Dataset Information

Expression of heat-resistant β-glucosidase in Escherichia coli and its application in the production of gardenia blue.

ABSTRACT: Gardenia blue is a natural blue pigment that is environmentally friendly, non-toxic, and stable. The hydrolysis of geniposide, catalyzed by β-glucosidase, is a critical step in the production process of gardenia blue. However, β-glucosidase is not resistant to high temperatures, limiting the production of gardenia blue. In this study, we investigated the effectiveness of a heat-resistant glucosidase obtained from Thermotoga maritima in the production of gardenia blue. The enzyme exhibited a maximum activity of 10.60 U/mL at 90 °C. Single-factor and orthogonal analyses showed that exogenously expressed heat-resistant glucosidase reacted with 470.3 μg/mL geniposide and 13.5 μg/mL glycine at 94.2 °C, producing a maximum yield of 26.2857 μg/mL of gardenia blue after 156.6 min. When applied to the dyeing of denim, gardenia blue produced by this method yielded excellent results; the best color-fastness was achieved when an iron ion mordant was used. This study revealed the feasibility and application potential of microbial production of gardenia blue.

SUBMITTER: Li W

PROVIDER: S-EPMC8390534 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Expression of heat-resistant β-glucosidase in <i>Escherichia coli</i> and its application in the production of gardenia blue.

Li Wenxi W Li Jielin J Xu Ying Y Huang Yan Y Xu Shuqi S Ou Zirui Z Long Xiaoli X Li Xinyu X Liu Xinyu X Xiao Zening Z Huang Jiaqi J Chen Weizhao W

Synthetic and systems biotechnology 20210824 3

Gardenia blue is a natural blue pigment that is environmentally friendly, non-toxic, and stable. The hydrolysis of geniposide, catalyzed by β-glucosidase, is a critical step in the production process of gardenia blue. However, β-glucosidase is not resistant to high temperatures, limiting the production of gardenia blue. In this study, we investigated the effectiveness of a heat-resistant glucosidase obtained from <i>Thermotoga maritima</i> in the production of gardenia blue. The enzyme exhibited ...[more]

PMID: 34504963

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Project description:Gardenia blue (GB) is a natural blue pigment widely used in textiles and the pharmaceutical industry. The geniposide in gardenia fruits can be hydrolyzed by β-glucosidase to form genipin, which reacts with amino acids to produce GB. In this study, a bacterial strain which secreted thermostable β-glucosidase (EC 3.2.1.21) was isolated from soil and identified as Bacillus altitudinis JYY-02. This strain could potentially be used for GB production from geniposide by fermentation. Optimal fermentation results were achieved at pH 6.5 or 8.0 at 45°C for 45 h with additional sucrose. To obtain a large amount of β-glucosidase, the whole genome of B. altitudinis JYY-02 was sequenced and annotated; it is 3,727,518 bp long and contains 3,832 genes. The gene encoding β-glucosidase (bgl) in B. altitudinis JYY-02 was screened from the genome and overexpressed in Escherichia coli BL21(DE3). The recombinant β-glucosidase was purified by affinity chromatography on a Ni Sepharose 6 fast flow (FF) column. The optimal temperature, pH, and Km values for the recombinant β-glucosidase were 60°C, pH 5.6, and 0.331 mM, respectively, when p-nitrophenyl-β-d-glucopyranoside (pNPG) was used as the substrate. The recombinant β-glucosidase catalyzed the deglycosylation reaction of geniposide, which was then used to produce GB. IMPORTANCE β-Glucosidases are enzymes capable of hydrolyzing β-glucosidic linkages present in saccharides and glycosides and have many agricultural and industrial applications. Although they are found in all domains of living organisms, commercial β-glucosidases are still expensive, limiting their application in industry. In the present study, a thermostable β-glucosidase-producing strain was obtained for GB production by fermentation, engineered bacteria were constructed for preparing recombinant β-glucosidase, and a one-step method to purify the recombinant enzyme was established. A large amount of purified β-glucosidase was easily obtained from the engineered bacteria for industrial applications such as GB production.

Project description:Backgroundβ-Glucosidase has attracted substantial attention in the scientific community because of its pivotal role in cellulose degradation, glycoside transformation and many other industrial processes. However, the tedious and costly expression and purification procedures have severely thwarted the industrial applications of β-glucosidase. Thus development of new strategies to express β-glucosidases with cost-effective and simple procedure to meet the increasing demands on enzymes for biocatalysis is of paramount importance.ResultsLight activated cassette YF1/FixJ and the SRRz lysis system were successfully constructed to produce Bgl1A(A24S/F297Y), a mutant β-glucosidase tolerant to both glucose and ethanol. By optimizing the parameters for light induction, Bgl1A(A24S/F297Y) activity reached 33.22 ± 2.0 U/mL and 249.92 ± 12.25 U/mL in 250-mL flask and 3-L fermentation tank, respectively, comparable to the controls of 34.02 ± 1.96 U/mL and 322.21 ± 10.16 U/mL under similar culture conditions with IPTG induction. To further simplify the production of our target protein, the SRRz lysis gene cassette from bacteriophage Lambda was introduced to trigger cell autolysis. As high as 84.53 ± 6.79% and 77.21 ± 4.79% of the total β-glucosidase were released into the lysate after cell autolysis in 250 mL flasks and 3-L scale fermentation with lactose as inducer of SRRz. In order to reduce the cost of protein purification, a cellulose-binding module (CBM) from Clostridium thermocellum was fused into the C-terminal of Bgl1A(A24S/F297Y) and cellulose was used as an economic material to adsorb the fusion enzyme from the lysate. The yield of the fusion protein could reach 92.20 ± 2.27% after one-hour adsorption at 25 °C.ConclusionsWe have developed an efficient and inexpensive way to produce β-glucosidase for potential industrial applications by using the combination of light induction, cell autolysis, and CBM purification strategy.

Project description:Decontamination practices, which often involve thermal treatments, are routinely performed in beef packing plants and have generally improved the safety of meat in North America. We investigated whether Escherichia coli in the beef production chain is becoming more heat resistant due to those treatments. Cattle isolates (n = 750) included seven serogroups (O157, O103, O111, O121, O145, O26, and O45) which were collected between 2002 and 2017. Beef plant isolates (n = 700) from carcasses, fabrication equipment, and beef products were included. Heat resistance was determined in Luria-Bertani broth at 60°C and by PCR screening for the locus of heat resistance (LHR). The decimal reduction for E. coli at 60°C (D60ºC values) ranged from 0 to 7.54 min, with 97.2% of the values being <2 min. The prevalence of E. coli with D60ºC values of >2 min was not significantly different (P > 0.05) among cattle and meat plant isolates. E. coli from equipment before sanitation (median, 1.03 min) was more heat resistant than that after sanitation (median, 0.9 min). No significant difference in D60ºC values was observed among E. coli isolates from different years, from carcasses before and after antimicrobial interventions, or from before and during carcass chilling. Of all isolates, 1.97% harbored LHR, and the LHR-positive isolates had greater median D60ºC values than the LHR-negative isolates (3.25 versus 0.96 min). No increase in heat resistance in E. coli was observed along the beef production chain or with time.IMPORTANCE The implementation of multiple hurdles in the beef production chain has resulted in substantial improvement in the microbial safety of beef in Canada. In this study, we characterized a large number of Escherichia coli isolates (n = 1,450) from various sources/stages of beef processing to determine whether the commonly used antimicrobial interventions would give rise to heat-resistant E. coli on meat, which in turn may require alternatives to the current control of pathogens and/or modifications to the current cooking recommendations for meat. The findings show that the degree and rate of heat resistance in E. coli did not increase along the production chain or with time. This furthers our understanding of man-made ecological niches that are required for the development of heat resistance in E. coli.

Dataset Information

Expression of heat-resistant β-glucosidase in Escherichia coli and its application in the production of gardenia blue.

Publications

Expression of heat-resistant β-glucosidase in <i>Escherichia coli</i> and its application in the production of gardenia blue.

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