**3. Enhancement of nitrogen circulation by soybean cultivation and soybean protein**

### **3.1. Evaluation of nitrogen circulation in soil environment**

The nitrogen cycle is illustrated in Figure 3. Organic forms of nitrogen such as protein are de‐ graded to peptides and amino acids by soil microorganisms, and these peptides and amino acids are then converted to NH4 + . Subsequently, NH4 + is further converted to NO2 and NO3 - (nitrification). NO2 is denitrified to N2 by denitrifying bacteria and this N2 is converted to NH4 + by the nitrogen fixing bacteria, and NH4 + is accumulated in the soil environment again.

**Figure 3.** The soil nitrogen cycle

The nitrification process is the rate limiting step in the nitrogen cycle [28]. To further investi‐ gate the soil nitrogen cycle, a new method for the evaluation of nitrogen circulation activity was constructed based on bacterial number, ammonium oxidizing activity (AOA), and ni‐ trite oxidizing activity (NOA) (Figure 4) [29]. These three indices were used to construct a radar chart of nitrogen circulation in the soil. The area of the radar chart was calculated, and then the value was treated as a nitrogen circulation activity (0–100 points).

### **3.2. Enhancement of nitrogen circulation**

A database of nitrogen circulation activity was constructed using 155 agricultural soils (Fig‐ ure 5). The nitrogen circulation activity of agricultural soil ranges from 0 to 99.6 points with an average of 26 points.

A: Un-fertile soil, B: fertile soil.

**3. Enhancement of nitrogen circulation by soybean cultivation and**

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

The nitrogen cycle is illustrated in Figure 3. Organic forms of nitrogen such as protein are de‐ graded to peptides and amino acids by soil microorganisms, and these peptides and amino

The nitrification process is the rate limiting step in the nitrogen cycle [28]. To further investi‐ gate the soil nitrogen cycle, a new method for the evaluation of nitrogen circulation activity was constructed based on bacterial number, ammonium oxidizing activity (AOA), and ni‐ trite oxidizing activity (NOA) (Figure 4) [29]. These three indices were used to construct a radar chart of nitrogen circulation in the soil. The area of the radar chart was calculated, and

A database of nitrogen circulation activity was constructed using 155 agricultural soils (Fig‐ ure 5). The nitrogen circulation activity of agricultural soil ranges from 0 to 99.6 points with

then the value was treated as a nitrogen circulation activity (0–100 points).

+

is denitrified to N2 by denitrifying bacteria and this N2 is converted to NH4

is further converted to NO2

is accumulated in the soil environment again.


and NO3


+

. Subsequently, NH4

+

**3.1. Evaluation of nitrogen circulation in soil environment**

+

**soybean protein**

Relationships

52

(nitrification). NO2

**Figure 3.** The soil nitrogen cycle

an average of 26 points.

**3.2. Enhancement of nitrogen circulation**

acids are then converted to NH4


by the nitrogen fixing bacteria, and NH4

**Figure 4.** Values of nitrogen circulation activity in soil environments

**Figure 5.** Database of nitrogen circulation activity in 155 agricultural soils

Soybean cultivation leads to nitrogen accumulation in the soil environment, and therefore nitrogen circulation activity should be enhanced by soybean cultivation. This enhancement was further analyzed (Figure 6) and activity was shown to be enhanced 26 to 95 points after soybean cultivation.

Soybean waste is also rich in nitrogen (Table 1), and is often used as an organic fertilizer. Soil nitrogen is increased by using soybean waste as fertilizer, and consequently nitrogen circulation is increased. Soybean waste is also rich in carbon (C/N values; 5.1), and therefore soil bacteria and bacterial activity may also be increased by the addition of soybean waste.

**Figure 6.** Effect of soybean cultivation on nitrogen circulation activity in soil


**Table 1.** Components of soybean meal

Soybean cultivation leads to nitrogen accumulation in the soil environment, and therefore nitrogen circulation activity should be enhanced by soybean cultivation. This enhancement was further analyzed (Figure 6) and activity was shown to be enhanced 26 to 95 points after

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

Soybean waste is also rich in nitrogen (Table 1), and is often used as an organic fertilizer. Soil nitrogen is increased by using soybean waste as fertilizer, and consequently nitrogen circulation is increased. Soybean waste is also rich in carbon (C/N values; 5.1), and therefore soil bacteria and bacterial activity may also be increased by the addition of soybean waste.

**Figure 6.** Effect of soybean cultivation on nitrogen circulation activity in soil

soybean cultivation.

Relationships

54

## **4. Bioactive peptides from soybean protein**

#### **4.1. Plant growth promoting peptides from soybean waste**

For efficient use of soybean waste, it is treated with an alkaline protease from *Bacillus circu‐ lans* HA12 (degraded soybean meal products; DSP) [4, 6]. Plant growth promotion by DSP has been investigated using various plant species [30]. The fresh weight of *Brassica rapa* was shown to be increased by 25% through the addition of DSP (12 mg-peptides/kg-soil) (Figure 7). The growth of *Solanum tuberosum* L., *Solanum lycopersicum*, and *Brassica juncea* were also promoted by addition of DSP. Moreover, DSP also produced thicker roots than a chemical fertilizer, indicating that DSP contains bioactive peptides for plant growth.

**Figure 7.** Plant growth-promoting effect of DSP, A: Chemical fertilizer, B; DSP.

#### **4.2. Root hair promoting peptide in DSP**

The number of root hairs in *B. rapa* was increased and each was elongated when DSP (30 µg/ml) was added (Figure 8) to the soil. In order to analyze the root hair promoting effect by DSP, the structure of the root hair promoting peptide (RHPP) in DSP was investigated [6]. Degraded products of Kunitz trypsin inhibitor (KTI) in soybean protein showed higher root hair promoting activity, and the RHPP was purified by several chromatographic steps from degraded products of KTI.

**Figure 8.** Root hair promoting effect of DSP, A: Root of *Brassica rapa* grown in plant growth medium, B: root of *B. rapa* grown with DSP in plant growth medium. Bar denotes 1 mm.

The molecular mass of RHPP was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [6]. The molecular weight of the bioac‐ tive peptide was 1,198.2 Da (Figure 9), and the molecular weight of the amino acid sequence in KTI was searched. Positions 27–38 in KTI (Gly-Gly-Ile-Arg-Ala-Ala-Pro-Thr-Gly-Asn-Glu-Arg) were identical to this molecular weight, and this peptide was thus designated as the RHPP (Figure 9). The RHPP that was chemically synthesized was also shown to have root hair promoting activity (data not shown).

**Figure 9.** Amino acid sequence of RHPP in Kunitz trypsin inhibitor, the RHPP amino acid sequence is shown by gray box.

## **5. Novel plant bioactive peptides from other legume**

Many other legumes form root-nodules with nitrogen fixing bacteria. The nitrogen fixing bacteria related to legume cultivation are classified into 13 genera (*Rhizobium*, *Ensifer*, *Meso‐ rhizobium*, *Bradyrhizobium*, *Methylobacterium*, *Azorhizobium*, *Devosia*, *Burkholderia*, *Phyllobacte‐ rium, Microvirga, Ochrobactrum, Cupriavidus,* and *Shinella*) and 98 species [31].

Legumes such as *Astragalus sinicus*, *Trifolium repens*, and *Arachis hypogaea* are cultivated as green manure for the improvement of soil fertility. The host specificity of the nitrogen fixing bacteria, *M. huakuii*, *R. trifolii*, and *Bradyrhizobium* sp., are very high, infecting *A. sinicus*, *T.* *repens*, and *A. hypogaea*, respectively [32]. These legumes are rich in proteins and form rootnodules via the same mechanisms as soybean.

In order to find novel bioactive peptides, attempts to degrade protein biomass from *A. hypo‐ gaea* by various proteases (thermolysin, subtilisin, proteinaseK, and trypsin) were made. Bio‐ activities of root hair and lateral root formation were found by degradation with proteinaseK (Figure 10). Degraded products of *A. hypogaea* by proteinase K (30 µg/ml) showed strong root hair promoting activity at the same level as DSP. Moreover, degraded products of *A. hypogaea* promoted lateral root growth in *B. rapa*, suggesting that degradation of legume proteins has a possibility to produce new bioactive peptides.

**Figure 10.** Bioactive effect of degraded products of *A. hypogaea* on root of *B. rapa*, A: Root of *Brassica rapa* grown in plant growth medium, B: root of *B. rapa* grown with degraded products of *A. hypogaea*. Bar denotes 1 mm.
