Cloning, expression and properties of trehalase from Pectobacterium cypripedii / 生物工程学报

Trehalase is widely used in industrial fermentation, food, medicine and other fields. There is a lack of industrial varieties of trehalase with excellent performance in China. Moreover, the applied research on trehalase was not well conducted. In this study, a strain of Pectobacterium cypripedii was screened from nature, and the gene PCTre encoding an acidic trehalase was cloned and expressed in E. coli BL21(DE3). The highest enzyme activity reached 4130 U/mL after fermenting in a 5 L fermenter for 28 h. The enzymatic properties study showed that PCTre hydrolyzed trehalose specifically. The optimum pH and temperature were 5.5 and 35 ℃, respectively. 80% of the enzyme activity was retained after being treated at pH 4.0, 4.5, and 5.0 for 8 h, showing good acid tolerance. Moreover, it has good tolerance to organic solvents, 60% enzyme activity was retained after being treated with 20% (V/V) ethanol solution for 24 h. Furthermore, trehalose could be completely hydrolyzed within 16 h in a simulated fermentation system containing 20% (V/V) ethanol and 7.5% trehalose, with 500 U/L PCTre added. This indicated a good application potential for industrial ethanol fermentation.

Differences of the structure, succession and function of Clostridial communities between jiupei and pit mud during Luzhou-flavour baijiu fermentation / 生物工程学报

Clostridia inhabiting in jiupei and pit mud plays key roles in the formation of flavour during the fermentation process of Luzhou-flavour baijiu. However, the differences of Clostridial communities between jiupei and pit mud remains unclear. Here, the species assembly, succession, and metabolic capacity of Clostridial communities between jiupei and pit mud were analysed by high-throughput sequencing and pure culture approaches. The ratio of Clostridial biomass to bacterial biomass in the pit mud was relatively stable (71.5%-91.2%) throughout the fermentation process. However, it varied widely in jiupei (0.9%-36.5%). The dominant Clostridial bacteria in jiupei were Clostridium (19.9%), Sedimentibacter (8.8%), and Hydrogenispora (7.2%), while Hydrogenispora (57.2%), Sedimentibacter (5.4%), and Caproiciproducens (4.9%) dominated in the Clostridial communities in pit mud. The structures of Clostridial community in pit mud and jiupei were significantly different (P=0.001) throughout fermentation. Isolated Clostridial strains showed different metabolic capacities of volatile fatty acids in pure culture. Spatial and temporal heterogeneity of Clostridial communities existed in the baijiu fermentation pit, which was closely related to the main flavour components of Luzhou-flavour baijiu.

Distribution of archaeal community in the mud pit during strong-flavor baijiu fermentation / 生物工程学报

Multi-species solid-state fermentation in a mud pit is one of the typical features of strong-flavor baijiu, in which archaea plays important roles, however, the archaeal community distribution and diversity during fermentation are still lack of research. The biomass, composition and succession of archaea communities in fermented grains and pit mud were analyzed by high throughput sequencing. The potential interaction between archaea and bacteria was analyzed by co-occurrence network. Results demonstrate that the average biomass of archaea in pit mud was about 200 times higher than that of fermented grains. There was no significant difference in archaeal community structure between fermented grains and pit mud (r=0.017, P=0.074), but succession patterns between them showed significant correlation (r=0.30, P=0.03). Methanobacterium was the most abundant archaea in fermented grains and pit mud, and other dominant groups included Methanosarcina, Methanocorpusculum, Methanoculleus, and Methanobrevibacter. The co-occurrence network analysis showed that Methanobacterium was positively correlated with most bacteria in fermented grains and pit mud, especially with Hydrogenispora and Caproiciproducens, the dominant bacteria in pit mud. Our results revealed the temporal and spatial distribution characteristics and potential functions of the archaeal community in the mud pit of strong-flavor baijiu.

Amino acid exporters and metabolic modification of Corynebacterium glutamicum - a review / 生物工程学报

Amino acids are important compounds with a wide range of applications in the food, medicine and chemical industries. Corynebacterium glutamicum is a powerful workhorse commonly used in industrial amino acid production, with the scale of more than one million tons. In addition to its efficient anabolism, the effective exporters also ensure the high amino acid production by C. glutamicum. In this review, the research progress of amino acid exporter of C. glutamicum is summarized, to provide the foundation for further improving amino acid production by C. glutamicum via metabolic engineering.

Effect of analgesia and sedation scheme for ICU delirium among patients with cardiac surgery / 中国实用护理杂志

Objective To investigate the effects of analgesia and sedation scheme on ICU delirium among patients with cardiac surgery. Methods A total of 235 patients undergoing mechanical ventilation were enrolled via simple random number sampling method from January to December,2016, and divided into the experimental group(117 cases)and the control group(118 cases).The experimental group received analgesia and sedation scheme designed by our department,and the control group received routine sedation protocol. The incidence of ICU delirium, the dosages of drugs, duration of mechanical ventilation, length of ICU stay were recorded. Results The compliance rate of shallow sedation in the experimental group was 87.17% (102/117), which was lower than 57.62% (68/118) in the control group with statistical difference(χ2=25.642,P<0.05).The incidence of ICU delirium in the experimental group was 17.09% (20/117), which was lower than 34.75% (41/118) in the control group with statistical difference (χ2=9.524, P<0.05). The dosages of sedation drugs were: dexmedetomidine, (269.46 ± 32.47) μg,disoprofol,(286.84±81.96)mg,the duration of mechanical ventilation was(14.31±1.43)h,the length of ICU stay was(16.02±1.25)h.Those data were all lower than those of the control group[(507.29±58.27) μg,(575.63±95.74)mg,(20.45±2.29)h,(22.82±2.45)h]with statistical difference(t=-5.529--4.371, P<0.05). Conclusion Analgesia and sedation scheme was worthy of popularization and application.

Nursing care of patients with lung transplantation patients receiving high flow nasal cannulae oxygen therapy during perioperative period / 中国实用护理杂志

Objective To summarize the experiences of postoperative care of 7 patients with lung transplantation patients receiving high flow nasal cannulae oxygen therapy. Methods The key points to ensure the success of operation were oxygen therapy waiting for lung transplantation,sequential treatment after tracheal intubation, assisted by fiberoptic bronchoscopy, nasal high flow oxygen therapy failed to switch to non-invasive treatment or re intubation. Results All 7 patients got through the intensive care period successful. Conclusions High flow nasal cannulae can improve the comfort and compliance of lung transplant patients,and easy to operate.

Application of standardized workflow in intra-hospital transport of human infections of avian influenza A(H7N9)virus / 中国实用护理杂志

Objective To summarize the successful application experience of intra-hospital transport of 13 H7N9 avian influenza patients from the general wards to the avian influenza ward. Methods Form the expert group, to determine the design target and principle of the standardized workflow and point out the operation points of standard workflow in intra-hospital transport of each link. Results The standardized workflow included the disposal of the transfer notice, condition assessment, department contact,patient preparation,object preparation,custody transfer personnel preparation,transit guardianship and transfer to the avian influenza ward,a total of eight procedures.Between January 2013 and March 2017,13 cases were successfully transfered.All patients safely arrived avian influenza ward. The process was quick and smooth.Nobody was died or rescued within 1 h after transport. Conclusions The main differences of intra-hospital transport between H7N9 avian influenza patients and general critically ill patients are the transit time control, the particularity of terminal disposal, transshipment arrangement and hospital infection management personnel involved in the whole process.

Application of oxygen therapy for treatment of human infections of avian influenza A (H7N9) virus / 中华护理杂志

This paper retrospectively analyzed nursing care of 20 critically ill patients with human infections of avian influenza A(H7N9) virus treated by oxygen therapy.According to the severity of hypoxia in patients admitted to the hospital,individualized oxygen therapy strategy was selected,such as humidified high flow nasal cannula or mechanical ventilation.Oxygen therapy strategy was adjusted in a timely manner according to patients' condition,such as prone position ventilation and extracorporeal membrane oxygenation.As a resuh,15 cases were transferred to the general ward when the virus associated test was negative,and 5 cases died.

Nursing care of 13 critically ill patients with extracorporeal membrane oxygenation for intra-hospital transfer / 中华护理杂志

This paper summarized nursing care in intra-hospital transfer of 13 critically ill patients with extracorporeal membrane oxygenation. The key points to guarantee safety of critically ill patients were establishing a well-trained professional team and developing standardized procedures,and applying checklist for ECMO Transfer. The key points in nursing were assessment and pretreatment,homogenized nursing during transfer and effective handover after transfer. As a result,six cases of avian influenza patients successfully completed CT ex-amination,five cases of lung re-transplant patients and two cases of lung transplant patients were successfully trans-ferred to the operating room.

Application of citrate anticoagulation with continuous renal replacement therapy at high risk of bleeding patients / 中国实用护理杂志

Objective To investigate the efficacy and safety of citrate anticoagulation for continuous renal placement therapy (CRRT) in patients at high risk of bleeding. Methods Forty-seven patients who needed to CRRT and were admitted into the department of critical care medicine from January 2015 to October 2016 were enrolled in this study. According to the patient′s actual condition, they were divided into citrate groups (24 cases) and saline group (23 cases). Patients in saline group were given saline wash. The efficacy and safety were compared between the two groups. The filter lifetime, after treatment the activated partial thromboplastin time (APTT), hemoglobin (Hb), blood gas indexes were compared between the two groups. Results The citrate group used 76 filters while the 0.9% sodium chloride group used 87 filters. The average filter lifetime in citrate group was (22.4 ± 12.7) h which was longer than (8.6±3.3) h in 0.9%sodium chloride group (t=9.79, P<0.01). The incidence of coagulation in extracorporeal circulation was 3.9%(3/76) which was lower than 16.1%(14/87) in 0.9%sodium chloride group(χ2=5.20, P<0.05). Conclusions Regional citrate anticoagulation is a safe and effective option for CRRT in patients at high risk of bleeding.

Effect of calcium on sporulation of Taiwanofungus camphoratus in submerged fermentation / 生物工程学报

Taiwanofungus camphoratus is a valuable and rare medicinal mushroom with various bioactivities, such as liver protection and anti-cancer. T. camphoratus can produce many arthroconidia at the end of submerged fermentation, but molecular mechanism underlying this submerged conidiation remains unknown. In this study, we found that Ca²⁺ concentration in culture medium significantly affected the arthroconidium production of T. camphoratus. Then, we identified two proteins (CaM and HSP90) involved in Ca²⁺/calmodulin signaling pathway and one protein (AbaA) involved in FluG-mediated conidiation pathway by two-dimensional electrophoresis analyses. Furthermore, we proposed a Ca²⁺/calmodulin- and FluG-mediated signaling pathway by bioinformatics analysis. By real-time quantitative PCR analyses of 23 key genes in the Ca²⁺/calmodulin- and FluG-mediated conidiation pathway, we found that expression levels of 7 genes (crz1, hsp90, flbB, brlA, abaA, wetA and fadA) showed significant responses to Ca²⁺ concentration in fermentation medium. Our research is beneficial for elucidating the underlying mechanism of submerged fermentation conidiation for T. camphoratus.

Mutation breeding of high 9α-hydroxy-androst-4-ene-3,17- dione transforming strains from phytosterols and their conversion process optimization / 生物工程学报

In order to improve transformation efficiency of phytosterols into 9α-hydroxylation of 4-androstene-3,17-dione (9α-OH-AD) by Mycobacterium sp. LY-1, we studied the strains breeding using atmospheric and room temperature plasma (ARTP) technology and optimized their conversion process. A high production strain named C33 with a good genetic stability was selected and the product molar yield reached to 15.5%, 34.8% higher than that of original strain with 15 g/L phytosterols. Furthermore, the fermentation medium was optimized through the design of orthogonal experiment. Besides, oil-water bidirectional transformation system was set up to improve the 9α-OH-AD molar yield of mutant strain C33. With adding 12 mL soybean oil to each 1 g phytosterols, the molar yield of 9α-OH-AD reached 47.0%, which increased twice than that of control (15.5%).

Application of next generation sequencing in studying food microorganisms-a review / 生物工程学报

Next generation sequencing technology has revolutionized studies in fermentation process, in particular, to explore the mechanism by which food microorganisms, including physiology, metabolic pathways, diversity and dynamic changes of microbial community. In addition, phylogenetic characteristics of different species or strains of the food microorganisms are disclosed. All these aspects will help explain how the microbes are interacting and responding to environmental factors. Bioinformatics analysis of genome and metagenome sequence data of food microorganisms could provide essential clues to improve fermentation process and function of microbes as well as control and prevention of foodborne disease outbreak. In this review, we summarized recent genomics and metagenomics studies on food microorganisms. The impact of next generation sequencing for the development and trends of food microorganism researches were discussed in details.

Optimization of 1,2,4-butanetriol synthetic pathway in Escherichia coli / 生物工程学报

1,2,4-Butanetriol (BT) is an important non-natural chemical with a variety of industrial applications. A recombinant Escherichia coli biosynthesizing BT from D-xylose was constructed by heterologously expressing xdh and mdlC, and knocking out competing pathway genes including xylA, xylB, yjhE, yagH and ycdW. To optimize BT synthesis pathway, the third catalytic step that catalyzes the decarboxylation reaction of 3-deoxy-D-glycero-pentulosonic acid was identified as a potential bottleneck. Consequently, 2-keto acid decarboxylases from three different microorganisms were screened, and the kivD gene from Lactococcus lactis was found to increase BT titer by 191%. The improved strain BW-025 reached a final BT titer of 2.38 g/L under optimized transformation conditions. Attempts on synthetic pathway optimization were also made by fine-tuning the expression levels of each enzyme involved in the whole pathway based on BW-025. As a result, an xdh overexpressed recombinant strain, BW-074 was finally generated, with 48.62% higher BT production than that of BW-025.

Overexpressing 3-ketosteroid-Δ1-dehydrogenase for degrading phytosterols into androst-1,4-diene-3,17-dione / 生物工程学报

We constructed plasmid pMTac to overexpress 3-ketosteroid-Δ1-dehydrogenase (KSDD) in Mycobacterium neoaurum JC-12 for improving androst-1,4-diene-3,17-dione (ADD) production. To construct pMTac, pACE promoter on pMF41 was replaced by tac promoter, and then four recombinants were constructed, which were M. neoaurum JC-12/pMF41-gfp, M. neoaurum JC-12/pMTac-gfp, M. neoaurum JC-12/pMF41-ksdd and M. neoaurum JC-12/pMTac-ksdd. Fluorescence detection results show that much more green fluorescent protein (GFP) was expressed in M. neoaurum JC-12/pMTac-ksdd than M. neoaurum JC-12/pMF41-ksdd. The activity of KSDD was 2.41 U/mg in M. neoaurum JC-12/pMTac-ksdd, 6.53-fold as that of M. neoaurum JC-12 and 4.36-fold as that of M. neoaurum JC-12/pMF41-ksdd. In shake flask fermentation, ADD production of M. neoaurum JC-12/pMTac-ksdd was 5.94 g/L, increased about 22.2% compared to the original strain M. neoaurum JC-12 and 12.7% to M. neoaurum JC-12/pMF41-ksdd. AD (4-androstene-3,17-dione) production of JC-12/pMTac-ksdd was 0.17 g/L, decreased 81.5% compared to M. neoaurum JC-12 and 71.2% to M neoaurum JC-12/pMF41-ksdd. In the 5 L fermenter, 20 g/L phytosterols was used as substrate, ADD production of M. neoaurum JC-12/pMTac-ksdd was improved to 10.28 g/L. pMTac is favorable for expressing KSDD in M. neoaurum JC-12, and overexpression of KSDD has beneficial effect on ADD producing, and it is the highest level ever reported using fermentation method in M. neoaurum.

Optimization of hydroxylating DHEA to 7alpha,15alpha-diOH-DHEA by compound mutation and fermentation optimization / 生物工程学报

Combined with method of ketoconazole resistance screening, a 7alpha,15alpha-diOH-DHEA high-producing mutant Colletotrichum lini ST-1 was obtained by compound mutation of NTG and low energy N+ ion beam implantation. With the substrate concentration of 10 g/L DHEA, the molar yield of 7alpha,15alpha-diOH-DHEA reached 34.2%, increased by 46.2% than that of the original strain. Then we optimized the medium. First, Plackett-Burman design was used to evaluate the effects of medium components on molar yield of the product. Results show that glucose, yeast extract and MgSO4 x 7H2O were the important parameters for the biotransformation process. Subsequently, the path of steepest ascent was used to approach the optimal levels. To obtain the optimal levels, central composite design and response surface analysis were carried out. The optimal medium was as follows (g/L): glucose 26.34, yeast extract 12.15, corn flour 3.00, FeSO4 x 7H2O 0.015, MgSO4 x 7H2O 0.14, KH2PO4 0.90. Under the optimal conditions, the molar yield of 7alpha,15alpha-diOH-DHEA reached 49.3%, which was 44.2% higher than that of using the medium before optimization.

Screening, identification and culture optimization of a newly isolated aromatic nitrilase-producing bacterium--Pseudomonas putida CGMCC3830 / 生物工程学报

Microbial nitrilases have attracted increasing attention in nitrile hydrolysis for carboxylic acid production in recent years. A bacterium with nitrilase activity was isolated and identified as Pseudomonas putida CGMCC3830 based on its morphology, physiological and biochemical characteristics, as well as 16S rRNA gene sequence. The nitrilase production was optimized by varying culture conditions using the one-factor-at-a-time method and response surface methodology. Glycerol 13.54 g/L, tryptone 11.59 g/L, yeast extract 5.21 g/L, KH2PO4 1 g/L, NaCl 1 g/L, urea 1 g/L, initial pH 6.0 and culture temperature 30 degrees C were proved to be the optimal culture conditions. It resulted in the maximal nitrilase production of 36.12 U/mL from 2.02 U/mL. Investigations on substrate specificity demonstrate P. putida nitrilase preferentially hydrolyze aromatic nitriles. When applied in nicotinic acid synthesis, 2 mg/mL P. putida cells completely hydrolyzed 20.8 g/L 3-cyanopyridine into nicotinic acid in 90 min. The results indicated P. putida CGMCC3830 displayed potential for industrial production of nicotinic acid.

Culture conditions optimization and high cell density fermentation of recombinant bacteria producing heparinase II from Flavobacterium heparinum / 生物工程学报

Heparinase II (Hep II) from Flavobacterium heparinum is an enzyme that could specifically cleave certain sequence of heparin and heparan sulfate. In this work, fermentation conditions of recombinant heparinase II (His-Hep II) producing bacteria were optimized, including initial induction time, inducer (IPTG) concentration, induction temperature and induction time. The optimum conditions were as follows: cultivating recombinant bacteria to exponential prophase under 37 degrees C, then adding IPTG to a final concentration of 0.3 g/L, finally cultivating recombinant bacteria under 20 degrees C for 10 h. The total crude enzyme activity reached 570 U/L. Based on these results, high cell density fermentation of recombinant bacteria was studied. The final OD600 could reach 98 and the total crude enzyme activity of His-Hep II increased to 9 436 U/L.

Effects of pH and oxygen supply on production of 2,3-butanediol from biodiesel-derived glycerol by Bacillus amyloliquefaciens / 生物工程学报

Bacillus amyloliquefaciens B10-127 was used to produce 2,3-butanediol (2,3-BD) from residual glycerol obtained from biodiesel synthesis. Important variables for 2,3-BD fermentation, pH and dissolved oxygen, were studied. When pH was maintained constant, the yield of 2,3-BD was inhibited. The highest 2,3-BD yields were achieved by fermentation without any pH control with an optimized initial pH 6.5. Batch fermentative production of 2,3-BD by B. amyloliquefaciens was investigated using various oxygen supply methods by changing agitation speed. Based on the analysis of three kinetic parameters including specific cell growth rate (micro), specific glucose consumption rate (q(s)) and specific 2,3-BD formation rate (q(p)), a three-stage agitation speed control strategy was proposed, aimed at achieving high concentration, high yield and high productivity of 2,3-BD. Maximum concentration of 2,3-BD reached 38.1 g/L, with the productivity of 1.06 g/(L x h), which were 14.8% and 63.1% over the best results from constant agitation speeds. In a pulse fed-batch fermentation, 2,3-BD concentration and productivity were significantly improved to 71.2 g/L and 0.99 g/(L x h), respectively. To our knowledge, these results were the highest for 2,3-BD production from biodiesel-derived glycerol.

Breeding of high 3beta,7alpha,15alpha-trihydroxy-5-androsten-17-one transforming strains and their conversion process optimization / 生物工程学报

In order to improve transformation efficiency of dehydroepiandrosterone (DHEA) into 3beta,7alpha,15alpha-trihydroxy-5-androsten-17-one (7alpha,15alpha-diOH-DHEA) by Gibberella intermedia CA3-1, we investigated the strains breeding and their conversion process optimization. G. intermedia CA3-1 strains were treated with 0.12 mg/mL 1-methyl-3-nitro-1-nitroso-guanidin (NTG) for 30 min and chosen by 350 micromol/L minimum inhibitory concentration ketoconazole resistance marker. The high production strain named M-10 with a good genetic stability was selected and the product molar yield achieved to 70.2%, which was 20% higher than that of original strain. Under the improved conversion process with the DHEA concentration of 5 g/L, the product molar yield of the mutant M-10 reached 75.6%, which was improved by 31.3% than that of original strain.