PoRgl4B（Rhamnogalacturonan lyase）

PoRgl4B

En-Rgl0339

(EC. 4.2.2.23)   Rhamnogalacturonan lyase

CAZy Family: PL4


PROPERTIES

1. ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~57 kDa)


Fig 1. Electrophoresis analysis of PoRgl4B. M, molecular weight marker (PageRuler Prestained Protein Ladder, Thermo Scientific); lane 1, culture lysate before methanol induction; lane 2, culture lysate after induction for 24 h; lane 3, culture lysate after induction for 48 h;lane 4, culture lysate after induction for 72 h;lane 5, PoRgl4B purified from Ni sepharose fast flow column.


2. SPECIFIC ACTIVITY

116.3 U/mg protein (on RG-I from Potato) at pH 5.0 and 37°C

One unit of RGL activity was defined as the amount of enzyme required to generate 1 μmol of 4,5-unsaturated galacturonic acid in 1 min.


3. RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Table 1 Substrate specificity of PoRGL4B

Substrate

Structural features

Relative activity (%)a

HG

HG

0.77±1.70

P-RG-I-PP

RG-I backbone

100±4.95

P-RG-I

RG-I

26.2±7.00

AT-RG-I

RG-I

62.14±9.33

PQ-RG-I

RG-I

5.41±1.98

PG-A

RG-I

6.48±3.25

PG-B

RG-I

12.53±5.09

PG-CP

Galactan

1.16±2.19

SA

Arabinan+RG-I backbone

9.91±4.17

a Activity toward P-RG-I-PP(potato) is taken as 100%


4. PHYSICOCHEMICAL PROPERTIES

pH Optima: 5.0

pH Stability: 4.0-7.0

Temperature Optima: 40°C

Temperature Stability: 30-50°C

Figure 2. Effect of temperature and pH on the activity and stability of recombinant PoRgl4B. (A) Optimum pH. Assays were carried out at 37°C for 5 min in buffers ranging in pH from 2.0 to 11.0. The activity at optimum pH was defifined as 100%. (B) pH stability. Assays were carried out after the incubation of the enzyme in buffers ranging from pH 2.0-11.0 at 4°C. (C) Optimum temperature. Activity was measured between 20°C and 90°C at pH 5.0 for 5 min. The activity at optimum temperature was defifined as 100%. (D) Thermostability. Assays were carried out in 20 mM HAc-NaAc buffer (pH 5.0) at 37°C for 5 min after the incubation of the enzyme at 30-70°C. The activity without heat treatment was defifined as 100%.

Figure 3. The mode of action of PoRgl4B. HPGPC analysis of the hydrolysis products of P-RG-I-PP(a) and P-RG-I(b) by PoRgl4B. MALDI-TOF-MS analysis the hydrolysis products of P-RG-I-PP(c) and P-RG-I(d) by PoRgl4B.

Figure 4. TLC analysis the hydrolysis products of AT-RG-I and P-RG-I by PoRgl4B.


5. STORAGE CONDITIONS

The enzyme should be stored at -20°C. For assay, this enzyme should be diluted in HAc-NaAc buffer (20 mM) pH 5.0. Swirl to mix the enzyme immediately prior to use.



6. REFERENCE

[1] 孟嘉仪. 草酸青霉中鼠李半乳糖醛酸酶的克隆表达及功能研究[D]. 东北师范大学, 2024.

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RGI果胶降解酶

PoRgl4B（Rhamnogalacturonan lyase）

PoRgl4B

En-Rgl0339

(EC. 4.2.2.23)   Rhamnogalacturonan lyase

CAZy Family: PL4


PROPERTIES

1. ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~57 kDa)


Fig 1. Electrophoresis analysis of PoRgl4B. M, molecular weight marker (PageRuler Prestained Protein Ladder, Thermo Scientific); lane 1, culture lysate before methanol induction; lane 2, culture lysate after induction for 24 h; lane 3, culture lysate after induction for 48 h;lane 4, culture lysate after induction for 72 h;lane 5, PoRgl4B purified from Ni sepharose fast flow column.


2. SPECIFIC ACTIVITY

116.3 U/mg protein (on RG-I from Potato) at pH 5.0 and 37°C

One unit of RGL activity was defined as the amount of enzyme required to generate 1 μmol of 4,5-unsaturated galacturonic acid in 1 min.


3. RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Table 1 Substrate specificity of PoRGL4B

Substrate

Structural features

Relative activity (%)a

HG

HG

0.77±1.70

P-RG-I-PP

RG-I backbone

100±4.95

P-RG-I

RG-I

26.2±7.00

AT-RG-I

RG-I

62.14±9.33

PQ-RG-I

RG-I

5.41±1.98

PG-A

RG-I

6.48±3.25

PG-B

RG-I

12.53±5.09

PG-CP

Galactan

1.16±2.19

SA

Arabinan+RG-I backbone

9.91±4.17

a Activity toward P-RG-I-PP(potato) is taken as 100%


4. PHYSICOCHEMICAL PROPERTIES

pH Optima: 5.0

pH Stability: 4.0-7.0

Temperature Optima: 40°C

Temperature Stability: 30-50°C

Figure 2. Effect of temperature and pH on the activity and stability of recombinant PoRgl4B. (A) Optimum pH. Assays were carried out at 37°C for 5 min in buffers ranging in pH from 2.0 to 11.0. The activity at optimum pH was defifined as 100%. (B) pH stability. Assays were carried out after the incubation of the enzyme in buffers ranging from pH 2.0-11.0 at 4°C. (C) Optimum temperature. Activity was measured between 20°C and 90°C at pH 5.0 for 5 min. The activity at optimum temperature was defifined as 100%. (D) Thermostability. Assays were carried out in 20 mM HAc-NaAc buffer (pH 5.0) at 37°C for 5 min after the incubation of the enzyme at 30-70°C. The activity without heat treatment was defifined as 100%.

Figure 3. The mode of action of PoRgl4B. HPGPC analysis of the hydrolysis products of P-RG-I-PP(a) and P-RG-I(b) by PoRgl4B. MALDI-TOF-MS analysis the hydrolysis products of P-RG-I-PP(c) and P-RG-I(d) by PoRgl4B.

Figure 4. TLC analysis the hydrolysis products of AT-RG-I and P-RG-I by PoRgl4B.


5. STORAGE CONDITIONS

The enzyme should be stored at -20°C. For assay, this enzyme should be diluted in HAc-NaAc buffer (20 mM) pH 5.0. Swirl to mix the enzyme immediately prior to use.



6. REFERENCE

[1] 孟嘉仪. 草酸青霉中鼠李半乳糖醛酸酶的克隆表达及功能研究[D]. 东北师范大学, 2024.