 각각 고정하고 이들의 Gel strength, 활성도 및 회분식 발효조건을 비교하였다. 그 결과 Alginate-Titanium hydroxide를 담체로 선정하여 고정화 C, glutamicum 융합주의 재사용성 및 안정성을 검토하였으며, Continuous-Flow Stirred-Tank Reactor를 구성하여 L-Lysine 의 연속발효를 시도하였다.

B. sphaericus의 sporeless ts-D1216 돌연변이체의 유전학적 특성을 방사성 동위원소를 이용하여 DNA의 합성과 RNA의 합성을 측정하였고 일반적 특성을 연구하였다. ts-D1216 돌연변이체의 대수증식기 세포를 3$0^{\circ}C$에서 제한온도 (42$^{\circ}C$)로 이동시켰을 때 RNA의 합성은 4-5시간 정상적으로 합성이 계속 일어났고, DNA의 합성은 60-100분까지는 정상적인 율로 일어나다가 그 후에 결정적으로 감소되었다. 또한, DNA합성이 멈춘후에도 세포수는 증가했고, 4$0^{\circ}C$에서 성장기간이 더 길면 길수록 DNA합성에의 회복능력이 상실되었다.

3$0^{\circ}C$에서 대수증식기 세포를 42$^{\circ}C$에서 1시간동안 배양시킨 다음 3$0^{\circ}C$로 다시 이동시켰을 때 DNA합성은 Chloramphenicol의 존재하에서도 재 개시되었다. 그리고 Chloramphenicol을 처리하였을 때 세포 생존력은 크게 증가하지 않았고, 세포의 모양은 정상세포보다 더 길어졌다.

A gene encoding alcohol dehydrogenase (ADH) in Zymomonas mobilis was cloned into E. coli JM 83 with plasmid pUC 9. The ADH produced by the E. coli transformant was purified bysonication, (NH$^4$)2SO4 fractionation, Affi-Gel blue and hydroxylapatite chromatography. The ADH produced by Z. mobilis was also purified by the same procedures. The two enzyme preparations were characterized and compared. It was found that the E. coli ADH was identical to one of two ADH isozymes of Z. mobilis. Analytical gel filtrations led to the conclusion that the molecule of E. coli ADH was composedv of four subunits having molecular weight of 40,000 (+1,000) dalton each The effect of metal ions on ADH activity and optimum pH were investigated.

5'-Xanthylic acid 생성균주인 Brevibacterium ammoniagenes ATCC 21263 R에 XMP에서 GMP로의 전환효소인 GMP synthetase활성을 부여하기 위해 동종간 세포융합을 시도하여 융합균주들을 얻었다. 이들 우량 융합균주들과 융합모균의 GMP synthetase 활성을 측정하여 상호 비교하였으며, pH 변화에 따른 GMP synthetase 활성과 GMP 생성량과의 관계를 검토하였다. 또한 최적 pH에서 균성장에 따른 당소모량과 GMP 생성량을 비교하였다.

In order to optimally induce sporulation and toxin formation in Bacillus thuringiensis, exhaustion of specific nutrients as well as resuspension experiments were tried. Sporulation and toxin formation was most abunduntly occurred when the growth was limited by carbon source. It was also occurred in a resuspension medium containing only distilled water. Various environmental and physiological factors affecting the efficiencies of spore and toxin formation were examined in chemically defined media. As a result of these studies, a batch fermentation resulted in higher spore and toxin yield than ever reported

A Screening test was carried out for chitin-decomposing bacteria. In 100 samples from soil, fesh water and sea water, 7 strains of Chitinolytic bacteria were isolated. 5-3K which exhibited the highest chitinase activity was identified as Aeromonas hydrophila and cultural conditions from maximum chitinase production were determined. Optimum Chitinase production was obtained at pH 7, 33eC and with chitin concentration greater tham 0.2% Under optimal conditions, high yields of Chitinase were obtained in 16-30 hours. Chitinase was purified by ammonium sulfate precipitation and sephadex G-100 gel-filtration from the culture filtrgte.

To extend the spectrum of enzyme utilization in the organic solvent system, C. rugosal lipase was selected as a model enzyme because its substrate is soluble to organic solvent. One of the serious disadvantages in this system was the deactivation of the lipase. The pattern of lipase deactivation was the biphasic model. The activation energies for the deactivation were 14.05${\times}$10$^4$ KJ/Kg mole in the first phase and 3.59 ${\times}$ 10$^4$ KJ/mole in the second phase. The several factors were studied for their influences on the pattern of deactivation. Iso-octane as organic solvent influenced more on the first phase than the second phase. Urea as the reagent affecting boty hydrophobic interaction and hydrogen bond of enzyme also influencea more on the first phase. And the optimum pH for the activity was not correlated to that of the stability.

The hydrolysis of olive oil was attempted with immobilized C. rugosa lipase in the reverse phase solvent system. (i.e. immobilized wet particles is dispersed in continuous phase olive oil or organic solvents containing olive oil). Sephadex LH-20 and LH-60 were used as the supports that can be used in organic solvents. The water content of wet particles of sephadex LH-20 and LH-60 were about 72% (w/w) and 85% (w/w), respectively Both swollen gels with 0.05M buffers adsorbed about 18% of lipase dissolved. They were easily dispersed in liquid olive oil or in organic solvents. The effects of organic solvents on the stability and catalytic activity of the lipase have been examined. The results revealed that isooctane is superior to the other solvents examined for enzymatic fat spliting in reverse phase system. Kinetics of enzymatic hydrolys of olive oil by immobilized lipase has been investigated in a batch reactor. Effects of pH and temperature on the lipase were studied. The substrate concentration was influenced positively on the thermal stability.

CMCase, a member of cellulose decomposing enzymes, hydrolyze cellulose up to cellobiose. Cellobiase splits cellobiose to glucose units. Therefore, a linkage of the twogenes coding for CMCase and cellobiase on the same plasmid is needed to produce a cellulase complex which can produce glucose from cellulose. A genetic operon in which the two structural genes are under the control of a single promoter would be ideal for this purpose. The present report is on the linking of the two cellulase genes in one plasmid as a preliminary step of the operon construction.

The Bacillus CMCase gene we have previously cloned in E. coli is contained in the 3.2 Kb chromosomal insert of the 7.5 Kb pBSl plasmid. We have also found that the CMCase produced by this gene has molecular weight of about 32,000 suggesting that the CMCase coding region lies on about 0.3 Kb fragment. The present report deals with a series of subclonings to localize more precisely the region coding for the CMCase production.

Carboxymethyl cellulase (CMCase) produced by cloned B. megaterium was found to contain 5.2% carbohydrate but no metal ion. The enzyme was isoelectric at pH 7.23 and was high is basic amino acids. The N-terminal of the enzyme was glutamic acid. The cellulolytic activity of this enzyme was extended to the small molecular substrates such as from cellotriose to cellopentaose. In additon, the enzyme showed transglycoslation activity. The pK values of the enzyme we estimated to be 4.4 and 6.7, andthat of the enzyme-substrate complex were 4.2 and 7.2, respectively. The enzyme was not affected by the treatment with iodoacetic acid, but the modification of enzyme with carbodiimide and diethyl pyrocarbonate resulted in a marked loss of the enzyme activity. These results suggest that the active site of enzyme essentially contains carboxylic and imidazole group of amino acid residues.

To clone ${\beta}$-glucosidase gene from Cellulomonas sp. a gene library was constructed using E. coli JM83 pUC9. Among 2,500 pseudotransformants obtained, 20 clones developed yellow color on the p-nitrophenyl- -D-glucopyranoside filter paper These 20 clones were classified into three groups based on the results of activity staining using nondenaturating polyacrylamide gel electrophoresis and restriction enzyme digestions. Among the three groups, only one group containing pCEl plasmid has specificity for cellobiose.

Cephalexin synthesizing enzyme (${\alpha}$ amino acid ester hydrolase) was partially purified from the culture broth of Acetobacter turbidans ATCC9325 through ammonium sulfate fractionation, DEAE, CM, and Sephacryl S-200 gel filtration. The enzyme has optimum pH 6.0 and temperature, 40$^{\circ}C$ respectively. From the analysis of reaction mixtures by thin layer chromatographic and high performance liquid chromatographic techniques, it was confirmed this enzyme catalyzed simultaneously the following reactions : 1) Synthesis of cephalexin from D-${\alpha}$-phenylglycine methylester (PGM) and 7-amino 3-deacetoxy-cetoxycephalosporanic acid (7-ADCA) 2) Hydrolysis of cephalexin to form 7-ADCA and phenylglycine (PG) 3) Hydrolysis of PGM to form PG and methanol. Base on the above experimental observations, the reaction model of this enzyme was identical with that of the enzyme from Xanthomonas citri.

For the analysis of fermentation characteristics and productivity of plasmid coded product, car-boxymethylcellulase in a recombinant DNA cell fermentation system, batch and continuous fermentations were carried out using a Bacillus megaterium ATCC 14945 transformed with a plasmid, pCK 108 haboring carboxymethyl cellulase gene. The effects of carbon and nitrogen sources and of temperature and pH on cell growth, product yield, plasmid stability, specific plasmid contents of cell, and gene expression efficiency were carefully studied. These experimental results will be discussed in some details.

세균내 glutathione의 동태를 연구하기 위한 일환으로 P. mirabilis로부터 cysteinylglycine 분해효소를 정제 검토하였다. 본 균이 생산하는 cysteinylglycine 분해효소의 정제는 무세포추출액에 비해 비활성이 10배 증가하였고 0.68%의 낮은 수율을 나타내었다. 본 호소는 (NH$_4$)$_2$ SO$_4$ 침전과정에서 활성을 크게 손실하는 등 정제과정에서 불안정하였으며 투석 중에 형성하는 불용성 침전물은 4% Trition X-100 처리에 의해 효과적으로 용해되었다.

P. mirabilis 로부터 정제한 cysteinylglycine 분해효소의 성질 및 세포내 국재성을 검토하였다. 본 효소의 일반적 성질은 pH가 7.3 온도 37$^{\circ}C$에서 최대 활성을 냐타내었으며 pH 8.0에서 안정하였고, 열안정성은 $50^{\circ}C$, 30분처리에 30%의 활성 손실을 보였다. 또한 $Mn^{+2}$이온과 $Mg^{+2}$ 이온에 의해 활성이 촉진되었으며 본 효소를 반응전 30분 Preincubation 하므로서 최대 활성을 보였다. 본 효소는 glutathione 일단계 분해효소인 ${\gamma}$-glutamyl transpeptidase와 마찬가지로 세포내의 periplasmic space에 존재하였다.

토양시료에서 분리한 250여종의 방선균과 500여종의 세균에 대한 L-methionine 생산능력을 조사한 결과 SL 140 분리균이 배양액 $m\ell$당 2 $\mu\textrm{g}$의 L-methionine 을 생산함으로서 가장 높은 생산량을 나타내었다. 따라서 본 연구에서는 우선 일차로 SL 140 균주의 형태적 내지는 생리적 특성과 세포벽 구성 성분등을 분석하여 균 동정을 실시한바 SL-140 분리균이 Streptomyces속에 속하는 방선균임을 알았다.

토양시료로부터 높은 활성의 extracellular inulase를 생산하는 균주를 분리하여 분리균의 형태적 내지는 생리적 성질을 조사한 결과 Pseudomonas species로 동정되었다. 또한 Pseudomonas species로 동정된 본 분리균은 initial pH가 7.0인 1% inulin, 1.5% cornsteepliquor 0.8% (NH$_4$)$_2$ HPO$_4$ 및 0.003% FeSO$_4$ 7$H_2O$의 조성을 지닌 배지, 배양온도 45$^{\circ}C$ 및 배양시간 72시간 등의 배양조건에서 효소활성이 배양액 $m\ell$ 당 6.1 units로서 가장 높은 효소생산량을 나타내었다.

Several factors predicted to affect the protoplast formation and regeneration were investigated. The optimum lysozyme, casamino acid and PVP concentration were 0.5 (mg/$m\ell$), 0.1 (%) and 1.5(%). In B. pumilus, Penicillin-G treatment concentration was 0.3 (U/$m\ell$) and optimum treatment period was transit log. phase. And in the case of Celm. fimi, 0.3 (U/$m\ell$) and initial log. phase. Osmotic stabilizer and di-cation for OSM medium of B.pumilus and Gelm .fimi were 25mM CaCl2, 0.5M sodium sucinate and 50mM MgCl$_2$, 100mM CaCl$_2$, 0.4M sodium succinate. The regeneration frequency of B.pumilus and Celm. fimi were 14.6(%) and 6.9(%).

Cellulose utilising hybrids between Cellulomonas fimi and Bacillus pumilus were isolated after PEG mediated protoplast fusion. 33% (w/v) PEG #6, 000 and 50mM $Ca^{++}$were optimum concentration. The intergeneric fusion frequency was 3.2$\times$10$^{-7}$ xtracellular CMCase and $\beta$-glucosidase activities were detected from one hydrid unlike only CMCase was detected from Cellulomonas fimi.

섬유소 분해효소를 생산하는 P. verruculosum으로부터 유도된 영 양요구성 돌연변이주의 원형질체 융합을 위한 조건을 검토하였다. 18-20시간 배양한 각 영양요구성 돌연변이주 균사체에 Novozym 234를 처리하여 원형질체를 추출할 수 있었으며, 원형질체 생성량은 각 영양요구성 돌연변이주의 균사체 40mg (dry weight) 당 2.4-3.0$\times$10/suo 7/수준이었고, 원형질체 재생 완전 배지상에서의 환원율은 26.6-42.4% 수준이었다. 원형질체 융합을 위한 Polyethylene glycol (PEG) 6000의 최적농도는 20%였으며, PEG 최적 처리시간은 10분, CaCl$_2$의 최적 첨가농도는 10mM, 최적 pH는 5.5였고, 원형질체 융합 최적조건 하에서의 융합율은 1.8$\times$$10^{-3}$ 수준으로 나타났다. 모균주와 융합체로부터 측정된 DNA 양의 차이로 보아 융합체의 염색체는 aneuploid 상태임을 알 수 있었으며, filter paper를 기질로 한 Cellulase 활성측정에서 융합체가 야생형 균주보다 2배이상, 모균주 보다는 1-3배 이상 향상되었다.

항생물질 발효 중 자주 오염을 일으키는 원인균을 분리하여 그 특성과 오염 방지를 위한 연구를 수행하였다. 오염원인균을 분리한 결과, 열저항성 포자를 형성하며, Gram 양성, Catalase 양성, 간균인 Bacillus sp. 이었다. 이 오염균은 여러종류의 항생물질에 대하여 내성을 가지고 있었으며, R-Plasmid는 갖고 있지 않았다. 항생물질 발효시 매 24시간마다 인위적으로 오염을 시켜본 결과 초기 2일내에 오염이 되었을 경우에는 항생물질 생산이 거의 이루어지지 않았으나, 발효 3일 이후 즉 항생물질 생성시기 (idiophase)에는 오염이 되었다 하더라도 항생물질 생성에 크게 영향을 못 미쳤다. 또한 초기 오염억제의 방법으로 낮은 농도의 젠타마이신을 발효 초기에 첨가한 결과 항생물질 발효에는 영향을 주지 않고서도 오염을 억제할 수 있었다.

For isolation of the CMCase gene of the alkalophilic Bacillus sp. strain N-4 to analyze their genetic information for the multicomponents of the cellulase, Bscherichia coli K12 and plasmid DNA pBR322 was used as host-vector system. After the digestion of purified chromosomal DNA and plasmid DNA pBR322 with HindIII, these were ligated. The ligated DND were transformed into Escherichia coli, and recombinant plasmid 107 carried the gene coding for CMCase was constructed. The CMCase produced by Escherichia coli cells containing plasmid DNA pYBC107 was found in the cells as intracellular enzyme and nearly 60% of the total CMCase activity was localized in cellular fraction. Also, the optimum pH for the reaction of CMCase produced by Escherichia coli was appeared at pH .8.0 and the enzyme was stable between pH 7.0 and pH 8.0.

pSp-Si (1.6kbp) was originally found in pediococcus halophilus to be a cryptic multicopy-plasmid. Hoping that the plasmid can also replicate in Bacillus subtilis, protoplast transformation of strain 207-25 (recE) was performed using pSP-Sl onto which was added the marker of tmrB8 (on 4.9 kbp EcoRI fragment ) or tmrB+ (on 0.9 kbp xbaI fragment) gene. Though the tmrB8 gene can expres tunicamycin-resistance at the single copy state, and the tmrB+ gene exerts the resistance only at the multicopy state, we could not confirm the replication of pSP-Sl (tmrB8) or pSP-Sl(tmrB+) in B. subtilis. During the experiment, however, we unexpectedly found that the circularized 0.9 kbp xgaI fragment (tmrB+) itself, which had no replication origin, could transform strain 207-25 to tunicamycin-resistant by protoplast transformation. Southern hybridization analyses with tmrB+ and other probes revealed the integration of the fragment at a single copy state into a position other than the homologous tmrB gene. This recE independent integration of another tmrB+ gene into the chromosome may contribute to the tunicamycinresistance in the transformants.

유산균발효에 이용하는 Streptococcus속과 Lactobacillus속 균주를 Sacharomyces cerevisiae KFCC 32017과 각각 대두유상에서 혼합배양하여 산생산이 좋은 균주 S. lactis KFCC 32406을 선별하였다. 혼합배양시 산생산성은 20시간 후 거의 정상치에 도달하였고 배양온도는 34$^{\circ}C$ 부근에서 가장 좋은 것으로 나타났다. Sucrose와 skim milk의 영향을 조사하기 위해 0.5 - 3%까지 각각 첨가한 결과 sucrose 1.5% 농도에서 2배 정도로 산도가 높아졌고 skim milk의 경우는 산도에 별다른 영향을 끼치지 않는 것으로 밝혀졌다. 또한 soywhey에서는 뚜렷한 차이를 보이지 않았지만 2.0-3.0% 부근의 skim milk 농도에서 산도의 증가를 나타내었다.

젖산균 Lactobacillus casei와 Saccharomyces uvarum을 사용하여 대두유중에서의 최적 젖산발효조건을 검토하였다. 젖산발효 중의 온도의 영향은 Saccharomyces uvarum과의 혼합배양에서 37-4$0^{\circ}C$가 가장 좋았다. 배양시간은 24시간일때, Lactobacillus casei 와 Saccharomyces uvarum 의 접종비는 2:1일때 산생성이 가장 좋았다. Sucrose와 skim milk의 혼합배양시 대두유의 젖산발효에 미치는 영향을 검토한 결과 sucrose의 경우 1.0% 첨가하였을 때 산생성은 높게 나타났으며 skim milk를 첨가했을 때는 첨가하지 않고 배양했을 때와 비교하여 별다른 차이를 보여주지 않았다.

L. acidophilus를 대두유에 젖산발효 시킬 때 단독배양과 효모인 K. fragilis와의 혼합배양에 대하여 검토하였다. 젖산의 생성량은 젖산균 단독배양보다는 효모와 혼합배양 하는 것이 2.6배나 많았으며 L. acidophilus를 단독배양 하였을 경우 균의 생육속도와 PH 강하속도는 K. fragilis를 단독배양한 것에 비교하여 늦었다. 그러나 혼합배양 함으로써 생육속도와 PH 강하속도는 K, fragilis를 단독배양한 것보다 빨랐다. 단독 또는 혼합젖산발효에 미치는 온도의 영향을 조사한 결과는 L. acidophilus.는 37$^{\circ}C$, K. frahilis는 3$0^{\circ}C$, 두 균의 혼합배양시는 35-37$^{\circ}C$ 범위에서 산생성이 가장 좋았다. 혼합배양 시 검토한 젖산발효 최적조건은 L. acidophilus와 K. fragilis의 접종비율이 1:5, sucrose 0.5-1.0% SKIMMILK 1.5%를 각각 첨 가한 것이 좋았다.

대두유중에서 L, acidophilus와 K. fragilis를 단독발효시키는 것 보다 혼합배양 함으로써 젖산의 생성이 촉진되었는데 균간의 어떠한 상호작용에 의하여 산의 생산이 증가되었는가를 연구하였다. 대두유와 Soywhey를 사용하였을 때의 젖산 생성속도는 L. acidophilus 단독보다는 K. fragilis 와 혼합배양하는 것이 빨랐다. 이들 균을 단독 또는 혼합배양하여 경시적으로 발효 대두유중의 당 성분을 HPLC로 분석하였다. 그 결과 L. acidophilus가 발효하기 어려운 sucrosed raffinose, stachyose를 K. fragilis가 생산하는 효소에 의하여 젖산균이 발효할 수 있는 glucose, fructose로 분해되기 때문에 혼합배양 함으로써 젖산 발효가 촉진된다는 것을 알았다.

유산균이 생성하는 polysaccharide 에 관한 연구의 일환으로서 L. casei YIT 9018 SKD-0007, L. bulgaricus SKD-0003, sir. faecalis SKD-1007, str. thermophilus SKD- 1005, str. thermophilus 510 SKD-1006등을 탈지유에 배양하면서 polysaccharide 생성에 의한 배양액의 점도 변화를 검토하였다. 이러한 다당류가 Antitumor로서의 활성과 제암효과를 가지며 인체내의 생리효과를 고양시킨다고 보고되어 있는바 다당류가 최대로 생성되는 조건을 찾는 것을 본 실험의 목적으로 하였다. 다당류의 생성을 측정하는 방법으로서 배지로는 10% 환원탈지유와 12% 환원전지유를 선택하였으며 Brookfield Viscometer를 사용하여 배양액의 점도를 경시적으로 측정하였다. 시험균 중에서 str. thermophilus 510 SKD-1006이 가장 높은 점도를 나타내었는데 41$^{\circ}C$ 배양 5일째에 5000 cps였고 다른균에 비해서 1000cps이상 높았다. 접종량이 증가할수록 점도도 증가하였으며 14%이상의 탈지유 농도에서는 7000cps를 나타냈다. 탈지유와 전지유 배양액 모두 의가소성 유체의 유동 양식을 나타내었는데 12% 환원전지유 배양액은 최대점도값이 2500 cps에 불과하였으며 배양 2일째에 최대점도 값에 도달하여 그 이후로 급격한 감소현상을 보였다.

Alkaline protease which is an important enzyme used in detergents, leather tanning and food industry was produced by alkalophilic bacterium, Bacillus sp. KCTC 1723 isolated from soil. The maximum productivity of the enzyme in alkaline medium containing 1% sodium bicarbonate was obtained by incubating for 3 days at 37$^{\circ}C$. The optimum pH of the enzyme was 11.5 and calcium ion was effective on stabilization of the enzyme at high temperature. The enzyme was not inhibited by metal chelating agent such as El)TA but inhibited by diisopropyl fluorophosphate. Purification of the enzyme was carried out DEAE- and CM-cellulose column chromatographies and molecular weight of the purified enzyme was determined