• Fisheries and Aquatic Sciences
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• 제16권4호
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• pp.311-318
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• 2013

The gene encoding an esterase from Photobacterium sp. MA1-3 was cloned in Escherichia coli using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (948 bp) corresponded to a protein of 315 amino acid residues with a molecular weight of 35 kDa and a pI of 6.06. The deduced protein showed 74% and 68% amino acid sequence identities with the putative esterases from Photobacterium profundum SS9 and Photobacterium damselae, respectively. Absence of a signal peptide indicated that it was a cell-bound protein. Sequence analysis showed that the protein contained the signature G-X-S-X-G included in most serine-esterases and lipases. The MA1-3 esterase was produced in both soluble and insoluble forms when E. coli cells harboring the gene were cultured at $18^{\circ}C$. The enzyme was a serine-esterase and was active against $C_2$, $C_4$, $C_8$ and $C_{10}$ p-nitrophenyl esters. The optimum pH and temperature for enzyme activity were pH 8.0 and $30^{\circ}C$, respectively. Relative activity remained up to 45% even at $5^{\circ}C$ with an activation energy of 7.69 kcal/mol, which indicated that it was a cold-adapted enzyme. Enzyme activity was inhibited by $Cd^{2+}$, $Cu^{2+}$, $Zn^{2+}$, and $Hg^{2+}$ ions.

• Journal of Microbiology and Biotechnology
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• 제27권2호
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• pp.277-288
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• 2017

Rhizomucor miehei NRRL 5282 and Rhizopus oryzae NRRL 1526 can produce lipases with high synthetic activities in wheat bran-based solid-state culture. In this study, the purification and biochemical characterization of the lipolytic activities of these lipases are presented. SDS-PAGE indicated a molecular mass of about 55 and 35 kDa for the purified R. miehei and Rh. oryzae enzymes, respectively. p-Nitrophenyl palmitate (pNPP) hydrolysis was maximal at $40^{\circ}C$ and pH 7.0 for the R. miehei lipase, and at $30^{\circ}C$ and pH 5.2 for the Rh. oryzae enzyme. The enzymes showed almost equal affinity to pNPP, but the $V_{max}$ of the Rh. oryzae lipase was about 1.13 times higher than that determined for R. miehei using the same substrate. For both enzymes, a dramatic loss of activity was observed in the presence of 5 mM $Hg^{2+}$, $Zn^{2+}$, or $Mn^{2+}$, 10 mM N-bromosuccinimide or sodium dodecyl sulfate, and 5-10% (v/v) of hexanol or butanol. At the same time, they proved to be extraordinarily stable in the presence of n-hexane, cyclohexane, n-heptane, and isooctane. Moreover, isopentanol up to 10% (v/v) and propionic acid in 1 mM concentrations increased the pNPP hydrolyzing activity of R. miehei lipase. Both enzymes had 1,3-regioselectivity, and efficiently hydrolyzed p-nitrophenyl (pNP) esters with C8-C16 acids, exhibiting maximum activity towards pNP-caprylate (R. miehei) and pNP-dodecanoate (Rh. oryzae). The purified lipases are promising candidates for various biotechnological applications.

• 한국표면공학회지
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• 제47권2호
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• pp.81-85
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• 2014

Ga doped ZnO (GZO)/Al bi-layered films were deposited on the glass substrate by RF and DC magnetron sputtering and then vacuum annealed at different temperatures of 100, 200 and $300^{\circ}C$ for 30 minutes to consider the effects of annealing temperature on the structural, electrical and optical properties of the films. For all depositions, the thicknesses of the GZO and Al films were kept constant at 95 and 5 nm, respectively, by controlling the deposition time. As-deposited GZO/Al bi-layered films showed a relatively low optical transmittance of 62%, while the films annealed at $300^{\circ}C$ showed a higher transmittance of 81%, compared to the other films. In addition, the electrical resistivity of the films was influenced by annealing temperature and the lowest resistivity of $9.8{\times}10^{-4}{\Omega}cm$ was observed in the films annealed at $300^{\circ}C$. Due to the increased carrier mobility, 2.35 $cm^2V^{-1}S^{-1}$ of the films. From the experimental results, it can be concluded that increasing the annealing temperature enhanced the optical and electrical properties of the GZO/Al films.

• 한국미생물·생명공학회지
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• 제8권3호
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• pp.199-206
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• 1980

전보에서 분리한 균을 사용하여 당밀과 가용성 전분을 발효기질로써 구연산생산에 관한 몇 가지 실험을 한 결과는 다음과 같다. 1) 가용성전분을 기질로 하여 분리균 M-80을 표면배양할 경우 soluble starch 120g, (N $H_4$)$_2$S $O_4$3g, K $H_2$P $O_4$2g, MgS $O_4$.7$H_2O$ 0.2g, F $e^{++}$ 1.5mg, $Zn^{++}$ 1mg, methanol 20m1, D. W. 1ι, initial pH 5.0 으로 하여 8일간 배양하였을 때 61.8mg/ml의 구연산이 생성되었다. 2) 분리균 M-315를 황혈염으로 전처리한 당밀을 기질로 하여 발효조건을 구한 결과, 표면배양은 당밀 250g, N $H_4$N $O_3$0.3g, K $H_2$P $O_4$0.05g, MgS $O_4$.7$H_2O$ 0.01g, methanol 30m1, D. W. 1ι, initial PH 5.0이었고, 액내배양의 경우는 당밀 250g, N $H_4$N $O_3$0.3g, K $H_2$P $O_4$0.1g, MgS $O_4$.7$H_2O$ 0.01g, methanol 30m1, D. W. 1ι, initial pH5.0이었으며 9일 배양으로 각각 69.4mg/ml, 39.6mg/ml의 구연산이 생성되었다.

• Journal of Microbiology and Biotechnology
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• 제25권11호
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• pp.1880-1893
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• 2015

In this study, Pseudomonas R0-14, which was isolated from Arctic soil samples, showed a clear halo when grown on M9 medium agarose plates containing olive oil-rhodamine B as substrate, suggesting that it expressed putative lipase(s). A putative lipase gene, lipR, was cloned from R0-14 by genome walking and Touchdown PCR. lipR encodes a 562-amino-acid polypeptide showing a typical α/β hydrolase structure with a catalytic triad consisting of Ser₁₅₃-Asp₂₀₂-His₂₆₀ and one α-helical lid (residues 103-113). A phylogenetic analysis revealed that LipR belongs to the lipase subfamily I.3. LipR was successfully expressed in Escherichia coli, purified, and biochemically characterized. Recombinant LipR exhibited its maximum activity towards p-nitrophenyl butyrate at pH 8.5 and 60℃ with a K_m of 0.37 mM and a k_cat of 6.42 s^-1. It retained over 90% of its original activity after incubation at 50℃ for 12 h. In addition, LipR was activated by Ca²⁺, Mg²⁺, Ba²⁺, and Sr²⁺, while strongly inhibited by Cu²⁺, Zn²⁺, Mn²⁺, and ethylenediaminetetraacetic acid. Moreover, it showed a certain tolerance to organic solvents, including acetonitrile, isopropanol, acetone, methanol, and tert-butanol. When algal oil was hydrolyzed by LipR for 24 h, there was an enrichment of n-3 long-chain polyunsaturated fatty acids, including eicosapentaenoic acid (1.22%, 1.65-fold), docosapentaenoic acid (21.24%, 2.04-fold), and docosahexaenoic acid (36.98%, 1.33-fold), and even a certain amount of diacylglycerols was also produced. As a result, LipR has great prospect in industrial applications, especially in food and/or cosmetics applications.

• Journal of Microbiology and Biotechnology
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• 제17권8호
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• pp.1271-1283
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• 2007

A fibrinolytic protease (PoFE) was purified from the cultured mycelia of the edible oyster mushroom Pleurotus ostreatus, using a combination of various chromatographies. The purification protocol resulted in an 876-fold purification of the enzyme, with a final yield of 6.5%. The apparent molecular mass of the purified enzyme was estimated to be 32 kDa by SDS-PAGE, fibrin-zymography, and size exclusion using FPLC. The optimal reaction pH value and temperature were pH 6.5 and $35^{\circ}C$, respectively. PoFE effectively hydrolyzed fibrinogen, preferentially digesting the $A{\alpha}$-chain and the $B{\beta}$-chain over the ${\gamma}$-chain. Enzyme activity was enhanced by the addition of $Ca^{2+},\;Zn^{2+},\;and\;Mg^{2+}$ ions. Furthermore, PoFE activity was potently inhibited by EDTA, and it was found to exhibit a higher specificity for the chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The first 19 amino acid residues of the N-terminal sequence were ALRKGGAAALNIYSVGFTS, which is extremely similar to the metalloprotease purified from the fruiting body of P. ostreatus. In addition, we cloned the PoFE protein, encoding gene, and its nucleotide sequence was determined. The cDNA of cloned PoFE is 867 nucleotides long and consists of an open reading frame encoding 288 amino acid residues. Its cDNA showed a high degree of homology with PoMEP from P. ostreatus fruiting body. The mycelia of P. ostreatus may thus represent a potential source of new therapeutic agents to treat thrombosis.

• 한국미생물·생명공학회지
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• 제41권2호
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• pp.145-152
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• 2013

소나무잔나비버섯(Fomitopsis pinicola MKACC 54347)의 균사체로부터 endoglucanase를 생산하기 위한 최적 배양조건을 조사하였다. 복합배지 중 MSM이 가장 높은 endoglucanase 활성을 나타내었으며, MSM의 성분과 농도를 각각 2.0% CMC, 2.0% yeast extract, 0.2% $KH_2PO_4$, 0.03% $MnSO_4$ 및 0.3% trace metal 용액으로 첨가하였을 때 효소활성이 가장 우수하였다. 따라서 F. pinicola로부터 endoglucanase를 생산하기 위한 최적 배지조건은 2.0% CMC, 2.0% yeast extract, 0.2% $KH_2PO_4$, 0.03% $MnSO_4$ 및 0.3% trace metal 용액이다. 이상의 배지를 사용하여 배양온도 $25^{\circ}C$에서 8일 동안 배양하였을 때 최대로 효소 활성이 나타내는 것을 확인하였다. CMC를 기질로 사용한 activity staining의 결과를 통해 F. pinicola 균사체의 endoglucanase 활성여부를 확인할 수 있었으며 그 분자량이 43-70 kDa 임을 확인하였고, 배양액 중의 효소활성의 최적 pH와 온도는 각각 pH 5.0과 $55^{\circ}C$인 것으로 나타났다.

• 한국식품영양과학회지
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• 제16권2호
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• pp.98-104
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• 1987

메뚜기 건조시료의 in vitro 소화율과 trypsin 비소화성물질을 정량하고, 아미노산 분석결과를 이용하여 이들 시료의 품질을 C-PER technique으로 평가하였다. 메뚜기 건조시료의 조단백질이 73% 이상(건물중량)으로 단백질원으로서의 가치가 인정되며 chitin질을 제거하면 순도가 높은 단백질을 얻을 수 있을 것이다. 특히 인(p)과 아연(zn)의 함량이 높게 나타났다. In vitro 소화율은 탈지시료가 일건 및 열풍건조 시료에 비하여 높게 나타나 열처리로 인한 소화율의 감소를 가져왔으며 이는 trypsin 비소화성물질의 증가와 밀접한 관계를 가진다. 아미노산 분석결과 lysine의 함량이 ANRC casein에 비하여 특히 낮았으며 tryptophan과 methionine의 함량이 낮았고 aspartic acid, glutamic acid, arginine등의 함량이 일건 및 열풍건조 시료에서 증가하는 경향을 나타내었다. 전반적인 단백효율비의 계산 결과 C-PER이 아미노산 분석결과로만 계산되는 DC-PER에 비해 높은 결과를 나타내었고, 예측소화율은 90%정도로 나타났다. 이상에서 메뚜기 일건 및 열건시료는 산화지질과의 반응으로 단백질의 손실을 초래하였고 TIS의 증가, 아미노산 조성의 변화로 in vitro 소화율저하를 초래하였다.

• 한국재료학회지
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• 제20권12호
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• pp.676-680
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• 2010

Most TCOs such as ITO, AZO(Al-doped ZnO), FTO(F-doped $SnO_2$) etc., which have been widely used in LCD, touch panel, solar cell, and organic LEDs etc. as transparent electrode material reveal n-type conductivity. But in order to realize transparent circuit, transparent p-n junction, and introduction of transparent p-type materials are prerequisite. Additional prerequisite condition is optical transparency in visible spectral region. Oxide based materials usually have a wide optical bandgap more than ~3.0 eV. In this study, single-phase transparent semiconductor of $SrCu_2O_2$, which shows p-type conductivity, have been synthesized by 2-step solid state reaction at $950^{\circ}C$ under $N_2$ atmosphere, and single-phase $SrCu_2O_2$ thin films of p-type TCOs have been deposited by RF magnetron sputtering on alkali-free glass substrate from single-phase target at $500^{\circ}C$, 1% $H_2$/(Ar + $H_2$) atmosphere. 3% $H_2$/(Ar + $H_2$) resulted in formation of second phases. Hall measurements confirmed the p-type nature of the fabricated $SrCu_2O_2$ thin films. The electrical conductivity, mobility of carrier and carrier density $5.27{\times}10^{-2}S/cm$, $2.2cm^2$/Vs, $1.53{\times}10^{17}/cm^3$ a room temperature, respectively. Transmittance and optical band-gap of the $SrCu_2O_2$ thin films revealed 62% at 550 nm and 3.28 eV. The electrical and optical properties of the obtained $SrCu_2O_2$ thin films deposited by RF magnetron sputtering were compared with those deposited by PLD and e-beam.

• Journal of Microbiology and Biotechnology
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• 제20권3호
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• pp.518-524
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• 2010

A mannanase gene (man26B) was obtained from a sea bacterium, Paenibacillus sp. BME-14, through the constructed genomic library and inverse PCR. The gene of man26B had an open reading frame of 1,428 bp that encoded a peptide of 475- amino acid residues with a calculated molecular mass of 53 kDa. Man26B possessed two domains, a carbohydrate binding module (CBM) belonging to family 6 and a family 26 catalytic domain (CD) of glycosyl hydrolases, which showed the highest homology to Cel44C of P. polymyxa (60% identity). The optimum pH and temperature for enzymatic activity of Man26B were 4.5 and $60^{\circ}C$, respectively. The activity of Man26B was not affected by $Mg^{2+}$ and $Co^{2+}$, but was inhibited by $Hg^{2+},\;Ca^{2+},\;Cu^{2+},\;Mn^{2+},\;K^+,\;Na^+$, and $\beta$-mercaptoethanol, and slightly enhanced by $Pb^{2+}$ and $Zn^{2+}$. EDTA did not affect the activity of Man26B, which indicates that it does not require divalent ions to function. Man26B showed a high specific activity for LBG and konjac glucomannan, with $K_m,\;V_{max}$, and $k_{cat}$ values of 3.80 mg/ml, 91.70 ${\mu}mol$/min/mg protein, and 77.08/s, respectively, being observed when LBG was the substrate. Furthermore, deletion of the CBM6 domain increased the enzyme stability while enabling it to retain 80% and 60% of its initial activity after treatment at $80^{\circ}C$ and $90^{\circ}C$ for 30 min, respectively. This finding will be useful in industrial applications of Man26B, because of the harsh circumstances associated with such processes.