Antarctic tundra soil metagenome as useful natural resources of cold-active lignocelluolytic enzymes

Han Na Oh,Doyoung Park,Hoon Je Seong,Dockyu Kim,Woo Jun Sul 한국미생물학회 2019 The journal of microbiology Vol.57 No.10

Lignocellulose composed of complex carbohydrates and aromatic heteropolymers is one of the principal materials for the production of renewable biofuels. Lignocellulose-degrading genes from cold-adapted bacteria have a potential to increase the productivity of biological treatment of lignocellulose biomass by providing a broad range of treatment temperatures. Antarctic soil metagenomes allow to access novel genes encoding for the cold-active lignocellulose-degrading enzymes, for biotechnological and industrial applications. Here, we investigated the metagenome targeting cold-adapted microbes in Antarctic organic matter-rich soil (KS 2-1) to mine lignolytic and celluloytic enzymes by performing single molecule, real-time metagenomic (SMRT) sequencing. In the assembled Antarctic metagenomic contigs with relative long reads, we found that 162 (1.42%) of total 11,436 genes were annotated as carbohydrate-active enzymes (CAZy). Actinobacteria, the dominant phylum in this soil’s metagenome, possessed most of candidates of lignocellulose catabolic genes like glycoside hydrolase families (GH13, GH26, and GH5) and auxiliary activity families (AA7 and AA3). The predicted lignocellulose degradation pathways in Antarctic soil metagenome showed synergistic role of various CAZyme harboring bacterial genera including Streptomyces, Streptosporangium, and Amycolatopsis. From phylogenetic relationships with cellular and environmental enzymes, several genes having potential for participating in overall lignocellulose degradation were also found. The results indicated the presence of lignocellulose-degrading bacteria in Antarctic tundra soil and the potential benefits of the lignocelluolytic enzymes as candidates for cold-active enzymes which will be used for the future biofuel-production industry.

Characterization of 5-Enolpyruvylshikimate-3-Phosphate Synthase from Colwellia psychrerythraea

Kim, Hak Jun The Korean Society for Microbiology and Biotechnol 2022 한국미생물·생명공학회지 Vol.50 No.2

Psychrophiles have evolved to produce cold-adapted enzymes to enable survival in low-temperature environments. In this study, the cold adaptation of 5-enolpyruvylshikimate-3-phosphate synthase (CpsEPSPS) from Colwellia psychrerythraea, a model psychrophile, was analyzed. The optimum temperature for the activity of CpsEPSPS was found to be 25℃, with 35% activity remaining at 5℃. However, the unfolding temperature of CpsEPSPS was 54℃. This phenomenon is frequently observed in cold-active enzymes. As is the cases for most cold-active enzymes, the K_m values of CpsEPSPS for its substrates were higher than those of Escherichia coli EPSPS. These results indicate that CpsEPSPS is cold-adapted, but not perfectly.

Identification of Proteolytic Bacteria from the Arctic Chukchi Sea Expedition Cruise and Characterization of Cold-active Proteases

박하주,이영미,김성희,위아람,한세종,김한우,김일찬,임정한,김덕규 한국미생물학회 2014 The journal of microbiology Vol.52 No.10

Following collection of seawater samples during an ArcticChukchi Sea expedition cruise of the Korean icebreakerAraon in 2012, a total of 15,696 bacteria were randomly isolatedfrom Marine Broth 2216 agar plates. Of these, 2,526(16%) showed proteolytic activity and were identified asmainly Alteromonas (31%), Staphylococcus (27%), and Pseudoalteromonas(14%). Among the proteolytic strains, sevenwere selected based on their significant ability to grow andproduce a halo on skim milk plates at low temperatures(<5°C) owing to cold-active proteases. These strains wereaffiliated with the genus Pseudoalteromonas and were dividedinto three groups based on phylogenetic analysis of the 16SrRNA genes. Profiling cell membrane fatty acids confirmedthe 16S rRNA-based differentiation and revealed the accordancebetween the two analyses. Seven genes for serine proteaseprecursors were amplified from the correspondingstrains, and based on sequence similarities, these genes weredivided into three groups that were identical to those identifiedby the 16S rRNA phylogenetic analysis. Three proteasegenes from the representative strains of each groupwere composed of 2,127–2,130 bp, encoding 708–709 aminoacids, and these genes yielded products with calculated molecularweights of approximately 72.3–72.8 kDa. Amino acidsequence analysis suggested that the precursors are membersof the subtilase serine endo- and exo-peptidase clan and containfour domains (signal peptide, N-terminal prosequence,catalytic domain, and two pre-peptidase C-terminal domains). Upon expression in E. coli, each recombinant protease exhibitedproteolytic activity on zymogram gels.

Multicatalytic Alkaline Serine Pretense from the Psychrotrophic Bacillus amyloliquefaciens S94

Son, Eui-Sun,Kim, Jong-Il The Microbiological Society of Korea 2003 The journal of microbiology Vol.41 No.1

An extracellular pretense of Bacillus amyloliquefaciens S94 was purified to apparent homogeneity. The enzyme activity was strongly inhibited by general inhibitor for serine protease, PMSF, suggesting that the enzyme is a serine pretense. The purified enzyme activity was inhibited by leucine peptidase inhibitor, bestatin, suggesting that the enzyme is a leucine endopeptidase. The maximum proteolytic activity against different protein substrates occurred at pH 10, 45$^{\circ}C$ (protein substrate) and pH 8, 45$^{\circ}C$ (synthetic substrate). The purified enzyme was specific in that it readily hydrolyBed substrates with Leu or Lys residues at P$_1$ site. The pretense had characteristics of a cold-adapted protein, which was more active for the hydrolysis of synthetic substrate in the range of 15$^{\circ}C$ to 45$^{\circ}C$, specially at low temperature.

Multicatalytic Alkaline Serine Protease from the Psychrotrophic Bacillus amyloliquefaciens S94

Eui-Sun Son,Jong-Il Kim 한국미생물학회 2003 The journal of microbiology Vol.41 No.1

An extracellular protease of Bacillus amyloliquefaciens S94 was purified to apparent homogeneity. The enzyme activity was strongly inhibited by general inhibitor for serine protease, PMSF, suggesting that the enzyme is a serine protease. The purified enzyme activity was inhibited by leucine peptidase inhibitor, bestatin, suggesting that the enzyme is a leucine endopeptidase. The maximum proteolytic activity against different protein substrates occurred at pH 10, 45oC (protein substrate) and pH 8, 45oC (synthetic substrate). The purified enzyme was specific in that it readily hydrolyzed substrates with Leu or Lys residues at P1 site. The protease had characteristics of a cold-adapted protein, which was more active for the hydrolysis of synthetic substrate in the range of 15oC to 45oC, specially at low temperature.

Cold plasma treatment advancements in food processing and impact on the physiochemical characteristics of food products

Salma Farooq,Aamir Hussain Dar,Kshirod Kumar Dash,Shivangi Srivastava,Vinay Kumar Pandey,Wani Suhana Ayoub,R. Pandiselvam,Sobiya Manzoor,Mandeep Kaur 한국식품과학회 2023 Food Science and Biotechnology Vol.32 No.5

Cold plasma processing is a nonthermal approach that maintains food quality while minimizing the effects of heat on its nutritious qualities. Utilizing activated, highly reactive gaseous molecules, cold plasma processing technique inactivates contaminating microorganisms in food and packaging materials. Pesticides and enzymes that are linked to quality degradation are currently the most critical issues in the fresh produce industry. Using cold plasma causes pesticides and enzymes to degrade, which is associated with quality deterioration. The product surface characteristics and processing variables, such as environmental factors, processing parameters, and intrinsic factors, need to be optimized to obtain higher cold plasma efficiency. The purpose of this review is to analyse the impact of cold plasma processing on qualitative characteristics of food products and to demonstrate the effect of cold plasma on preventing microbiological concerns while also improving the quality of minimally processed products.

HvIRIP 과발현 유채 형질전환체의 내한성 증진

노경희 ( Kyung Hee Roh ),박종석 ( Jong Sug Park ),강한철 ( Han Chul Kang ),김종범 ( Jong Bum Kim ),장영석 ( Young Suk Jang ),김광수 ( Kwang Soo Kim ),이한길 ( Hankuil Yi ) 한국응용생명화학회 2015 Journal of Applied Biological Chemistry (J. Appl. Vol.58 No.4

Rapeseed (Brassica napus) is now the second largest oilseed crop after soybean. Cold temperature tolerance is an important agronomic trait in winter rapeseed that determines the plant`s ability to control below freezing temperatures. To improve cold tolerance of rapeseed plants, an expression vector containing an Barley Ice recrystallization inhibition protein (HvIRIP) cDNA driven by a cauliflower mosaic virus 35S promoter was transferred into rapeseed plants. Transgenic expression of HvIRIP was proved by southern- and northern-blot analyses. The level of freezing tolerance of transgenic T3 plants was found to be significantly greater than that of wild-type rapeseed plants by freezing assay. Proline accumulation during cold stress was also highly induced in the transgenic rapeseed plants. The transgenic plants exhibited considerable tolerance against oxidative damage induced by cold stress. Our results indicated that heterologous HvIRIP expression in transgenic rapeseed plants may induce several oxidative-stress responsive genes to protect from cold stress.

Diversity and Physiological Characteristics of Culturable Bacteria from Marine Sediments of Ross Sea, Antarctica

이영미,정유정,홍순규,김지희,이홍금,Lee, Yung Mi,Jung, You-Jung,Hong, Soon Gyu,Kim, Ji Hee,Lee, Hong Kum The Microbiological Society of Korea 2014 미생물학회지 Vol.50 No.2

남극 로스해의 퇴적물로부터 배양을 통해 분리한 균주의 분류 및 생리학적 특성 분석을 수행하였다. 분리 세균 63균주의 16S rRNA 유전자 염기서열을 이용한 계통분류학적 분석 결과, 이들은 Actinobacteria, Bacteroidetes, Alphaproteobacteria 및 Gammaproteobacteria 내의 21개의 파일로타입(phylotypes)에 속하였다. 98.65% 염기서열 유사도를 기준으로, 약 49%의 균주가 잠재적으로 신종 또는 신속 후보인 것으로 나타났다. 분리된 균주 중, 각각 46%, 25% 및 32%의 균주가 세포외 단백질분해효소, 지질분해효소 및 외부다당체 생성에 대한 활성을 나타냈다. 43개의 균주는 최소 1개의 세포외 분비 물질을 생산하였고, 이들 중 21개 균주는 최소 2개의 세포외 단백질분해효소, 지질분해효소 또는/및 세포외다당체를 생성하였다. 이러한 결과는 남극 로스해 퇴적물 내의 배양된 세균 군집이 해당 환경에서 탄소와 질소와 관련된 유기물질의 가수분해에 영향을 미치고 있다는 것을 시사한다. The affiliations and physiological characteristics of culturable bacteria isolated from the sediments of Ross Sea, Antarctica were investigated. Sixty-three isolates obtained by cultivation were grouped into 21 phylotypes affiliated with the phyla Actinobacteria and Bacteroidetes and with the classes Alphaproteobacteria and Gammaproteobacteria by phylogenetic analysis of 16S rRNA gene sequences. Based on phylogenetic analysis (<98.65% sequence similarity), approximately 49% of total isolates represented potentially novel species or genus. Among them, extracellular protease, lipase, and exopolysaccharide activities at $10^{\circ}C$ or $20^{\circ}C$ were detected in approximately 46%, 25%, and 32% of the strains, respectively. Forty-three isolates produced at least one type of extracellular material and 21 of them produced at least two extracellular protease, lipase, and/or exopolysaccharides. Our findings indicate that culturable bacterial diversity present within the marine sediments of Ross Sea, Antarctica may contribute to the hydrolysis of the major organic constituents which is closely related with carbon and nitrogen cycling in this environment.

Comparative Analysis of Arthrobacter spp. Provide Insight into Their Elucidating the Role of CAZyme in a Cold Environment

So-Ra HAN,Tae-Jin OH 한국생물공학회 2022 한국생물공학회 학술대회 Vol.2022 No.9

Construction of Pseudoalteromonas - Escherichia coli shuttle vector based on a small plasmid from the marine organism Pseudoalteromonas

김덕규,박하주,박현,Kim, Dockyu,Park, Ha Ju,Park, Hyun The Microbiological Society of Korea 2016 미생물학회지 Vol.52 No.1

남극 해양세균 Pseudoalteromonas sp. PAMC 21150에서 분리한 소형 플라스미드(small plasmid, pDK4)의 크기는 3,480bp이고 G+C 함량은 41.64%이며, 3개의 open reading frames(ORFs)을 포함하고 있다. 3개의 ORF는 replication initiation protein (RepA), conjugative mobilization protein (Mob), 그리고 기능이 밝혀지지 않은 단백질을 코팅하고 있다. PCR 반응으로 증폭한 pDK4를 Escherichia coli high-copy pUC19 클로닝 벡터에 삽입하여 fusion vector (pDOC153)를 제작하였고, pDOC 153에 chloramphenicol 저항성 유전자를 삽입하여 ampicillin/chloramphenicol 저항성 Pseudoalteromonas - Escherichia coli 셔틀 벡터(shuttle vector; 7,216 bp 크기; pDOC155)를 제작하였다. 북극 해양세균 P. issachenkonii PAMC 22718이 보유한 2개의 유전자(TonB-dependent receptor gene, chi22718_IV, and exochitinase gene, chi22718_III)를 pDOC155에 삽입하여 두 개의 pDOC155 변형체(pDOC158, pDOC165)를 제작하였다. pDOC158 혹은 pDOC165을 이용하여 triparental mating 방법에 의해 플리스미드 미보유 해양세균인 Pseudoalteromonas sp. PAMC 22137를 형질전환하였다. PCR을 이용한 유전자 증폭실험을 통해서, pDOC158와 pDOC165에 삽입된 유전자들은 Pseudoalteromonas sp. PAMC 22137와 E. coli $DH5{\alpha}$ 내에서 안정적으로 유지되는 것을 확인하였다. 위의 결과는 셔틀 벡터 pDOC155는 Pseudoalteromonas spp. 유래 유전자들을 다른 Pseudoalteromonas spp. 세포 안으로 전달할 수 있는 새로운 유전자 전달시스템으로 이용될 수 있음을 보여주었다. A small plasmid (pDK4) from the Antarctic marine organism Pseudoalteromonas sp. PAMC 21150, was purified, sequenced and analyzed. pDK4 was determined to be 3,480 bp in length with a G+C content of 41.64% and contains three open reading frames encoding a replication initiation protein (RepA), a conjugative mobilization protein (Mob) and a hypothetical protein. PCR-amplified pDK4 was cloned in high-copy pUC19 to yield the fusion vector pDOC153. The chloramphenicol resistance gene was inserted into pDOC153 to give an ampicillin and chloramphenicol-resistant, Pseudoalteromonas - Escherichia coli shuttle vector (7,216 bp; pDOC155). The TonB-dependent receptor (chi22718_IV ) and exochitinase (chi22718_III ) genes from Arctic marine P. issachenkonii PAMC 22718 were cloned into pDOC155 to produce pDOC158 and pDOC165, respectively. Both vector derivatives were transferred into plasmid-free Pseudoalteromonas sp. PAMC 22137 by the triparental mating method. PCR experiments showed that the genes were stably maintained both in Pseudoalteromonas sp. PAMC 22137 and E. coli $DH5{\alpha}$ cells, indicating the potential use of pDOC155 as a new gene transfer system into marine Pseudoalteromonas spp.