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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2019, Vol. 39 Issue (4): 38-51    DOI: 10.13523/j.cb.20190406
    
The Effective of Additives on the Immobilization of Lipase by Microporous Absorbent Resin
Hai-jiao LIN1,2,Ji-fu ZHANG3,Yun ZHANG1,Ai-jun SUN1,Yun-feng HU1**()
1 CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301,China
2 University of Chinese Academy of Sciences,Beijing 100049,China
3 Guangdong Provincial Hospital of Chinese Medicine, Guangzhou 510120, China
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Abstract  

Macroporous absorbent resin DA-201 was adopted as a carrier to immobilize marine-derived lipase, and the influence of additives on the immobilization process was explored. NH4Cl, mannose and glycine were used as the additives, and the conditions were optimized by a combination of single factor and orthogonal experiments. The results showed that the optimal conditions with additive NH4Cl were: citric acid-sodium citrate buffer pH6.0, immobilization temperature 30℃, carrier quantity 0.5g, NH4Cl concentration 25mmol/L, immobilized time 3.0h; enzyme activity reached 115.27U/g, which was 47.42% higher than that of the immobilized enzyme without additive. The optimal conditions with additive mannose were: potassium dihydrogen phosphate-sodium hydroxide buffer pH7.0, immobilized temperature 35℃, carrier quantity 0.5g, mannose concentration 10mmol/L, the immobilized time 4.5h; the enzyme activity reached 122.75U/g, which was 6.50% higher than that of the immobilized enzyme without additive. The optimal conditions with glycine additive: potassium dihydrogen phosphate-sodium hydroxide buffer pH7.0, immobilized temperature 20℃, carrier quantity 0.5g, the glycine concentration 25mmol/L, the immobilized time 7.5h; the enzyme activity reached 141.69U/g, which was 26.12% higher than that of the immobilized enzyme without additive. The addition of different additives exhibited great effects on the immobilization through absorbtion by macroporous absorbent resin DA-201 and could greatly improve the adsorption efficiency. Additionally, buffer type, pH, temperature, additive concentration and immobilization time were found to have great influence on the adsorption of lipase by resin DA-201, which provides good reference for subsequent research of the immobilization of industrial enzymes.

Key words： Macroporous absorbent resin      Marine lipase      Immobilization      Additives     
Received: 01 November 2018      Published: 08 May 2019
ZTFLH:  Q814.2  
Corresponding Authors: Yun-feng HU     E-mail: yunfeng.hu@scsio.ac.cn
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Hai-jiao LIN
Ji-fu ZHANG
Yun ZHANG
Ai-jun SUN
Yun-feng HU
Cite this article:

Hai-jiao LIN,Ji-fu ZHANG,Yun ZHANG,Ai-jun SUN,Yun-feng HU. The Effective of Additives on the Immobilization of Lipase by Microporous Absorbent Resin. China Biotechnology, 2019, 39(4): 38-51.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20190406     OR     https://manu60.magtech.com.cn/biotech/Y2019/V39/I4/38

序号 pH 温度
(℃)		 载体量
(g)		 浓度
(mmol/L)		 时间
(h)
1 5.0 20 0.5 10 1.5
2 5.5 25 1.0 15 3.0
3 6.0 30 1.5 20 4.5
4 6.5 35 2.0 25 6.0
Table 1 Orthogonal test factors and levels of the optimization of immobilization in the presence of NH4Cl
序号 pH 温度
(℃)		 载体量
(g)		 浓度
(mmol/L)		 时间
(h)
1 6.0 25 0.5 2 1.5
2 6.5 30 1.0 5 3.0
3 7.0 35 1.5 10 4.5
4 7.5 40 2.0 15 6.0
Table 2 Orthogonal test factors and levels of the optimization of immobilization in the presence of ammonium mannose
序号 pH 温度
(℃)		 载体量
(g)		 浓度
(mmol/L)		 时间
(h)
1 6.0 20 0.5 10 3.0
2 6.5 25 1.0 15 4.5
3 7.0 30 1.5 20 6.0
4 7.5 35 2.0 25 7.5
Table 3 Orthogonal test factors and evels of the optimization of immobilization in the presence of ammonium glycine
Fig.1 Screening of carriers and effects of additives on the adsorption immobilization of lipase (a)Screening of macroporous adsorption resin (b) Effect of inorganic salt additives on the immobilization of lipase (c) Effect of carbohydrate additives on the immobilization of lipase (d) Effect of amino acid additives on the immobilization of lipase
Fig.2 Optimization of lipase immobilization in the presence of NH4Cl (a) Effect of buffers on the immobilization of lipase (b) Effect of pH on the immobilization of lipase (c) Effect of temperature on the immobilization of lipase (d) Effect of the quantity of kieselguhr on the immobilization of lipase (e) Effect of NH4Cl additive concentration on the immobilization of lipase (f) Effect of immobilizing time on the immobilization of lipase
序号 pH 温度(℃) 载体量(g) 浓度(mmol/L) 时间(h) 相对酶活力(%)
1 5.0 20 0.5 10 1.5 72.62±0.97
2 5.0 25 1.0 15 3.0 76.87±0.86
3 5.0 30 1.5 20 4.5 66.82±0.73
4 5.0 35 2.0 25 6.0 55.87±0.96
5 5.5 20 1.0 20 6.0 62.74±1.40
6 5.5 25 0.5 25 4.5 91.14±0.51
7 5.5 30 2.0 10 3.0 61.77±1.03
8 5.5 35 1.5 15 1.5 44.82±1.41
9 6.0 20 1.5 25 3.0 61.55±1.34
10 6.0 25 2.0 20 1.5 55.21±0.43
11 6.0 30 0.5 15 6.0 100.00±1.28
12 6.0 35 1.0 10 4.5 66.62±0.61
13 6.5 20 2.0 15 4.5 55.36±0.63
14 6.5 25 1.5 10 6.0 65.41±1.11
15 6.5 30 1.0 25 1.5 71.53±0.43
16 6.5 35 0.5 20 3.0 91.06±1.36
K1 272.17 252.27 354.82 266.41 244.18
K2 260.48 288.63 277.75 277.05 291.25
K3 283.38 300.12 238.60 275.83 279.94
K4 283.36 258.37 228.22 280.10 284.02
R 22.90 47.85 126.60 13.68 47.07
优势水平 3 3 1 4 2
优势组合 pH6.0, 温度30℃, 载体量0.5g, 浓度25mmol/L, 时间3.0h
主次因素 载体量>温度>时间>pH>浓度
Table 4 Optimization of orthogonal experimental results and analysis of immobilization conditions through adsorption in the presence of NH4Cl
Fig.3 Optimization of lipase immobilization in the presence of mannose (a) Effect of buffers on the immobilization of lipase (b) Effect of pH on the immobilization of lipase (c) Effect of temperature on the immobilization of lipase (d) Effect of the quantity of kieselguhr on the immobilization of lipase (e) Effect of mannose additive concentration on the immobilization of lipase (f) Effect of immobilizing time on the immobilization of lipase
序号 pH 温度(℃) 载体量(g) 浓度(mmol/L) 时间(h) 相对酶活力(%)
1 6.0 25 0.5 2 1.5 80.57±1.23
2 6.0 30 1.0 5 3.0 55.17±2.50
3 6.0 35 1.5 10 4.5 71.18±1.97
4 6.0 40 2.0 15 6.0 50.25±2.13
5 6.5 25 1.0 10 6.0 82.37±1.91
6 6.5 30 0.5 15 4.5 93.74±0.49
7 6.5 35 2.0 2 3.0 44.10±1.86
8 6.5 40 1.5 5 1.5 54.56±1.79
9 7.0 25 1.5 15 3.0 61.60±1.50
10 7.0 30 2.0 10 1.5 47.96±0.50
11 7.0 35 0.5 5 6.0 100.00±0.81
12 7.0 40 1.0 2 4.5 70.73±2.32
13 7.5 25 2.0 5 4.5 46.93±1.38
14 7.5 30 1.5 2 6.0 48.55±0.79
15 7.5 35 1.0 15 1.5 65.13±0.87
16 7.5 40 0.5 10 3.0 79.81±0.50
K1 257.18 271.47 354.12 243.95 248.23
K2 274.77 245.42 273.40 256.66 240.69
K3 280.29 280.42 235.90 281.32 282.58
K4 240.42 255.34 189.24 270.73 281.16
R 39.87 35.00 164.89 37.37 41.89
优势水平 3 3 1 3 3
优势组合 pH7.0, 温度35℃, 载体量0.5g, 浓度10mmol/L, 时间4.5h
主次因素 载体量>时间>pH>浓度>温度
Table 5 Optimization of orthogonal experimental results and analysis of immobilization conditions through adsorption in the presence of mannose
Fig.4 Optimization of lipase immobilization in the presence of glycine (a) Effect of buffers on the immobilization of lipase (b) Effect of pH on the immobilization of lipase (c) Effect of temperature on the immobilization of lipase (d) Effect of the quantity of kieselguhr on the immobilization of lipase (e) Effect of glycine additive concentration on the immobilization of lipase (f) Effect of immobilizing time on the immobilization of lipase
序号 pH 温度(℃) 载体量(g) 浓度(mmol/L) 时间(h) 相对酶活力(%)
1 6.0 20 0.5 10 3.0 89.55±0.52
2 6.0 25 1.0 15 4.5 70.73±1.11
3 6.0 30 1.5 20 6.0 52.93±0.52
4 6.0 35 2.0 25 7.5 50.92±0.51
5 6.5 20 1.0 20 7.5 78.02±2.12
6 6.5 25 0.5 25 6.0 86.82±1.59
7 6.5 30 2.0 10 4.5 48.21±0.54
8 6.5 35 1.5 15 3.0 50.09±0.82
9 7.0 20 1.5 25 4.5 75.86±1.08
10 7.0 25 2.0 20 3.0 66.10±1.57
11 7.0 30 0.5 15 7.5 100.00±1.42
12 7.0 35 1.0 10 6.0 72.46±0.57
13 7.5 20 2.0 15 6.0 59.18±1.06
14 7.5 25 1.5 10 7.5 61.70±0.77
15 7.5 30 1.0 25 3.0 76.22±0.89
16 7.5 35 0.5 20 4.5 81.37±0.88
K1 264.13 302.61 357.74 271.92 281.97
K2 263.15 285.36 297.43 280 276.17
K3 314.42 277.36 240.58 278.41 271.39
K4 278.47 254.84 224.42 289.83 290.64
R 51.27 47.76 133.33 17.91 19.25
优势水平 3 1 1 4 4
优势组合 pH7.0温度20℃载体量0.5浓度25时间7.5h
主次因素 载体量>pH>温度>时间>浓度
Table 6 Optimization of orthogonal experimental results and analysis of immobilization conditions through adsorption in the presence of glycine
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