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The selection of alkaline protease-producing yeasts from marine environments and evaluation of their bioactive peptide production

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chinese Journal of Oceanology and Limnology 2009年第4期27卷 753-761页

作者：李静池振明王祥红彭莹池哲 unesco chinese center of marine biotechnology Ocean University of China

A total of 400 yeast strains from seawater, sediments, saltern mud, marine fish guts, and marine algae were obtained. The protease activity of the yeast cultures was estimated, after which four strains （HN3.11, Nllb, YF04C and HN4.9） capable of secreting extracellular alkaline protease were isolated. The isolated strains were identified as Aureobasidium pullulans, Yarrowia lipolytica, lssatchenkia orientalis and Cryptococcus cf. aureus. The optimal pH of the protease activity produced by strains HN3.11, YF04C, and HN4.9 was 9.0, while that of the protease produced by strain N1 lb was 10.0. The optimal temperature for protease activity was 45℃for strains HN3.11, N11b, and YF04C, and 50℃ for strain HN4.9. After digestion of shrimp （Penaeus vannamei） protein and spirulina （Arthospira platens＆） protein with the four crude alkaline proteases, the filtrate from spirulina （Arthrospira platensis） powder digested by the crude alkaline protease of strain HNYl 1 was found to have the highest antioxidant activity （61.4%） and the highest angiotensin I converting enzyme （ACE）-inhibitory activities （68.4%）. The other filtrates had much lower antioxidant activity and ACE-inhibitory activities.

关键词： marine yeasts alkaline protease yeast diversity phylogenetic analysis bioactive peptide

A Carboxymethyl Cellulase from a marine Yeast(Aureobasidium pullulans 98): Its Purification, Characterization, Gene Cloning and Carboxymethyl Cellulose Digestion

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Journal of Ocean University of China 2015年第5期14卷 913-921页

作者： RONG Yanjun ZHANG Liang CHI Zhenming WANG Xianghong unesco chinese center of marine biotechnology Ocean University of China

We have reported that A. pullulans 98 produces a high yield of cellulase. In this study, a carboxymethyl cellulase （CMCase） in the supematant of the culture ofA. pullulans 98 was purified to homogeneity, and the maximum production of CMCase was 4.51 U （mg protein）-1. The SDS-PAGE analysis showed that the molecular mass of the purified CMCase was 67.0kDa. The optimal temperature of the purified enzyme with considerable thermosensitivity was 40℃, much lower than that of the CMCases from other ftmgi. The optimal pH of the enzyme was 5.6, and the activity profile was stable in a range of acidity （pH 5,0-6.0）. The enzyme was activated by Na＋, Mg2＋, Ca2＋, K＋, Fe2＋ and Cu2＋, however, it was inhibited by Fe3＋, Ba2＋, Zn2＋, Mn2＋ and Ag＋. Km and Vmax values of the purified enzyme were 4.7mgmL-1 and 0.57 pmol L-1 min-1 （mg protein）-1, respectively. Only oligosaccharides with different sizes were released from carboxymethylcellulose （CMC） after hydrolysis with the purified CMCase. The putative gene encoding CMCase was cloned from A. pullulans 98, which contained an open reading flame of 954bp （EU978473）. The protein deduced contained the conserved domain of cellulase superfamily （glucosyl hydrolase family 5）. The N-terminal amino acid sequence of the purified CMCase was M-A-P-H-A-E-P-Q-S-Q-T-T-E-Q-T-S-S-G-Q-F, which was consistent with that deduced from the cloned gene. This suggested that the purified CMCase was indeed encoded by the cloned CMCase gene in this yeast.

关键词： marine yeast Aureobasidium pullulans gene cloning carboxymethyl cellulase purification characterization

Screening of protease-producing marine yeasts for production of the bioactive peptides

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Acta Oceanologica Sinica 2008年第2期27卷 116-125页

作者： NI Xiumei CHI Zhenming LIU Zhiqiang YUE Lixi unesco chinese center of marine biotechnology Ocean University of China Qingdao 266003 China

Over 400 yeast strains from seawater and sediments were obtained, but only five strains named HN2 -3, N13d, N13C, Mb5 and HN3 - 2 among them could form clear zones around their colonies on the double plates with 2.0% casein. Peptides in the hydrolysate produced by the proteases from strains HN2 -3 and N13d had higher angiotensin I-converting-enzyme （ACE）-inhibitory activity. The two marine yeast strains were identified to be Aureobasidium pullulans according to the results of routine yeast identification and molecular methods. After purification of the proteases from the two marine yeast strains, it was found that the optimal pH for them was both 9.0, both of them were serine alkaline protease. However, the optimal temperature for the protease from the strain HN2 -3 was 52℃ while that from strain N13d was 48℃. ACE-inhibitory activity of the peptides in the hydrolysate of shrimp protein produced by the purified protease from the strain HN2 -3 was the highest while antioxidant activity in the hydrolysate of spirulina protein produced by the purified protease from the strain N13d was the highest.

关键词： marine yeasts alkaline protease bioactive peptide ACE inhibitory activity antioxidant activity

Isolation and identification of a marine killer yeast strain YF07b and cloning of the gene encoding killer toxin from the yeast

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Acta Oceanologica Sinica 2007年第6期26卷 101-108页

作者： WANG Xianghong CHI Zhenming LI Jing unesco chinese center of marine biotechnology Ocean University of China Qingdao 266003 China

It was found that the marine yeast strain YF07b could secrete a large amount of killer toxin against a pathogenic yeast strain WCY which could cause milky disease in Portunus trituberculcttus. The marine yeast strain YF07b was identified to be Pichia anomala according to the results of routine yeast identification and 18S rDNA and ITS sequences. The gene encoding killer toxin in the marine yeast strain YF07b was amplified by PCR technology. After sequencing, the results show that an open reading frame, consisting of 1 281 bp, encoded a presumed protein of 427 amino acids. The sequence of the cloned gene was found to have 99% match with that of the gene encoding killer toxin in Pichia anomalas strain K. A signal peptide including 17 amino acids appeared in the N-terminal domain of the killer toxin. Therefore, the mature protein consisted of 410 amino acids, its molecular mass was estimated to be 47.4 ku and its isoelctronic point was 4.5.

关键词： pathogenic yeast crab marine killer yeast identification killer toxin gene cloning

Optimization of medium and cultivation conditions for enhanced exopolysaccharide yield by marine Cyanothece sp. 113

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chinese Journal of Oceanology and Limnology 2007年第4期25卷 411-417页

作者：苏传东池振明卢伟东 unesco chinese center of marine biotechnology Ocean University of China

Cyanothece sp. 113 is a unicellular, aerobic, diazotrophic and photosynthetic marine cyanobacterium. The optimal medium for exopolysaccharide yield by the strain was 70.0 g/L of NaCl, and 0.9 g/L of MgSO4 based on the modified F/2 medium for cultivation of marine algae. The optimal cultivation condition for exopolysaccharide yield by this cyanobacterial strain was 29℃, aeration, and continuous illumination at 86.0 μE/M^2/S. Under the optimal conditions, over 18.4 g/L of exopolysaccharide was produced within 12 days. This was so far the highest exopolysaccharide yield produced with strains of Cyanothece sp. obtained.

关键词： Cyanothece exopolysaccharide N2 fixation photosynthesis marine cyanobacteria

Occurrence and Diversity of marine Yeasts in Antarctica Environments

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Journal of Ocean University of China 2012年第1期11卷 70-74页

作者： ZHANG Xue HUA Mingxia SONG Chunli CHI Zhenming unesco chinese center of marine biotechnology Ocean University of ChinaQingdao 266003P.R.China

A total of 28 yeast strains were obtained from the sea sediment of *** to the results of routine identi-fication and molecular characterization,the strains belonged to species of Yarrowia lipolytica,Debaryomyces hansenii,Rhodotorula slooffiae,Rhodotorula mucilaginosa,Sporidiobolus salmonicolor,Aureobasidium pullulans,Mrakia frigida and Guehomyces pullu-lans,*** Antarctica yeasts have wide potential applications in biotechnology,for some of them can produce b-galactosidase and killer toxins.

关键词： Antarctica Psychrotolerant yeasts identification b-galactosidase killer toxin

Amylase Production by the marine Yeast Aureobasidium pullulans N13d

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Journal of Ocean University of China 2007年第1期6卷 60-65页

作者： LI Haifeng CHI Zhenming WANG Xiaohong a MA Chunling unesco chinese center of marine biotechnology Ocean University of ChinaQingdao 266003P.R.China

The marine yeast strain N13d, producing an extracellular amylase, was isolated from the deep sea sediments of the Pa-cific Ocean. This strain was identified to be Aureobasidium pullulans by 18S rRNA gene sequence analysis and routine yeast identi-fication methods. The optimal sea water medium for amylase production by this yeast strain was 1.0% peptone and 1.0% soluble starch with pH 4.0. The optimal conditions for amylase production by this yeast strain were with temperature 28 ℃, aeration rate 6 Lmin-1 and agitation speed 250 rmin-1. Under these conditions, 58.5 units of amylase activity per mg protein were produced within 56 h of fermentation.

关键词： extracellular amylase marine yeast Aureobasidium pullulans fermentation

Enhanced Carotenoid Production by a Mutant of the marine Yeast Rhodotorula sp. hidai

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Journal of Ocean University of China 2007年第1期6卷 66-71页

作者： CONG Li CHI Zhenming LI Jing WANG Xianghong unesco chinese center of marine biotechnology Ocean University of ChinaQingdao 266003P.R.China

After a serial of UV, EMS and NTG mutagenesis, a mutant named MM of the red marine yeast strain Rhodotorula sp. hidai was obtained. The mutant MM could produce 603.93 μg g-1 of carotenoid within 5 days in the medium containing 4.0 g sucrose, 1.5 g yeast extract, 0.1 g MgSO4, and 100 mL of sea water, with pH 6.0 and at 30 ℃, while only 213.18 μg g-1 of carotenoid was pro-duced by the wild type under the same conditions.

关键词： red marine yeasts Rhodotorula sp.hidai carotenoids fermentation

Exopolysaccharide Production by Four Cyanobacterial Isolates and Preliminary Identification of These Isolates

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Journal of Ocean University of China 2007年第2期6卷 147-152页

作者： ZHANG Yunyi CHI Zhenming LU Weidong unesco chinese center of marine biotechnology Ocean University of China Qingdao 266003 P. R. China

Four marine cyanobacterial isolates, named 104, 109, 113 and 115, from marine water off China’s coast can release a large amount of exopolysaccharide (EPS) to *** effects of different components in medium on EPS production by the four isolates were investigated. Under the optimal condition, the EPS released by isolates 104, 109, 113 and 115 reached 7.48 g L-1, 8.33 g L-1, 18.26 g L-1, and 6.78 g L-1 within 14 d, respectively. Based on the conventional identification methods for cyanobacteria, these isolates were assigned to genus Cyanothece.

关键词： cyanobacteria Cyanothece exopolysaccharide phylogenetic tree